Luís F. Moita

Exosome biogenesis is poorly understood. The small GTPases Rab27a and Rab27b and their effectors, Slp4 and Slac2b, control exosome secretion at different steps by regulating the peripheral localization, retention and docking of exosomal precursors, the multivesicular endosomes. Exosomes are secreted membrane vesicles that share structural and biochemical characteristics with intraluminal vesicles of multivesicular endosomes (MVEs). Exosomes could be involved in intercellular communication and in the pathogenesis of infectious and degenerative diseases. The molecular mechanisms of exosome biogenesis and secretion are, however, poorly understood. Using an RNA interference (RNAi) screen, we identified five Rab GTPases that promote exosome secretion in HeLa cells. Among these, Rab27a and Rab27b were found to function in MVE docking at the plasma membrane. The size of MVEs was strongly increased by Rab27a silencing, whereas MVEs were redistributed towards the perinuclear region upon Rab27b silencing. Thus, the two Rab27 isoforms have different roles in the exosomal pathway. In addition, silencing two known Rab27 effectors, Slp4 (also known as SYTL4, synaptotagmin-like 4) and Slac2b (also known as EXPH5, exophilin 5), inhibited exosome secretion and phenocopied silencing of Rab27a and Rab27b, respectively. Our results therefore strengthen the link between MVEs and exosomes, and introduce ways of manipulating exosome secretion in vivo.

We have identified 242 Anopheles gambiae genes from 18 gene families implicated in innate immunity and have detected marked diversification relative to Drosophila melanogaster . Immune-related gene families involved in recognition, signal modulation, and effector systems show a marked deficit of orthologs and excessive gene expansions, possibly reflecting selection pressures from different pathogens encountered in these insects' very different life-styles. In contrast, the multifunctional Toll signal transduction pathway is substantially conserved, presumably because of counterselection for developmental stability. Representative expression profiles confirm that sequence diversification is accompanied by specific responses to different immune challenges. Alternative RNA splicing may also contribute to expansion of the immune repertoire.

Anopheles mosquitoes are major vectors of human malaria in Africa. Large variation exists in the ability of mosquitoes to serve as vectors and to transmit malaria parasites, but the molecular mechanisms that determine vectorial capacity remain poorly understood. We report that the hemocyte-specific complement-like protein TEP1 from the mosquito Anopheles gambiae binds to and mediates killing of midgut stages of the rodent malaria parasite Plasmodium berghei. The dsRNA knockdown of TEP1 in adults completely abolishes melanotic refractoriness in a genetically selected refractory strain. Moreover, in susceptible mosquitoes this knockdown increases the number of developing parasites. Our results suggest that the TEP1-dependent parasite killing is followed by a TEP1-independent clearance of dead parasites by lysis and/or melanization. Further elucidation of the molecular mechanisms of TEP1-mediated parasite killing will be of great importance for our understanding of the principles of vectorial capacity in insects.

Abstract During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer in vivo validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context. Cancer Res; 72(19); 4920–30. ©2012 AACR.

We characterize a novel hemocyte-specific acute phase glycoprotein from the malaria vector, Anopheles gambiae. It shows substantial structural and functional similarities, including the highly conserved thioester motif, to both a central component of mammalian complement system, factor C3, and to a pan-protease inhibitor, alpha2-macroglobulin. Most importantly, this protein serves as a complement-like opsonin and promotes phagocytosis of some Gram-negative bacteria in a mosquito hemocyte-like cell line. Chemical inactivation by methylamine and depletion by double-stranded RNA knockout demonstrate that this function is dependent on the internal thioester bond. This evidence of a complement-like function in a protostome animal adds substantially to the accumulating evidence of a common ancestry of immune defenses in insects and vertebrates.

Innate immune cells detect pathogens via pattern recognition receptors (PRRs), which signal for initiation of immune responses to infection. Studies with Dectin-1, a PRR for fungi, have defined a novel innate signaling pathway involving Syk kinase and the adaptor CARD9, which is critical for inducing Th17 responses to fungal infection. We show that another C-type lectin, Dectin-2, also signals via Syk and CARD9, and contributes to dendritic cell (DC) activation by fungal particles. Unlike Dectin-1, Dectin-2 couples to Syk indirectly, through association with the FcRgamma chain. In a model of Candida albicans infection, blockade of Dectin-2 did not affect innate immune resistance but abrogated Candida-specific T cell production of IL-17 and, in combination with the absence of Dectin-1, decreased Th1 responses to the organism. Thus, Dectin-2 constitutes a major fungal PRR that can couple to the Syk-CARD9 innate signaling pathway to activate DCs and regulate adaptive immune responses to fungal infection.

Anopheles gambiae, the major vector of human malaria parasite, is an important insect model to study vector-parasite interactions. Here, we developed a simple in vivo double-stranded RNA (dsRNA) knockout approach to determine the function of the mosquito antimicrobial peptide gene Defensin. We injected dsRNA into adults and observed efficient and reproducible silencing of Defensin. Analysis of the knockdown phenotype revealed that this peptide is required for the mosquito antimicrobial defense against Gram-positive bacteria. In contrast, in mosquitoes infected by Plasmodium berghei, no loss of mosquito viability and no significant effect on the development and morphology of the parasite midgut stages were observed in the absence of Defensin. We conclude that this peptide is not a major antiparasitic factor in A. gambiae in vivo. Our results open new perspectives for the study of mosquito gene function in vivo and provide a basis for genome-scale systematic functional screens by targeted gene silencing.

Interleukin-1β (IL-1β) represents one of the most important mediators of inflammation and host responses to infection. Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, induces IL-1β secretion at the site of infection, but the underlying mechanism(s) are poorly understood. In this work we show that Mtb infection of macrophages stimulates caspase-1 activity and promotes the secretion of IL-1β. This stimulation requires live intracellular bacteria expressing a functional ESX-1 secretion system. ESAT-6, an ESX-1 substrate implicated in membrane damage, is both necessary and sufficient for caspase-1 activation and IL-1β secretion. ESAT-6 promotes the access of other immunostimulatory agents such as AG85 into the macrophage cytosol, indicating that this protein may contribute to caspase-1 activation largely by perturbing host cell membranes. Using a high-throughput shRNA-based screen we found that numerous NOD-like receptors (NLRs) and CARD domain-containing proteins (CARDs) were important for IL-1β secretion upon Mtb infection. Most importantly, NLRP3, ASC and caspase-1 form an infection-inducible inflammasome complex that is essential for IL-1β secretion. In summary, we show that recognition of Mtb infection by the NLRP3 inflammasome requires the activity of the bacterial virulence factor ESAT-6, and the subsequent IL-1β response is regulated by a number of NLR/CARD proteins.

Disease tolerance is a defence strategy that functions by improving tissue repair or by reducing the detrimental effect of inflammatory signals to prevent the harmful effects of pathogens. This Review describes the mechanisms underlying disease tolerance to infections and how these can be targeted therapeutically. The immune system probably evolved to limit the negative effects exerted by pathogens on host homeostasis. This defence strategy relies on the concerted action of innate and adaptive components of the immune system, which sense and target pathogens for containment, destruction or expulsion. Resistance to infection refers to these immune functions, which reduce the pathogen load of an infected host as the means to preserve homeostasis. Immune-driven resistance to infection is coupled to an additional, and arguably as important, defence strategy that limits the extent of dysfunction imposed on host parenchymal tissues during infection, without exerting a direct negative effect on pathogens. This defence strategy, known as disease tolerance, relies on tissue damage control mechanisms that prevent the deleterious effects of pathogens and that uncouples immune-driven resistance mechanisms from immunopathology and disease. In this Review, we provide a unifying view of resistance and disease tolerance in the framework of immunity to infection.

Antigen (Ag) crosspresentation by dendritic cells (DCs) involves the presentation of internalized Ags on MHC class I molecules to initiate CD8+ T cell-mediated immunity in response to certain pathogens and tumor cells. Here, we identify the SNARE Sec22b as a specific regulator of Ag crosspresentation. Sec22b localizes to the ER-Golgi intermediate compartment (ERGIC) and pairs to the plasma membrane SNARE syntaxin 4, which is present in phagosomes (Phgs). Depletion of Sec22b inhibits the recruitment of ER-resident proteins to Phgs and to the vacuole containing the Toxoplasma gondii parasite. In Sec22b-deficient DCs, crosspresentation is compromised after Ag phagocytosis or endocytosis and after invasion by T. gondii. Sec22b silencing inhibited Ag export to the cytosol and increased phagosomal degradation by accelerating lysosomal recruitment. Our findings provide insight into an intracellular traffic pathway required for crosspresentation and show that Sec22b-dependent recruitment of ER proteins to Phgs critically influences phagosomal functions in DCs.

<h2>Summary</h2> A unique subpopulation of spleen dendritic cells (DCs) that express the CD8 surface marker efficiently present phagocytosed antigens to CD8⁺ T lymphocytes in a process called "crosspresentation," which initiates cytotoxic immune responses. We now show that the small GTPase Rac2 plays a critical role in antigen crosspresentation selectively in this DC subpopulation. In CD8⁺ DCs, Rac2 determines the subcellular assembly of the NADPH oxidase complex (NOX2) to phagosomes, whereas in CD8⁻ DCs, Rac1 mediates the assembly of NOX2 at the plasma membrane. In the absence of Rac2, the production of reactive oxygen species (ROS) in DC-phagosomes was abolished, the phagosomal pH dropped, and the efficiency of antigen crosspresentation was reduced. We conclude that the activity of Rac1 and 2 control crosspresentation in DC subpopulations through the regulation of phagosomal oxidation and pH.

During inflammation, the lymph node stromal compartment is shown to accommodate high numbers of infiltrating lymphocytes by relaxing the cytoskeleton of fibroblastic reticular cells, allowing the latter to stretch and the lymph node to expand. Lymph nodes are dynamic structures that must respond rapidly to large cellular influxes provoked by local inflammation. However, how the lymph node stromal compartment reacts to accommodate lymph node expansion remains unclear. This study shows that the lymph node stromal compartment can accommodate large numbers of infiltrating lymphocytes by relaxing the cytoskeleton of fibroblastic reticular cells, allowing the cells to stretch and the lymph node to expand. This lymph node remodelling reaction is driven by the interaction of CLEC-2 protein on incoming antigen-presenting dendritic cells with podoplanin on fibroblastic reticular cells. After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion1,2. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes3 also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion—driven by lymphocyte influx and proliferation—that is the critical hallmark of adaptive immunity.

