Amal O. Amer

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Missense mutations in the CIAS1 gene cause three autoinflammatory disorders: familial cold autoinflammatory syndrome, Muckle-Wells syndrome and neonatal-onset multiple-system inflammatory disease. Cryopyrin (also called Nalp3), the product of CIAS1, is a member of the NOD-LRR protein family that has been linked to the activation of intracellular host defence signalling pathways. Cryopyrin forms a multi-protein complex termed 'the inflammasome', which contains the apoptosis-associated speck-like protein (ASC) and caspase-1, and promotes caspase-1 activation and processing of pro-interleukin (IL)-1beta (ref. 4). Here we show the effect of cryopyrin deficiency on inflammasome function and immune responses. Cryopyrin and ASC are essential for caspase-1 activation and IL-1beta and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of tumour-necrosis factor-alpha and IL-6, as well as activation of NF-kappaB and mitogen-activated protein kinases (MAPKs) were unaffected by cryopyrin deficiency. Furthermore, we show that Toll-like receptors and cryopyrin control the secretion of IL-1beta and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes.

Viral infection induces the production of interleukin (IL)-1β and IL-18 in macrophages through the activation of caspase-1, but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. In this report, we have identified a signaling pathway leading to caspase-1 activation that is induced by double-stranded RNA (dsRNA) and viral infection that is mediated by Cryopyrin/Nalp3. Stimulation of macrophages with dsRNA, viral RNA, or its analog poly(I:C) induced the secretion of IL-1β and IL-18 in a cryopyrin-dependent manner. Consistently, caspase-1 activation triggered by poly(I:C), dsRNA, and viral RNA was abrogated in macrophages lacking cryopyrin or the adaptor ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) but proceeded normally in macrophages deficient in Toll-like receptor 3 or 7. We have also shown that infection with Sendai and influenza viruses activates the cryopyrin inflammasome. Finally, cryopyrin was required for IL-1β production in response to poly(I:C) in vivo. These results identify a mechanism mediated by cryopyrin and ASC that links dsRNA and viral infection to caspase-1 activation resulting in IL-1β and IL-18 production. Viral infection induces the production of interleukin (IL)-1β and IL-18 in macrophages through the activation of caspase-1, but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. In this report, we have identified a signaling pathway leading to caspase-1 activation that is induced by double-stranded RNA (dsRNA) and viral infection that is mediated by Cryopyrin/Nalp3. Stimulation of macrophages with dsRNA, viral RNA, or its analog poly(I:C) induced the secretion of IL-1β and IL-18 in a cryopyrin-dependent manner. Consistently, caspase-1 activation triggered by poly(I:C), dsRNA, and viral RNA was abrogated in macrophages lacking cryopyrin or the adaptor ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) but proceeded normally in macrophages deficient in Toll-like receptor 3 or 7. We have also shown that infection with Sendai and influenza viruses activates the cryopyrin inflammasome. Finally, cryopyrin was required for IL-1β production in response to poly(I:C) in vivo. These results identify a mechanism mediated by cryopyrin and ASC that links dsRNA and viral infection to caspase-1 activation resulting in IL-1β and IL-18 production. Innate immunity is the initial line of host defense against microbial pathogens, including viral infection. The early recognition of viruses by the host initiates signaling pathways leading to the induction of anti-viral responses, including the secretion of type I interferons (IFNs) 5The abbreviations used are: IFN, interferon; NLR, NOD-like receptor; TLR, Toll-like receptor; NF, nuclear factor; TNF, tumor necrosis factor; ssRNA, single-stranded RNA; dsRNA, double-stranded RNA; IL, interleukin; IKK, IkB kinase; FHV, flock house virus; ERK, extracellular signal-regulated kinase; ELISA, enzyme-linked immunosorbent assay; WT, wild-type; MAPK, mitogen-activated protein kinase. α and β and pro-inflammatory cytokines (1Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2715) Google Scholar, 3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1434) Google Scholar). Recognition of pathogens including viruses by the host immune system relies on the detection of conserved molecular structures that are shared by large pathogens (pathogen-associated molecular patterns or PAMPs). In the case of viruses, genomic DNA and single-stranded RNA (ssRNA) or double-stranded RNA (dsRNA) produced during viral replication are sensed by host cells to induce anti-viral responses (3Kawai T. Akira S. Nat. Immunol. 2006; 7: 131-137Crossref PubMed Scopus (1434) Google Scholar). Viral DNA, ssRNA, and dsRNA are recognized by a subfamily of Toll-like receptors (TLRs 3, 7, 8, and 9) in endosomes after the endocytosis of viral particles in mammalian cells (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar, 9Krug A. French A.R. Barchet W. Fischer J.A. Dzionek A. Pingel J.T. Orihuela M.M. Akira S. Yokoyama W.M. Colonna M. Immunity. 2004; 21: 107-119Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). Upon activation, these TLRs recruit the adaptor proteins MyD88 or TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing interferon-β) to activate the IkB kinase (IKK) complex and the IKK-related kinases TBK1 and IKKϵ (1Takeda K. Akira S. Int. Immunol. 2005; 17: 1-14Crossref PubMed Scopus (2715) Google Scholar, 10Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar). Viral dsRNA is recognized by TLR3 and the two cytosolic RNA helicases, the retinoic acid inducible gene I (RIG-I), and MDA5 (which induces activation of IKK and TBK1/IKKϵ) (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar, 11Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar, 12Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3122) Google Scholar). Activation of the IKK complex that includes the catalytic subunits IKKα and IKKβ as well as the regulatory subunit IKKγ/NEMO mediates NFκB activation, whereas that of TBK1 and IKKϵ induces phosphorylation and activation of IRF3 or IRF7 (10Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar, 13Ishitani T. Takaesu G. Ninomiya-Tsuji J. Shibuya H. Gaynor R.B. Matsumoto K. EMBO J. 2003; 22: 6277-6288Crossref PubMed Scopus (219) Google Scholar, 14Jin G. Klika A. Callahan M. Faga B. Danzig J. Jiang Z. Li X. Stark G.R. Harrington J. Sherf B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 2028-2033Crossref PubMed Scopus (70) Google Scholar). The nuclear translocation of NFκB and IRF3/IRF7 mediates the transcriptional activation of interferon and cytokine genes that limit viral replication and promote adaptive immune responses (15McWhirter S.M. Fitzgerald K.A. Rosains J. Rowe D.C. Golenbock D.T. Maniatis T. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 233-238Crossref PubMed Scopus (451) Google Scholar, 17Sharma S. tenOever B.R. Grandvaux N. Zhou G.P. Lin R. Hiscott J. Science. 2003; 300: 1148-1151Crossref PubMed Scopus (1366) Google Scholar). Signaling pathways other than NFκB or IFN that are activated upon viral recognition and mediate anti-viral responses are poorly understood. Previous studies have shown that infection of macrophages with certain viruses including influenza A and Sendai virus induce interleukin (IL)-1β and IL-18 secretion (18Pirhonen J. Sareneva T. Kurimoto M. Julkunen I. Matikainen S. J. Immunol. 1999; 162: 7322-7329PubMed Google Scholar, 19Julkunen I. Sareneva T. Pirhonen J. Ronni T. Melen K. Matikainen S. Cytokine Growth Factor Rev. 2001; 12: 171-180Crossref PubMed Scopus (287) Google Scholar), but the mechanism by which host cells sense viruses to induce caspase-1 activation is unknown. Both IL-1β and IL-18 are synthesized as inactive cytoplasmic precursors that are processed into biologically active mature forms in response to various pro-inflammatory stimuli, including viruses by caspase-1, a cysteine protease (18Pirhonen J. Sareneva T. Kurimoto M. Julkunen I. Matikainen S. J. Immunol. 1999; 162: 7322-7329PubMed Google Scholar, 20Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4233) Google Scholar, 21Pirhonen J. Sareneva T. Julkunen I. Matikainen S. Eur. J. Immunol. 2001; 31: 726-733Crossref PubMed Scopus (65) Google Scholar). Caspase-1 is synthesized as an inactive zymogen that becomes activated by cleavage at aspartic residues to generate an enzymatically active heterodimer composed of a 10- and 20-kDa chain (20Martinon F. Burns K. Tschopp J. Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (4233) Google Scholar). Recent studies have implicated members of the NOD-like receptor (NLR) family of proteins (also called NOD-LRR (nucleotide binding oligomerization domain-(leucine-rich repeat) or CATERPILLER) in the regulation of caspase-1 activation in response to microbial pathogens (22Inohara N. Chamaillard M. McDonald C. Núñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (812) Google Scholar, 23Ting J.P. Davis B.K. Annu. Rev. Immunol. 2005; 23: 387-414Crossref PubMed Scopus (299) Google Scholar). The NLR family is composed of 23 cytosolic proteins, including Nod1, Nod2, Cryopyrin/Nalp3, and Ipaf. The structure of NLRs include an amino-terminal effector binding region that consists of protein-protein interaction domains, such as caspase-recruitment domains or pyrin (a central nucleotide binding oligomerization domain that acts to oligomerize these proteins) and carboxyl-terminal leucine-rich repeats that are required to detect specific PAMPs (22Inohara N. Chamaillard M. McDonald C. Núñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (812) Google Scholar). Cryopyrin forms an endogenous multiprotein complex containing ASC (apoptosis-associated speck-like protein containing a caspase-activating and recruitment domain) and caspase-1 dubbed “the inflammasome,” which promotes caspase activation and processing of pro-IL-1β (24Martinon F. Agostini L. Meylan E. Tschopp J. Curr. Biol. 2004; 14: 1929-1934Abstract Full Text Full Text PDF PubMed Scopus (465) Google Scholar, 25Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar). Notably, missense mutations in the CIAS1 gene that encodes Cryopyrin cause three autoinflammatory disorders characterized by deregulated production of IL-1β (26Stojanov S. Kastner D.L. Curr. Opin. Rheumatol. 2005; 17: 586-599Crossref PubMed Scopus (308) Google Scholar). Cryopyrin senses bacterial RNA, synthetic anti-viral purine analogs, and monosodium urate or calcium pyrophosphate dehydrate crystals (27Kanneganti T.D. Ozoren N. Body-Malapel M. Amer A. Park J.H. Franchi L. Whitfield J. Barchet W. Colonna M. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Akira S. Núñez G. Nature. 2006; 440: 233-236Crossref PubMed Scopus (902) Google Scholar, 28Martinon F. Petrilli V. Mayor A. Tardivel A. Tschopp J. Nature. 2006; 440: 237-241Crossref PubMed Scopus (3790) Google Scholar). In addition, other results indicate that Cryopyrin regulates caspase-1 activation in response to factors that induce intracellular K+ efflux, such as certain toxins and high concentrations of extracellular ATP (29Mariathasan S. Weiss D.S. Newton K. McBride J. O'Rourke K. Roose-Girma M. Lee W.P. Weinrauch Y. Monack D.M. Dixit V.M. Nature. 2006; 440: 228-232Crossref PubMed Scopus (2348) Google Scholar, 30Sutterwala F.S. Ogura Y. Szczepanik M. Lara-Tejero M. Lichtenberger G.S. Grant E.P. Bertin J. Coyle A.J. Galan J.E. Askenase P.W. Flavell R.A. Immunity. 2006; 24: 317-327Abstract Full Text Full Text PDF PubMed Scopus (784) Google Scholar). The genome of poxviruses encode Pyrin-containing proteins that interact with components of the inflammasome and inhibit caspase-1 activation and the processing of IL-1β and IL-18 induced by diverse stimuli (31Johnston J.B. Barrett J.W. Nazarian S.H. Goodwin M. Ricuttio D. Wang G. McFadden G. Immunity. 2005; 23: 587-598Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). These results suggest that certain viruses target components of caspase-1 activation pathways to circumvent host anti-viral responses. However, the signaling pathways that link viral infection to caspase-1 activation and IL-1β/IL-18 are unknown. Here we show that viral dsRNA and its analog poly(I:C) as well as viral infection activate caspase-1 through cryopyrin, resulting in the production of active IL-1β and IL-18. The signaling pathway stimulated by dsRNA and cryopyrin was independent of TLR3 or TLR7. These results identify a novel TLR-independent signaling pathway that is mediated by cryopyrin and ASC and leads to the secretion of pro-inflammatory cytokines in response to viral infection. Mice—Cryopyrin, ASC, and TLR7 knock-out mice have been described previously (27Kanneganti T.D. Ozoren N. Body-Malapel M. Amer A. Park J.H. Franchi L. Whitfield J. Barchet W. Colonna M. Vandenabeele P. Bertin J. Coyle A. Grant E.P. Akira S. Núñez G. Nature. 2006; 440: 233-236Crossref PubMed Scopus (902) Google Scholar, 32Ozoren N. Masumoto J. Franchi L. Kanneganti T.D. Body-Malapel M. Erturk I. Jagirdar R. Zhu L. Inohara N. Bertin J. Coyle A. Grant E.P. Núñez G. J. Immunol. 2006; 176: 4337-4342Crossref PubMed Scopus (156) Google Scholar, 33Hemmi H. Kaisho T. Takeuchi O. Sato S. Sanjo H. Hoshino K. Horiuchi T. Tomizawa H. Takeda K. Akira S. Nat. Immunol. 2002; 3: 196-200Crossref PubMed Scopus (2072) Google Scholar). TLR3 knock-out mice (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar) were obtained from The Jackson Laboratory. Macrophages—Bone marrow was prepared from the leg bones of 5–20-week-old mice. The legs were dissected and the bone marrow flushed out. Bone marrow cells were cultured with Iscove's modified Dulbecco's medium supplemented with 30% L929 supernatant containing macrophage-stimulating factor, glutamine, sodium pyruvate, 10% heat-inactivated fetal bovine serum (Invitrogen), 50 μg/ml penicillin, and 50 μg/ml streptomycin at 37 °C in 5% CO2 for 5 days (bone marrow differentiation medium). Bone marrow macrophages were then harvested with rubber scrapers and seeded. Peritoneal macrophages were elicited to the peritoneum of mice and isolated 4 days after the injection of 4% thioglycolate broth. After 1 day, non-adherent cells were removed, and the remaining macrophages were incubated in Iscove's modified Dulbecco's medium supplemented with 10% heat-inactivated fetal bovine serum, 50 μg/ml penicillin, and 50 μg/ml streptomycin. In Vitro RNA Transcription, Viruses, and Viral RNA—The dsRNA for hsp90 and LacZ were made according to the protocol described in Ref. 34Kampmueller K.M. Miller D.J. J. Virol. 2005; 79: 6827-6837Crossref PubMed Scopus (80) Google Scholar. To prepare flock house virus (FHV) dsRNA, the plasmids pFHV2(0,0) and p2VHF(2,0) (35Ball L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12443-12447Crossref PubMed Scopus (49) Google Scholar) were used to generate viral (+) and (–) ssRNAs, respectively. Plasmids were linearized with RsrII, in vitro transcripts were synthesized using T7 polymerase and an Ambion Megascript kit per the manufacturer's instructions, and RNA was purified by phenol-chloroform extraction and ethanol precipitation and quantitated by spectrophotometry. Equal amounts of (+) and (–) RNA transcripts were mixed in RNase-free water, heated to 75 °C for 30 min, and allowed to cool gradually at room temperature to form dsRNA. Viral RNA integrity and formation of dsRNA were assessed by non-denaturing agarose gel electrophoresis. Murine Sendai virus (strain Cantell) was purchased from the American Type Culture Collection and influenza A/Puerto Rico/8/34 virus (H1N1) was a gift from James R. Becker, Jr. (University of Michigan). Rotavirus (strain SA11–4F) dsRNA was isolated by phenol-chloroform extraction from virus grown in monkey kidney (MA014) cells and purified by CsCl centrifugation (36Patton J.T. Wentz M. Xiaobo J. Ramig R.F. J. Virol. 1996; 70: 3961-3971Crossref PubMed Google Scholar). Microbial Ligands and Antibodies—Pam2CGDPKHPKSF (FSL-1), Pam3CSK4, poly(I:C), and CpG oligonucleotide (5′-TCCATGACGTTCCTGACGTT-3′) were purchased from Invivogen. poly(A), (C), (G), and (U) ssRNA and poly(I)·poly(C) dsRNA were from Sigma. Purified total RNA from Escherichia coli was purchased from Ambion. Bacillus anthracis protective antigen and lethal factor were obtained from List Biologicals Laboratories and were used at 1 μg/ml concentration. Aliquots of RNA samples were incubated with RNases (Ambion) as suggested by the manufacturer. Rabbit anti-mouse caspase-1 was a generous gift of P. Vandenabeele (Ghent University, Ghent, Belgium). The antibodies for mouse IκBα, phospho-IκBα, p38, phospho-p38, ERK, and phospho-ERK were from Cell Signaling Technologies. Immunoblotting—Cells were washed twice with phosphate-buffered saline and scraped in lysis buffer solution (150 mm NaCl, 10 mm Tris, pH 7.4, 5 mm EDTA, 1 mm EGTA, 0.1% Nonidet P-40) supplemented with 1× protease inhibitor mixture (Roche Diagnostics). For analysis of caspase-1 activation, macrophages were cultured with stimuli for 1–3 h and then with medium containing 5 mm ATP (Sigma) for 30 min. Extracts were prepared from cells and culture supernatants by adding lysis buffer containing 1% Nonidet P-40 supplemented with complete protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany) and 2 mm dithiothreitol. Samples were clarified, denatured with SDS buffer, and boiled for 5 min, separated by SDS-PAGE, and transferred to nitrocellulose membranes. The membranes were immunoblotted with primary antibodies and proteins detected with appropriate secondary anti-rabbit antibody conjugated to horseradish peroxidase followed by enhanced chemiluminescence. Measurements of Cytokines— Macrophages were stimulated with various microbial and synthetic ligands for 24 h and the supernatants were analyzed for IL-1β, IL-18, TNFα, and IL-6 secretion. The ligand concentrations used were FLS-1 and Pam3CSK4 (at 1μg/ml) and poly(I:C) (at 2.5 μg/ml). Mouse cytokines were measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN). Cryopyrin Is Required for IL-1β and IL-18 Secretion in Response to the dsRNA Analog Poly(I:C)—To assess a role for cryopyrin in immune responses induced by viruses, macrophages from wild-type (WT) and cryopyrin-deficient mice were stimulated with polyinosinic-polycytidylic acid (poly(I:C), a synthetic dsRNA analog that mimics viral infection and a synthetic diacylated lipopeptide (FSL-1, TLR2 agonist) or E. coli RNA as controls. Incubation of wild-type macrophages with poly(I:C) induced IL-1β, but this response was abolished in cryopyrin-null macrophages (Fig. 1, A and B). Consistent with previous results, production of IL-1β in response to E. coli RNA (but not FSL-1) was deficient in cryopyrinnull macrophages (Fig. 1A). Additionally, cryopyrin was required for IL-18 induced by poly(I:C), but dispensable for IFNα production (Fig. 1, C and D). These results indicate that cryopyrin is specifically required for IL-1β/IL-18 secretion induced by poly(I:C). Cryopyrin-dependent IL-1β Secretion by Poly(I:C) Is Independent of NFκB and MAPK Activation—Stimulation of macrophages with poly(I:C) induces the secretion of several pro-inflammatory cytokines (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar). We found that cryopyrin was dispensable for the production of TNFα and IL-6 induced by poly(I:C) (Fig. 2, A and B). In contrast, production of TNFα and IL-6 required TLR3 (Fig. 2, C and D), as previously reported (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar). The induction of IL-1β secretion is thought to involve the up-regulation of pro-IL-1β through transcriptional mechanisms via NFκB and then a second stimulus that leads to the activation of caspase-1, processing of pro-IL-1β, and release of mature IL-1β (37Perregaux D.G. McNiff P. Laliberte R. Conklyn M. Gabel C.A. J. Immunol. 2000; 165: 4615-4623Crossref PubMed Scopus (212) Google Scholar, 38Dinarello C.A. Ann. N. Y. Acad. Sci. 1998; 856: 1-11Crossref PubMed Scopus (422) Google Scholar). Stimulation with poly(I:C) induced comparable levels of NFκB, ERK, and p38 activation in WT and cryopyrin–/– macrophages (Fig. 2E). By contrast, the activation of NFκB and MAPKs was abolished in TLR3-deficient macrophages (Fig. 2F). These results demonstrate that cryopyrin-mediated IL-1β secretion induced by poly(I:C) is independent of NFκB and MAPK as well as TLR3. Cryopyrin (but Not Nod2/TLR3/TLR7) Is Essential for Activation of Caspase-1 in Response to Poly(I:C)— Proteolytic activation of procaspase-1 is a critical step in the induction of IL-1β secretion (39Cerretti D.P. Kozlosky C.J. Mosley B. Nelson N. Van Ness K. Greenstreet T.A. March C.J. Kronheim S.R. Druck T. Cannizzaro L.A. Huebner K. Black R.A. Science. 1992; 256: 97-100Crossref PubMed Scopus (999) Google Scholar, 40Thornberry N.A. Bull H.G. Calaycay J.R. Chapman K.T. Howard A.D. Kostura M.J. Miller D.K. Molineaux S.M. Weidner J.R. Aunins J. Elliston K.O. Ayala J.M. Casano F.J. Chin J. Ding G.J.F. Egger L.A. Gaffney E.P. Limjuco G. Palyha O.C. Raju S.M. Rolando A.M. Salley J.P. Yamin T.T. Lee T.D. Shively J.E. MacCross M. Mumford R.A. Schmidt J.A. Tocci M.J. Nature. 1992; 356: 768-774Crossref PubMed Scopus (2210) Google Scholar). Importantly, processing of pro-caspase-1 was induced rapidly (by 1 h) and in a dose-dependent manner after stimulation of WT macrophages with poly(I:C), as determined by the detection of the mature 20-kDa subunit of caspase-1 (Fig. 3, A and B). Such activation of caspase-1 was abrogated in macrophages lacking cryopyrin (Fig. 3, A and B) or ASC (Fig. 3C), an adaptor that links cryopyrin to caspase-1 (25Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar, 41Dowds T.A. Masumoto J. Zhu L. Inohara N. Núñez G. J. Biol. Chem. 2004; 279: 21924-21928Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). In contrast, activation of caspase-1 induced by poly(I:C) was unimpaired in Nod2- and TLR3- or TLR7-deficient macrophages (Fig. 3, D–F). These results demonstrate that cryopyrin is essential for caspase-1 processing in response to poly(I:C). Furthermore, TLR3 is required for NFκB and MAPK activation but dispensable for caspase-1 activation. Cryopyrin-dependent IL-1β and IL-18 Secretion and Caspase-1 Activation by Poly(I:C) Requires dsRNA Structure—We next tested several ssRNA and dsDNA compounds to determine the structural requirements for IL-1β and IL-18 secretion and caspase-1 activation induced by poly(I:C). Importantly, the synthetic ssRNA analogs (polycytidylic acid (poly(C), polyuridylic acid (poly(U), or polyinosinic (poly(I)) neither induce IL-1β and IL-18 secretion (Fig. 4, A and B) nor caspase-1 activation (Fig. 4C). Moreover, the dsDNA analogs polydeoxyinosinic-deoxycytidylic acid (poly(dI: dC)) and polydeoxyinosinic-deoxcytidylic acid (poly(dG:dC)) did not have any effect on IL-1β secretion or caspase-1 activation (Fig. 4, A–C). To further verify these results, we treated poly(I:C) with a panel of RNases that cleave ssRNA, dsRNA, or both. Digestion of poly(I:C) with RNase A and RNase T1 that are specific for ssRNA had little or no effect on caspase-1 activation induced by poly(I:C) (Fig. 4D). In contrast, treatment of poly(I:C) with RNase V1 that cleaves dsRNA or with benzonase that digests both ssRNA and dsRNA abolished its ability to induce processing of caspase-1 (Fig. 4D). Together, these results indicate that the dsRNA structure of poly(I:C) is essential for cryopyrin-dependent induction of IL-1β secretion and caspase-1 activation in macrophages. Viral and Non-viral dsRNA Produced in Vitro Induce Cryopyrin-dependent Caspase-1 Activation— Poly(I:C) does not represent all viral and non-viral dsRNA. To assess whether in vitro transcribed dsRNAs induce caspase-1 activation, we produced ∼700 bp dsRNA fragments from the 5′ coding region of the lacZ and Drosophila Hsp83 genes using the in vitro transcription system (34Kampmueller K.M. Miller D.J. J. Virol. 2005; 79: 6827-6837Crossref PubMed Scopus (80) Google Scholar, 35Ball L.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12443-12447Crossref PubMed Scopus (49) Google Scholar). In addition, complementary plus sense (+) and minus sense (–) ssRNAs of FHV were produced by in vitro transcription and annealed to form viral dsRNA. Stimulation of macrophages with FHV dsRNA as well as non-viral dsRNAs induced caspase-1 activation as effectively as the poly(I:C) that was used as a control (Fig. 5A). As expected, neither the (+) ssRNA nor the (–) ssRNA used in preparing FHV dsRNA was able to induce processing of caspase-1 (Fig. 5B). As observed with poly(I:C), digestion of FHV dsRNA with RNases that digest dsRNA (but not ssRNA) abolished their ability to induce caspase-1 activation (Fig. 5, C and D). Additionally, treatment of dsRNAs with E. coli RNase III, an endoribonuclease that cleaves dsRNA into 10–18-bp dsRNA fragments, had no effect on their ability to induce caspase-1 activation (Fig. 5E). Cryopyrin/ASC-dependent Activation of Caspase-1 by Naturally Produced Viral dsRNA—We next tested the ability of genomic dsRNA purified from rotavirus grown in monkey kidney cells to induce caspase-1 activation. Incubation of macrophages with naturally produced rotavirus dsRNA induced caspase-1 activation in WT but not cryopyrin-deficient cells (Fig. 6A). Similarly, cryopyrin was required for the activation of caspase-1 induced by viral dsRNA purified from plant cells infected with brome mosaic virus as well as by in vitro produced brome mosaic viral dsRNA. (Fig. 6B). In contrast, caspase-1 activation triggered by the lethal toxin of B. anthracis, consisting of the protective antigen and lethal factor that depends on Nalp1b (42Boyden E.D. Dietrich W.F. Nat. Genet. 2006; 38: 240-244Crossref PubMed Scopus (655) Google Scholar), proceeded normally in the absence of cryopyrin (Fig. 6A). Consistent with the results obtained with poly(I:C), activation of caspase-1 induced by rotavirus dsRNA required ASC but not TLR3 or TLR7 (Fig. 6, D–F). Furthermore, treatment of naturally produced rotavirus and dsRNA with RNase V1 (but not RNase III) abolished their ability to induce caspase-1 activation (Fig. 6G). Infection of Macrophages with Sendai and Influenza Viruses Induce Cryopyrin-dependent Caspase-1 Activation—We next tested the ability of Sendai and influenza A virus, two viruses known to stimulate IL-1β secretion in human macrophages (18Pirhonen J. Sareneva T. Kurimoto M. Julkunen I. Matikainen S. J. Immunol. 1999; 162: 7322-7329PubMed Google Scholar, 21Pirhonen J. Sareneva T. Julkunen I. Matikainen S. Eur. J. Immunol. 2001; 31: 726-733Crossref PubMed Scopus (65) Google Scholar), to induce caspase-1 activation in WT and cryopyrin-deficient macrophages. Macrophages were infected with Sendai or influenza A virus at a low multiplicity of infection, and cell extracts were examined for pro-caspase-1 processing by immunoblotting. The analysis revealed that both viruses induced caspase-1 activation in WT but not cryopyrin-deficient macrophages (Fig. 7A). As it was observed with viral dsRNA, activation of caspase-1 triggered by viral infection was independent of TLR3 and TLR7 (Fig. 7B). Cryopyrin Is Required for Production of IL-1β and IL-18 after Administration of Poly(I:C) in Vivo—We next examined whether cryopyrin plays a role in the production of pro-inflammatory cytokines in the animal. Intraperitoneal administration of poly(I:C) induced the production of IL-1β, IL-18, TNFα, and IL-6 in the serum of wild-type mice. Similarly, the bacterial lipopeptide Pam3CSK4 (TLR2 agonist) also induced the production of IL-1β, TNFα, and IL-6 in the serum of wild-type mice. (Fig. 8). In contrast, the serum levels of IL-1β, IL-18, and to lesser extent IL-6 were greatly reduced, whereas those of TNFα were unimpaired in cryopyrin knock-out mice after injection with poly(I:C) (Fig. 8, A–D and G, and supplemental Fig. 1). The reduced levels of IL-6 detected in the serum of cryopyrinnull mice after stimulation with poly(I:C) is presumably due to the induction of IL-6 by IL-1β in vivo (43Netea M.G. Kullberg B.J. Verschueren I. Van Der Meer J.W. Eur. J. Immunol. 2000; 30: 3057-3060Crossref PubMed Scopus (102) Google Scholar). Notably, the production of pro-inflammatory cytokines including IL-1β was unimpaired in cryopyrin-deficient mice after intraperitoneal administration of Pam3CSK4, demonstrating the specific role of cryopyrin in regulating poly(I:C) responses in vivo. Infection of monocytes and macrophages with a variety of viruses is known to induce the secretion of IL-1β and IL-18 through the activation of caspase-1, but the molecular mechanisms involved have remained largely unknown. These studies demonstrate that cryopyrin plays an essential role in the secretion of IL-1β and IL-18 by sensing viral dsRNA and inducing the activation of caspase-1. Viral dsRNA also triggers the production of type I IFNs, TNFα, and IL-6 through TLR3 and the RIG-1 stimulation (4Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4936) Google Scholar, 11Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1110) Google Scholar), but these immune responses were independent of cryopyrin. The latter responses are mediated through the activation of the transcriptional factors IRF3/IRF7 and NFκB (16Sato M. Suemori H. Hata N. Asagiri M. Ogasawara K. Nakao K. Nakaya T. Katsuki M. Noguchi S. Tanaka N. Taniguchi T. Immunity. 2000; 13: 539-548Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 44Takaoka A. Taniguchi T. Cancer Sci. 2003; 94: 405-411Crossref PubMed Scopus (66) Google Scholar). Thus, dsDNA induces at least three distinct defense-signaling pathways in host cells to limit viral infection. IL-1β and IL-18 are induced by a variety of microbial stimuli through the activation of caspase-1. IL-1β is considered to be a master cytokine in that it mediates several innate and adaptive immune responses directly or through the induction of other cytokines such as IL-6 (43Netea M.G. Kullberg B.J. Verschueren I. Van Der Meer J.W. Eur. J. Immunol. 2000; 30: 3057-3060Crossref PubMed Scopus (102) Google Scholar). In addition, IL-1β is a potent pyrogen that is involved in the development of fever in response to pathogen infection (45Dinarello C.A. Wolff S.M. N. Engl. J. Med. 1993; 328: 106-113Crossref PubMed Scopus (949) Google Scholar). However, there is little evidence for a role of IL-1β in host defense against viral infection. In contrast, IL-18, a cytokine that stimulates nature killer cells and CD8+ T cells and is potently synergistic with IL-12 for this function (46Micallef M.J. Ohtsuki T. Kohno K. Tanabe F. Ushio S. Namba M. Tanimoto T. Torigoe K. Fujii M. Ikeda M. Fukuda S. Kurimoto M. Eur. J. Immunol. 1996; 26: 1647-1651Crossref PubMed Scopus (560) Google Scholar), is known to play an important role in viral clearance (47Liu B. Mori I. Hossain M.J. Dong L. Takeda K. Kimura Y. J. Gen. Virol. 2004; 85: 423-428Crossref PubMed Scopus (116) Google Scholar, 48Gherardi M.M. Ramirez J.C. Esteban M. J. Gen. Virol. 2003; 84: 1961-1972Crossref PubMed Scopus (59) Google Scholar). Furthermore, administration of IL-18 can protect the host against infection with herpes simplex and vaccinia virus (49Fujioka N. Akazawa R. Ohashi K. Fujii M. Ikeda M. Kurimoto M. J. Virol. 1999; 73: 2401-2409Crossref PubMed Google Scholar, 50Tanaka-Kataoka M. Kunikata T. Takayama S. Iwaki K. Ohashi K. Ikeda M. Kurimoto M. Cytokine. 1999; 11: 593-599Crossref PubMed Scopus (85) Google Scholar). A role for IL-18 in viral infection is also supported by the finding that the genome of several poxviruses encodes proteins that bind and inactivate IL-18 (51Xiang Y. Moss B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11537-11542Crossref PubMed Scopus (167) Google Scholar). Functional analyses of RNA compounds and enzymatic studies revealed that dsRNA (but not ssRNA) activate caspase-1 through cryopyrin. The ability of cryopyrin to discriminate between dsRNA and ssRNA provides a mechanism to sense viral RNA and to avoid harmful activation of the cryopyrin inflammasome by endogenous RNA. Treatment of 700 bp dsRNA fragments with RNase III, an endoribonuclease that cleaves both synthetic and natural dsRNA into small duplex products averaging 10–18 bp in length (52Dunn J.J. Gene. 1982; 245: 213-221Google Scholar), had no effect on its ability to induce caspase-1 activation. It is known that mammalian RNase III enzymes, such as Dicer, process dsRNA into 21–24-nt dsRNAs that can induce degradation of homologous mRNAs and specific gene silencing (53Carmell M.A. Hannon G.J. Nat. Struct. Mol. Biol. 2004; 11: 214-218Crossref PubMed Scopus (311) Google Scholar). It is also known that certain siRNAs can induce IFN responses and toxic effects in mammalian cells (54Sledz C.A. Holko M. de Veer M.J. Silverman R.H. Williams B.R. Nat. Cell Biol. 2003; 5: 834-839Crossref PubMed Scopus (1221) Google Scholar). Our results suggest both long viral and endogenous dsRNA fragments or their shorter dsRNA products generated by RNase III enzymes might lead to cryopyrin activation and IL-1β/IL-18 secretion. There are several mechanisms by which dsRNA could induce the activation of caspase-1 through cryopyrin. Viral dsRNA is produced during viral infection in the cytosol of host cells and could be sensed directly by cryopyrin. There is evidence that several NLR proteins, including Nod1, Nod2, Ipaf, and cryopyrin, can recognize microbial structures (22Inohara N. Chamaillard M. McDonald C. Núñez G. Annu. Rev. Biochem. 2005; 74: 355-383Crossref PubMed Scopus (812) Google Scholar, 23Ting J.P. Davis B.K. Annu. Rev. Immunol. 2005; 23: 387-414Crossref PubMed Scopus (299) Google Scholar). However, there is no evidence that the microbial products physically interact with the leucine-rich repeats of these NLR proteins (55Mackey D. Holt B.F. II I Wiig A. Dangl J.L. Cell. 2002; 108: 743-754Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar). The sensing of dsRNA by cryopyrin is most likely to be indirect, as it has been proposed for the recognition of microbial components by plant NLR homologs (55Mackey D. Holt B.F. II I Wiig A. Dangl J.L. Cell. 2002; 108: 743-754Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar, 56Mackey D. Belkhadir Y. Alonso J.M. Ecker J.R. Dangl J.L. Cell. 2003; 112: 379-389Abstract Full Text Full Text PDF PubMed Scopus (727) Google Scholar). Cryopyrin has been shown to form a large multiprotein complex containing ASC that promotes the activation of caspase-1 in cell-free systems (25Agostini L. Martinon F. Burns K. McDermott M.F. Hawkins P.N. Tschopp J. Immunity. 2004; 20: 319-325Abstract Full Text Full Text PDF PubMed Scopus (1389) Google Scholar). Thus, a possible model is that the leucine-rich repeats of cryopyrin sense dsRNA produced during viral infection, which induces the oligomerization of cryopyrin and recruitment of caspase-1 via ASC and possibly other factors involved in caspase-1 activation. In summary, our results have shown a novel role for cryopyrin in the activation of inflammasome by dsRNA/viral RNA and by viral infection. These findings provide important insight into the role of NLR proteins in the host response to dsRNA. The possibility that certain siRNAs might activate the cryopyrin inflammasome warrant further investigation, as such siRNAs are being explored for their potential therapeutic use (57Jacque J.M. Triques K. Stevenson M. Nature. 2002; 418: 435-438Crossref PubMed Scopus (768) Google Scholar, 58Gitlin L. Karelsky S. Andino R. Nature. 2002; 418: 430-434Crossref PubMed Scopus (519) Google Scholar). We thank Anthony Coyle, Ethan Grant, and John Bertin (Millennium Pharmaceuticals) and Shizuo Akira (Osaka University) for the generous supply of mutant mice, P. Vandenabeele for anti-caspase-1 antibody, C. Kao for Brome mosaic viral RNA, and the Cellular Immunology Core Facility of the University of Michigan Cancer Center for technical support. Download .pdf (.11 MB) Help with pdf files