Mouse CD8α(+) dendritic cells (DCs) in lymphoid organs and CD103(+) CD11b(-) DCs in nonlymphoid tissues share phenotypic and functional similarities, as well as a unique shared developmental dependence on the transcription factor Batf3. Human DCs resembling mouse CD8α(+) DCs in phenotype and function have been identified in human blood, spleen, and tonsil. However, it is not clear whether such cells are also present in human nonlymphoid organs, and their equivalence to mouse CD8α(+) DC has recently been questioned. Furthermore, the identification of "CD8α(+) DC-like" cells across different tissues and species remains problematic because of the lack of a unique marker that can be used to unambiguously define lineage members. Here we show that mouse CD8α(+) DCs and CD103(+) CD11b(-) DCs can be defined by shared high expression of DNGR-1 (CLEC9A). We further show that DNGR-1 uniquely marks a CD11b(-) human DC population present in both lymphoid and nonlymphoid tissues of humans and humanized mice. Finally, we demonstrate that knockdown of Batf3 selectively prevents the development of DNGR-1(+) human DCs in vitro. Thus, high expression of DNGR-1 specifically and universally identifies a unique DC subset in mouse and humans. Evolutionarily conserved Batf3 dependence justifies classification of DNGR-1(hi) DCs as a distinct DC lineage.

The identification of new bioactive compounds derived from medicinal plants with significant therapeutic properties has attracted considerable interest in recent years. Such is the case of the Tripterygium wilfordii, an herb used in Chinese medicine. Clinical trials performed so far using its root extracts have shown impressive therapeutic properties, but also revealed substantial gastrointestinal side-effects. The most promising bioactive compound obtained from Tripterygium wilfordii is celastrol. During the last decade, an increasing number of studies were published highlighting the medicinal usefulness of celastrol in diverse clinical areas. Here we systematically review the mechanism of action and the therapeutic properties of celastrol in inflammatory diseases, namely rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel diseases, osteoarthritis and allergy, as well as in cancer, neurodegenerative disorders and other diseases, such as diabetes, obesity, atherosclerosis and hearing loss. We will also focus in the toxicological profile and limitations of celastrol formulation, namely solubility, bioavailability and dosage issues that still limit its further clinical application and usefulness.

The transition from naïve to activated T cells is marked by alternative splicing of pre-mRNA encoding the transmembrane phosphatase CD45. Using a short hairpin RNA interference screen, we identified heterogeneous ribonucleoprotein L-like (hnRNPLL) as a critical inducible regulator of CD45 alternative splicing. HnRNPLL was up-regulated in stimulated T cells, bound CD45 transcripts, and was both necessary and sufficient for CD45 alternative splicing. Depletion or overexpression of hnRNPLL in B and T cell lines and primary T cells resulted in reciprocal alteration of CD45RA and RO expression. Exon array analysis suggested that hnRNPLL acts as a global regulator of alternative splicing in activated T cells. Induction of hnRNPLL during hematopoietic cell activation and differentiation may allow cells to rapidly shift their transcriptomes to favor proliferation and inhibit cell death.