<i>Legionella pneumophila</i> is an intracellular bacterium that causes an acute form of pneumonia called Legionnaires' disease. After infection of human macrophages, the <i>Legionella</i>-containing phagosome (LCP) avoids fusion with the lysosome allowing intracellular replication of the bacterium. In macrophages derived from most mouse strains, the LCP is delivered to the lysosome resulting in <i>Legionella</i> degradation and restricted bacterial growth. Mouse macrophages lacking the NLR protein Ipaf or its downstream effector caspase-1 are permissive to intracellular <i>Legionella</i> replication. However, the mechanism by which Ipaf restricts <i>Legionella</i> replication is not well understood. Here we demonstrate that the presence of flagellin and a competent type IV secretion system are critical for <i>Legionella</i> to activate caspase-1 in macrophages. Activation of caspase-1 in response to <i>Legionella</i> infection also required host Ipaf, but not TLR5. In the absence of Ipaf or caspase-1 activation, the LCP acquired endoplasmic reticulum-derived vesicles, avoided fusion with the lysosome, and allowed <i>Legionella</i> replication. Accordingly a <i>Legionella</i> mutant lacking flagellin did not activate caspase-1, avoided degradation, and replicated in wild-type macrophages. The regulation of phagosome maturation by Ipaf occurred within 2 h after infection and was independent of macrophage cell death. <i>In vivo</i> studies confirmed that flagellin and Ipaf play an important role in the control of <i>Legionella</i> clearance. These results reveal that Ipaf restricts <i>Legionella</i> replication through the regulation of phagosome maturation, providing a novel function for NLR proteins in host defense against an intracellular bacterium.

Endotoxin administration recapitulates many of the host responses to sepsis. Inhibitors of the cysteine protease caspase 1 have long been sought as a therapeutic because mice lacking caspase 1 are resistant to LPS-induced endotoxic shock. According to current thinking, caspase 1-mediated shock requires the proinflammatory caspase 1 substrates IL-1β and IL-18. We show, however, that mice lacking both IL-1β and IL-18 are normally susceptible to LPS-induced splenocyte apoptosis and endotoxic shock. This finding indicates the existence of another caspase 1-dependent mediator of endotoxemia. Reduced serum high mobility group box 1 (HMGB1) levels in caspase 1-deficient mice correlated with their resistance to LPS. A critical role for HMGB1 in endotoxemia was confirmed when mice deficient for IL-1β and IL-18 were protected from a lethal dose of LPS by pretreatment with HMGB1-neutralizing Abs. We found that HMGB1 secretion from LPS-primed macrophages required the inflammasome components apoptotic speck protein containing a caspase activation and recruitment domain (ASC), caspase 1 and Nalp3, whereas HMGB1 secretion from macrophages infected in vitro with Salmonella typhimurium was dependent on caspase 1 and Ipaf. Thus, HMGB1 secretion, which is critical for endotoxemia, occurs downstream of inflammasome assembly and caspase 1 activation.

After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10-30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages' processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.

Legionella pneumophila (L. pneumophila), the causative agent of a severe form of pneumonia called Legionnaires' disease, replicates in human monocytes and macrophages. Most inbred mouse strains are restrictive to L. pneumophila infection except for the A/J, Nlrc4−/− (Ipaf−/−), and caspase-1−/− derived macrophages. Particularly, caspase-1 activation is detected during L. pneumophila infection of murine macrophages while absent in human cells. Recent in vitro experiments demonstrate that caspase-7 is cleaved by caspase-1. However, the biological role for caspase-7 activation downstream of caspase-1 is not known. Furthermore, whether this reaction is pertinent to the apoptosis or to the inflammation pathway or whether it mediates a yet unidentified effect is unclear. Using the intracellular pathogen L. pneumophila, we show that, upon infection of murine macrophages, caspase-7 was activated downstream of the Nlrc4 inflammasome and required caspase-1 activation. Such activation of caspase-7 was mediated by flagellin and required a functional Naip5. Remarkably, mice lacking caspase-7 and its macrophages allowed substantial L. pneumophila replication. Permissiveness of caspase-7−/− macrophages to the intracellular pathogen was due to defective delivery of the organism to the lysosome and to delayed cell death during early stages of infection. These results reveal a new mechanism for caspase-7 activation downstream of the Nlrc4 inflammasome and present a novel biological role for caspase-7 in host defense against an intracellular bacterium.

Cystic fibrosis (CF) is the most common inherited lethal disease of Caucasians which results in multi organ dysfunction. However, 85% of the deaths are due to pulmonary infections. Infection by Burkholderia cenocepacia (B. cepacia) is a particularly lethal threat to CF patients because it causes severe and persistent lung inflammation and is resistant to nearly all available antibiotics. In CFTR ΔF508 mouse macrophages, B. cepacia persists in vacuoles that do not fuse with the lysosomes and mediates increased production of IL-1β. It is believed that intracellular bacterial survival contributes to the persistence of the bacterium. Here we show for the first time that in wild-type macrophages but not in ΔF508 macrophages, many B. cepacia reside in autophagosomes that fuse with lysosomes at later stages of infection. Accordingly, association and intracellular survival of B. cepacia are higher in CFTR-ΔF508 (ΔF508) macrophages than in WT macrophages. An autophagosome is a compartment that engulfs non-functional organelles and parts of the cytoplasm then delivers them to the lysosome for degradation to produce nutrients during periods of starvation or stress. Furthermore, we show that B. cepacia downregulates autophagy genes in WT and ΔF508 macrophages. However, autophagy dysfunction is more pronounced in ΔF508 macrophages since they already have compromised autophagy activity. We demonstrate that the autophagy-stimulating agent, rapamycin markedly decreases B. cepacia infection in vitro by enhancing the clearance of B. cepacia via induced autophagy. In vivo, Rapamycin decreases bacterial burden in the lungs of CF mice and drastically reduces signs of lung inflammation. Together, our studies reveal that if efficiently activated, autophagy can control B. cepacia infection and ameliorate the associated inflammation. Therefore, autophagy is a novel target for new drug development for CF patients to control B. cepacia infection and accompanying inflammation.

Inflammasomes are multiprotein complexes that include members of the NLR (nucleotide-binding domain leucine-rich repeat containing) family and caspase-1. Once bacterial molecules are sensed within the macrophage, the inflammasome is assembled, mediating the activation of caspase-1. Caspase-11 mediates caspase-1 activation in response to lipopolysaccharide and bacterial toxins, and yet its role during bacterial infection is unknown. Here, we demonstrated that caspase-11 was dispensable for caspase-1 activation in response to Legionella, Salmonella, Francisella, and Listeria. We also determined that active mouse caspase-11 was required for restriction of L. pneumophila infection. Similarly, human caspase-4 and caspase-5, homologs of mouse caspase-11, cooperated to restrict L. pneumophila infection in human macrophages. Caspase-11 promoted the fusion of the L. pneumophila vacuole with lysosomes by modulating actin polymerization through cofilin. However, caspase-11 was dispensable for the fusion of lysosomes with phagosomes containing nonpathogenic bacteria, uncovering a fundamental difference in the trafficking of phagosomes according to their cargo.

The intracellular Gram-negative bacterium Francisella tularensis causes the disease tularemia and is known for its ability to subvert host immune responses. Previous work from our laboratory identified the PI3K/Akt pathway and SHIP as critical modulators of host resistance to Francisella. Here, we show that SHIP expression is strongly down-regulated in monocytes and macrophages following infection with F. tularensis novicida (F.n.). To account for this negative regulation we explored the possibility that microRNAs (miRs) that target SHIP may be induced during infection. There is one miR that is predicted to target SHIP, miR-155. We tested for induction and found that F.n. induced miR-155 both in primary monocytes/macrophages and in vivo. Using luciferase reporter assays we confirmed that miR-155 led to down-regulation of SHIP, showing that it specifically targets the SHIP 3′UTR. Further experiments showed that miR-155 and BIC, the gene that encodes miR-155, were induced as early as four hours post-infection in primary human monocytes. This expression was dependent on TLR2/MyD88 and did not require inflammasome activation. Importantly, miR-155 positively regulated pro-inflammatory cytokine release in human monocytes infected with Francisella. In sharp contrast, we found that the highly virulent type A SCHU S4 strain of Francisella tularensis (F.t.) led to a significantly lower miR-155 response than the less virulent F.n. Hence, F.n. induces miR-155 expression and leads to down-regulation of SHIP, resulting in enhanced pro-inflammatory responses. However, impaired miR-155 induction by SCHU S4 may help explain the lack of both SHIP down-regulation and pro-inflammatory response and may account for the virulence of Type A Francisella.

Abstract The nucleotide binding oligomerization domain-like receptor (NLR) family of pattern recognition molecules is involved in a diverse array of processes required for host immune responses against invading pathogens. Unlike TLRs that mediate extracellular recognition of microbes, several NLRs sense pathogens in the cytosol and upon activation induce host defense signaling pathways. Although TLRs and NLRs differ in their mode of pathogen recognition and function, they share similar domains for microbial sensing and cooperate to elicit immune responses against the pathogen. Genetic variation in several NLR genes is associated with the development of inflammatory disorders or increased susceptibility to microbial infection. Further understanding of NLRs should provide critical insight into the mechanisms of host defense and the pathogenesis of inflammatory diseases.

Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2−/− and Nod2−/− macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2−/− mice. Tlr2−/− and Nod2−/− cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes. Listeria monocytogenes is a facultative intracellular pathogen that invades both phagocytic and non-phagocytic cells. Recent studies have shown that L. monocytogenes infection activates the autophagy pathway. However, the innate immune receptors involved and the downstream signaling pathways remain unknown. Here, we show that macrophages deficient in the TLR2 and NOD/RIP2 pathway display defective autophagy induction in response to L. monocytogenes. Inefficient autophagy in Tlr2−/− and Nod2−/− macrophages led to a defect in bacteria colocalization with the autophagosomal marker GFP-LC3. Consequently, macrophages lacking TLR2 and NOD2 were found to be more susceptible to L. monocytogenes infection, as were the Rip2−/− mice. Tlr2−/− and Nod2−/− cells showed perturbed NF-κB and ERK signaling. However, autophagy against L. monocytogenes was dependent selectively on the ERK pathway. In agreement, wild-type cells treated with a pharmacological inhibitor of ERK or ERK-deficient cells displayed inefficient autophagy activation in response to L. monocytogenes. Accordingly, fewer bacteria were targeted to the autophagosomes and, consequently, higher bacterial growth was observed in cells deficient in the ERK signaling pathway. These findings thus demonstrate that TLR2 and NOD proteins, acting via the downstream ERK pathway, are crucial to autophagy activation and provide a mechanistic link between innate immune receptors and induction of autophagy against cytoplasm-invading microbes, such as L. monocytogenes.

Summary Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen of macrophages. Intracellular pattern recognition receptors in macrophages such as nucleotide-binding oligomerization domain (NOD) proteins regulate pro-inflammatory cytokine production. NOD2-mediated signalling pathways in response to M.tb have been studied primarily in mouse models and cell lines but not in primary human macrophages. Thus we sought to determine the role of NOD2 in regulating cytokine production and growth of virulent M.tb and attenuated Mycobacterium bovis BCG (BCG) in human macrophages. We examined NOD2 expression during monocyte differentiation and observed a marked increase in NOD2 transcript and protein following 2–3 days in culture. Pre-treatment of human monocyte-derived and alveolar macrophages with the NOD2 ligand muramyl dipeptide enhanced production of TNF-α and IL-1β in response to M.tb and BCG in a RIP2-dependent fashion. The NOD2-mediated cytokine response was significantly reduced following knock-down of NOD2 expression by using small interfering RNA (siRNA) in human macrophages. Finally, NOD2 controlled the growth of both M.tb and BCG in human macrophages, whereas controlling only BCG growth in murine macrophages. Together, our results provide evidence that NOD2 is an important intracellular receptor in regulating the host response to M.tb and BCG infection in human macrophages.

Abstract Burkholderia cenocepacia is an opportunistic pathogen that causes chronic infection and induces progressive respiratory inflammation in cystic fibrosis patients. Recognition of bacteria by mononuclear cells generally results in the activation of caspase-1 and processing of IL-1β, a major proinflammatory cytokine. In this study, we report that human pyrin is required to detect intracellular B. cenocepacia leading to IL-1β processing and release. This inflammatory response involves the host adapter molecule ASC and the bacterial type VI secretion system (T6SS). Human monocytes and THP-1 cells stably expressing either small interfering RNA against pyrin or YFP–pyrin and ASC (YFP–ASC) were infected with B. cenocepacia and analyzed for inflammasome activation. B. cenocepacia efficiently activates the inflammasome and IL-1β release in monocytes and THP-1. Suppression of pyrin levels in monocytes and THP-1 cells reduced caspase-1 activation and IL-1β release in response to B. cenocepacia challenge. In contrast, overexpression of pyrin or ASC induced a robust IL-1β response to B. cenocepacia, which correlated with enhanced host cell death. Inflammasome activation was significantly reduced in cells infected with T6SS-defective mutants of B. cenocepacia, suggesting that the inflammatory reaction is likely induced by an as yet uncharacterized effector(s) of the T6SS. Together, we show for the first time, to our knowledge, that in human mononuclear cells infected with B. cenocepacia, pyrin associates with caspase-1 and ASC forming an inflammasome that upregulates mononuclear cell IL-1β processing and release.

Aberrant NLRP3 inflammasome activation contributes to the development of endotoxemia. The importance of negative regulation of NLRP3 inflammasomes remains poorly understood. Here, we show that the E3 ubiquitin ligase Cbl-b is essential for preventing endotoxemia induced by a sub-lethal dose of LPS via a caspase-11/NLRP3–dependent manner. Further studies show that NLRP3 undergoes both K63- and K48-linked polyubiquitination. Cbl-b binds to the K63-ubiquitin chains attached to the NLRP3 leucine-rich repeat domain (LRR) via its ubiquitin-associated region (UBA) and then targets NLRP3 at K496 for K48-linked ubiquitination and proteasome-mediated degradation. We also identify RNF125 as an additional E3 ubiquitin ligase that initiates K63-linked ubiquitination of the NLRP3 LRR domain. Therefore, NLRP3 is sequentially ubiquitinated by K63- and K48-linked ubiquitination, thus keeping the NLRP3 inflammasomes in check and restraining endotoxemia.

Autophagy is a biological process characterized by self-digestion and involves induction of autophagosome formation, leading to degradation of autophagic cargo. Aging is associated with the reduction of autophagy activity leading to neurodegenerative disorders, chronic inflammation, and susceptibility to infection; however, the underlying mechanism is unclear. DNA methylation by DNA methyltransferases reduces the expression of corresponding genes. Since macrophages are major players in inflammation and defense against infection we determined the differences in methylation of autophagy genes in macrophages derived from young and aged mice. We found that promoter regions of Atg5 and LC3B are hypermethylated in macrophages from aged mice and this is accompanied by low gene expression. Treatment of aged mice and their derived macrophages with methyltransferase inhibitor (2)-epigallocatechin-3-gallate (EGCG) or specific DNA methyltransferase 2 (DNMT2) siRNA restored the expression of Atg5 and LC3 in vivo and in vitro. Our study builds a foundation for the development of novel therapeutics aimed to improve autophagy in the elderly population and suggests a role for DNMT2 in DNA methylation activities.

Legionella pneumophila (L. pneumophila) is an opportunistic waterborne pathogen and the causative agent for Legionnaires' disease, which is transmitted to humans via inhalation of contaminated water droplets. The bacterium is able to colonize a variety of man-made water systems such as cooling towers, spas, and dental lines and is widely distributed in multiple niches, including several species of protozoa In addition to survival in planktonic phase, L. pneumophila is able to survive and persist within multi-species biofilms that cover surfaces within water systems. Biofilm formation by L. pneumophila is advantageous for the pathogen as it leads to persistence, spread, resistance to treatments and an increase in virulence of this bacterium. Furthermore, Legionellosis outbreaks have been associated with the presence of L. pneumophila in biofilms, even after the extensive chemical and physical treatments. In the microbial consortium-containing L. pneumophila among other organisms, several factors either positively or negatively regulate the presence and persistence of L. pneumophila in this bacterial community. Biofilm-forming L. pneumophila is of a major importance to public health and have impact on the medical and industrial sectors. Indeed, prevention and removal protocols of L. pneumophila as well as diagnosis and hospitalization of patients infected with this bacteria cost governments billions of dollars. Therefore, understanding the biological and environmental factors that contribute to persistence and physiological adaptation in biofilms can be detrimental to eradicate and prevent the transmission of L. pneumophila. In this review, we focus on various factors that contribute to persistence of L. pneumophila within the biofilm consortium, the advantages that the bacteria gain from surviving in biofilms, genes and gene regulation during biofilm formation and finally challenges related to biofilm resistance to biocides and anti-Legionella treatments.

Significance We report the discovery of fundamental roles for the noncanonical inflammasome molecule Caspase-4/11 in promoting pathological inflammatory and prothrombotic pathways in severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) infections. Our work demonstrates that Caspase-11 has a broader role in immune responses beyond its previously appreciated effects in bacterial infections. Further, we show that Caspase-11–deficient mice infected with SARS–CoV-2 fare significantly better in terms of overall illness, lung inflammation, and thrombosis than wild-type (WT) mice, thus implicating Caspase-11 as a new therapeutic target for preventing or treating COVID-19.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Macrophages protect their host by engulfing foreign bodies within phagosomes that rapidly develop into microbicidal organelles. Numerous pathogens, such as species of Toxoplasma, Leishmania, Mycobacterium, Salmonella and Legionella, thrive in human macrophages, sometimes with disastrous effects. Defining the survival tactics of intracellular parasites is one approach to understanding macrophage function. Here, we briefly review phagosome maturation, then discuss how particular microbes may target particular host factors to short-circuit membrane traffic in macrophages. Recent studies support a new paradigm in which pathogens evade lysosomal degradation by entering macrophages within specialized lipid microdomains of the plasma membrane.

Similar to Ipaf and caspase-1, the Nod-like receptor protein Naip5 restricts intracellular proliferation of Legionella pneumophila, the causative agent of a severe form of pneumonia known as Legionnaires' disease. Thus, Naip5 has been suggested to regulate Legionella replication inside macrophages through the activation of caspase-1. In this study, we show that cytosolic delivery of recombinant flagellin activated caspase-1 in A/J macrophages carrying a mutant Naip5 allele, and in C57BL/6 (B6) macrophages congenic for the mutant Naip5 allele (B6-Naip5(A/J)), but not in Ipaf(-/-) cells. In line with these results, A/J and B6-Naip5(A/J) macrophages induced high levels of caspase-1 activation and IL-1beta secretion when infected with Legionella. In addition, transgenic expression of a functional Naip5 allele in A/J macrophages did not alter Legionella-induced caspase-1 activation and IL-1beta secretion. Notably, defective Naip5 signaling renders B6-Naip5(A/J) macrophages permissive for Legionella proliferation despite normal caspase-1 activation. These results indicate that the restriction of intracellular Legionella replication is more complex than previously appreciated and requires both Ipaf-dependent caspase-1 activation as well as functional Naip5 signaling.

A controlled field trial of a serogroup A meningococcal polysaccharide vaccine was conducted at three locations in Egypt during the winter cerebrospinal meningitis (CSM) season of 1971-72. The study population consisted of schoolchildren 6-15 years of age. No cases of serogroup A meningococcal CSM occurred in the group of students vaccinated with the test vaccine whereas 8 cases occurred in the control group vaccinated with tetanus toxoid, and 151 cases occurred in an unvaccinated contrast group. The case rate was significantly different between the test and control groups as well as between the test and contrast groups but was similar between the control and contrast groups. The previously demonstrated safety of the vaccine was confirmed. A significant serological response was elicited in the majority of the vaccinated students.

Cystic fibrosis (CF) is the most common lethal inherited genetic disorder affection Caucasians. Even with medical advances, CF is life-shortening with patients typically surviving only to age 38. Infection of the CF lung by Burkholderia cenocepacia presents exceptional challenges to medical management of these patients as clinically this microbe is resistant to virtually all antibiotics, is highly transmissible and infection of CF patients with this microbe renders them ineligible for lung transplant, often the last lifesaving option. Here we have targeted two abundant components of the B. cenocepacia biofilm for immune intervention: extracellular DNA and DNABII proteins, the latter of which are bacterial nucleic acid binding proteins. Treatment of B. cenocepacia biofilms with antiserum directed at one of these DNABII proteins (integration host factor or IHF) resulted in significant disruption of the biofilm. Moreover, when anti-IHF mediated destabilization of a B. cenocepacia biofilm was combined with exposure to traditional antibiotics, B. cenocepacia resident within the biofilm and thereby typically highly resistant to the action of antibiotics, were now rendered susceptible to killing. Pre-incubation of B. cenocepacia with anti-IHF serum prior to exposure to murine CF macrophages, which are normally unable to effectively degrade ingested B. cenocepacia, resulted in a statistically significant increase in killing of phagocytized B. cenocepacia. Collectively, these findings support further development of strategies that target DNABII proteins as a novel approach for treatment of CF patients, particularly those whose lungs are infected with B. cenocepacia.

Burkholderia cenocepacia is a virulent pathogen that causes significant morbidity and mortality in patients with cystic fibrosis (CF), survives intracellularly in macrophages, and uniquely causes systemic infections in CF. Autophagy is a physiologic process that involves engulfing non-functional organelles and proteins and delivering them for lysosomal degradation, but also plays a role in eliminating intracellular pathogens, including B. cenocepacia. Autophagy is defective in CF but can be stimulated in murine CF models leading to increased clearance of B. cenocepacia, but little is known about autophagy stimulation in human CF macrophages. IFN-γ activates macrophages and increases antigen presentation while also inducing autophagy in macrophages. We therefore, hypothesized that treatment with IFN-γ would increase autophagy and macrophage activation in patients with CF. Peripheral blood monocyte derived macrophages (MDMs) were obtained from CF and non-CF donors and subsequently infected with B. cenocepacia. Basal serum levels of IFN-γ were similar between CF and non-CF patients, however after B. cenocepacia infection there is deficient IFN-γ production in CF MDMs. IFN-γ treated CF MDMs demonstrate increased co-localization with the autophagy molecule p62, increased autophagosome formation, and increased trafficking to lysosomes compared to untreated CF MDMs. Electron microscopy confirmed IFN-γ promotes double membrane vacuole formation around bacteria in CF MDMs, while only single membrane vacuoles form in untreated CF cells. Bacterial burden is significantly reduced in autophagy stimulated CF MDMs, comparable to non-CF levels. IL-1β production is decreased in CF MDMs after IFN-γ treatment. Together, these results demonstrate that IFN-γ promotes autophagy-mediated clearance of B. cenocepacia in human CF macrophages.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. Macroautophagy/autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their macrophages exhibit limited autophagy activity. The study of microRNAs (Mirs), and other noncoding RNAs, continues to offer new therapeutic targets. The objective of this study was to elucidate the role of Mirs in dysregulated autophagy-related genes in CF macrophages, and then target them to restore this host-defense function and improve CFTR channel function. We identified the Mirc1/Mir17-92 cluster as a potential negative regulator of autophagy as CF macrophages exhibit decreased autophagy protein expression and increased cluster expression when compared to wild-type (WT) counterparts. The absence or reduced expression of the cluster increases autophagy protein expression, suggesting the canonical inverse relationship between Mirc1/Mir17-92 and autophagy gene expression. An in silico study for targets of Mirs that comprise the cluster suggested that the majority of the Mirs target autophagy mRNAs. Those targets were validated by luciferase assays. Notably, the ability of macrophages expressing mutant F508del CFTR to transport halide through their membranes is compromised and can be restored by downregulation of these inherently elevated Mirs, via restoration of autophagy. In vivo, downregulation of Mir17 and Mir20a partially restored autophagy expression and hence improved the clearance of Burkholderia cenocepacia. Thus, these data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.