Article19 July 2013free access SAMHD1-dependent retroviral control and escape in mice Jan Rehwinkel Corresponding Author Jan Rehwinkel Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Jonathan Maelfait Jonathan Maelfait Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Anne Bridgeman Anne Bridgeman Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Rachel Rigby Rachel Rigby Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Bruce Hayward Bruce Hayward Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds, UK Search for more papers by this author Rachel A Liberatore Rachel A Liberatore Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Paul D Bieniasz Paul D Bieniasz Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Luis F Moita Luis F Moita Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Yanick J Crow Yanick J Crow Manchester Centre for Genomic Medicine, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK Search for more papers by this author David T Bonthron David T Bonthron Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds, UK Search for more papers by this author Caetano Reis e Sousa Corresponding Author Caetano Reis e Sousa Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Jan Rehwinkel Corresponding Author Jan Rehwinkel Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Jonathan Maelfait Jonathan Maelfait Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Anne Bridgeman Anne Bridgeman Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Rachel Rigby Rachel Rigby Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK Search for more papers by this author Bruce Hayward Bruce Hayward Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds, UK Search for more papers by this author Rachel A Liberatore Rachel A Liberatore Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Paul D Bieniasz Paul D Bieniasz Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Greg J Towers Greg J Towers Division of Infection and Immunity, University College London, London, UK Search for more papers by this author Luis F Moita Luis F Moita Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal Search for more papers by this author Yanick J Crow Yanick J Crow Manchester Centre for Genomic Medicine, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK Search for more papers by this author David T Bonthron David T Bonthron Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds, UK Search for more papers by this author Caetano Reis e Sousa Corresponding Author Caetano Reis e Sousa Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK Search for more papers by this author Author Information Jan Rehwinkel 1,2, Jonathan Maelfait2, Anne Bridgeman2, Rachel Rigby2, Bruce Hayward3, Rachel A Liberatore4, Paul D Bieniasz4, Greg J Towers5, Luis F Moita6, Yanick J Crow7, David T Bonthron3 and Caetano Reis e Sousa 1 1Immunobiology Laboratory, Cancer Research UK, London Research Institute, London, UK 2Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, UK 3Leeds Institute of Molecular Medicine, University of Leeds, St James's University Hospital, Leeds, UK 4Laboratory of Retrovirology, Aaron Diamond AIDS Research Center, Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA 5Division of Infection and Immunity, University College London, London, UK 6Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal 7Manchester Centre for Genomic Medicine, Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK *Corresponding authors. Medical Research Council Human Immunology Unit, Radcliffe Department of Medicine, Medical Research Council Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS, UK. Tel.:+44 (0)1865 222362; Fax:+44 (0)1865 222502; E-mail: [email protected] Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3LY, UK. Tel.:+44 (0)20 72692832; Fax:+44 (0)20 72692833; E-mail: [email protected] The EMBO Journal (2013)32:2454-2462https://doi.org/10.1038/emboj.2013.163 There is a Have you seen? (September 2013) associated with this Article. 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 SAMHD1 is a host restriction factor for human immunodeficiency virus 1 (HIV-1) in cultured human cells. SAMHD1 mutations cause autoimmune Aicardi-Goutières syndrome and are found in cancers including chronic lymphocytic leukaemia. SAMHD1 is a triphosphohydrolase that depletes the cellular pool of deoxynucleoside triphosphates, thereby preventing reverse transcription of retroviral genomes. However, in vivo evidence for SAMHD1's antiviral activity has been lacking. We generated Samhd1 null mice that do not develop autoimmune disease despite displaying a type I interferon signature in spleen, macrophages and fibroblasts. Samhd1−/− cells have elevated deoxynucleoside triphosphate (dNTP) levels but, surprisingly, SAMHD1 deficiency did not lead to increased infection with VSV-G-pseudotyped HIV-1 vectors. The lack of restriction is likely attributable to the fact that dNTP concentrations in SAMHD1-sufficient mouse cells are higher than the KM of HIV-1 reverse transcriptase (RT). Consistent with this notion, an HIV-1 vector mutant bearing an RT with lower affinity for dNTPs was sensitive to SAMHD1-dependent restriction in cultured cells and in mice. This shows that SAMHD1 can restrict lentiviruses in vivo and that nucleotide starvation is an evolutionarily conserved antiviral mechanism. Introduction Human dendritic cells (DCs) and other myeloid cells in culture are largely refractory to infection with human immunodeficiency virus 1 (HIV-1) (Manel et al, 2010). The closely related virus HIV-2 encodes the protein Vpx, which can relieve restriction of HIV-1 in trans by targeting a host factor for proteasomal degradation (Goujon et al, 2007; Kaushik et al, 2009). Recent work has identified SAMHD1 as the cellular protein targeted by Vpx (Hrecka et al, 2011; Laguette et al, 2011). SAMHD1 has an N-terminal sterile alpha motif and a C-terminal HD domain that hydrolyses deoxynucleoside triphosphates (dNTPs), generating nucleosides and inorganic triphosphate (Goldstone et al, 2011; Powell et al, 2011). Experiments in vitro suggest that SAMHD1-mediated reduction of cellular dNTP concentrations may block HIV-1 reverse transcription and, consequently, restrict virus infection (Kim et al, 2012; Lahouassa et al, 2012). Consistent with that model, SAMHD1 depletion by Vpx delivery or RNA interference renders many human myeloid cells and resting T cells more permissive to HIV-1 infection (Berger et al, 2011; Hrecka et al, 2011; Laguette et al, 2011; Baldauf et al, 2012; Descours et al, 2012; Kim et al, 2012; Lahouassa et al, 2012). Moreover, monocytes and resting T cells from SAMHD1-deficient individuals more efficiently support HIV-1 replication (Berger et al, 2011; Baldauf et al, 2012; Descours et al, 2012). Collectively, these in vitro observations define SAMHD1 as a host restriction factor for HIV-1 in cultured human cells (Ayinde et al, 2012). SAMHD1 deficiency in humans results in Aicardi-Goutières syndrome (AGS), a hereditary autoimmune encephalopathy that mimics congenital virus infection and is characterized by type I interferon (IFN) production (Crow and Livingston, 2008; Rice et al, 2009). AGS can also be caused by mutations in the TREX1, RNASEH2A-C or ADAR1 genes (Crow and Livingston, 2008; Rice et al, 2012). TREX1-deficient mice develop spontaneous and IFN-dependent multi-organ autoimmunity, particularly of the heart (Morita et al, 2004; Stetson et al, 2008; Gall et al, 2012). Interestingly, TREX1-deficient cells display cytoplasmic accumulation of DNA from endogenous retroviruses or after HIV-1 infection (Stetson et al, 2008; Yan et al, 2010). This DNA then triggers an STING-dependent innate antiviral pathway that culminates in IRF3-dependent IFN induction (Stetson et al, 2008; Yan et al, 2010; Gall et al, 2012). Disease in TREX1-deficient mice can be ameliorated by administering reverse transcriptase (RT) inhibitors (Beck-Engeser et al, 2011). As such, it appears that a failure to control endogenous and exogenous retroviruses and to degrade their nucleic acid products underlies autoimmunity caused by TREX1 deficiency. However, it is unclear whether SAMHD1 behaves similarly and whether its loss-of-function precipitates disease by permitting accumulation of retroviral nucleic acid products that chronically trigger IFN induction pathways. To further study the molecular basis of AGS and the in vivo relevance of SAMHD1 as an antiviral host factor, we generated SAMHD1-deficient mice. Here, we show that such mice do not develop autoimmunity, even though they show evidence of spontaneous IFN production in selected tissues and cells. The levels of all four dNTPs were increased in Samhd1−/− cells, but infection with VSV-G-pseudotyped HIV-1 vectors was comparable in wild-type and Samhd1−/− DCs, macrophages and other cells, as well as in SAMHD1-deficient and control mice. These data can be explained by the fact that baseline dNTP concentrations in SAMHD1-sufficient mouse DCs were 10-fold higher than those reported for human cells and exceeded the KM of HIV-1 RT for dNTPs. Consistent with this notion, introduction of a point mutation into the viral polymerase that lowers its affinity for dNTPs revealed potent SAMHD1-dependent restriction of the resulting attenuated HIV-1 vector in cells and in vivo. Therefore, nucleotide restriction is an evolutionarily conserved mechanism for defense from retroviruses. Our data also suggest that HIV-1 has evolved a polymerase that is active at low dNTP concentrations, thereby partly circumventing some of the effect of SAMHD1 restriction. Results Samhd1−/− mice are healthy, although they display an IFN signature in some tissues and cells We generated SAMHD1-deficient mice in a mixed or a pure C57BL/6 background (Supplementary Figure S1A–E). Independent of background, Samhd1−/− animals were born at Mendelian ratios (Figure 1A; Supplementary Figure S1F) and developed normally. They were indistinguishable from their wild-type or heterozygous littermates, were fertile and did not show signs of disease even after ageing for over 24 months. Unlike serum samples from AGS patients (Goutieres et al, 1998), sera from SAMHD1-deficient and littermate control mice did not contain detectable IFN as measured by a sensitive bioassay based on induction of the IFN-stimulated gene (ISG), IFIT1 (Figure 1B). Transcript levels of the ISG IFIT2 or of the pro-inflammatory cytokine TNFα were also not increased in lung, kidney and heart from Samhd1−/− mice when compared to tissues from wild-type mice (Figure 1C). However, in spleen, IFIT2 and TNFα mRNA levels were upregulated seven- and four-fold, respectively (Figure 1C). Next, we analysed whether Samhd1−/− cells in culture produce IFN spontaneously. mRNA levels for ISGs were two- to four-fold increased in SAMHD1-deficient bone marrow-derived macrophages (BM-DMs) (Figure 1D). Similarly, SAMHD1-deficient primary mouse embryonic fibroblasts (MEFs) showed a 10-fold increase in IFIT1 mRNA levels when compared to control Samhd1+/− MEFs, which we found to express SAMHD1 mRNA and protein (Figure 1E; Supplementary Figure S1C). In summary, although SAMHD1-deficient mice do not display detectable amounts of circulating IFN or ISG upregulation in most tissues, an IFN signature is evident in Samhd1−/− spleens, macrophages and fibroblasts. Nevertheless, these mice are healthy and do not develop autoimmune disease. Figure 1.Spontaneous IFN production by cells and mice lacking SAMHD1. (A) Numbers (N) and percentages (%) of Samhd1+/+, Samhd1+/− and Samhd1−/− offspring of heterozygous Samhd1+/− breedings. Data are pooled from 5D6 (pure B6 background) and 1F8 (mixed S6/B6 background) mice. See Supplementary Figure S1F for further details. (B) Serum samples were obtained from Samhd1+/+, Samhd1+/− and Samhd1−/− littermates by tail bleeding and were analysed for the presence of IFN by bioassay. Samples were added to NIH3T3 cells and induction of the IFN-stimulated mRNA IFIT1 was assessed by RT Q-PCR. Data from samples of 5D6 and 1F8 mice were pooled and the differences between SAMHD1-sufficient and SAMHD1-deficient mice were not statistically significant. This was also the case when 5D6 and 1F8 samples were analysed separately. IFN-A/D was used as a positive control at the indicated concentrations; bars represent the mean of duplicate measurements and error bars show the range. (C) Littermate animals of the indicated Samhd1 genotypes were sacrificed at the age of 9 months and RNA was extracted from lung, kidney, heart or spleen. IFIT2 and TNFα mRNA levels were determined by RT Q-PCR. Circles and squares correspond to individual animals (duplicate measurement). (D) IFIT1, IFIT2 and IFI-44 mRNA levels in BM-DMs of the indicated Samhd1 genotypes cultured for 12 days were determined by RT Q-PCR. Bars represent the mean of four BM-DM cultures from independent mice and error bars show the standard deviation. *P<0.05 (unpaired t-test). (E) Primary MEFs of the indicated Samhd1 genotypes were obtained by crossing Samhd1+/− and Samhd1−/− animals. The expression levels of SAMHD1 (one primer in exon 2) and IFIT1 mRNAs were determined by RT Q-PCR. Bars correspond to cell lines derived from individual embryos and show the mean of triplicate measurements; error bars show the standard deviation. The number in square brackets corresponds to the cell line identification code. (B–E) Relative expression levels compared to GAPDH mRNA are shown. (C–E) Data from 5D6 mice and cells (pure B6 background) are shown. Download figure Download PowerPoint Nucleic acids and viruses induce normal IFN responses in Samhd1−/− cells and mice Next, we induced IFN by treating SAMHD1-deficient cells and mice with nucleic acids or by infecting them with viruses. IFN induction in Samhd1−/− and control MEFs was similar in response to transfection of in vitro-transcribed RNA, poly dA:dT or E. coli DNA (Figure 2A). Similarly, SAMHD1-sufficient and SAMHD1-deficient BM-DMs produced comparable amounts of IFNα when stimulated with in vitro-transcribed RNA or cyclic diguanylate monophosphate, an STING agonist (Figure 2B). The induction of IFNβ and IFIT1 transcripts and of mRNAs for the pro-inflammatory factors IL-6, TNFα and IL-1β was similar in SAMHD1-depleted and control BM-derived DCs (BM-DCs) in response to transfected E. coli DNA (Supplementary Figure S2). When assessed in vivo, Samhd1−/− mice produced normal levels of IFNα after encephalomyocarditis virus (EMCV) infection when compared to controls (Figure 2C). These data suggest that SAMHD1 is either redundant or not involved in the pathways that trigger IFN in response to nucleic acids or virus infection. Figure 2.Cells and mice lacking SAMHD1 produce normal levels of IFN in response to nucleic acid or viral challenge. (A) Primary MEFs (Figure 1E) were transfected with 2000, 400, 80 or 16 ng of in vitro-transcribed RNA (IVT-RNA), poly dA:dT or E. coli DNA. After overnight incubation, IFN in culture supernatants was quantified by LL171 bioassay. *None detectable. (B) BM-DMs from 5D6 mice of the indicated Samhd1 genotypes were transfected with 1000 or 100 ng of IVT-RNA or cyclic diguanylate monophosphate (c-di-GMP). Six hours later, IFNα in culture supernatants was analysed by ELISA. Error bars show the standard deviation of triplicate measurements. (C) Mice of the indicated genotypes were infected with EMCV by intraperitoneal injection. Serum IFNα was determined by ELISA 24 h later. Data from samples of 5D6 and 1F8 mice were pooled and the differences between Samhd+/− and Samhd1−/− mice were not statistically significant. This was also the case when 5D6 and 1F8 samples were analysed separately. Download figure Download PowerPoint Infection of SAMHD1-deficient cells and mice with retroviruses and retroviral vectors Human SAMHD1 restricts HIV-1 in vitro by degrading dNTPs (Goldstone et al, 2011; Hrecka et al, 2011; Laguette et al, 2011; Powell et al, 2011; Baldauf et al, 2012; Kim et al, 2012; Lahouassa et al, 2012). Using a primer extension assay (Diamond et al, 2004), we found increased concentrations of all four dNTPs in Samhd1−/− BM-DCs (Figure 3A–D). Similarly, dTTP levels were elevated in SAMHD1-deficient BM-DMs (Figure 3E). We independently confirmed these observations using a different fluorescence-based assay (Wilson et al, 2011) (Figure 3F and data not shown). These data substantiate earlier biochemical findings with human SAMHD1 (Goldstone et al, 2011; Powell et al, 2011) and show that dNTP hydrolysis is an evolutionarily conserved function of the protein. To test whether SAMHD1 and dNTP hydrolysis impacts infection with murine retroviruses, we infected neonatal mice with the gamma-retrovirus Moloney murine leukaemia virus (Mo-MLV) and determined viral titers in the serum 2 weeks after infection. We were unable to detect differences in viral load between Samhd1−/− animals and heterozygous littermates (Figure 4). However, we cannot exclude that SAMHD1 controls viral titers at earlier or later time points. SAMHD1 deficiency also had no impact on RNA levels of the MusD, IAP, Mu-ERV-L and m-poly-MLV retroelements in BM-DCs and MEFs, or in lung and spleen tissue (Figure 5A and B). Similarly, we did not detect increased staining of Samhd1−/− spleen cells with an antibody reacting with the envelope glycoproteins of many classes of endogenous murine leukaemia viruses (Figure 5C). Thus, SAMHD1 appears to be dispensable or redundant for in vivo control of replication of exogenous Mo-MLV and for restricting RNA or protein expression from the endogenous retroelements we tested. Nevertheless, it remains possible that SAMHD1 controls other retroelements and/or prevents cDNA synthesis. Figure 3.SAMHD1 degrades dNTPs. (A) dATP levels in cell extracts from one million BM-DCs of the indicated Samhd1 genotypes were assessed using a primer extension assay that incorporates a single dATP nucleotide. dATP was used as a positive control and water, dCTP, dGTP and dTTP as negative controls. (B) As (A) for dCTP. (C) As (A) for cGTP. (D) As (A) for dTTP. (E) dTTP levels in cell extracts from BM-DMs were determined as in A. (F) Cell extracts as in A were assessed for dTTP with a fluorescence-based assay. On the basis of a dTTP standard and the cell volume of BM-DCs (2244 μm3, see Supplementary Materials and methods), dTTP concentrations were determined. Horizontal lines indicate the average of four independent samples and error bars depict the standard deviation. (A–F) Cells were on the B6 background (5D6). *P<0.05 (unpaired t-test). Download figure Download PowerPoint Figure 4.Infection of SAMHD1-deficient mice with Mo-MLV. Neonatal mice from Samhd1+/− × Samhd1−/− matings were infected with Mo-MLV by intraperitoneal injection. Fourteen days later, animals were sacrificed. MLV serum titers are shown as focus forming units (FFU) per ml. DNA extracted from tail samples was used for genotyping. Data from two experiments with pure B6 background mice (5D6) were pooled. The differences between the two groups are not statistically significant. Download figure Download PowerPoint Figure 5.Loss of SAMHD1 does not result in increased RNA and envelope glycoprotein expression of selected endogenous retroelements. (A) RNA from BM-DC cultures (left) or primary MEFs (right) was analysed by RT Q-PCR for the expression of retroelement transcripts. Some cells were treated overnight with 1000 U/ml IFN-A/D as indicated (left). Each bar corresponds to one DC culture or MEF cell line from an individual embryo; numbers in square brackets refer to the cell line identifier (right). Average relative expression levels compared to GAPDH mRNA from two measurements are shown; error bars represent the range. (B) Littermate animals of the indicated Samhd1 genotypes were sacrificed at the age of 9 months and RNA was extracted from lung and spleen. Retroelement transcripts were analysed as in A. Each bar corresponds to samples from an individual animal. (C) Cultured RAW264.7 cells and freshly isolated splenocytes of the indicated Samhd1 genotypes were stained with monoclonal antibody 83A25 and secondary PE-conjugated α-rat antibody and then analysed by flow cytometry. As a control, an aliquot of cells was stained with PE-α-rat alone. 83A25 reacts with the envelope glycoproteins of endogenous murine leukaemia viruses expressed in RAW264.7 cells. (A–C) Cells and mice were on the B6 background (5D6). Download figure Download PowerPoint We extended the analysis to infection with single-round, VSV-G-pseudotyped HIV-1 vectors encoding GFP (referred to as HIV-1-GFP below). These minimal, replication-defective lentivirus vectors transduce quiescent cells, which have lower dNTP concentrations than the cycling cells targeted by Mo-MLV (Baldauf et al, 2012; Lahouassa et al, 2012), and have internal promoters driving the GFP reporter. We infected BM-DCs or BM-DMs in vitro after their differentiation with GM-CSF or M-CSF, respectively, at a time when the cells are no longer cycling. Surprisingly, for both myeloid cell types, the efficiency of transduction with HIV-1-GFP was independent of SAMHD1 (Figure 6A). Similarly, transduction with a vector encoding the HIV-1 accessory proteins Vif, Vpr, Vpu and Nef was unchanged in Samhd1−/− BM-DCs (Figure 6B). IFN was not induced to detectable levels upon HIV-1-GFP infection in either wild-type or SAMHD1-deficient BM-DCs (Supplementary Figure S3). Next, we infected freshly isolated non-dividing splenocytes with HIV-1- and MLV-based vectors and identified DCs in the mixture by CD11c staining. Similar numbers of CD11c+ cells were transduced by both vectors independently of Samhd1 genotype (Supplementary Figure S4A). We also infected fresh bone marrow and then cultured the transduced cells with GM-CSF to promote DC differentiation. Transduction efficiencies were identical between SAMHD1-deficient and control DC precursors (Supplementary Figure S4B) and were similarly comparable in SAMHD1-sufficient and SAMHD1-deficient immortalized MEFs (Supplementary Figure S4C). Collectively, these data suggest that SAMHD1 is largely ineffective at restricting reverse transcription of HIV-1 in mouse cells. Figure 6.SAMHD1 inhibits pseudotyped HIV-1 in vitro. (A) BM-DMs and BM-DCs of the indicated Samhd1 genotypes were infected with VSV-G-pseudotyped HIV-1-GFP (pCSGW/p8.91, MOI=1). Virus stocks were titrated in 293T cells and multiplicities of infection (MOIs) were calculated using 293T infectious units. Twenty-four hours after infection, the fraction of GFP-expressing cells was determined by flow cytometry. Averages from two different bone marrow donors are shown and error bars represent the range. (B) BM-DCs of the indicated Samhd1 genotypes were infected with VSV-G-pseudotyped HIV-1-GFP (pRRLsin.eGFP/pCMVΔ8.2) and with a vector that has a single amino-acid substitution in the RT (pRRLsin.eGFP/pCMVΔ8.2 V148I). Twenty-four hours after infection, DCs were identified by CD11c staining and percentages of GFP-expressing cells were determined by flow cytometry as further detailed in Supplementary Figure S5. Averages from three DC cultures per group from independent mice are shown and error bars represent the standard deviation. (C) Primary MEFs of the indicated Samhd1 genotypes were infected with the viruses from B. Twenty-four hours after infection, GFP expression was analysed by flow cytometry. Averages from three infections are shown and error bars represent the standard deviation. (A–C) Cells were on the B6 background (5D6). *P<0.05 (unpaired t-test). The differences between the samples infected with wild-type RT HIV-1-GFP were not statistically significant. Download figure Download PowerPoint SAMHD1 can restrict an RT mutant HIV-1 in vitro and in vivo One possible explanation for the above results is that baseline dNTP concentrations in mouse cells are high enough to support HIV-1 reverse transcription, even in the presence of SAMHD1. Consistent with this idea, we found that wild-type BM-DCs contained around 0.5 μM dTTP (Figure 3F), while the KM of HIV-1 RT for dTTP is 0.07 μM (Diamond et al, 2004). We therefore tested a modified HIV-1-GFP encoding a mutant RT with lower affinity for dNTPs (V148I) (Diamond et al, 2003; Lahouassa et al, 2012). Infection of differentiated BM-DCs with HIV-1-GFPV148I resulted in about five-fold increased transduction in Samhd1−/− cells compared to SAMHD1-sufficient control cells (Figure 6B; Supplementary Figure S5). Similarly, HIV-1-GFPV148I transduction rates were three- to five-fold higher in Samhd1−/− primary MEFs (Figure 6C). To test whether the same is true in vivo, we infected SAMHD1-deficient and wild-type animals with the different VSV-G-pseudotyped HIV-1-GFP vectors by intravenous injection. We analysed transduction of splenocytes 5–6 days after infection by flow cytometry. In mice infected with wild-type RT HIV-1-GFP, transduction efficiency was equivalent between control and SAMHD1-deficient animals and resulted in around 1% of all cells in the spleen expressing GFP (Figure 7A and B; Supplementary Figure S6). Similarly, when we infected mice with HIV-1-GFP derived from a different construct that does not encode the accessory proteins, transduction efficiencies of total splenocytes were not significantly different between Samhd1+/+ and Samhd1−/− mice (Supplementary Figure S7). We then identified individual cell populations in the spleen by cell surface marker staining, including lymphoid cells (B cells (B220+, CD11c−), T cells (CD3+, NK1.1−), NK cells (NK1.1+, CD3−)) and myeloid cells (DCs (CD11c+, MHCII+), plasmacytoid DCs (CD11c+, B220+), macrophages (F4/80+, autofluorescence+), neutrophils (CD11bhigh, Gr1high) and inflammatory monocytes (CD11bint, Gr1int)) (gating strategies in Supplementary Figure S6). A percentage of cells between 0.2% (neutrophils) and 36% (macrophages) of these populations were transduced in both wild-type and Samhd1−/− animals with no differences observable between the genotypes (Figure 7C and D; Supplementary Figure S7). In contrast, in vivo infection with HIV-1-GFPV148I resulted in an eight-fold increase in transduction of Samhd1−/− splenocytes compared to control mice (Figure 7A and B). Moreover, transduction of all myeloid and lymphoid cell populations that we analysed, as well as of non-haematopoietic cells (CD45−), was much greater in SAMHD1-deficient mice (Figure 7C–F). These data show that SAMHD1 can restrict infection by some lentiviruses in a plethora of cell types in vivo. Given that the