Summary Autophagy is originally described as the main catabolic pathway responsible for maintaining intracellular nutritional homeostasis that involves the formation of a unique vacuole, the autophagosome, and the interaction with the endosome‐lysosome pathways. This conserved machinery plays a key role in immune‐protection against different invaders, including pathogenic bacteria, intracellular parasites, and some viruses like herpes simplex and hepatitis C virus. Importantly, autophagy is linked to a number of human diseases and disorders including neurodegenerative disease, Crohn's disease, type II diabetes, tumorigenesis, cardiomyopathy, and fatty liver disease. On the other hand, inflammasomes are multiprotein platforms stimulated upon several environmental conditions and microbial infection. Once assembled, the inflammasomes mediate the maturation of pro‐inflammatory cytokines and promote phagosome‐lysosome fusion to sustain an innate immune response. The intersections between autophagy and inflammasome have been observed in various diseases and microbial infections. This review highlights the molecular aspects involved in autophagy and inflammasome interactions during different medical conditions and microbial infections.

Gout is characterized by attacks of arthritis with hyperuricemia and monosodium urate (MSU) crystal-induced inflammation within joints. Innate immune responses are the primary drivers for tissue destruction and inflammation in gout. MSU crystals engage the Nlrp3 inflammasome leading to the activation of caspase-1 and production of IL-1β and IL-18 within gout-affected joints, promoting the influx of neutrophils and monocytes. Here we show that caspase-11-/- mice and their derived macrophages produce significantly reduced levels of gout-specific cytokines including IL-1β, TNFα, IL-6, and KC, while others like IFNγ and IL-12p70 are not altered. IL-1β induces the expression of caspase-11 in an IL-1 receptor-dependent manner in macrophages. In neutrophils, the absence of caspase-11 reduced the ability of neutrophils to migrate in response to exogenously injected KC in vivo. Notably, in vitro, caspase-11-/- neutrophils displayed random migration in response to a KC gradient when compared to their WT counterparts. This phenotype was associated with altered cofilin phosphorylation. Unlike their wild-type counter parts, caspase-11-/- neutrophils also failed to produce neutrophil extracellular traps (NETs) when treated with MSU. Together, this is the first report demonstrating that caspase-11 promotes neutrophil directional trafficking and function in an acute model of gout. Caspase-11 also governs the production of inflammasome-dependent and -independent cytokines from macrophages. Our results offer new, previously unrecognized functions for caspase-11 in macrophages and neutrophils that may apply to other neutrophil-mediated disease conditions besides gout.

CASP4/caspase-11-dependent inflammasome activation is important for the clearance of various Gram-negative bacteria entering the host cytosol. Additionally, CASP4 modulates the actin cytoskeleton to promote the maturation of phagosomes harboring intracellular pathogens such as Legionella pneumophila but not those enclosing nonpathogenic bacteria. Nevertheless, this non-inflammatory role of CASP4 regarding the trafficking of vacuolar bacteria remains poorly understood. Macroautophagy/autophagy, a catabolic process within eukaryotic cells, is also implicated in the elimination of intracellular pathogens such as Burkholderia cenocepacia. Here we show that CASP4-deficient macrophages exhibit a defect in autophagosome formation in response to B. cenocepacia infection. The absence of CASP4 causes an accumulation of the small GTPase RAB7, reduced colocalization of B. cenocepacia with LC3 and acidic compartments accompanied by increased bacterial replication in vitro and in vivo. Together, our data reveal a novel role of CASP4 in regulating autophagy in response to B. cenocepacia infection.

The prevalence of asthma has been rising steadily for several decades, and continues to be a major public health and global economic burden due to both direct and indirect costs. Asthma is defined as chronic heterogeneous inflammatory diseases characterized by airway obstruction, mucus production and bronchospasm. Different endotypes of asthma are being recognized based on the distinct pathophysiology, genetic predisposition, age, prognosis, and response to remedies. Mucosal innate response to environmental triggers such as pollen, cigarette smoke, fragrances, viral infection, and house dust mite (HDM) are now recognized to play an important role in allergic asthma. HDM are the most pervasive allergens that co-habitat with us, as they are ubiquitous in-house dusts, mattress and bedsheets, and feed on a diet of exfoliated human skin flakes. Dermatophagoides pteronyssinus, is one among several HDM identified up to date. During the last decade, extensive studies have been fundamental in elucidating the interactions between HDM allergens, the host immune systems and airways. Moreover, the paradigm in the field of HDM-mediated allergy has been shifted away from being solely a Th2-geared to a complex response orchestrated via extensive crosstalk between the epithelium, professional antigen presenting cells (APCs) and components of the adaptive immunity. In fact, HDM have several lessons to teach us about their allergenicity, the complex interactions that stimulate innate immunity in initiating and perpetuating the lung inflammation. Herein, we review main allergens of Dermatophagoides pteronyssinus and their interactions with immunological sentinels that promote allergic sensitization and activation of innate immunity, which is critical for the development of the Th2 biased adaptive immunity to HDM allergens and development of allergic asthma.

Autophagy is a proposed route of amyloid-β (Aβ) clearance by microglia that is halted in Alzheimer's Disease (AD), though mechanisms underlying this dysfunction remain elusive. Here, primary microglia from adult AD (5xFAD) mice were utilized to demonstrate that 5xFAD microglia fail to degrade Aβ and express low levels of autophagy cargo receptor NBR1. In 5xFAD mouse brains, we show for the first time that AD microglia express elevated levels of microRNA cluster Mirc1/Mir17-92a, which is known to downregulate autophagy proteins. By in situ hybridization in post-mortem AD human tissue sections, we observed that the Mirc1/Mir17-92a cluster member miR-17 is also elevated in human AD microglia, specifically in the vicinity of Aβ deposits, compared to non-disease controls. We show that NBR1 expression is negatively correlated with expression of miR-17 in human AD microglia via immunohistopathologic staining in human AD brain tissue sections. We demonstrate in healthy microglia that autophagy cargo receptor NBR1 is required for Aβ degradation. Inhibiting elevated miR-17 in 5xFAD mouse microglia improves Aβ degradation, autophagy, and NBR1 puncta formation in vitro and improves NBR1 expression in vivo. These findings offer a mechanism behind dysfunctional autophagy in AD microglia which may be useful for therapeutic interventions aiming to improve autophagy function in AD.

Influenza virus activates cellular inflammasome pathways, which can be both beneficial and detrimental to infection outcomes. Here, we investigate the function of the inflammasome-activated, pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice (Gsdmd-/-) significantly attenuates influenza virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected Gsdmd-/- mice exhibit decreased inflammatory gene signatures shown by lung transcriptomics. Among these, diminished neutrophil gene activation signatures are corroborated by decreased detection of neutrophil elastase and myeloperoxidase in KO mouse lungs. Indeed, directly infected neutrophils are observed in vivo and infection of neutrophils in vitro induces release of DNA and tissue-damaging enzymes that is largely dependent on GSDMD. Neutrophil depletion in infected WT mice recapitulates the reductions in mortality, lung inflammation, and lung dysfunction observed in Gsdmd-/- animals, while depletion does not have additive protective effects in Gsdmd-/- mice. These findings implicate a function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a therapeutic avenue for treating severe influenza.

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.

The ability of Legionella pneumophila to cause pneumonia is determined by its capability to evade the immune system and grow within human monocytes and their derived macrophages. Human monocytes efficiently activate caspase-1 in response to Salmonella but not to L. pneumophila. The molecular mechanism for the lack of inflammasome activation during L. pneumophila infection is unknown. Evaluation of the expression of several inflammasome components in human monocytes during L. pneumophila infection revealed that the expression of the apoptosis-associated speck-like protein (ASC) and the NOD-like receptor NLRC4 are significantly down-regulated in human monocytes. Exogenous expression of ASC maintained the protein level constant during L. pneumophila infection and conveyed caspase-1 activation and restricted the growth of the pathogen. Further depletion of ASC with siRNA was accompanied with improved NF-κB activation and enhanced L. pneumophila growth. Therefore, our data demonstrate that L. pneumophila manipulates ASC levels to evade inflammasome activation and grow in human monocytes. By targeting ASC, L. pneumophila modulates the inflammasome, the apoptosome, and NF-κB pathway simultaneously.

Legionella pneumophila ( L . pneumophila ) is an intracellular bacterium of human alveolar macrophages that causes Legionnaires’ disease. In contrast to humans, most inbred mouse strains are restrictive to L . pneumophila replication. We demonstrate that autophagy targets L . pneumophila vacuoles to lysosomes and that this process requires ubiquitination of L . pneumophila vacuoles and the subsequent binding of the autophagic adaptor p62/ SQSTM 1 to ubiquitinated vacuoles. The L . pneumophila leg A 9 encodes for an ankyrin‐containing protein with unknown role. We show that the leg A 9 mutant replicate in WT mice and their bone marrow‐derived macrophages. This is the first L . pneumophila mutant to be found to replicate in WT bone marrow‐derived macrophages other than the F la mutant. Less leg A 9 mutant‐containing vacuoles acquired ubiquitin labeling and p62/ SQSTM 1 staining, evading autophagy uptake and avoiding lysosomal fusion. Thus, we describe a bacterial protein that targets the L . pneumophila ‐containing vacuole for autophagy uptake.

Autophagy and inflammasome complex assembly are physiological processes that control homeostasis, inflammation, and immunity. Autophagy is a ubiquitous pathway that degrades cytosolic macromolecules or organelles, as well as intracellular pathogens. Inflammasomes are multi-protein complexes that assemble in the cytosol of cells upon detection of pathogen- or danger-associated molecular patterns. A critical outcome of inflammasome assembly is the activation of the cysteine protease caspase-1, which activates the pro-inflammatory cytokine precursors pro-IL-1β and pro-IL-18. Studies on chronic inflammatory diseases, heart diseases, Alzheimer's disease, and multiple sclerosis revealed that autophagy and inflammasomes intersect and regulate each other. In the context of infectious diseases, however, less is known about the interplay between autophagy and inflammasome assembly, although it is becoming evident that pathogens have evolved multiple strategies to inhibit and/or subvert these pathways and to take advantage of their intricate crosstalk. An improved appreciation of these pathways and their subversion by diverse pathogens is expected to help in the design of anti-infective therapeutic interventions.

Cystic fibrosis (CF), one of the most common human genetic diseases worldwide, is caused by a defect in the CF transmembrane conductance regulator (CFTR). Patients with CF are highly susceptible to infections caused by opportunistic pathogens (including Burkholderia cenocepacia), which induce excessive lung inflammation and lead to the eventual loss of pulmonary function. Abundant neutrophil recruitment into the lung is a key characteristic of bacterial infections in CF patients. In response to infection, inflammatory neutrophils release reactive oxygen species and toxic proteins, leading to aggravated lung tissue damage in patients with CF. The present study shows a defect in reactive oxygen species production by mouse Cftr-/- , human F508del-CFTR, and CF neutrophils; this results in reduced antimicrobial activity against B. cenocepacia Furthermore, dysregulated Ca2+ homeostasis led to increased intracellular concentrations of Ca2+ that correlated with significantly diminished NADPH oxidase response and impaired secretion of neutrophil extracellular traps in human CF neutrophils. Functionally deficient human CF neutrophils recovered their antimicrobial killing capacity following treatment with pharmacological inhibitors of Ca2+ channels and CFTR channel potentiators. Our findings suggest that regulation of neutrophil Ca2+ homeostasis (via CFTR potentiation or by the regulation of Ca2+ channels) can be used as a new therapeutic approach for reestablishing immune function in patients with CF.

Innate immune cells, epithelial cells, and many other cell types are capable of detecting infection or tissue injury, thus mounting regulated immune response. Inflammasomes are highly sophisticated and effective orchestrators of innate immunity. These oligomerized multiprotein complexes are at the center of various innate immune pathways, including modulation of the cytoskeleton, production and maturation of cytokines, and control of bacterial growth and cell death. Inflammasome assembly often results in caspase-1 activation, which is an inflammatory caspase that is involved in pyroptotic cell death and release of inflammatory cytokines in response to pathogen patterns and endogenous danger stimuli. However, the nature of stimuli and inflammasome components are diverse. Caspase-1 activation mediated release of mature IL-1β and IL-18 in response to canonical stimuli initiated by NOD-like receptor (NLR), and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). On the other hand, caspase-11 delineates a non-canonical inflammasome that promotes pyroptotic cell death and non-pyroptotic functions in response to non-canonical stimuli. Caspase-11 in mice and its homologues in humans (caspase-4/5) belong to caspase-1 family of cysteine proteases, and play a role in inflammation. Knockout mice provided new genetic tools to study inflammatory caspases and revealed the role of caspase-11 in mediating septic shock in response to lethal doses of lipopolysaccharide (LPS). Recognition of LPS mediates caspase-11 activation, which promotes a myriad of downstream effects that include pyroptotic and non-pyroptotic effector functions. Therefore, the physiological functions of caspase-11 are much broader than its previously established roles in apoptosis and cytokine maturation. Inflammation induced by exogenous or endogenous agents can be detrimental and, if excessive, can result in organ and tissue damage. Consequently, the existence of sophisticated mechanisms that tightly regulate the specificity and sensitivity of inflammasome pathways provides a fine-tuning balance between adequate immune response and minimal tissue damage. In this review, we summarize effector functions of caspase-11.

Abstract Burkholderia cenocepacia ( B. cenocepacia ) is an opportunistic bacterium; causing severe life threatening systemic infections in immunocompromised individuals including cystic fibrosis patients. The lack of gasdermin D (GSDMD) protects mice against endotoxin lipopolysaccharide (LPS) shock. On the other hand, GSDMD promotes mice survival in response to certain bacterial infections. However, the role of GSDMD during B. cenocepacia infection is not yet determined. Our in vitro study shows that GSDMD restricts B. cenocepacia replication within macrophages independent of its role in cell death through promoting mitochondrial reactive oxygen species (mROS) production. mROS is known to stimulate autophagy, hence, the inhibition of mROS or the absence of GSDMD during B. cenocepacia infections reduces autophagy which plays a critical role in the restriction of the pathogen. GSDMD promotes inflammation in response to B. cenocepacia through mediating the release of inflammasome dependent cytokine (IL-1β) and an independent one (CXCL1) (KC). Additionally, different B. cenocepacia secretory systems (T3SS, T4SS, and T6SS) contribute to inflammasome activation together with bacterial survival within macrophages. In vivo study confirmed the in vitro findings and showed that GSDMD restricts B. cenocepacia infection and dissemination and stimulates autophagy in response to B. cenocepacia . Nevertheless, GSDMD promotes lung inflammation and necrosis in response to B. cenocepacia without altering mice survival. This study describes the double-edged functions of GSDMD in response to B. cenocepacia infection and shows the importance of GSDMD-mediated mROS in restriction of B. cenocepacia.

Abnormal macrophage function caused by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) is a critical contributor to chronic airway infections and inflammation in people with cystic fibrosis (PWCF). Elexacaftor/tezacaftor/ivacaftor (ETI) is a new CFTR modulator therapy for PWCF. Host-pathogen and clinical responses to CFTR modulators are poorly described. We sought to determine how ETI impacts macrophage CFTR function, resulting effector functions and relationships to clinical outcome changes.Clinical information and/or biospecimens were obtained at ETI initiation and 3, 6, 9 and 12 months post-ETI in 56 PWCF and compared with non-CF controls. Peripheral blood monocyte-derived macrophages (MDMs) were isolated and functional assays performed.ETI treatment was associated with increased CF MDM CFTR expression, function and localisation to the plasma membrane. CF MDM phagocytosis, intracellular killing of CF pathogens and efferocytosis of apoptotic neutrophils were partially restored by ETI, but inflammatory cytokine production remained unchanged. Clinical outcomes including increased forced expiratory volume in 1 s (+10%) and body mass index (+1.0 kg·m-2) showed fluctuations over time and were highly individualised. Significant correlations between post-ETI MDM CFTR function and sweat chloride levels were observed. However, MDM CFTR function correlated with clinical outcomes better than sweat chloride.ETI is associated with unique changes in innate immune function and clinical outcomes.

Macrophages activate autophagy as an immediate response to Legionella pneumophila infection, but what marks the pathogen phagosome as a target for the autophagy machinery is not known. Because a variety of bacteria, parasites, viruses, and toxins that associate with the endoplasmic reticulum enter host cells by a cholesterol-dependent route, we tested the hypothesis that autophagy is triggered when microbes engage components of lipid raft domains. As the intracellular respiratory pathogen L. pneumophila or the extracellular uropathogen FimH+ Escherichia coli entered macrophages by a cholesterol-sensitive mechanism, they immediately resided in vacuoles rich in glycosylphosphatidylinositol moieties and the autophagy enzyme Atg7. As expected for autophagosomes, the vacuoles sequentially acquired the endoplasmic reticulum protein BiP, the autophagy markers Atg8 and monodansyl-cadaverine, and the lysosomal protein LAMP-1. A robust macrophage response to the pathogens was cholesterol-dependent, since fewer Atg7-rich vacuoles were observed when macrophages were pre-treated with methyl-beta-cyclodextrin or filipin. A model in which macrophages exploit autophagy to capture pathogens within the lipid raft pathway for antigen presentation prior to disposal in lysosomes is discussed.

Cystic fibrosis (CF) is accompanied with heightened inflammation worsened by drug resistant Burkholderia cenocepacia. Human CF macrophage responses to B. cenocepacia are poorly characterized and variable in the literature. Therefore, we examined human macrophage responses to the epidemic B. cenocepacia J2315 strain in order to identify novel anti-inflammatory targets. Peripheral blood monocyte derived macrophages were obtained from 23 CF and 27 non-CF donors. Macrophages were infected with B. cenocepacia J2315 and analyzed for cytokines, cytotoxicity, and microscopy. CF macrophages demonstrated significant increases in IL-1β, IL-10, MCP-1, and IFN-γ production in comparison to non-CF controls. CF patients on prednisone exhibited globally diminished cytokines compared to controls and other CF patients. CF macrophages also displayed increased bacterial burden and cell death. In conclusion, CF macrophages demonstrate exaggerated IL-1β, IL-10, MCP-1, and IFN-γ production and cell death during B. cenocepacia infection. Treatment with corticosteroids acutely suppressed cytokine responses.

Burkholderia cenocepacia is an opportunistic pathogen that survives intracellularly in macrophages and causes serious respiratory infections in patients with cystic fibrosis. We have previously shown that bacterial survival occurs in bacteria-containing membrane vacuoles (BcCVs) resembling arrested autophagosomes. Intracellular bacteria stimulate IL-1β secretion in a caspase-1-dependent manner and induce dramatic changes to the actin cytoskeleton and the assembly of the NADPH oxidase complex onto the BcCV membrane. A Type 6 secretion system (T6SS) is required for these phenotypes but surprisingly it is not required for the maturation arrest of the BcCV. Here, we show that macrophages infected with B. cenocepacia employ the NLRP3 inflammasome to induce IL-1β secretion and pyroptosis. Moreover, IL-1β secretion by B. cenocepacia-infected macrophages is suppressed in deletion mutants unable to produce functional Type VI, Type IV, and Type 2 secretion systems (SS). We provide evidence that the T6SS mediates the disruption of the BcCV membrane, which allows the escape of proteins secreted by the T2SS into the macrophage cytoplasm. This was demonstrated by the activity of fusion derivatives of the T2SS-secreted metalloproteases ZmpA and ZmpB with adenylcyclase. Supporting this notion, ZmpA and ZmpB are required for efficient IL-1β secretion in a T6SS dependent manner. ZmpA and ZmpB are also required for the maturation arrest of the BcCVs and bacterial intra-macrophage survival in a T6SS-independent fashion. Our results uncover a novel mechanism for inflammasome activation that involves cooperation between two bacterial secretory pathways, and an unanticipated role for T2SS-secreted proteins in intracellular bacterial survival.