The increase in antibiotic drug resistance and the low number of new antibacterial drugs approved in the last few decades requires the development of new antimicrobial strategies. Antimicrobial peptides (AMPs) are very promising molecules to fight microbial infection since they kill quickly bacteria and, in some cases, target bacterial membrane. Although some AMPs may be stable against proteolytic degradation by chemical modification, in general, low AMP activity and stability in the presence of serum and proteolytic enzymes as well as their cytotoxicity have impaired their clinical translation. Here, we describe a one-step methodology to generate AMP-conjugated gold nanoparticles (Au NPs), with a high concentration of AMPs (CM-SH) (≈240 AMPs per NP), controlled size (14 nm) and low polydispersity. AMP-conjugated Au NPs demonstrated higher antimicrobial activity and stability in serum and in the presence of non-physiological concentrations of proteolytic enzymes than soluble AMP, as well as low cytotoxicity against human cells. Moreover, the NPs demonstrated high antimicrobial activity after in vivo administration in a chronic wound and in an animal model of systemic infection.

Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fancony Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

Abstract On the path to successful immunotherapy of hematopoietic tumors, γδ T cells offer great promise because of their human leukocyte antigen (HLA)–unrestricted targeting of a wide variety of leukemias/lymphomas. However, the molecular mechanisms underlying lymphoma recognition by γδ T cells remain unclear. Here we show that the expression levels of UL16-binding protein 1 (ULBP1) determine lymphoma susceptibility to γδ T cell–mediated cytolysis. Consistent with this, blockade of NKG2D, the receptor for ULBP1 expressed on all Vγ9+ T cells, significantly inhibits lymphoma cell killing. Specific loss-of-function studies demonstrate that the role of ULBP1 is nonredundant, highlighting a thus far unique physiologic relevance for tumor recognition by γδ T cells. Importantly, we observed a very wide spectrum of ULBP1 expression levels in primary biopsies obtained from lymphoma and leukemia patients. We suggest this will impact on the responsiveness to γδ T cell–based immunotherapy, and therefore propose ULBP1 to be used as a leukemia/lymphoma biomarker in upcoming clinical trials.

We have previously reported an increase in interleukin (IL)-1β and IL-17 levels, and a continuous activation of caspase-1 in early rheumatoid arthritis (RA) patients. These results suggest that drugs targeting IL-1β regulatory pathways, in addition to tumor necrosis factor (TNF), may constitute promising therapeutic agents in early RA. We have recently used a THP-1 macrophage-like cell line to screen 2320 compounds for those that down-regulate both IL-1β and TNF secretion. Celastrol was one of the most promising therapeutic candidates identified in that study. Our main goal in the present work was to investigate whether administration of celastrol is able to attenuate inflammation in a rat model of adjuvant-induced arthritis (AIA). Moreover, since IL-1β is known to play a role in the polarization of Th17 cells, we also investigate whether administration of digoxin, a specific inhibitor of Th17 cells polarization, is able to attenuate inflammation in the same rat model. We found that celastrol administration significantly suppressed joint inflammation. The histological and immunohistochemical evaluation revealed that celastrol-treated rats had a normal joint structure with complete abrogation of the inflammatory infiltrate and cellular proliferation. In contrast, we observed that digoxin administration significantly ameliorated inflammation but only if administrated in the early phase of disease course (after 4days of disease induction), and it was not efficient at inhibiting the infiltration of immune cells within the joint and in preventing damage. Thus, our results suggest that celastrol has significant anti-inflammatory and anti-proliferative properties and can constitute a potential anti-inflammatory drug with therapeutic efficacy in the treatment of immune-mediated inflammatory diseases such as RA. Furthermore, we find that early inhibition of Th17 cells polarization ameliorates arthritis but it is not as effective as celastrol.

Drugs that activate integrins inhibit leukocyte recruitment to sites of inflammation.

The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3β, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.

Genetic variations in the ITGAM gene (encoding CD11b) strongly associate with risk for systemic lupus erythematosus (SLE). Here we have shown that 3 nonsynonymous ITGAM variants that produce defective CD11b associate with elevated levels of type I interferon (IFN-I) in lupus, suggesting a direct link between reduced CD11b activity and the chronically increased inflammatory status in patients. Treatment with the small-molecule CD11b agonist LA1 led to partial integrin activation, reduced IFN-I responses in WT but not CD11b-deficient mice, and protected lupus-prone MRL/Lpr mice from end-organ injury. CD11b activation reduced TLR-dependent proinflammatory signaling in leukocytes and suppressed IFN-I signaling via an AKT/FOXO3/IFN regulatory factor 3/7 pathway. TLR-stimulated macrophages from CD11B SNP carriers showed increased basal expression of IFN regulatory factor 7 (IRF7) and IFN-β, as well as increased nuclear exclusion of FOXO3, which was suppressed by LA1-dependent activation of CD11b. This suggests that pharmacologic activation of CD11b could be a potential mechanism for developing SLE therapeutics.

Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a−/−) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a−/− animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available. Podocyte injury is an early event in diabetic kidney disease and is a hallmark of glomerulopathy. MicroRNA-146a (miR-146a) is highly expressed in many cell types under homeostatic conditions, and plays an important anti-inflammatory role in myeloid cells. However, its role in podocytes is unclear. Here, we show that miR-146a expression levels decrease in the glomeruli of patients with type 2 diabetes (T2D), which correlates with increased albuminuria and glomerular damage. miR-146a levels are also significantly reduced in the glomeruli of albuminuric BTBR ob/ob mice, indicating its significant role in maintaining podocyte health. miR-146a-deficient mice (miR-146a−/−) showed accelerated development of glomerulopathy and albuminuria upon streptozotocin (STZ)-induced hyperglycemia. The miR-146a targets, Notch-1 and ErbB4, were also significantly up-regulated in the glomeruli of diabetic patients and mice, suggesting induction of the downstream TGFβ signaling. Treatment with a pan-ErbB kinase inhibitor erlotinib with nanomolar activity against ErbB4 significantly suppressed diabetic glomerular injury and albuminuria in both WT and miR-146a−/− animals. Treatment of podocytes in vitro with TGF-β1 resulted in increased expression of Notch-1, ErbB4, pErbB4, and pEGFR, the heterodimerization partner of ErbB4, suggesting increased ErbB4/EGFR signaling. TGF-β1 also increased levels of inflammatory cytokine monocyte chemoattractant protein-1 (MCP-1) and MCP-1 induced protein-1 (MCPIP1), a suppressor of miR-146a, suggesting an autocrine loop. Inhibition of ErbB4/EGFR with erlotinib co-treatment of podocytes suppressed this signaling. Our findings suggest a novel role for miR-146a in protecting against diabetic glomerulopathy and podocyte injury. They also point to ErbB4/EGFR as a novel, druggable target for therapeutic intervention, especially because several pan-ErbB inhibitors are clinically available.

While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive for reasons that remain unclear. Here, we show that Linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of T helper-17 cell effector function in vitro, showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromised the integrity of the electron transport chain. Ultimately, this led to deficient oxidative phosphorylation, diminishing nicotinamide adenine dinucleotide concentrations and impairing cytokine production in differentiating T cells. In accordance, mice lacking mEF-G1 in T cells were protected from experimental autoimmune encephalomyelitis, demonstrating that this pathway is crucial in maintaining T cell function and pathogenicity.

Significance GDF15, a hormone produced by a wide variety of cells undergoing different types of stress, acts on a receptor in the brain where it transmits signals that are perceived by the organism as aversive. We now report an action of GDF15, whereby it activates the endocrine stress response and increases circulating levels of the principal glucocorticoid (a “stress” steroid). By studying mice genetically deficient in GDF15, we also demonstrate that GDF15 is a key signal through which damage due to toxic chemicals activates the steroid stress response. GDF15 is currently being explored as an antiobesity drug and examination of the degree and duration of the steroid effect will need to be incorporated into any human trials.

<h2>Summary</h2> Several classes of antibiotics have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here, we report that tetracycline antibiotics, which target the mitoribosome, protected against sepsis without affecting the pathogen load. Mechanistically, we found that mitochondrial inhibition of protein synthesis perturbed the electron transport chain (ETC) decreasing tissue damage in the lung and increasing fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a liver-specific partial and acute deletion of <i>Crif1</i>, a critical mitoribosomal component for protein synthesis, we found that mice were protected against sepsis, an observation that was phenocopied by the transient inhibition of complex I of the ETC by phenformin. Together, we demonstrate that mitoribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a mechanism for the induction of disease tolerance.

Alpha-Synuclein (aSyn) misfolding and aggregation is common in several neurodegenerative diseases, including Parkinson’s disease and dementia with Lewy bodies, which are known as synucleinopathies. Accumulating evidence suggests that secretion and cell-to-cell trafficking of pathological forms of aSyn may explain the typical patterns of disease progression. However, the molecular mechanisms controlling aSyn aggregation and spreading of pathology are still elusive. In order to obtain unbiased information about the molecular regulators of aSyn oligomerization, we performed a microscopy-based large-scale RNAi screen in living cells. Interestingly, we identified nine Rab GTPase and kinase genes that modulated aSyn aggregation, toxicity and levels. From those, Rab8b, Rab11a, Rab13 and Slp5 were able to promote the clearance of aSyn inclusions and rescue aSyn induced toxicity. Furthermore, we found that endocytic recycling and secretion of aSyn was enhanced upon Rab11a and Rab13 expression in cells accumulating aSyn inclusions. Overall, our study resulted in the identification of new molecular players involved in the aggregation, toxicity, and secretion of aSyn, opening novel avenues for our understanding of the molecular basis of synucleinopathies.

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55Gag is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4+ T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55Gag membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55Gag with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

Checkpoint kinase 1 (CHK1) is a key component of the ATR (ataxia telangiectasia-mutated and Rad3-related)-dependent DNA damage response pathway that protect cells from replication stress, a cell intrinsic phenomenon enhanced by oncogenic transformation. Here, we show that CHK1 is overexpressed and hyperactivated in T-cell acute lymphoblastic leukemia (T-ALL). CHEK1 mRNA is highly abundant in patients of the proliferative T-ALL subgroup and leukemia cells exhibit constitutively elevated levels of the replication stress marker phospho-RPA32 and the DNA damage marker γH2AX. Importantly, pharmacologic inhibition of CHK1 using PF-004777736 or CHK1 short hairpin RNA-mediated silencing impairs T-ALL cell proliferation and viability. CHK1 inactivation results in the accumulation of cells with incompletely replicated DNA, ensuing DNA damage, ATM/CHK2 activation and subsequent ATM- and caspase-3-dependent apoptosis. In contrast to normal thymocytes, primary T-ALL cells are sensitive to therapeutic doses of PF-004777736, even in the presence of stromal or interleukin-7 survival signals. Moreover, CHK1 inhibition significantly delays in vivo growth of xenotransplanted T-ALL tumors. We conclude that CHK1 is critical for T-ALL proliferation and viability by downmodulating replication stress and preventing ATM/caspase-3-dependent cell death. Pharmacologic inhibition of CHK1 may be a promising therapeutic alternative for T-ALL treatment.

Significance Sepsis remains a leading cause of death. New insights into its pathophysiology are likely to be key to the development of effective therapeutic strategies against sepsis. Given the role of GDF15 in metabolism regulation and in cachexia during late stages of cancer, features that also occur in sepsis, elucidation of the possible mechanistic role of GDF15 in sepsis is of great importance. We find that septic patients have very high levels of GDF15 in the peripheral blood, which correlate with clinical outcomes. Using Gdf15 -deficient mice, we show that GDF15 plays a causal role in sepsis by delaying the local control of infection. These findings suggest GDF15 as a potential therapeutic target in sepsis secondary to a bacterial infection.

Sepsis is a potentially lethal syndrome resulting from a maladaptive response to infection. Upon infection, glucocorticoids are produced as a part of the compensatory response to tolerate sepsis. This tolerance is, however, mitigated in sepsis due to a quickly induced glucocorticoid resistance at the level of the glucocorticoid receptor. Here, we show that defects in the glucocorticoid receptor signaling pathway aggravate sepsis pathophysiology by lowering lactate clearance and sensitizing mice to lactate-induced toxicity. The latter is exerted via an uncontrolled production of vascular endothelial growth factor, resulting in vascular leakage and collapse with severe hypotension, organ damage, and death, all being typical features of a lethal form of sepsis. In conclusion, sepsis leads to glucocorticoid receptor failure and hyperlactatemia, which collectively leads to a lethal vascular collapse.

Phospholipid-enveloped biodegradable polymer microparticles and nanoparticles synthesized by an emulsion–solvent evaporation process were characterized by confocal and cryoelectron microscopies to show that the lipid envelope exhibits two-dimensional fluidity and can be configured into 'shell', 'onion', or 'flower' nanostructures, depending on the quantity and composition of lipids employed in the synthesis.

The aetiology of acute appendicitis (AA), the most frequent abdominal surgical emergency, is still unclarified. Recent epidemiologic, clinical and laboratorial data point to an allergic component in the pathophysiology of AA. Mastocytes participate in the Th2 immune response, releasing inflammatory mediators from their granules upon stimulation by IgE-specific antigens. Among the well-known mediators are histamine, serotonin and tryptase, which are responsible for the clinical manifestations of allergies. We conducted a prospective single-centre study to measure histamine and serotonin (commercial ELISA kit) and tryptase (ImmunoCAP System) concentrations in appendicular lavage fluid (ALF) and serum. Consecutive patients presenting to the emergency department with a clinical diagnosis of AA were enrolled: 22 patients with phlegmonous AA and 24 with gangrenous AA The control group was composed of 14 patients referred for colectomy for colon malignancy. Appendectomy was performed during colectomy. Tryptase levels were strikingly different between histological groups, both in ALF and serum (p &lt; 0.001); ALF levels were higher than serum levels. Tryptase concentrations in ALF were 109 times higher in phlegmonous AA (APA) (796.8 (194.1–980.5) pg/mL) and 114 times higher in gangrenous AA (AGA) (837.4 (272.6–1075.1) pg/mL) than in the control group (7.3 (4.5–10.3) pg/mL. For the diagnosis of AA, the discriminative power of serum tryptase concentration was good (AUC = 0.825), but discriminative power was weak (AUC = 0.559) for the differential diagnosis between APA and AGA. Mastocytes are involved in AA during clinical presentations of both phlegmonous and gangrenous appendicitis, and no significant differences in concentration were found. No differences were found in serum and ALF concentrations of histamine and serotonin between histological groups. Due to their short half-lives, these might have elapsed by the time the samples were collected. In future research, these determinations should be made immediately after appendectomy. Our findings confirm the hypersensitivity type I reaction as an event occurring in the pathogenesis of AA: tryptase levels in ALF and serum were higher among patients with AA when compared to the control group, which is in line with a Th2 immune response and supports the concept of the presence of an allergic reaction in the pathogenesis of acute appendicitis. Our results, if confirmed, may have clinical implications for the treatment of AA.