Abstract Inflammasomes are multiprotein complexes that include members of the NOD-like receptor family and caspase-1. Caspase-1 is required for the fusion of the Legionella vacuole with lysosomes. Caspase-11, independently of the inflammasome, also promotes phagolysosomal fusion. However, it is unclear how these proteases alter intracellular trafficking. Here, we show that caspase-11 and caspase-1 function in opposing manners to phosphorylate and dephosphorylate cofilin, respectively upon infection with Legionella . Caspase-11 targets cofilin via the RhoA GTPase, whereas caspase-1 engages the Slingshot phosphatase. The absence of either caspase-11 or caspase-1 maintains actin in the polymerized or depolymerized form, respectively and averts the fusion of pathogen-containing vacuoles with lysosomes. Therefore, caspase-11 and caspase-1 converge on the actin machinery with opposing effects to promote vesicular trafficking.

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional nuclear magnetic resonance (NMR) spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipid mixtures is currently hampered by limited spectral resolution and the absence of a customized lipid NMR database along with user-friendly spectral analysis tools. We introduce a new two-dimensional (2D) NMR metabolite database “COLMAR Lipids” that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H heteronuclear single quantum coherence (HSQC) chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the (semi)automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, nonuniform sampling in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server, also in combination with mass spectrometry, as is demonstrated for Caco-2 cell and lung tissue cell extracts.

Significance We report that suppression of the serine protease elastase reshapes innate responses induced by injected vaccines containing alum adjuvant. This reprogramming improves the induction of protective antibodies in the bloodstream and stimulates innate signals, which support the development of antibody responses in mucosal tissues. Our findings identify elastase as the innate regulator that blunts the adjuvant activity of alum. They also demonstrate that vaccination via mucosal routes is not an absolute requirement for antibody responses in mucosal tissues and secretions. Supplementation of an alum-based vaccine containing SARS-CoV-2 spike protein subunit 1 as antigen increased anti–SARS-CoV-2 immunity in the blood and mucosal secretions in mice. Thus, this strategy could help in the development of future protein-based vaccines against SARS-CoV-2.

The integral membrane protein WecA mediates the transfer of N-acetylglucosamine (GlcNAc) 1-phosphate to undecaprenyl phosphate (Und-P) with the formation of a phosphodiester bond. Bacteria employ this reaction during the biosynthesis of enterobacterial common antigen as well as of many O-specific lipopolysaccharides (LPSs). Alignment of a number of prokaryotic and eukaryotic WecA-homologous sequences identified a number of conserved aspartic acid (D) residues in putative cytoplasmic loops II and III of the inner-membrane protein. Site-directed mutagenesis was used to study the role of the conserved residues D90, D91 (loop II), D156 and D159 (loop III). As controls, D35, D94 and D276 were also mutagenized. The resulting WecA derivatives were assessed for function by complementation analysis of O-antigen biosynthesis, by the ability to incorporate radiolabelled precursor to a biosynthetic intermediate, by detection of the terminal GlcNAc residue in LPS and by a tunicamycin competition assay. It was concluded from these analyses that the conserved aspartic acid residues are functionally important, but also that they participate differently in the transfer reaction. Based on these results it is proposed that D90 and D91 are important in forwarding the reaction product to the next biosynthetic step, while D156 and D159 are a part of the catalytic site of the enzyme.

HLA‐A, B and DR antigen frequencies were determined in three groups of periodontally diagnosed subjects: 49 patients with rapidly progressive periodontitis, 40 elderly subjects with minimal disease (considered as a resistant group) and 30 young subjects with minimal disease. The relative risk for HLA‐A9 (previously reported to be associated with periodontal disease) was 15.5. HLA‐A9 was present in 36.7% of the patients and 2.5% of the resistant group. HLA‐A10 showed a significantly increased incidence in the resistant group (30.0%) compared to a non‐periodontally diagnosed control population (9.0%), and was absent from the patient group. These findings provide additional evidence for the involvement of HLA‐A9 in susceptibility to periodontitis, and suggest that A10 may play a role in resistance to the disease.

Burkholderia cenocepacia infections in CF patients involve heightened inflammation, fatal sepsis, and high antibiotic resistance. Proinflammatory IL-1β secretion is important in airway inflammation and tissue damage. However, little is known about this pathway in macrophages upon B. cenocepacia infection. We report here that murine macrophages infected with B. cenocepacia K56-2 produce proinflammatory cytokine IL-1β in a TLR4 and caspase-1-mediated manner. We also determined that the OPS (O antigen) of B. cenocepacia LPS contributes to IL-1β production and pyroptotic cell death. Furthermore, we showed that the malfunction of the CFTR channel augmented IL-1β production upon B. cenocepacia infection of murine macrophages. Taken together, we identified eukaryotic and bacterial factors that contribute to inflammation during B. cenocepacia infection, which may aid in the design of novel approaches to control pulmonary inflammation.

Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

The apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) is an adaptor molecule that mediates inflammatory and apoptotic signals. Legionella pneumophila is an intracellular bacterium and the causative agent of Legionnaire's pneumonia. L. pneumophila is able to cause pneumonia in immuno-compromised humans but not in most inbred mice. Murine macrophages that lack the ability to activate caspase-1, such as caspase(-1-/-) and Nlrc4(-/-) allow L. pneumophila infection. This permissiveness is attributed mainly to the lack of active caspase-1 and the absence of its down stream substrates such as caspase-7. However, the role of Asc in control of L. pneumophila infection in mice is unclear. Here we show that caspase-1 is moderately activated in Asc(-/-) macrophages and that this limited activation is required and sufficient to restrict L. pneumophila growth. Moreover, Asc-independent activation of caspase-1 requires bacterial flagellin and is mainly detected in cellular extracts but not in culture supernatants. We also demonstrate that the depletion of Asc from permissive macrophages enhances bacterial growth by promoting L. pneumophila-mediated activation of the NF-κB pathway and decreasing caspase-3 activation. Taken together, our data demonstrate that L. pneumophila infection in murine macrophages is controlled by several mechanisms: Asc-independent activation of caspase-1 and Asc-dependent regulation of NF-κB and caspase-3 activation.

Members of the Burkholderia cepacia complex are virulent, multi-drug resistant pathogens that survive and replicate intracellularly in patients with cystic fibrosis (CF). We have discovered that B. cenocepacia cannot be cleared from CF macrophages due to defective autophagy, causing continued systemic inflammation and infection. Defective autophagy in CF is mediated through constitutive reactive oxygen species (ROS) activation of transglutaminase-2 (TG2), which causes the sequestration (accumulation) of essential autophagy initiating proteins. Cysteamine is a TG2 inhibitor and proteostasis regulator with the potential to restore autophagy. Therefore, we sought to examine the impact of cysteamine on CF macrophage autophagy and bacterial killing. Human peripheral blood monocyte-derived macrophages (MDMs) and alveolar macrophages were isolated from CF and non-CF donors. Macrophages were infected with clinical isolates of relevant CF pathogens. Cysteamine caused direct bacterial growth killing of live B. cenocepacia, B. multivorans, P. aeruginosa and MRSA in the absence of cells. Additionally, B. cenocepacia, B. multivorans, and P. aeruginosa invasion were significantly decreased in CF MDMs treated with cysteamine. Finally, cysteamine decreased TG2, p62, and beclin-1 accumulation in CF, leading to increased Burkholderia uptake into autophagosomes, increased macrophage CFTR expression, and decreased ROS and IL-1β production. Cysteamine has direct anti-bacterial growth killing and improves human CF macrophage autophagy resulting in increased macrophage-mediated bacterial clearance, decreased inflammation, and reduced constitutive ROS production. Thus, cysteamine may be an effective adjunct to antibiotic regimens in CF.

Article21 October 2019Open Access Transparent process Caspase-11 counteracts mitochondrial ROS-mediated clearance of Staphylococcus aureus in macrophages Kathrin Krause Kathrin Krause Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Kylene Daily Kylene Daily Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Shady Estfanous Shady Estfanous Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Kaitlin Hamilton Kaitlin Hamilton Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Asmaa Badr Asmaa Badr Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Arwa Abu Khweek Arwa Abu Khweek Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Department of Biology and Biochemistry, Birzeit University, Birzeit, West Bank, Palestine Search for more papers by this author Rana Hegazi Rana Hegazi Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Midhun NK Anne Midhun NK Anne Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Brett Klamer Brett Klamer Center for Biostatistics, Ohio State University, Columbus, OH, USA Search for more papers by this author Xiaoli Zhang Xiaoli Zhang Center for Biostatistics, Ohio State University, Columbus, OH, USA Search for more papers by this author Mikhail A Gavrilin Mikhail A Gavrilin Department of Internal Medicine, Ohio State University, Columbus, OH, USA Search for more papers by this author Vijay Pancholi Vijay Pancholi Department of Pathology, Ohio State University, Columbus, OH, USA Search for more papers by this author Amal O Amer Corresponding Author Amal O Amer [email protected] orcid.org/0000-0003-4487-7536 Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Kathrin Krause Kathrin Krause Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Kylene Daily Kylene Daily Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Shady Estfanous Shady Estfanous Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Kaitlin Hamilton Kaitlin Hamilton Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Asmaa Badr Asmaa Badr Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Arwa Abu Khweek Arwa Abu Khweek Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Department of Biology and Biochemistry, Birzeit University, Birzeit, West Bank, Palestine Search for more papers by this author Rana Hegazi Rana Hegazi Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Midhun NK Anne Midhun NK Anne Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Brett Klamer Brett Klamer Center for Biostatistics, Ohio State University, Columbus, OH, USA Search for more papers by this author Xiaoli Zhang Xiaoli Zhang Center for Biostatistics, Ohio State University, Columbus, OH, USA Search for more papers by this author Mikhail A Gavrilin Mikhail A Gavrilin Department of Internal Medicine, Ohio State University, Columbus, OH, USA Search for more papers by this author Vijay Pancholi Vijay Pancholi Department of Pathology, Ohio State University, Columbus, OH, USA Search for more papers by this author Amal O Amer Corresponding Author Amal O Amer [email protected] orcid.org/0000-0003-4487-7536 Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA Search for more papers by this author Author Information Kathrin Krause1, Kylene Daily1, Shady Estfanous1, Kaitlin Hamilton1, Asmaa Badr1, Arwa Abu Khweek1,2, Rana Hegazi1, Midhun NK Anne1, Brett Klamer3, Xiaoli Zhang3, Mikhail A Gavrilin4, Vijay Pancholi5 and Amal O Amer *,1 1Department of Microbial Infection and Immunity, Infectious Diseases Institute, Ohio State University, Columbus, OH, USA 2Department of Biology and Biochemistry, Birzeit University, Birzeit, West Bank, Palestine 3Center for Biostatistics, Ohio State University, Columbus, OH, USA 4Department of Internal Medicine, Ohio State University, Columbus, OH, USA 5Department of Pathology, Ohio State University, Columbus, OH, USA *Corresponding author. Tel: +1 614 247 1566; Fax: +1 614 292 9616; E-mail: [email protected] EMBO Reports (2019)20:e48109https://doi.org/10.15252/embr.201948109 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Methicillin-resistant Staphylococcus aureus (MRSA) is a growing health concern due to increasing resistance to antibiotics. As a facultative intracellular pathogen, MRSA is capable of persisting within professional phagocytes including macrophages. Here, we identify a role for CASP11 in facilitating MRSA survival within murine macrophages. We show that MRSA actively prevents the recruitment of mitochondria to the vicinity of the vacuoles they reside in to avoid intracellular demise. This process requires CASP11 since its deficiency allows increased association of MRSA-containing vacuoles with mitochondria. The induction of mitochondrial superoxide by antimycin A (Ant A) improves MRSA eradication in casp11−/− cells, where mitochondria remain in the vicinity of the bacterium. In WT macrophages, Ant A does not affect MRSA persistence. When mitochondrial dissociation is prevented by the actin depolymerizing agent cytochalasin D, Ant A effectively reduces MRSA numbers. Moreover, the absence of CASP11 leads to reduced cleavage of CASP1, IL-1β, and CASP7, as well as to reduced production of CXCL1/KC. Our study provides a new role for CASP11 in promoting the persistence of Gram-positive bacteria. Synopsis Staphylococcus aureus exploits caspase-11 to dissociate MRSA-containing vacuoles from mitochondria. Caspase-11 also promotes inflammasome activation and secretion of IL-1α, IL-1β, and CXCL1/KC in response to MRSA infection in macrophages. Methicillin-resistant Staphylococcus aureus (MRSA) exploits caspase-11 to promote its persistence. Caspase-11 is required for the dissociation of mitochondria from MRSA-containing vacuoles. Mitochondrial ROS contribute to MRSA clearance in the absence of caspase-11. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) refers to a group of Gram-positive cocci that have developed a resistance to most β-lactam antibiotics due to the expression of a penicillin-binding protein (PBP2a) 1. As an opportunistic pathogen, S. aureus exhibits a broad repertoire of virulence factors and can cause a variety of clinical manifestations, ranging from localized mild skin and soft tissue infections to severe invasive diseases with potentially fatal outcomes such as pneumonia, endocarditis, and sepsis 2, 3. Genetically diverse MRSA isolates can be found in healthcare facilities as well as communities all over the world, and resistances against antibiotics of last resort, such as vancomycin, have emerged 4. Alternative treatment strategies are therefore necessary to overcome multidrug-resistant MRSA infections. Inflammatory caspase-11/caspase-4 (CASP11) contributes to non-canonical NLRP3 inflammasome activation and subsequent inflammation 5. CASP11 is not expressed in healthy tissue unless induced by infection or other pathologic stress 6-9. Until recently, appreciated functions of CASP11 were the recognition of cytosolic LPS followed by the activation of CASP1, cleavage of gasdermin D (GSDMD), pro-inflammatory cytokine secretion, and cell death 5, 9-11. Additionally, the role of CASP11 is dependent on the infectious agent. While CASP11 deficiency has been shown to protect mice from LPS-induced endotoxemia due to reduced release of the inflammatory mediators IL-1α, IL-1β, and CXCL1/KC 5, 9, 12, the absence of CASP11 in the context of Gram-negative bacterial infections promotes bacterial replication and dissemination in mice 8, 9, 13, 14. Furthermore, CASP11 was shown to modulate the intracellular trafficking of pathogens, such as L. pneumophila and B. cenocepacia, leading to their degradation within lysosomes 8, 9, 13. In contrast, little is known about the role of CASP11 in the immune defense against Gram-positive bacteria. Recently, purified lipoteichoic acid (LTA), a cell wall component from Gram-positive bacteria, was reported to induce CASP11 activity via NLRP6 15. However, unlike mice infected with Gram-negative bacteria, mice deficient of CASP11 exhibit improved survival and efficient bacterial clearance in response to Gram-positive pathogens such as Listeria monocytogenes and S. aureus 15. The study by Hara et al demonstrated that increased production of IL-18 in WT mice impairs clearance of L. monocytogenes. However, during S. aureus infection, others have shown that the neutralization of IL-1β or IL-18 does not influence survival or pulmonary burdens of mice 16. Therefore, the mechanism behind reduced susceptibility of casp11−/− mice to Gram-positive bacteria is not completely understood and may vary according to the pathogen. Mitochondria are the main source for cellular ATP production. While mitochondrial reactive oxygen species (mtROS), such as superoxide and hydrogen peroxide, are generally considered byproducts of oxidative phosphorylation at the inner mitochondrial membrane, there is increasing evidence that mtROS can also augment the bactericidal capacity of macrophages 17, 18. The recruitment of mitochondria to phagosomes containing intracellular bacteria and subsequent elevated mtROS production have been shown to mediate the antibacterial response against Salmonella enterica serovar Typhimurium 17. Likewise, TNF-induced mtROS facilitate clearance of Mycobacterium tuberculosis from macrophages 19. Co-localization of internalized S. aureus with mitochondria was documented for both α-hemolysin (Hla)-deficient strains and in response to chemical inhibition of NLRP3, resulting in bacterial clearance by mtROS 16. Here, we propose a role for CASP11 in facilitating MRSA evasion from mtROS-mediated killing. We report that CASP11 deficiency leads to an increased association of MRSA with mitochondria, which is accompanied by elevated mtROS production and decreased inflammasome activation, thereby promoting more efficient clearance from murine macrophages. Antimycin A (Ant A) treatment, which inhibits complex III of the electron transport chain (ETC) thus raising mitochondrial superoxide production, further improves the bactericidal capacity of casp11−/− macrophages against MRSA. In WT macrophages, the inhibition of the actin cytoskeleton via cytochalasin D (Cyto D) prevents the dissociation of phagocytosed MRSA from mitochondria and hence restores Ant A-induced bacterial killing. Together, these results provide a novel role for CASP11 in the pathogenesis of Gram-positive bacteria. Results CASP11 contributes to MRSA-induced inflammasome activation in murine macrophages Staphylococcus aureus activates CASP1 through the NLRP3 inflammasome, leading to the secretion of IL-1β and cell death 20-23. While CASP11 was long believed to solely recognize cytosolic LPS from Gram-negative bacteria, leading to non-canonical NLRP3 inflammasome activation 6, 7, LTA derived from Gram-positive bacteria has been shown to promote CASP11 cleavage and activation 15. Since resting cells exhibit low levels of CASP11, we infected bone marrow-derived macrophages (BMDMs) from WT, casp11−/−, and casp1−/− mice with the MRSA strain USA300 at MOI 5:1 to determine whether intracellular MRSA stimulates CASP11 protein expression. At 24 h post-infection, we found a significant up-regulation of CASP11 in cell lysates of infected WT as well as casp1−/− macrophages (Fig 1A). To further elucidate whether CASP11 plays a role in inflammasome activation in response to MRSA, we evaluated cleavage of CASP1 and IL-1β in cell culture supernatants from WT and casp11−/− macrophages via Western blot analysis at 24 h post-infection (MOI 20:1). BMDMs from casp1−/− and gsdmd−/− mice were included to serve as a control for absent CASP1 or IL-1β secretion, respectively. In addition, we also added BMDMs deficient of the pseudokinase MLKL, which is a known effector protein for the necroptosis pathway. The inhibition of MLKL was demonstrated to reduce MRSA-induced IL-1β secretion from THP-1 macrophages 24, suggesting potential crosstalk between pyroptosis and necroptosis during MRSA infection. Compared to corresponding WT cells, cleavage and secretion of CASP1 and IL-1β were significantly reduced in supernatants from MRSA-infected casp11−/−, gsdmd−/−, and casp1−/− but not mlkl−/− macrophages (Fig 1B and C). Since CASP1 has been shown to activate CASP7 25, 26, we also analyzed the processing of CASP7 in response to MRSA. Similar to CASP1 and IL-1β, we found significantly lower amounts of CASP7 cleavage products in the supernatants from infected casp11−/−, gsdmd−/−, and casp1−/− but not mlkl−/− macrophages (Fig EV1A). In accordance with decreased secretion of CASP1, IL-1β, and CASP7, there were also lower amounts of the enzyme lactate dehydrogenase (LDH) in cell culture supernatants from MRSA-infected casp11−/−, gsdmd−/−, and casp1−/− macrophages when compared to WT or mlkl−/− cells (Fig EV1B). Interestingly, gsdmd−/− macrophages demonstrated mildly increased levels of secreted IL-1β and CASP7 compared to their casp11−/− and casp1−/− counterparts (Fig 1B), suggesting GSDMD-independent mechanisms contributing to the secretion of cleaved IL-1β and CASP7. Reduced secretion of IL-1α and CXCL1/KC was found only in casp11−/− macrophages (Fig 1C). We previously reported decreased CXCL1/KC production in casp11−/− macrophages and mice in response to B. cenocepacia infection 9. Here, our data with MRSA support the idea of a general defect in CXCL1/KC production in the absence of CASP11. No difference in the secretion of the inflammasome independent cytokine TNF could be observed among all five macrophage genotypes. Together, these results demonstrate that CASP11 but not MLKL contributes to MRSA-induced activation of CASP1 and subsequent processing of IL-1β and CASP7 in murine macrophages. Figure 1. CASP11 contributes to MRSA-induced inflammasome activation in murine macrophages Immunoblot analysis of CASP11 from WT, casp11−/−, and casp1−/− BMDMs infected with MRSA (MOI 5:1) at 24 h post-infection. Densitometry analysis represents mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using two-way ANOVA. **P ≤ 0.01, NT = no treatment. Immunoblot analysis of cleaved CASP1 and IL-1β in supernatants from WT, casp11−/−, gsdmd−/−, casp1−/−, and mlkl−/− BMDMs infected with MRSA (MOI 20:1) at 24 h post-infection. Densitometry analysis represents mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using two-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, NT = no treatment. Cytokine release from WT, casp11−/−, gsdmd−/−, casp1−/−, and mlkl−/− BMDMs infected with MRSA (MOI 20:1) at 24 h post-infection. Data represent mean ± SEM (n = 8 biological replicates). Statistical analysis was performed using one-way ANOVA. **P ≤ 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. CASP11 promotes MRSA-induced inflammasome activation and MRSA persistence in macrophages Immunoblot analysis of cleaved CASP7 in supernatants from WT, casp11−/−, gsdmd−/−, casp1−/−, and mlkl−/− BMDMs infected with MRSA (MOI 20:1) at 24 h post-infection. Densitometry analysis represents mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using two-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, NT = no treatment. MRSA-induced LDH released in supernatants from WT, casp11−/−, gsdmd−/−, casp1−/−, and mlkl−/− BMDMs at 24 h post-infection (MOI 20:1). Data represent mean ± SEM (n = 8 biological replicates). Statistical analysis was performed using one-way ANOVA. *P ≤ 0.05. Intracellular CFU of MRSA (MOI 5:1) in WT and casp11−/− BMDMs at 1 h post-infection. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using two-tailed Student's t-test. Intracellular survival of MRSA (MOI 5:1) in WT and casp11−/− BMDMs. Data represent mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using a linear mixed effects model. Intracellular survival of MRSA (MOI 0.5:1 and 20:1) in WT and casp11−/− BMDMs. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using a linear mixed effects model. ***P ≤ 0.001. Download figure Download PowerPoint Human CASP4 and CASP5, homologs of murine CASP11, are involved in IL-1β secretion and cell death induced by MRSA in THP-1 macrophages To determine whether CASP4 and CASP5, the human homologs of murine CASP11, contribute to MRSA-induced inflammasome activity in human THP-1 macrophages, we used corresponding knockout cells generated using the CRISPR/Cas system (a generous gift of Dr. Seth Masters, Walter and Eliza Hall Institute of Medical Research, Australia) 27. Single CASP1−/− and Cas9 THP-1 cells (from Dr. Masters) were included as controls. The CRISPR system was induced by incubating the cells with doxycycline. Then, THP-1 cells were differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA) and infected with MRSA at MOI 5:1 for 24 h. As compared to Cas9 control cells, cleavage products of IL-1β and CASP1 were significantly reduced in the supernatants of infected CASP4/5−/− cells (Fig 2A). CASP4/5−/− cells also exhibited reduced processing of pro-CASP1 in cell lysates (Fig 2B) and a decreased release of LDH in response to MRSA (Fig 2C). In addition, the absence of both CASP4 and CASP5 resulted in lower amounts of IL-1α and IL-8 (human homolog of murine CXCL1/KC) in the supernatants of infected cells (Fig 2D). Similar to murine macrophages, we observed no difference in the release of TNF among all 5 genotypes. These results thus indicate that, similar to murine CASP11, both human CASP4 and CASP5 contribute to MRSA-induced cleavage of CASP1 and IL-1β and subsequent cell death. Figure 2. CASP4/5 mediate MRSA-induced CASP1 activation and IL-1β release in human THP-1 macrophages Immunoblot analysis of cleaved IL-1β and CASP1 (p20) in supernatants from Cas9 control, CASP4−/−, CASP5−/−, CASP4/5−/−, and CASP1−/− THP-1 cells infected with MRSA at 24 h post-infection. Densitometry analysis represents mean ± SEM (n = 4 biological replicates). Statistical analysis was performed using two-way ANOVA. **P ≤ 0.01, ***P ≤ 0.001, NT = no treatment. Immunoblot analysis of pro-CASP1 (p45) in lysates from Cas9 control, CASP4−/−, CASP5−/−, CASP4/5−/−, and CASP1−/− THP-1 cells infected with MRSA at 24 h post-infection. Densitometry analysis represents mean ± SEM (n = 5 biological replicates). Statistical analysis was performed using two-way ANOVA. *P ≤ 0.05, ***P ≤ 0.001, NT = no treatment. MRSA-induced LDH released in supernatants from Cas9 control, CASP4−/−, CASP5−/−, CASP4/5−/−, and CASP1−/− THP-1 cells at 24 h post-infection. Data represent mean ± SEM (n = 15 biological replicates). Statistical analysis was performed using one-way ANOVA. *P ≤ 0.05, ***P ≤ 0.001. Cytokine release from Cas9 control, CASP4−/−, CASP5−/−, CASP4/5−/−, and CASP1−/− THP-1 cells infected with MRSA at 24 h post-infection. Data represent mean ± SEM (n = 10 biological replicates). Statistical analysis was performed using one-way ANOVA. *P ≤ 0.05, **P ≤ 0.01. Download figure Download PowerPoint CASP11 deficiency improves clearance of intracellular MRSA Although earlier reports indicated that CASP11 has no role in protection against L. monocytogenes infection 28, casp11−/− mice were recently demonstrated to have improved survival rates and lower bacterial loads in response to intravenously delivered L. monocytogenes as well as S. aureus 8325-4 15, indicating that the absence of CASP11 helps with the clearance of some Gram-positive bacteria. To investigate the impact of CASP11 on bacterial pulmonary loads, we intratracheally infected WT and casp11−/− mice with 2.5 × 108 CFU of MRSA and determined the bacterial burden at 96 h post-infection. We found significantly reduced numbers of MRSA in the lungs of casp11−/− mice as compared to corresponding WT mice (Fig 3A). Since MRSA was also shown to invade and persist in murine and human macrophages 29, 30, we next elucidated the role of CASP11 for the intracellular survival of MRSA. BMDMs from WT, casp11−/−, and casp1−/− mice were infected with MRSA, and intracellular bacterial numbers were determined at 2.5 and 24 h post-infection. As shown in Fig 3B, the intracellular load of MRSA was significantly lower at 24 h compared to 2.5 h for all three genotypes, indicating bacterial clearance. However, compared to WT or casp1−/− cells, the reduction of intracellular MRSA was more pronounced in macrophages lacking CASP11. LDH release was similar between all three genotypes (Fig 3C). No difference in the intracellular burden of MRSA in WT and casp11−/− macrophages could be observed at earlier time points (Fig EV1C and D). Testing different MOIs of 0.5:1 and 20:1 revealed increased restriction of MRSA at 24 h post-infection in the absence of CASP11 as well (Fig EV1E). In contrast, while the inhibition of NLRP3 was reported to promote clearance of MRSA from human macrophages 16, we did not find improved killing of MRSA in nlrp3−/− BMDMs (Fig 3D). Since GSDMD is directly cleaved by CASP11, we also determined intracellular survival of MRSA in gsdmd−/− macrophages. Yet, no difference in intracellular bacterial numbers could be found between WT and gsdmd−/− macrophages (Fig 3E). Together, these in vivo and in vitro data suggest that the macrophage response against MRSA is more effective in the absence of CASP11. Figure 3. CASP11 deficiency promotes MRSA clearance in vitro and in vivo In vivo CFUs from lungs of WT and casp11−/− mice intratracheally infected with MRSA (2.5 × 108 CFUs/mouse) at 96 h post-infection (n = 4 biological replicates). Boxplot with whiskers from minimum to maximum. Horizontal bands represent the median, and “+” represents the mean. Statistical analysis was performed using two-tailed Student's t-test. *P ≤ 0.05. Intracellular survival of MRSA in WT, casp11−/−, and casp1−/− BMDMs (MOI 5:1). Data represent mean ± SEM (n = 12 biological replicates). Statistical analysis was performed using a linear mixed effects model. **P ≤ 0.01, ***P ≤ 0.001. MRSA-induced LDH release in supernatants from WT, casp11−/−, and casp1−/− BMDMs (MOI 5:1) at 24 h post-infection. Data represent mean ± SEM (n = 6 biological replicates). Statistical analysis was performed using one-way ANOVA. Intracellular survival of MRSA in WT and nlrp3−/− BMDMs (MOI 5:1). Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using a linear mixed effects model. ***P ≤ 0.001. Intracellular survival of MRSA in WT and gsdmd−/− BMDMs (MOI 5:1). Data represent mean ± SEM (n = 6 biological replicates). Statistical analysis was performed using a linear mixed effects model. ***P ≤ 0.001. Download figure Download PowerPoint CASP11 deficiency facilitates MRSA clearance through mtROS It has been shown that mitochondrial ROS production drives the bactericidal activity of macrophages against MRSA 31 and that Hla-induced NLRP3 inflammasome activation negatively affects the association of phagosomes containing MRSA with mitochondria 16. To test whether mitochondria play a role in the clearance of MRSA in the absence of CASP11, we analyzed co-localization events between MRSA and mitochondria either stained with MitoTracker Deep Red or an anti-Tom20 antibody in WT and casp11−/− macrophages. As indicated in Fig 4A–D, casp11−/− macrophages display a higher percentage of bacteria co-localized with MitoTracker or Tom20 than corresponding WT cells. Electron microscopy revealed bacteria in proximity to mitochondria in casp11−/− but not WT macrophages (Fig 4E). Additionally, to elucidate whether only physical proximity to the mitochondria allows bacterial killing or whether mtROS production itself is increased in casp11−/− macrophages, we measured mitochondrial superoxide production using the MitoSOX Red reagent. Ant A, an inhibitor of complex III of the mitochondrial ETC, served as positive control for superoxide induction, and either the general antioxidant N-acetylcysteine (NAC) or the mtROS-quenching agent MitoQ was used to inhibit mtROS production. The chemicals had no direct bactericidal activity against MRSA (Fig EV2A and B). Compared to non-infected control cells, MRSA suppressed superoxide levels in WT macrophages (Fig 5A). Macrophages lacking CASP11 exhibited higher MitoSOX fluorescence in response to MRSA than corresponding WT cells. The addition of Ant A led to a marked increase in MitoSOX fluorescence in both non-infected and MRSA-infected cells. These results suggest that mtROS production was increased in the absence of CASP11. To determine whether increased mtROS production triggered by Ant A further improves bacterial clearance of intracellular MRSA, we treated MRSA-infected WT and casp11−/− macrophages with Ant A for 24 h and enumerated intracellular bacterial numbers. In contrast to WT cells, wherein no effect could be detected, Ant A treatment significantly reduced the burden of intracellular MRSA in casp11−/− macrophages (Fig 5B). LDH release was significantly increased in response to Ant A, yet no difference in Ant A-induced cytotoxicity could be found between WT and casp11−/− macrophages (Fig EV2C). There was also no increased extracellular bacterial growth in the presence of Ant A, indicating that gentamicin exclusion was still effective (Fig EV2D). In addition, treatment with the mtROS scavenger MitoQ elevated bacterial numbers in casp11−/− but not WT macrophages (Fig EV2E). This suggests that enhanced proximity to mitochondria contributes to MRSA elimination in response to Ant A-induced mitochondrial superoxide generation in casp11−/− macrophages. Figure 4. Association of the MRSA-containing vacuole with mitochondria is increased in casp11−/− macrophages MitoTracker Deep Red immunofluorescence assay of MRSA-infected WT and casp11−/− BMDMs at 4 h post-infection. White arrows indicate co-localization of MRSA with MitoTracker. Tom20 immunofluorescence assay of MRSA-infected WT and casp11−/− BMDMs at 4 h post-infection. White arrows indicate co-localization of MRSA with Tom20. Quantification of MRSA co-localized with MitoTracker Deep Red. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using two-tailed Student's t-test. *P ≤ 0.05. Quantification of MRSA co-localized with Tom20. Data represent mean ± SEM (n = 3 biological replicates). Statistical analysis was performed using two-tailed Student's t-test. *P ≤ 0.05. Qualitative transmission electron microscopy images of MRSA-infected WT and casp11−/− macrophages at 4 h post-infection. Yellow arrows indicate mitochondria. Data information: Scale bars, 10 μm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Role of inhibitors affecting mtROS production, autophagy, and F-actin polymerization on MRSA growth and macrophage viability MRSA growth in TSB in the presence of Ant A, FCCP, Cyto D, BafA1, rapamycin, and wortmannin (n = 3 biological replicates). Statistical analysis was performed using two-way ANOVA. *P ≤ 0.05, ***P ≤ 0.001, NT = no treatment. MRSA growth in TSB in the presence of NAC and MitoQ (n = 9 biological replicates). Statistical analysis was performed using two-way ANOVA. NT = no treatment. MRSA-induced LDH release in supernatants from WT and casp11−/− BMDMs (MOI 5:1) treated with Ant A or Cyto D at 24 h post-infection (n = 8 biological replicates). Statistical analysis was performed using two-way ANOVA. **P ≤ 0.01. Extracellular growth of MRSA in cell culture supernatants from WT and casp11−/− BMDMs (MOI 5:1) treated with Ant A or Cyto D at 24 h post-infection (n = 8 biological replicates). Statistical analysis was performed using two-way ANOVA. NT = no treatment. Intracellular CFU of MRSA in WT and casp11−/− macrophages treated with MitoQ at 24 h post-infection (MOI 5:1). Data represent mean ± SEM (n = 8 biological replicates

Cystic fibrosis (CF) human and mouse macrophages are defective in their ability to clear bacteria such as Burkholderia cenocepacia . The autophagy process in CF (F508del) macrophages is halted, and the underlying mechanism remains unclear. Furthermore, the role of CFTR in maintaining the acidification of endosomal and lysosomal compartments in CF cells has been a subject of debate. Using 3D reconstruction of z-stack confocal images, we show that CFTR is recruited to LC3-labeled autophagosomes harboring B. cenocepacia. Using several complementary approaches, we report that CF macrophages display defective lysosomal acidification and degradative function for cargos destined to autophagosomes, whereas non-autophagosomal cargos are effectively degraded within acidic compartments. Notably, treatment of CF macrophages with CFTR modulators (tezacaftor/ivacaftor) improved the autophagy flux, lysosomal acidification and function, and bacterial clearance. In addition, CFTR modulators improved CFTR function as demonstrated by patch-clamp. In conclusion, CFTR regulates the acidification of a specific subset of lysosomes that specifically fuse with autophagosomes. Therefore, our study describes a new biological location and function for CFTR in autophago-lysosomes and clarifies the long-standing discrepancies in the field.

The Gram-negative intracellular pathogen Francisella tularensis is known for its ability to dampen host immune responses. We recently performed a microarray analysis comparing human monocyte responses to the highly virulent F. tularensis tularensis Schu S4 strain (F.t.) versus the less virulent F. tularensis novicida (F.n.).(1) Many groups of genes were affected, including those involved with autophagy and with the regulation of autophagy. Here, we discuss the implications in the context of Francisella virulence and host cell response, then conclude with potential future experiments.

Abstract Purpose: Activation of Toll-like receptors (TLR) 7 and 8 by engineered agonists has been shown to aid in combating viruses and tumors. Here, we wished to test the effect of TLR7/8 activation on monocyte Fcγ receptor (FcγR) function, as they are critical mediators of antibody therapy. Experimental Design: The effect of the TLR7/8 agonist R-848 on cytokine production and antibody-dependent cellular cytotoxicity by human peripheral blood monocytes was tested. Affymetrix microarrays were done to examine genomewide transcriptional responses of monocytes to R-848 and Western blots were done to measure protein levels of FcγR. Murine bone marrow–derived macrophages from WT and knockout mice were examined to determine the downstream pathway involved with regulating FcγR expression. The efficacy of R-848 as an adjuvant for antibody therapy was tested using a CT26-HER2/neu solid tumor model. Results: Overnight incubation with R-848 increased FcγR-mediated cytokine production and antibody-dependent cellular cytotoxicity in human peripheral blood monocytes. Expression of FcγRI, FcγRIIa, and the common γ-subunit was increased. Surprisingly, expression of the inhibitory FcγRIIb was almost completely abolished. In bone marrow–derived macrophage, this required TLR7 and MyD88, as R-848 did not increase expression of the γ-subunit in TLR7−/− nor MyD88−/− cells. In a mouse solid tumor model, R-848 treatment superadditively enhanced the effects of antitumor antibody. Conclusions: These results show an as-yet-undiscovered regulatory and functional link between the TLR7/8 and FcγR pathways. This suggests that TLR7/8 agonists may be especially beneficial during antibody therapy. Clin Cancer Res; 16(7); 2065–75. ©2010 AACR.

Francisella tularensis infects macrophages and escapes phago-lysosomal fusion to replicate within the host cytosol, resulting in host cell apoptosis. Here we show that the Fas-mediated death pathway is activated in infected cells and correlates with escape of the bacterium from the phagosome and the bacterial burden. Our studies also demonstrate that constitutive activation of Akt, or deletion of SHIP, promotes phago-lysosomal fusion and limits bacterial burden in the host cytosol, and the subsequent induction of Fas expression and cell death. Finally, we show that phagosomal escape/intracellular bacterial burden regulate activation of the transcription factors sp1/sp3, leading to Fas expression and cell death. These data identify for the first time host cell signaling pathways that regulate the phagosomal escape of Francisella, leading to the induction of Fas and subsequent host cell death.

Legionella pneumophila has become a model system to decipher the non-apoptotic functions of caspases and their role in immunity. In permissive cells, the L. pneumophila-containing vacuole evades endosomal traffic and is remodelled by the endoplasmic reticulum. Evasion of the endosomes is mediated by the Dot/Icm type IV secretion system. Upon L. pneumophila infection of genetically restrictive cells such as wild-type (WT) C57Bl/6J murine macrophages, flagellin is sensed by the NOD-like receptor Nlrc4 leading to caspase-1 activation by the inflammasome complex. Then, caspase-7 is activated downstream of the Nlrc4 inflammasome, promoting non-apoptotic functions such as L. pneumophila-containing phagosome maturation and bacterial degradation. Interestingly, caspase-3 is activated in permissive cells during early stages of infection. However, caspase-3 activation does not lead to apoptosis until late stages of infection because it is associated with potent Dot/Icm-mediated anti-apoptotic stimuli that render the infected cells resistant to external apoptotic inducers. Therefore, the role of caspase-1 and non-apoptotic functions of executioner caspases are temporally and spatially modulated during infection by L. pneumophila, which determine permissiveness to intracellular bacterial proliferation. This review will examine the novel activation pathways of caspases by L. pneumophila and discuss their role in genetic restriction and permissiveness to infection.

Nlrc4 is a member of the Nod-like receptors (NLRs), a family of cytosolic receptors involved in sensing bacterial molecules. NLRs are a group of proteins containing spans of leucine-rich repeats that senses bacterial factors within the eukaryotic cytosol. The recognition of bacterial factors provokes the formation of the inflammasome complex which includes specific NLRs. The inflammasome is responsible for caspase-1 activation which leads to the cleavage and maturation of inflammatory cytokines such as IL-1β and IL-18. Nlrc4 was considered to be a devoted flagellin sensor in eukaryotic cells. However, studies using a variety of pathogens such as Salmonella, Legionella, Shigella and Pseudomonas at high bacterial burdens revealed that Nlrc4 can mediate caspase-1 activation independent of bacterial flagellin. On the other hand, new reports showed that Nlrc4 can restrict bacterial infection independently of caspase-1. Therefore, Nlrc4 maybe involved in sensing more than one bacterial molecule and may participate in several immune complexes.