<h2>Abstract</h2> <i>Pseudomonas aeruginosa</i> is an opportunistic pathogen of great medical relevance, although the mechanisms involved in chronic <i>P. aeruginosa</i> infection are unclear. Tomlinson et al. have now shown that systemic and local pathogen-induced ketone bodies (KBs) select strains that preserve respiratory integrity by failing to substantially increase glycolysis, which drives immunopathology resulting from resistance mechanisms.

Effective responses against severe systemic infection require coordination between two complementary defense strategies that minimize the negative impact of infection on the host: resistance, aimed at pathogen elimination, and disease tolerance, which limits tissue damage and preserves organ function. Resistance and disease tolerance mostly rely on divergent metabolic programs that may not operate simultaneously in time and space. Due to evolutionary reasons, the host initially prioritizes the elimination of the pathogen, leading to dominant resistance mechanisms at the potential expense of disease tolerance, which can contribute to organ failure. Here, we summarize our current understanding of the role of physiological perturbations resulting from infection in immune response dynamics and the metabolic program requirements associated with resistance and disease tolerance mechanisms. We then discuss how insight into the interplay of these mechanisms could inform future research aimed at improving sepsis outcomes and the potential for therapeutic interventions.

U2AF is a heterodimeric splicing factor composed of a large (U2AF 65 ) and a small (U2AF 35 ) subunit. In humans, alternative splicing generates two U2AF 35 variants, U2AF 35 a and U2AF 35 b. Here, we used RNA interference to specifically ablate the expression of each isoform in HeLa cells. Our results show that knockdown of the major U2AF 35 a isoform reduced cell viability and impaired mitotic progression, leading to accumulation of cells in prometaphase. Microarray analysis revealed that knockdown of U2AF 35 a affected the expression level of ∼500 mRNAs, from which &gt;90% were underrepresented relative to the control. Among mRNAs underrepresented in U2AF 35 a-depleted cells we identified an essential cell cycle gene, Cdc27, for which there was an increase in the ratio between unspliced and spliced RNA and a significant reduction in protein level. Furthermore, we show that depletion of either U2AF 35 a or U2AF 35 b altered the ratios of alternatively spliced isoforms of Cdc25B and Cdc25C transcripts. Taken together our results demonstrate that U2AF 35 a is essential for HeLa cell division and suggest a novel role for both U2AF 35 protein isoforms as regulators of alternative splicing of a specific subset of genes.

The gene encoding protein kinase WNK2 was recently identified to be silenced by promoter hypermethylation in gliomas and meningiomas, suggesting a tumour-suppressor role in these brain tumours. Following experimental depletion in cell lines, WNK2 was further found to control GTP-loading of Rac1, a signalling guanosine triphosphatase involved in cell migration and motility. Here we show that WNK2 promoter methylation also occurs in 17.5% (29 out of 166) of adult gliomas, whereas it is infrequent in its paediatric forms (1.6%; 1 out of 66). Re-expression of WNK2 in glioblastoma cells presenting WNK2 gene silencing reduced cell proliferation in vitro, tumour growth in vivo and also cell migration and invasion, an effect correlated with reduced activation of Rac1. In contrast, when endogenous WNK2 was depleted from glioblastoma cells with unmethylated WNK2 promoter, changes in cell morphology, an increase in invasion and activation of Rac1 were observed. Together, these results validate the WNK2 gene as a recurrent target for epigenetic silencing in glia-derived brain tumours and provide first mechanistic evidence for a tumour-suppressing role of WNK2 that is related to Rac1 signalling and tumour cell invasion and proliferation.

Pathogen recognition, signaling transduction pathways, and effector mechanisms are necessary steps of innate immune responses that play key roles in the early phase of defense and in the stimulation of the later specific response of adaptive immunity. Here, we argue that in addition to the direct recognition of conserved common structural and functional molecular signatures of microorganisms using pattern recognition receptors, hosts can mount an immune response following the sensing of disruption in homeostasis as proximal reporters for infections. Surveillance of disruption of core cellular activities leading to defense responses is a flexible strategy that requires few additional components and that can effectively detect relevant threats. It is likely to be evolutionarily very conserved and ancient because it is operational in organisms that lack pattern recognition triggered immunity. A homeostasis disruption model of immune response initiation and modulation has broad implications for pathophysiology and treatment of disease and might constitute an often overlooked but central component of a comprehensive conceptual framework for innate immunity.

Obesity is the result of positive energy balance. It can be caused by excessive energy consumption but also by decreased energy dissipation, which occurs under several conditions including when the development or activation of brown adipose tissue (BAT) is impaired. Here we evaluated whether iRhom2, the essential cofactor for the Tumour Necrosis Factor (TNF) sheddase ADAM17/TACE, plays a role in the pathophysiology of metabolic syndrome.We challenged WT versus iRhom2 KO mice to positive energy balance by chronic exposure to a high fat diet and then compared their metabolic phenotypes. We also carried out ex vivo assays with primary and immortalized mouse brown adipocytes to establish the autonomy of the effect of loss of iRhom2 on thermogenesis and respiration.Deletion of iRhom2 protected mice from weight gain, dyslipidemia, adipose tissue inflammation, and hepatic steatosis and improved insulin sensitivity when challenged by a high fat diet. Crucially, the loss of iRhom2 promotes thermogenesis via BAT activation and beige adipocyte recruitment, enabling iRhom2 KO mice to dissipate excess energy more efficiently than WT animals. This effect on enhanced thermogenesis is cell-autonomous in brown adipocytes as iRhom2 KOs exhibit elevated UCP1 levels and increased mitochondrial proton leak.Our data suggest that iRhom2 is a negative regulator of thermogenesis and plays a role in the control of adipose tissue homeostasis during metabolic disease.

Mosquitoes use effective immune responses, including phagocytosis, to fight microbial infection. Here we show that in an Anopheles gambiae immune responsive cell line, RGD recognizing receptors play an important role in the phagocytic response, suggesting overlap between molecular components implicated in adhesion and phagocytosis. Integrins are a major class of adhesive receptors that recognize ligands containing an RGD motif. We have cloned a gene encoding a new β integrin, BINT2, and demonstrated its involvement in Escherichia coli engulfment. Based on molecular modeling, we propose a structural reason for the role of BINT2, but not BINT1, on phagocytosis of Gram-negative bacteria. Using bioinformatic tools, we have identified and compared the complete A. gambiae integrin repertoire as a prelude to a future systematic functional study.

TET1 is a member of the recently identified family of epigenetic regulators, TET1-3 which catalyze the enzymatic conversion of the methyl mark on cytosine (methylcytosine, mC) to the hydroxymethyl mark (hmC). The functions of hmC are required for stem cell maintenance and for controlling differentiation and reprogramming. So far, no roles for TET proteins have been identified in cells of the immune system. Here we show that TET1 is a negative regulator of IL-1β transcription following an inflammatory stimulus and negatively modulates IL-1β secretion in THP-1 cells. In addition, TET1 expression is regulated during inflammation both in THP-1 and in primary dendritic cells. Importantly, other highly induced pro-inflammatory genes are also regulated by TET1, including cytokines, chemokines and adhesion molecules. The other member of the TET family with known roles in stem cell regulation, TET2, is also regulated in THP-1 cells following the inflammatory stimulus and may also participate in IL-1β regulation, according to our observations. Our results suggest a TET1-dependent anti-inflammatory pathway, which may include TET2. In particular, IL-1β transcriptional regulation is likely to depend on TET1-regulated chromatin domains. This work highlights the contribution of epigenetic mechanisms to the efficient organization of inflammatory responses.

We have previously reported a continuous activation of caspase-1 and increased interleukin (IL)-1β levels in early rheumatoid arthritis (RA). These observations raised the hypothesis that drugs targeting the IL-1β pathway, in addition to tumour necrosis factor (TNF), may be particularly effective for early RA treatment. We have recently identified gambogic acid as a promising therapeutic candidate to simultaneously block IL-1β and TNF secretion. Our main goal here was to investigate whether gambogic acid administration was able to attenuate inflammation in antigen-induced arthritis (AIA) rats.Gambogic acid was administered to AIA rats in the early and late phases of arthritis. The inflammatory score, ankle perimeter, and body weight were evaluated during the period of treatment. Rats were sacrificed after 19 days of disease progression and paw samples were collected for histological and immunohistochemical evaluation.We found that inflammation in joints was significantly suppressed following gambogic acid administration. Histological and immunohistochemical evaluation of treated rats revealed normal joint structures with complete abrogation of the inflammatory infiltrate and cellular proliferation.Our results suggest that gambogic acid has significant anti-inflammatory properties and can possibly constitute a prototype anti-inflammatory drug with therapeutic efficacy in the treatment of inflammatory diseases such as RA.

HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA) library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

The immune system relies on the plasticity of its components to produce appropriate responses to frequent environmental challenges. Dendritic cells (DCs) are critical initiators of innate immunity and orchestrate the later and more specific adaptive immunity. The generation of diversity in transcriptional programs is central for effective immune responses. Alternative splicing is widely considered a key generator of transcriptional and proteomic complexity, but its role has been rarely addressed systematically in immune cells. Here we used splicing-sensitive arrays to assess genome-wide gene- and exon-level expression profiles in human DCs in response to a bacterial challenge. We find widespread alternative splicing events and splicing factor transcriptional signatures induced by an E. coli challenge to human DCs. Alternative splicing acts in concert with transcriptional modulation, but these two mechanisms of gene regulation affect primarily distinct functional gene groups. Alternative splicing is likely to have an important role in DC immunobiology because it affects genes known to be involved in DC development, endocytosis, antigen presentation and cell cycle arrest.

Background Rheumatoid arthritis (RA) is a chronic immune-mediated inflammatory disease characterized by cellular infiltration into the joints, hyperproliferation of synovial cells and bone damage. Available treatments for RA only induce remission in around 30% of the patients, have important adverse effects and its use is limited by their high cost. Therefore, compounds that can control arthritis, with an acceptable safety profile and low production costs are still an unmet need. We have shown, in vitro, that celastrol inhibits both IL-1β and TNF, which play an important role in RA, and, in vivo, that celastrol has significant anti-inflammatory properties. Our main goal in this work was to test the effect of celastrol in the number of sublining CD68 macrophages (a biomarker of therapeutic response for novel RA treatments) and on the overall synovial tissue cellularity and joint structure in the adjuvant-induced rat model of arthritis (AIA). Methods Celastrol was administered to AIA rats both in the early (4 days after disease induction) and late (11 days after disease induction) phases of arthritis development. The inflammatory score, ankle perimeter and body weight were evaluated during treatment period. Rats were sacrificed after 22 days of disease progression and blood, internal organs and paw samples were collected for toxicological blood parameters and serum proinflammatory cytokine quantification, as well as histopathological and immunohistochemical evaluation, respectively. Results Here we report that celastrol significantly decreases the number of sublining CD68 macrophages and the overall synovial inflammatory cellularity, and halted joint destruction without side effects. Conclusions Our results validate celastrol as a promising compound for the treatment of arthritis.

Objective Rheumatoid arthritis (RA) is characterised by chronic inflammation leading to articular bone and cartilage damage. Despite recent progress in RA management, adverse effects, lack of efficacy and economic barriers to treatment access still limit therapeutic success. Therefore, safer and less expensive treatments that control inflammation and bone resorption are needed. We have previously shown that celastrol is a candidate for RA treatment. We have observed that it inhibits both interleukin (IL)-1β and tumor necrosis factor (TNF) in vitro, and that it has anti-inflammatory properties and ability to decrease synovial CD68+ macrophages in vivo. Herein our goal was to evaluate the effect of celastrol in local and systemic bone loss. Methods Celastrol was administrated intraperitoneally at a dose of 1 µg/g/day to female Wistar adjuvant-induced arthritic rats. Rats were sacrificed after 22 days of disease progression, and blood, femurs, tibiae and paw samples were collected for bone remodelling markers quantification, 3-point bending test, micro-CT analysis, nanoindentation and Fourier transform infrared spectroscopy measurements, and immunohistochemical evaluation. Results We have observed that celastrol preserved articular structures and decreased the number of osteoclasts and osteoblasts present in arthritic joints. Moreover, celastrol reduced tartrate-resistant acid phosphatase 5b, procollagen type 1 amino-terminal propeptide and C terminal crosslinked telopeptide of type II collagen serum levels. Importantly, celastrol prevented bone loss and bone microarchitecture degradation. Celastrol also preserved bone nanoproperties and mineral content. Additionally, animals treated with celastrol had less fragile bones, as depicted by an increase in maximum load and yield displacement. Conclusions These results suggest that celastrol reduces both bone resorption and cartilage degradation, and preserves bone structural properties.

Plasmodium sporozoites are transmitted by Anopheles mosquitoes and infect hepatocytes, where a single sporozoite replicates into thousands of merozoites inside a parasitophorous vacuole. The nature of the Plasmodium-host cell interface, as well as the interactions occurring between these two organisms, remains largely unknown. Here we show that highly dynamic hepatocyte actin reorganization events occur around developing Plasmodium berghei parasites inside human hepatoma cells. Actin reorganization is most prominent between 10 to 16 hours post infection and depends on the actin severing and capping protein, gelsolin. Live cell imaging studies also suggest that the hepatocyte cytoskeleton may contribute to parasite elimination during Plasmodium development in the liver.

Purpose: Metabolic syndrome and cardiovascular disease are strongly associated with obstructive sleep apnea syndrome (OSAS), which causes substantial changes to normal circadian physiological functions, including metabolic pathways. Because core clock genes are known to be modulated by sleep/vigilance cycles, we asked whether the expression level of mRNA coding for clock genes is altered in non-treated OSAS patients and if it can be corrected by standard positive pressure treatment. Methods: Peripheral blood was collected from male patients diagnosed with severe OSAS (AHI≥30/hr) before and after treatment initiation. qPCR was used to measure mRNA levels of genes associated with the central circadian pacemaker including CLOCK, Bmal1, Cry1, Cry2 and three Period genes (Per 1, 2, 3) in peripheral blood mononuclear cells (PBMCs). Results: We found statistically significant differences for CLOCK (p-value=0.022) expression in PBMCs of OSAS patients that were not reverted by treatment with standard positive pressure treatment. We have also found a substantial decrease in the slow wave sleep (SWS) content in OSAS patients (p-value<0.001) that, contrary to REM sleep, was not corrected by CPAP (p=0.875). Conclusions: CPAP treatment does not correct substantial changes in expression of core clock genes in OSAS patients. Because CPAP treatment is also unable to normalize the SWS in these patients, it is likely that additional therapeutic interventions that increase SWS content and complement the benefits of CPAP are required to more effectively reduce the known increased cardiovascular risk associated with OSAS patients.

The accurate replication and repair of DNA is central to organismal survival. This process is challenged by the many factors that can change genetic information such as replication errors and direct damage to the DNA molecule by chemical and physical agents. DNA damage can also result from microorganism invasion as an integral step of their life cycle or as collateral damage from host defense mechanisms against pathogens. Here we review the complex crosstalk of DNA damage response and immune response pathways that might be evolutionarily connected and argue that DNA damage response pathways can be explored therapeutically to induce disease tolerance through the activation of tissue damage control processes. Such approach may constitute the missing pillar in the treatment of critical illnesses caused by multiple organ failure, such as sepsis and septic shock.