EDITORIAL article Front. Cell. Infect. Microbiol., 19 June 2014Sec. Molecular Bacterial Pathogenesis Volume 4 - 2014 | https://doi.org/10.3389/fcimb.2014.00083

As the eighth leading cause of annual mortality in the USA, influenza A viruses are a major public health concern. In 20% of patients, severe influenza progresses to acute lung injury (ALI). However, pathophysiological mechanisms underlying ALI development are poorly defined. We reported that, unlike wild-type (WT) C57BL/6 controls, influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator (HETs) did not develop ALI. This effect was associated with higher IL-6 and alveolar macrophages (AMs) at 6 days postinfection (d.p.i.) in HET bronchoalveolar lavage fluid (BALF). In the present study, we found that HET AMs were an important source of IL-6 at 6 d.p.i. Infection also induced TGF-β production by HET but not WT mice at 2 d.p.i. TGF-β neutralization at 2 d.p.i. (TGF-N) significantly reduced BALF IL-6 in HETs at 6 d.p.i. Neither TGF-N nor IL-6 neutralization at 4 d.p.i. (IL-6-N) altered postinfection weight loss or viral replication in either mouse strain. However, both treatments increased influenza A virus-induced hypoxemia, pulmonary edema, and lung dysfunction in HETs to WT levels at 6 d.p.i. TGF-N and IL-6-N did not affect BALF AM and neutrophil numbers but attenuated the CXCL-1/keratinocyte chemokine response in both strains and reduced IFN-γ production in WT mice. Finally, bone marrow transfer experiments showed that HET stromal and myeloid cells are both required for protection from ALI in HETs. These findings indicate that TGF-β-dependent production of IL-6 by AMs later in infection prevents ALI development in influenza A virus-infected HET mice.

IntroductionCystic fibrosis (CF) is a multi-organ disorder characterized by chronic sino-pulmonary infections and inflammation. Many patients with CF suffer from repeated pulmonary exacerbations that are predictors of worsened long-term morbidity and mortality. There are no reliable markers that associate with the onset or progression of an exacerbation or pulmonary deterioration. Previously, we found that the Mirc1/Mir17–92a cluster which is comprised of 6 microRNAs (Mirs) is highly expressed in CF mice and negatively regulates autophagy which in turn improves CF transmembrane conductance regulator (CFTR) function. Therefore, here we sought to examine the expression of individual Mirs within the Mirc1/Mir17–92 cluster in human cells and biological fluids and determine their role as biomarkers of pulmonary exacerbations and response to treatment.MethodsMirc1/Mir17–92 cluster expression was measured in human CF and non-CF plasma, blood-derived neutrophils, and sputum samples. Values were correlated with pulmonary function, exacerbations and use of CFTR modulators.ResultsMirc1/Mir17–92 cluster expression was not significantly elevated in CF neutrophils nor plasma when compared to the non-CF cohort. Cluster expression in CF sputum was significantly higher than its expression in plasma. Elevated CF sputum Mirc1/Mir17–92 cluster expression positively correlated with pulmonary exacerbations and negatively correlated with lung function. Patients with CF undergoing treatment with the CFTR modulator Ivacaftor/Lumacaftor did not demonstrate significant change in the expression Mirc1/Mir17–92 cluster after six months of treatment.ConclusionsMirc1/Mir17–92 cluster expression is a promising biomarker of respiratory status in patients with CF including pulmonary exacerbation.

L. pneumophila is the causative agent of Legionnaires' disease, a human illness characterized by severe pneumonia. In contrast to those derived from humans, macrophages derived from most mouse strains restrict L. pneumophila replication. The restriction of L. pneumophila replication has been shown to require bacterial flagellin, a component of the type IV secretion system as well as the cytosolic NOD-like receptor (NLR) Nlrc4/ Ipaf. These events lead to caspase-1 activation which, in turn, activates caspase-7. Following caspase-7 activation, the phagosome-containing L. pneumophila fuses with the lysosome, resulting in the restriction of L. pneumophila growth. The LegS2 effector is injected by the type IV secretion system and functions as a sphingosine 1-phosphate lyase. It is homologous to the eukaryotic sphingosine lyase (SPL), an enzyme required in the terminal steps of sphingolipid metabolism. Herein, we show that mice Bone Marrow-Derived Macrophages (BMDMs) and human Monocyte-Derived Macrophages (hMDMs) are more permissive to L. pneumophila legS2 mutants than wild-type (WT) strains. This permissiveness to L. pneumophila legS2 is neither attributed to abolished caspase-1, caspase-7 or caspase-3 activation, nor due to the impairment of phagosome-lysosome fusion. Instead, an infection with the legS2 mutant resulted in the reduction of some inflammatory cytokines and their corresponding mRNA; this effect is mediated by the inhibition of the nuclear transcription factor kappa-B (NF-κB). Moreover, BMDMs infected with L. pneumophila legS2 mutant showed elongated mitochondria that resembles mitochondrial fusion. Therefore, the absence of LegS2 effector is associated with reduced NF-κB activation and atypical morphology of mitochondria.

Autophagy is a highly regulated, biological process that provides energy during periods of stress and starvation. This conserved process also acts as a defense mechanism and clears microbes from the host cell. Autophagy is impaired in Cystic Fibrosis (CF) patients and CF mice, as their cells exhibit low expression levels of essential autophagy molecules. The genetic disorder in CF is due to mutations in the cystic fibrosis transmembrane conductance regulator (cftr) gene that encodes for a chloride channel. CF patients are particularly prone to infection by pathogens that are otherwise cleared by autophagy in healthy immune cells including Burkholderia cenocepacia (B. cenocepacia). The objective of this study is to determine the mechanism underlying weak autophagic activity in CF macrophages and find therapeutic targets to correct it. Using reduced representation bisulfite sequencing (RRBS) to determine DNA methylation profile, we found that the promoter regions of Atg12 in CF macrophages are significantly more methylated than in the wild-type (WT) immune cells, accompanied by low protein expression. The natural product epigallocatechin-3-gallate (EGCG) significantly reduced the methylation of Atg12 promoter improving its expression. Accordingly, EGCG restricted B. cenocepacia replication within CF mice and their derived macrophages by improving autophagy and preventing dissemination. In addition, EGCG improved the function of CFTR protein. Altogether, utilizing RRBS for the first time in the CF field revealed a previously unrecognized mechanism for reduced autophagic activity in CF. Our data also offers a mechanism by which EGCG exerts its positive effects in CF.

Objectives Gout is the most prevalent inflammatory arthritis. To study the effects of regular physical activity and exercise intensity on inflammation and clinical outcome, we examined inflammatory pathogenesis in an acute model of murine gout and analyzed human gout patient clinical data as a function of physical activity. Methods NF-κB-luciferase reporter mice were organized into four groups and exercised at 0 m/min (non-exercise), 8 m/min (low-intensity), 11 m/min (moderate-intensity), and 15 m/min (high-intensity) for two weeks. Mice subsequently received intra-articular monosodium urate (MSU) crystal injections (0.5mg) and the inflammatory response was analyzed 15 hours later. Ankle swelling, NF-κB activity, histopathology, and tissue infiltration by macrophages and neutrophils were measured. Toll-like receptor (TLR)2 was quantified on peripheral monocytes/neutrophils by flow cytometry and both cytokines and chemokines were measured in serum or synovial aspirates. Clinical data and questionnaires accessing overall physical activity levels were collected from gout patients. Results Injection of MSU crystals produced a robust inflammatory response with increased ankle swelling, NF-κB activity, and synovial infiltration by macrophages and neutrophils. These effects were partially mitigated by low and moderate-intensity exercise. Furthermore, IL-1β was decreased at the site of MSU crystal injection, TLR2 expression on peripheral neutrophils was downregulated, and expression of CXCL1 in serum was suppressed with low and moderate-intensity exercise. Conversely, the high-intensity exercise group closely resembled the non-exercised control group by nearly all metrics of inflammation measured in this study. Physically active gout patients had significantly less flares/yr, decreased C-reactive protein (CRP) levels, and lower pain scores relative to physically inactive patients. Conclusions Regular, moderate physical activity can produce a quantifiable anti-inflammatory effect capable of partially mitigating the pathologic response induced by intra-articular MSU crystals by downregulating TLR2 expression on circulating neutrophils and suppressing systemic CXCL1. Low and moderate-intensity exercise produces this anti-inflammatory effect to varying degrees, while high-intensity exercise provides no significant difference in inflammation compared to non-exercising controls. Consistent with the animal model, gout patients with higher levels of physical activity have more favorable prognostic data. Collectively, these data suggest the need for further research and may be the foundation to a future paradigm-shift in conventional exercise recommendations provided by Rheumatologists to gout patients.

Pregnant women are highly susceptible to infection by the bacterial pathogen Listeria monocytogenes, leading to miscarriage, premature birth, and neonatal infection. L. monocytogenes is thought to breach the placental barrier by infecting trophoblasts at the maternal/fetal interface. However, the fate of L. monocytogenes within chorionic villi and how infection reaches the fetus are unsettled. Hofbauer cells (HBCs) are fetal placental macrophages and the only leukocytes residing in healthy chorionic villi, forming a last immune barrier protecting fetal blood from infection. Little is known about the HBCs' antimicrobial responses to pathogens. Here, we studied L. monocytogenes interaction with human primary HBCs. Remarkably, despite their M2 anti-inflammatory phenotype at basal state, HBCs phagocytose and kill non-pathogenic bacteria like Listeria innocua and display low susceptibility to infection by L. monocytogenes. However, L. monocytogenes can exploit HBCs to spread to surrounding placental cells. Transcriptomic analyses by RNA sequencing revealed that HBCs undergo pro-inflammatory reprogramming upon L. monocytogenes infection, similarly to macrophages stimulated by the potent M1-polarizing agents lipopolysaccharide (LPS)/interferon gamma (IFN-γ). Infected HBCs also express pro-inflammatory chemokines known to promote placental infiltration by maternal leukocytes. However, HBCs maintain the expression of a collection of tolerogenic genes and secretion of tolerogenic cytokines, consistent with their tissue homeostatic role in prevention of fetal rejection. In conclusion, we propose a previously unrecognized model in which HBCs promote the spreading of L. monocytogenes among placental cells and transition to a pro-inflammatory state likely to favor innate immune responses, while maintaining the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity. IMPORTANCE Infection of the placental/fetal unit by the facultative intracellular pathogen Listeria monocytogenes results in severe pregnancy complications. Hofbauer cells (HBCs) are fetal macrophages that play homeostatic anti-inflammatory functions in healthy placentas. HBCs are located in chorionic villi between the two cell barriers that protect fetal blood from infection: trophoblast cells at the maternal interface (in contact with maternal blood), and fetal endothelial cells at the fetal interface (in contact with fetal blood). As the only leukocytes residing in chorionic villi, HBCs form a critical immune barrier protecting the fetus from infection. Here, we show that although HBCs display low susceptibility to L. monocytogenes, the bacterium still replicates intracellularly and can spread to other placental and fetal cells. We propose that HBCs are permissive to L. monocytogenes transplacental propagation and can repolarize toward a pro-inflammatory phenotype upon infection. However, consistent with their placental homeostatic functions, repolarized HBCs maintain the expression of tolerogenic factors known to prevent maternal anti-fetal adaptive immunity, at least at early stages of infection.

WecA, an integral membrane protein that belongs to a family of polyisoprenyl phosphate N-acetylhexosamine-1-phosphate transferases, is required for the biosynthesis of O-specific LPS and enterobacterial common antigen in Escherichia coli and other enteric bacteria. WecA functions as an UDP-N-acetylglucosamine (GlcNAc):undecaprenyl-phosphate GlcNAc-1-phosphate transferase. A conserved short sequence motif (His-Ile-His-His; HIHH) and a conserved arginine were identified in WecA at positions 279-282 and 265, respectively. This region is located within a predicted cytosolic segment common to all bacterial homologues of WecA. Both HIHH279-282 and the Arg265 are reminiscent of the HIGH motif (His-Ile-Gly-His) and a nearby upstream lysine, which contribute to the three-dimensional architecture of the nucleotide-binding site among various enzymes displaying nucleotidyltransferase activity. Thus, it was hypothesized that these residues may play a role in the interaction of WecA with UDP-GlcNAc. Replacement of the entire HIHH motif by site-directed mutagenesis produced a protein that, when expressed in the E. coli wecA mutant MV501, did not complement the synthesis of O7 LPS. Membrane extracts containing the mutated protein failed to transfer UDP-GlcNAc into a lipid-rich fraction and to bind the UDP-GlcNAc analogue tunicamycin. Similar results were obtained by individually replacing the first histidine (H279) of the HIHH motif as well as the Arg265 residue. The functional importance of these residues is underscored by the high level of conservation of H279 and Arg265 among bacterial WecA homologues that utilize several different UDP-N-acetylhexosamine substrates.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious and often fatal form of pneumonia. The susceptibility to L. pneumophila arises from the ability of this intracellular pathogen to multiply in human alveolar macrophages and monocytes. L. pneumophila also replicates in several professional and non-professional phagocytic human-derived cell lines. With the exception of the A/J mouse strain, most mice strains are restrictive, thus they do not support L. pneumophila replication. Mice lacking the NOD-like receptor Nlrc4 or caspase-1 are also susceptible to L. pneumophila. On the other hand, in the susceptible human hosts, L. pneumophila utilizes several strategies to ensure intracellular replication and protect itself against the host immune system. Most of these strategies converge to prevent the fusion of the L. pneumophila phagosome with the lysosome, inhibiting host cell apoptosis, activating survival pathways, and sequestering essential nutrients for replication and pathogenesis. In this review, we summarize survival mechanisms employed by L. pneumophila to maintain its replication in human cells. In addition, we highlight different human-derived cell lines that support the multiplication of this intracellular bacterium. Therefore, these in vitro models can be applicable and are reproducible when investigating L. pneumophila/phagocyte interactions at the molecular and cellular levels in the human host.

Seasonal and pandemic influenza are significant public health concerns. Influenza stimulates respiratory epithelial Cl(-) secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). The purpose of this study was to determine the contribution of this effect to influenza pathogenesis in mice with reduced CFTR activity.C57BL/6-congenic mice heterozygous for the F508del CFTR mutation (HET) and wild-type (WT) controls were infected intranasally with 10 000 focus-forming units of influenza A/WSN/33 (H1N1) per mouse. Body weight, arterial O2 saturation, and heart rate were monitored daily. Pulmonary edema and lung function parameters were derived from ratios of wet weight to dry weight and the forced-oscillation technique, respectively. Levels of cytokines and chemokines in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay.Relative to WT mice, influenza virus-infected HET mice showed significantly delayed mortality, which was accompanied by attenuated hypoxemia, cardiopulmonary dysfunction, and pulmonary edema. However, viral replication and weight loss did not differ. The protective HET phenotype was correlated with exaggerated alveolar macrophage and interleukin 6 responses to infection and was abrogated by alveolar macrophage depletion, using clodronate liposomes.Reduced CFTR expression modulates the innate immune response to influenza and alters disease pathogenesis. CFTR-mediated Cl(-) secretion is therefore an important host determinant of disease, and CFTR inhibition may be of therapeutic benefit in influenza.

Bacterial infections of hip and knee implants are rare but devastating complications of orthopedic surgery. Despite a widespread appreciation of the considerable financial, physical, and emotional burden associated with the development of a prosthetic joint infection, the establishment of bacteria in the synovial joint remains poorly understood.

This Pillars of Immunology article is a commentary on “Cutting Edge: CATERPILLER: A large family of mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains,” a pivotal article written by J. A. Harton, M. W. Linhoff, J. Zhang, and J. P.-Y. Ting,” and published in The Journal of Immunology, in 2002. https://doi.org/10.4049/jimmunol.169.8.4088.

Abstract Alzheimer’s disease (AD) is the sixth leading cause of death in the USA. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release pro-inflammatory products such as IL-1β which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed reduced representation bisulfite sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed promotes the generation of IL-1β and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4 and CASP11, which is encoded by mouse Caspase-4 (5xFAD/ Casp4 −/− ). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-β (Aβ) and increased microglial production of IL-1β in 5xFAD mice. Utilizing RNA-sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1β from macrophages in response to cytosolic Aβ through cleavage of downstream effector Gasdermin D (GSDMD). Therefore, here we unravel the role for CASP11 and GSDMD in the generation of IL-1β in response to Aβ and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential DNA methylation in AD microglia contributes to the progression of AD pathobiology. Thus, we identify CASP4 as a potential target for immunotherapies for the treatment and prevention of AD.

ABSTRACT The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N -acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

Legionella pneumophila remains a major health concern, especially for hospitalized patients. L. pneumophila in the environment can survive extracellular or as protozoan parasite within amoeba. After human infection it efficiently replicates in alveolar macrophages without activating inflammasome assembly and cleavage of caspase-1. In contrast murine macrophages actively recognize intracellular L. pneumophila via inflammasome components which initiate pro-inflammatory cytokine secretion, phagosomal maturation and pyroptotic cell death thereby leading to bacterial restriction. During this process flagellin-dependent and -independent signaling pathways trigger the canonical as well as the non-canonical inflammasome. This review describes the current knowledge about L. pneumophila-induced inflammasome pathways in permissive and restrictive host cells.

Neuroinflammation and accumulation of Amyloid Beta (Aβ) accompanied by deterioration of special memory are hallmarks of Alzheimer's disease (AD). Effective preventative and treatment options for AD are still needed. Microglia in AD brains are characterized by elevated levels of microRNA-17 (miR-17), which is accompanied by defective autophagy, Aβ accumulation, and increased inflammatory cytokine production. However, the effect of targeting miR-17 on AD pathology and memory loss is not clear. To specifically inhibit miR-17 in microglia, we generated mannose-coated lipid nanoparticles (MLNPs) enclosing miR-17 antagomir (Anti-17 MLNPs), which are targeted to mannose receptors readily expressed on microglia. We used a 5XFAD mouse model (AD) that recapitulates many AD-related phenotypes observed in humans. Our results show that Anti-17 MLNPs, delivered to 5XFAD mice by intra-cisterna magna injection, specifically deliver Anti-17 to microglia in vivo and in vitro. Anti-17 MLNPs downregulated miR-17 expression in microglia but not in neurons, astrocytes, and oligodendrocytes. Anti-17 MLNPs attenuated inflammation, improved autophagy, and reduced Aβ burdens in the brains. Additionally, Anti-17 MLNPs reduced the deterioration in spatial memory and decreased anxiety-like behavior in 5XFAD mice. Therefore, targeting miR-17 using MLNPs is a viable strategy to prevent several AD pathologies. This selective targeting strategy delivers specific agents to microglia without the adverse off-target effects on other cell types. Additionally, this approach can be used to deliver other molecules to microglia and other immune cells in other organs.

Autophagy has emerged as a significant innate immune response to pathogens. Typically, autophagosomes deliver their contents to lysosomes for degradation. Some pathogens such as Salmonella enterica serovar Typhimurium succumb to autophagy and are transported to lysosomes for degradation. Yet, many professional pathogens, including Legionella pneumophila and Burkholderia cenocepacia, subvert this pathway exploiting autophagy to their advantage.

An examination was made of blastogenic response to phytohaemagglutinin (PHA) in HLA-B8 and/or DR3 positive subjects and B8/DR3 negative individuals. Both B8 and DR3 antigens were associated with a depression of the response at all three doses of PHA used. The possession of both these antigens did not lead to a further depression of the response.

Human lungs continuously handle various pollutants, microbes, and allergens. The pulmonary innate immune response eliminates most of these foreign particles while maintaining a sterile environment within the lung. This response is tightly regulated in order to minimize inflammation and protect the host. This review focuses on the major pulmonary innate immune components that respond to infectious agents, including alveolar macrophages and their major receptors and surfactant, and its ability to regulate the host response to certain infectious pathogens. Finally, potential therapeutic applications relevant to these innate immune determinants will be discussed.

Abstract Influenza virus activates cellular inflammasome pathways, which can be either beneficial or detrimental to infection outcomes. Here, we investigated the role of the inflammasome-activated pore-forming protein gasdermin D (GSDMD) during infection. Ablation of GSDMD in knockout (KO) mice significantly attenuated virus-induced weight loss, lung dysfunction, lung histopathology, and mortality compared with wild type (WT) mice, despite similar viral loads. Infected GSDMD KO mice exhibited decreased inflammatory gene signatures revealed by lung transcriptomics, which also implicated a diminished neutrophil response. Importantly, neutrophil depletion in infected WT mice recapitulated the reduced mortality and lung inflammation observed in GSDMD KO animals, while having no additional protective effects in GSDMD KOs. These findings reveal a new function for GSDMD in promoting lung neutrophil responses that amplify influenza virus-induced inflammation and pathogenesis. Targeting the GSDMD/neutrophil axis may provide a new therapeutic avenue for treating severe influenza.

The role of apoptosis-associated speck-Like protein (ASC) in the assembly of the inflammasome complex within macrophages has been elucidated in several studies. In this particular role, ASC functions as an adaptor protein by linking nod-like receptors (NLRs) and procaspase-1, thereby leading to the activation of caspase-1 to cleave inflammatory cytokines IL-1β and IL-18 and inducing pyroptosis. It has been noted that ASC maintains inflammasome-independent roles, including but not limited to controlling the expression of Dock2 and mitogen-activated protein kinases (MAPK/ERK2) and regulating the NF-κB pathway. This paper will emphasize the major roles of ASC during pathogen infection, the mechanisms by which it mediates inflammation, and discuss its more recently discovered functions.

Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages.Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients.We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia.The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

Marijuana consumption is on the rise in the US but the health benefits of cannabis smoking are controversial and the impact of cannabis components on lung homeostasis is not well-understood. Lung function requires a fine regulation of the ion channel CFTR, which is responsible for fluid homeostasis and mucocilliary clearance. The goal of this study was to assess the effect that exposure to Δ9-tetrahydrocannabinol (THC), the psychoactive substance present in marijuana, has on CFTR expression and function. Cultures of human bronchial epithelial cell line 16HBE14o- and primary human airway epithelial cells were exposed to THC. The expression of CFTR protein was determined by immunoblotting and CFTR function was measured using Ussing chambers. We also used specific pharmacological inhibitors of EGFR and ERK to determine the role of this pathway in THC-induced regulation of CFTR. THC decreased CFTR protein expression in primary human bronchial epithelial cells. This decrease was associated with reduced CFTR-mediated short-circuit currents. THC also induced activation of the ERK MAPK pathway via activation of EGFR. Inhibition of EGFR or MEK/ERK prevented THC-induced down regulation of CFTR protein expression. THC negatively regulates CFTR and this is mediated through the EGFR/ERK axis. This study provides the first evidence that THC present in marijuana reduces the expression and function of CFTR in airway epithelial cells.

The innate immune system is a critical component of host defense against invading microbial pathogens. It is responsible for mounting proper inflammatory and repair responses that contribute to the elimination of the invading pathogen and for instructing the adaptive immune system to develop a prolonged immunity against microbial pathogens. This is accomplished through the regulation of transcriptional and posttranslational programs that culminate in the production of proinflammatory cytokines and chemokines, the induction of type I and II interferon responses and autophagy responses, and the induction of programmed cell death modes that eliminate infected host cells and expose intracellular pathogens to surveillance by the immune system. This issue includes eight published papers which are discussing the following issues.

Abstract Background Autophagy is a conserved homeostatic cellular process for clearing dysfunctional organelles, pathogens, and protein aggregates. Neurons in patients with Alzheimer’s disease (AD) exhibit accumulation of immature autophagic vacuoles, which is thought to contribute to amyloid‐beta (Aβ) plaque build‐up and neurotoxicity. However, it is still unclear what causes autophagic dysfunction in neurons and how it contributes to disease progression. Moreover, the role of autophagy in microglia, the innate immune cell and phagocyte of the CNS, has not been explored. We hypothesized that autophagy in microglia in AD is dysfunctional, leading to the inability of microglia to effectively clear Aβ plaque protein aggregates. Method We purify microglia from adult mice of an AD mouse model (5xFAD – 5 familial mutations of AD). We are utilizing a novel method developed in our lab to quantify Aβ internalization and degradation which does not require radioactive or fluorophore labeling. Additionally, we are able to visualize Aβ autophagic trafficking utilizing microglia from a mouse with GFP+ LC3, a key autophagy protein involved in the elongation of the autophagosome. Lastly, to understand the components at play in autophagy processing of Aβ in microglia, we are able to reduce expression of various autophagy proteins and microRNAs which target autophagy utilizing a magnetic nanoparticle transfection reagent. Result Microglia from AD mice and age‐matched wild‐type littermates internalize the same amount of Aβ, but AD microglia do not efficiently degrade Aβ in vitro . When stimulating autophagy using rapamycin, degradation of Aβ in AD microglia improves. We are able to see that microglia traffic Aβ to an LC3+ autophagosome, and that this trafficking and subsequent Aβ‐degradation is dependent on the expression of various autophagy molecules. Furthermore, we have reduced expression of microRNAs which downregulate autophagy and are overexpressed in AD, and recovered the degradative capacity of AD microglia in vitro . Conclusion Together these results indicate that Aβ is degraded via autophagy and that autophagy is defective in AD microglia. Our projects in the lab are seeking to establish additional consequences beyond failure of Aβ‐clearance, such as inability to regulate expression of inflammatory cytokines, and mechanisms behind dysregulated autophagy, such as epigenetic changes.

Spontaneous proliferation of T cells, B cells and unseparated lymphocytes was studied in patients with severe periodontal disease and control subjects. In the patient group only, spontaneous lymphocyte proliferation was reduced, whereas B cell proliferation was enhanced. The findings offer further support for the existence of a disturbance in immune regulation in patients with severe periodontal disease.

Asthma is an inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, and bronchial hyper-responsiveness. House dust mites (HDM) are the most prevalent cause of allergic sensitization. Canonical and noncanonical inflammasomes are multiprotein complexes that assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). Murine caspase-11 engages the noncanonical inflammasome. We addressed the role of caspase-11 in mediating host responses to HDM and subsequent allergic inflammation using caspase-11-/- mice, which lack caspase-11 while express caspase-1. We found that HDM induce caspase-11 expression in vitro. The presence of IL-4 and IL-13 promote caspase-11 expression. Additionally, caspase-11-/- macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, HDM sensitization of caspase-11-/- mice resulted in similar levels of IgE responses and hypothermia in response to nasal HDM challenge compared to WT. However, analysis of cell numbers and cytokines in bronchiolar alveolar lavage fluid (BALF) and histopathology of representative lung segments demonstrate altered inflammatory responses and reduced neutrophilia in the airways of the caspase-11-/- mice. These findings indicate that caspase-11 regulates airway inflammation in response to HDM exposure.

Abstract SARS-CoV-2 is a worldwide health concern, and new treatment strategies are needed 1 . Targeting inflammatory innate immunity pathways holds therapeutic promise, but effective molecular targets remain elusive. Here, we show that human caspase-4 (CASP4), and its mouse homologue, caspase-11 (CASP11), are upregulated in SARS-CoV-2 infections, and that CASP4 expression correlates with severity of SARS-CoV-2 infection in humans. SARS-CoV-2-infected Casp11 -/- mice were protected from severe weight loss and lung pathology, including blood vessel damage, compared to wild-type (WT) and gasdermin-D knock out ( Gsdmd -/- ) mice. GSDMD is a downstream effector of CASP11 and CASP1. Notably, viral titers were similar in the three genotypes. Global transcriptomics of SARS-CoV-2-infected WT, Casp11 -/- and Gsdmd -/- lungs identified restrained expression of inflammatory molecules and altered neutrophil gene signatures in Casp11 -/- mice. We confirmed that protein levels of inflammatory mediators IL-1β, IL6, and CXCL1, and neutrophil functions, were reduced in Casp11 -/- lungs. Additionally, Casp11 -/- lungs accumulated less von Willebrand factor, a marker for endothelial damage, but expressed more Kruppel-Like Factor 2, a transcription factor that maintains vascular integrity. Overall, our results demonstrate that CASP4/11, promotes detrimental SARS-CoV-2-associated inflammation and coagulopathy, largely independently of GSDMD, identifying CASP4/11 as a promising drug target for treatment and prevention of severe COVID-19.

Plants are an important natural source of compounds used in cancer therapy. Pancratium maritimum contains potential anti-cancer agents such as alkaloids. In this study, we investigated the anti-proliferative effects of P. maritimum extracts on MDA-MB-231 human epithelial adenocarcinoma cell line and on normal lymphocytes in vitro.Leaves, flowers, roots, and bulbs of P. maritimum were collected and their contents were extracted and diluted to different concentrations that were applied on MDA-MB-231 cells and normal human lymphocytes cell in vitro for different intervals. Cells viability, proliferation, cell cycle distribution, apoptosis, and growth were evaluated by flow cytometry and microscopy. Parametric unpaired t-test was used to compare effects of plant extracts on treated cell cultures with untreated control cell cultures. IC50 was also calculated.P. maritimum extract had profound effects on MDA-MB-321 cells. It inhibited cell proliferation in a dose- and time-dependent manner. The IC50 values were 0.039, 0.035, and 0.026 mg/ml after 48, 72, and 96 hours of treatment with 0.1 mg/ml concentration of bulb extract, respectively. Those values were 0.051 and 0.03 mg/ml after 72 and 96 hours for root extract, respectively, and 0.048 mg/ml after 96 hours for flower extract. There were no significant effects of P. maritimum bulb extracts on normal lymphocytes proliferation.P. maritimum extract has anti-proliferative effects on MDA-MB-231 cell line in vitro. The effects imply the involvement of mechanisms that inhibits cell growth and arresting cells at S and G2/M phases. Cyclin B1, Bcl-2, and Ki67 expression was also affected.

Methods: 16 Pa strains from CF patients were selected based on (a) clinical laboratory sensitivity to ceftazidime (Cef ) and tobramycin (Tob); (b) biofilm formation in vitro; (c) sensitivity to the phage cocktail in conventional plaque assay.Bacteria were inoculated into 96 well plates and incubated static for 24 hours to allow adherent biofilms to form.Cef and Tob (both at 2 × MIC) were then applied together, with and without the further addition of a cocktail of four phages for a further 24 hours.A fluorescent resazurin assay was used to estimate cell viability and wells were stained with crystal violet to assess biomass.Results: Treatments were described as percent reduction in cell viability and biomass compared to negative control wells (no treatment).Compared with the dual antibiotics, the phage cocktail resulted in a further mean 1.3fold reduction in cell viability ( p < 0.001) and 1.7-fold reduction in biomass ( p < 0.001).Notably, in 4 biofilms which were largely resistant to the antibiotic mix, the addition of phage conferred a 50 ± 15% reduction in cell viability and 60 ± 12% reduction in biomass (95% CI).Conclusion: Our results show a convincing reduction in pre-formed Pa biofilms when a lytic phage cocktail was added to standard antibiotic therapy.The most striking benefits seen were in the 4 clinical strains with the least response to conventional antibiotics.In an era of increased bacterial resistance, bacteriophage is a promising adjunct to standard clinical care.

Mammalian cells are frequently exposed to mechanical and biochemical stressors resulting in plasma membrane injuries. Repair mechanisms reseal the plasma membrane to restore homeostasis and prevent cell death. In the present work, a silencing RNA screen was performed to uncover plasma membrane repair mechanisms of cells exposed to a pore-forming toxin (listeriolysin O). This screen identified molecules previously known to repair the injured plasma membrane such as annexin A2 (ANXA2) as well as novel plasma membrane repair candidate proteins. Of the novel candidates, we focused on septin 7 (SEPT7) because the septins are an important family of conserved eukaryotic cytoskeletal proteins. Using diverse experimental approaches, we established for the first time that SEPT7 plays a general role in plasma membrane repair of cells perforated by pore-forming toxins and mechanical wounding. Remarkably, upon cell injury, the septin cytoskeleton is extensively redistributed in a Ca 2+ -dependent fashion, a hallmark of plasma membrane repair machineries. The septins reorganize into subplasmalemmal domains arranged as knob and loop (or ring) structures containing F-actin, myosin II, and annexin A2 (ANXA2) and protrude from the cell surface. Importantly, the formation of these domains correlates with the plasma membrane repair efficiency. Super-resolution microscopy shows that septins and actin are arranged in intertwined filaments associated with ANXA2. Silencing SEPT7 expression prevented the formation of the F-actin/myosin II/ANXA2 domains, however, silencing expression of ANXA2 had no observable effect on their formation. These results highlight the key structural role of the septins in remodeling the plasma membrane and in the recruitment of the repair molecule ANXA2. Collectively, our data support a novel model in which the septin cytoskeleton acts as a scaffold to promote the formation of plasma membrane repair domains containing contractile F-actin and annexin A2.

Abstract Asthma is a long-term recurring inflammatory lung disorder characterized by mucus hypersecretion, cellular infiltration, chronic airway inflammation and bronchial hyper-responsiveness. House dust mites (HDM), such as Dermatophagoides pteronyssinus, are an unsurpassed cause of atopic sensitization and the major causes of allergic asthma worldwide. The multiprotein complexness of the canonical and noncanonical inflammasomes assemble in response to pathogen or danger-associated molecular patterns (PAMPs or DAMPs). We have shown that caspase-11 expression is induced in vitro in response to HDM. Treatment with TH2 cytokines such as IL-4 and IL-13 mediates caspase-11 expression. Additionally, caspase-11−/−macrophages show reduced release of IL-6, IL-12, and KC, and express lower levels of costimulatory molecules (e.g., CD40, CD86 and MHCII) in response to HDM stimulation. Notably, similar level of IgE responses and similar degree of hypothermia in response to nasal HDM challenge were seen in WT and caspase-11−/−mice following HDM sensitization. However, altered inflammatory responses and reduced neutrophilia in bronchiolar alveolar lavage fluid (BALF) and in representative histopathological lung tissues were seen in the caspase-11−/−mice. Therefore, caspase-11 is a regulator of airway inflammation in response to HDM exposure.

Alzheimer's Disease (AD) is the 6th leading cause of death in the US. It is established that neuroinflammation contributes to the synaptic loss, neuronal death, and symptomatic decline of AD patients. Accumulating evidence suggests a critical role for microglia, innate immune phagocytes of the brain. For instance, microglia release proinflammatory products such as IL-1β which is highly implicated in AD pathobiology. The mechanisms underlying the transition of microglia to proinflammatory promoters of AD remain largely unknown. To address this gap, we performed Reduced Representation Bisulfite Sequencing (RRBS) to profile global DNA methylation changes in human AD brains compared to no disease controls. We identified differential DNA methylation of CASPASE-4 (CASP4), which when expressed, can be involved in generation of IL-1β and is predominantly expressed in immune cells. DNA upstream of the CASP4 transcription start site was hypomethylated in human AD brains, which was correlated with increased expression of CASP4. Furthermore, microglia from a mouse model of AD (5xFAD) express increased levels of CASP4 compared to wild-type (WT) mice. To study the role of CASP4 in AD, we developed a novel mouse model of AD lacking the mouse ortholog of CASP4, CASP11, which is encoded by mouse Caspase-4 (5xFAD/Casp4-/-). The expression of CASP11 was associated with increased accumulation of pathologic protein aggregate amyloid-β (Aβ) and increased microglial production of IL-1β in 5xFAD mice. Utilizing RNA sequencing, we determined that CASP11 promotes unique transcriptomic phenotypes in 5xFAD mouse brains, including alterations of neuroinflammatory and chemokine signaling pathways. Notably, in vitro, CASP11 promoted generation of IL-1β from macrophages in response to cytosolic Aβ through cleavage of downstream effector Gasdermin D (G SDMD). We describe a role for CASP11 and GSDMD in the generation of IL-1β in response to Aβ and the progression of pathologic inflammation in AD. Overall, our results demonstrate that overexpression of CASP4 due to differential methylation in AD microglia contributes to the progression of AD pathobiology, thus identifying CASP4 as a potential target for immunotherapies for the treatment of AD.

As SARS-CoV-2 variants of concern (VoCs) that evade immunity continue to emerge, next-generation adaptable COVID-19 vaccines which protect the respiratory tract and provide broader, more effective, and durable protection are urgently needed. Here, we have developed one such approach, a highly efficacious, intranasally delivered, trivalent measles-mumps-SARS-CoV-2 spike (S) protein (MMS) vaccine candidate that induces robust systemic and mucosal immunity with broad protection. This vaccine candidate is based on three components of the MMR vaccine, a measles virus Edmonston and the two mumps virus strains [Jeryl Lynn 1 (JL1) and JL2] that are known to provide safe, effective, and long-lasting protective immunity. The six proline-stabilized prefusion S protein (preS-6P) genes for ancestral SARS-CoV-2 WA1 and two important SARS-CoV-2 VoCs (Delta and Omicron BA.1) were each inserted into one of these three viruses which were then combined into a trivalent “MMS” candidate vaccine. Intranasal immunization of MMS in IFNAR1 −/− mice induced a strong SARS-CoV-2-specific serum IgG response, cross-variant neutralizing antibodies, mucosal IgA, and systemic and tissue-resident T cells. Immunization of golden Syrian hamsters with MMS vaccine induced similarly high levels of antibodies that efficiently neutralized SARS-CoV-2 VoCs and provided broad and complete protection against challenge with any of these VoCs. This MMS vaccine is an efficacious, broadly protective next-generation COVID-19 vaccine candidate, which is readily adaptable to new variants, built on a platform with a 50-y safety record that also protects against measles and mumps.

Abstract Background Under normal conditions, amyloid (Aβ) is released extracellularly by neurons as a neuroprotective molecule while healthy microglia ingest and degrade Aβ to maintain a balance between production and clearance. One of the hallmarks of Alzheimer’s Disease (AD) is the presence of plaques composed of aggregated, fibrillar amyloid beta (Aβ) that usually precedes the deposition of Tau. Aggregated Aβ can be degraded within healthy microglia largely by autophagy. Most vacuoles containing Aβ acquire the autophagy marker LC3 in primary WT microglia but not in AD microglia. It is still unclear why microglia in the AD brain fail to clear Aβ. Microglia in human AD brain sections and in the 5XFAD (AD) mouse model congregate around Aβ plaques and express high levels of miR‐17 which we found targets autophagy effectors. Method We examined the effect of targeting microRNA miR‐17 in vitro and in vivo using designed mannose‐labeled nanoparticles for the delivery of antagomirs exclusively to microglia. Result Isolated primary microglia from adult AD mouse fail to degrade Aβ in comparison to wild‐type (WT) microglia. The expression of autophagy effectors Nbr1, Atg5 and Atg7, which are targets of miR‐17, are significantly reduced in AD mouse microglia (CD11b + ) and not in CD11b − fraction which contains astrocytes and neuronal cells. This is the first report of significant increase of miR‐17 in the microglia of AD humans and mice. The increased expression of miR‐17 in AD microglia reduces the expression of autophagy effectors leading to defective autophagy in AD microglia. We show that reducing the expression of miR‐17 using specific antagomir (Anti‐17) in AD microglia in vitro, is sufficient to significantly improve their ability to degrade Aβ. Importantly, we successfully injected live AD mice with Anti‐17 enclosed within mannose‐labeled nanoparticles (Man‐NPs) which specifically target the mannose receptor mainly expressed on microglia. This delivery method significantly and explicitly reduced the elevated expression of miR‐17 in microglia, reduced the expression of Aβ in the brain and reduced hyperactivity behavior in live AD mice. Conclusion Together, we describe Man‐NPs for the delivery of antagomirs specifically to microglia. We show that targeting miR‐17 in microglia is sufficient to prevent AD pathobiology.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cystic fibrosis (CF) is a fatal, genetic disorder that critically affects the lungs and is directly caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in defective CFTR function. In epithelial cells, the CFTR channel conducts anions and plays a critical role in regulating the volume and composition of airway surface liquid. This thin layer of aqueous fluid and mucus covering the airway surface facilitates mucociliary clearance, bacterial killing, and epithelial cell homeostasis. The importance of the CFTR channel in macrophages was revealed in recent work that demonstrated that defective CFTR function is accompanied by impaired innate immune responses to specific infections. Notably, most CF-associated infections are caused by microbes that are cleared by autophagy in healthy cells. Autophagy is a highly regulated biological process that provides energy during periods of stress and starvation. Autophagy clears pathogens, inflammatory molecules, and dysfunctional protein aggregates within macrophages. However, this process is impaired in CF patients and CF mice, as their cells exhibit limited autophagy activity. The mechanisms linking a malfunctioning ion channel function to the defective autophagy remains unclear. In this chapter, we describe and discuss the recent findings indicating the presence of several mechanisms leading to defective autophagy in CF cells. Thus, these novel data advance our understanding of mechanisms underlying the pathobiology of CF and provide a new therapeutic platform for restoring CFTR function and autophagy in patients with CF.