Article11 September 2020Open Access Transparent process Zinc inhibits lethal inflammatory shock by preventing microbe-induced interferon signature in intestinal epithelium Jolien Souffriau Jolien Souffriau Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Steven Timmermans Steven Timmermans Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tineke Vanderhaeghen Tineke Vanderhaeghen Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Charlotte Wallaeys Charlotte Wallaeys Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Kelly Van Looveren Kelly Van Looveren Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsy Aelbrecht Lindsy Aelbrecht Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sylviane Dewaele Sylviane Dewaele Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Vandewalle Jolien Vandewalle orcid.org/0000-0003-1844-6476 Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Evy Goossens Evy Goossens Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Serge Verbanck Serge Verbanck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Filip Boyen Filip Boyen Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Melanie Eggermont Melanie Eggermont Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsey De Commer Lindsey De Commer Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Michiel De Bruyne Michiel De Bruyne orcid.org/0000-0002-1276-1857 Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Raul Tito Raul Tito Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Marlies Ballegeer Marlies Ballegeer Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sofie Vandevyver Sofie Vandevyver Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tiago Velho Tiago Velho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Luis Ferreira Moita Luis Ferreira Moita Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Tino Hochepied Tino Hochepied Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Karolien De Bosscher Karolien De Bosscher VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Filip Van Immerseel Filip Van Immerseel Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Rudi Beyaert Rudi Beyaert orcid.org/0000-0002-5704-582X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Corresponding Author Claude Libert [email protected] orcid.org/0000-0001-6408-036X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Souffriau Jolien Souffriau Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Steven Timmermans Steven Timmermans Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tineke Vanderhaeghen Tineke Vanderhaeghen Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Charlotte Wallaeys Charlotte Wallaeys Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Kelly Van Looveren Kelly Van Looveren Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsy Aelbrecht Lindsy Aelbrecht Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sylviane Dewaele Sylviane Dewaele Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Jolien Vandewalle Jolien Vandewalle orcid.org/0000-0003-1844-6476 Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Evy Goossens Evy Goossens Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Serge Verbanck Serge Verbanck Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Filip Boyen Filip Boyen Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Melanie Eggermont Melanie Eggermont Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Lindsey De Commer Lindsey De Commer Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Riet De Rycke Riet De Rycke Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Michiel De Bruyne Michiel De Bruyne orcid.org/0000-0002-1276-1857 Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium Search for more papers by this author Raul Tito Raul Tito Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Marlies Ballegeer Marlies Ballegeer Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Sofie Vandevyver Sofie Vandevyver Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Tiago Velho Tiago Velho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Luis Ferreira Moita Luis Ferreira Moita Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Tino Hochepied Tino Hochepied Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Karolien De Bosscher Karolien De Bosscher VIB Center for Medical Biotechnology, Ghent, Belgium Department of Biochemistry, Ghent University, Ghent, Belgium Search for more papers by this author Jeroen Raes Jeroen Raes Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium VIB-KU Leuven Center for Microbiology, Leuven, Belgium Search for more papers by this author Filip Van Immerseel Filip Van Immerseel Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Search for more papers by this author Rudi Beyaert Rudi Beyaert orcid.org/0000-0002-5704-582X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Claude Libert Corresponding Author Claude Libert [email protected] orcid.org/0000-0001-6408-036X Center for Inflammation Research, VIB, Ghent, Belgium Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium Search for more papers by this author Author Information Jolien Souffriau1,2,‡, Steven Timmermans1,2,‡, Tineke Vanderhaeghen1,2, Charlotte Wallaeys1,2, Kelly Van Looveren1,2, Lindsy Aelbrecht1,2, Sylviane Dewaele1,2, Jolien Vandewalle1,2, Evy Goossens3, Serge Verbanck3, Filip Boyen3, Melanie Eggermont1,2, Lindsey De Commer4,5, Riet De Rycke6,7, Michiel De Bruyne6,7, Raul Tito4,5, Marlies Ballegeer1,2, Sofie Vandevyver1,2, Tiago Velho8, Luis Ferreira Moita8, Tino Hochepied1,2, Karolien De Bosscher9,10, Jeroen Raes4,5, Filip Van Immerseel3, Rudi Beyaert1,2 and Claude Libert *,1,2 1Center for Inflammation Research, VIB, Ghent, Belgium 2Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium 3Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium 4Department of Microbiology and Immunology, Rega Institute, KU Leuven, Leuven, Belgium 5VIB-KU Leuven Center for Microbiology, Leuven, Belgium 6Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University, Ghent, Belgium 7VIB Center for Inflammation Research and BioImaging Core, VIB, Ghent, Belgium 8Instituto Gulbenkian de Ciência, Oeiras, Portugal 9VIB Center for Medical Biotechnology, Ghent, Belgium 10Department of Biochemistry, Ghent University, Ghent, Belgium ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +32 9 3313700; E-mail: [email protected] EMBO Mol Med (2020)12:e11917https://doi.org/10.15252/emmm.201911917 The authors want to dedicate this paper to the memory of Prof. Walter Fiers (1931–2019), who passed away on 31 July 2019, and who was mentor and strong supporter of this project and research team. 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 The cytokine TNF drives inflammatory diseases, e.g., Crohn's disease. In a mouse model of TNF-induced systemic inflammatory response syndrome (SIRS), severe impact on intestinal epithelial cells (IECs) is observed. Zinc confers complete protection in this model. We found that zinc no longer protects in animals which lack glucocorticoids (GCs), or express mutant versions of their receptor GR in IECs, nor in mice which lack gut microbiota. RNA-seq studies in IECs showed that zinc caused reduction in expression of constitutive (STAT1-induced) interferon-stimulated response (ISRE) genes and interferon regulatory factor (IRF) genes. Since some of these genes are involved in TNF-induced cell death in intestinal crypt Paneth cells, and since zinc has direct effects on the composition of the gut microbiota (such as several Staphylococcus species) and on TNF-induced Paneth cell death, we postulate a new zinc-related anti-inflammatory mechanism. Zinc modulates the gut microbiota, causing less induction of ISRE/IRF genes in crypt cells, less TNF-induced necroptosis in Paneth cells, and less fatal evasion of gut bacteria into the system. Synopsis This study provides genetic and biochemical evidence that the intestinal health promoting effects of zinc supplementation is based on a specific, direct effect on members of the microbial community in the ileum. The cytokine TNF has dramatic effects on the viability of ileal crypt Paneth cells The TNF effect depends on expression of Interferon Stimulated Response Element (ISRE) and Interferon Regulatory Factor (IRF) genes which are induced by gut microbes By killing bacteria such as several Staphylococci species, zinc reduces this ISRE/IRF expression, preventing Paneth cell death during inflammation Since glucocorticoids, like dexamethasone, reduce ISRE/IRF on the transcriptional level, zinc works in concert with such steroids The paper explained Problem Zinc has therapeutic effects, mainly in intestinal infections and diarrhea. The mechanism of this zinc effect is poorly known. Zinc also protects against intestinal effects induced by the cytokine tumor necrosis factor (TNF), which plays a key role in Crohn's disease. Since zinc was previously shown to protect in the TNF model in mice, we applied this model to investigate zinc's mode of action. Results By applying RNA-seq in ileum biopsies in mice treated with water or ZnSO4 in the drinking water, we found that zinc causes strong downregulation of a large group of genes, known as ISRE/IRF genes. These are thought to (confirmed here) be induced in intestinal epithelium by gut microbes. Zinc lowers these genes, by actively and directly modulating the composition of the gut microbes, and our data suggest that mainly Staphylococci would be targeted by zinc. The reduction in ISRE/IRF genes is important, because several of these genes play an important role in killing certain epithelial cells (known as Paneth cells), when TNF is present. Because of the effects of zinc on bacteria and ISRE/IRF genes in these cells, they resist killing by TNF. How this leads to survival of the animals is not yet clear, but since zinc also reduces the escape of bacteria from the gut to other organs, the impact of zinc on the gut microbes or on the Paneth cell's death appears responsible as well. Mice with extremely high ISRE/IRF gene expression (e.g., because they have no adrenals) or extremely low ISRE/IRF genes, because they have no gut microbes, are not susceptible to the beneficial effects of zinc. Impact Now that the mechanism of zinc is unfolded, more precise zinc targeting or zinc replacing strategies in intestinal diseases where TNF is implicated (Crohn's disease) can be considered. Introduction The cytokine TNF is a central player in inflammatory diseases and infections. TNFR1 (Puimege et al, 2014) is the main pathogenic TNF receptor and is strongly stimulated by acute and chronic expressed TNF. The systemic effects of TNF have been studied in detail. It is striking that the intestinal epithelium is sensitive to TNF-induced damage: Transgenic TNF overexpression, via a myriad of mechanisms, clearly leads to inflammatory bowel disease and/or arthritis, and injection of TNF in mammals has shown that intestinal toxicity is a major dose-limiting issue (Piguet et al, 1998; Kontoyiannis et al, 1999). Cell death of IECs as well as reduced expression of tight junctions appears essential in the increase in gut permeability induced by TNF. This reduced barrier between the homeostatic body and the gut luminal content is crucial (Van Hauwermeiren et al, 2015). In the lumen, the microbiota, consisting of many billions of microbes, predominantly bacteria, is present (Vrancken et al, 2019). Most of these are harmless if they remain in the gut lumen. TNF has been shown to lead to outflow of bacteria from the lumen and colonization of draining mesenteric lymph nodes (MLNs) and spleen (Van Hauwermeiren et al, 2015). Hence, broad-spectrum antibiotics confer significant protection against TNF-induced lethal systemic inflammatory response syndrome (SIRS; Van Hauwermeiren et al, 2015). Since IEC-specific depletion of TNFR1 leads to a similar protective effect as an antibiotic treatment (Van Hauwermeiren et al, 2013), it appears conceivable that TNF, via TNFR1, induces IEC damage, followed by a bacterial contamination of organs. The damage that is caused by TNF to IECs is incompletely understood, but is of utmost importance (Van Assche et al, 2010), as it may play a role in Crohn's disease. TNF induces cell death of IECs, shrinkage of villi and erosion (Piguet et al, 1998), and death of goblet cells as well as Paneth cells (Van Hauwermeiren et al, 2015). Glucocorticoids (GCs; e.g., the synthetic dexamethasone (Dex)), which function by binding to the glucocorticoid receptor (GR), confer protection against Crohn's diseases (Van Assche et al, 2010) and against TNF-induced intestinal damage and lethal shock, when given prior to TNF. From a therapeutic viewpoint, this preventive therapy is not relevant, but this effect can help elucidating essential mechanisms. It was also shown that removal of adrenals which produce endogenous GCs drastically sensitizes to TNF and that GR mutant mice have sensitized IECs toward TNF (Ballegeer et al, 2018). The mechanism by which GCs/GR dampens TNF effects in the gut is thought to relate to the gut commensal flora: The microbes were recently shown to chronically induce, in the ileum, interferon-stimulated response element (ISRE) genes, and interferon regulatory factor (IRF) genes, some of which are involved in necroptosis (Ripk3, Mlkl, and Zbp1; Ballegeer et al, 2018). When GCs or GR was absent, the IECs lost control over these genes, and hence, these genes underwent high expression. Under such conditions, low doses of TNF sufficed to cause induction of necroptosis followed by death of the animals. Interestingly, this necroptosis was confined to crypt cells known as Paneth cells (Ballegeer et al, 2018). In humans, 25% of the global mortalities are caused by microbial infections. Many of these problems are due to the development of resistance against antibiotics. Investigation of interesting alternative antimicrobial strategies has become mandatory. Zinc has a huge potential in this respect, since zinc has been shown to increase the resistance of vulnerable patients (children, elderly, underfed) against diarrhea and gastrointestinal infections (Souffriau & Libert, 2018). We have previously published that a pretreatment of mice with ZnSO4 in the drinking water for 1 week leads to strong protection against TNF-induced SIRS (Waelput et al, 2001; Van Molle et al, 2007), but the mechanism of protection remained unclear. Zinc is essential for a healthy intestinal homeostasis and is known to be essential for Paneth cell function and survival (Jouppila et al, 1976; Podany et al, 2016). In these cells, zinc is actively adsorbed by transporters and shuttled into the secretory vesicles, which contain anti-bacterial defensins, some of which are activated by zinc-dependent proteases, the most important of which being matrix metalloproteinase-7 (MMP7; Wilson et al, 1999). Zinc has been shown to activate a specific transcription factor, metal transcription factor 1 (MTF1) by direct binding (Gunther et al, 2012), leading to numerous transcriptional changes, e.g., induction of a zinc transporter known as ZnT2 and coded by Slc30a2. ZnT2 is of importance in the intracellular zinc transport in Paneth cells (Podany et al, 2016). Zn has also been shown to have profound effects on the composition of the gut microbiota (Li et al, 2016; Zackular et al, 2016), but whether this is via direct anti-bacterial effects, or via Paneth cells or other mechanisms is not clear. In this paper, we studied the mechanism of protection of zinc against TNF-induced lethal SIRS. We studied the cross-talk of zinc with GCs/GR and found that zinc is unable to protect against TNF when GR is mutated or absent in the IECs or when corticosterone is absent. Zinc appeared to reduce the ISRE/IRF-dependent genes in the IECs, thereby protecting these cells (particularly Paneth cells) against TNF-induced necroptosis and bacterial influx from the gut lumen into the system. The mechanism by which zinc reduces ISRE/IRF genes was investigated and appears to relate to direct anti-bacterial effects of zinc against certain bacterial taxa, such as Staphylococcus sciuri and Staphylococcus nepalensis. The elucidation of the action mechanism of zinc may lead to better understanding of zinc's effects in the treatment of intestinal infections and diarrhea in humans and farm animals. Results Glucocorticoids and GR dimers play an essential role in the zinc protection against TNF-induced lethality Endogenous and synthetic GCs and their receptor GR are crucial for the survival of mice against TNF-induced SIRS (Vandevyver et al, 2012; Ballegeer et al, 2018). We investigated whether GCs are involved in the zinc-induced protection against TNF. We used adrenalectomized (Adx) mice, which are unable to produce corticosterone. Adx mice were pretreated for 7 days with 25 mM ZnSO4 via the drinking water, a protocol that was published to confer optimal protection in the TNF model in normal mice (Waelput et al, 2001). In contrast to naïve C57BL/6J mice, Adx mice were not protected by ZnSO4 against a lethal intraperitoneal (i.p.) injection of TNF (Fig 1A). Serum corticosterone levels were indeed very low in Adx mice and were not increased upon 1-week treatment of mice with ZnSO4 (Fig 1B). GRDim mice, which express a point-mutant GR protein that forms less efficient GR homodimers and DNA interaction, were previously found to be extremely sensitive for TNF-induced SIRS (Reichardt et al, 1998; Van Bogaert et al, 2011; Vandevyver et al, 2012; Ballegeer et al, 2018). ZnSO4 was unable to confer protection against TNF in these mice (Fig 1C). Increasing the zinc dose from 25 to 75 mM (the maximal tolerated dose for mice) for a week had no protective effect in GRDim mice either. Therefore, we conclude that the zinc protection against TNF requires the presence of corticosterone and functional GR dimers, i.e., involves GR-mediated gene regulation. Figure 1. ZnSO4 fails to protect against a lethal TNF injection in Adx, GRDim, and GRVillKO mice A. Survival curves of C57BL/6J naïve and adrenalectomized (Adx) mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 30 μg (naïve) or 2 μg (Adx) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 72 h after TNF injection. Results represent combined data of three experiments. B. Corticosterone levels in the serum of C57BL/6J naïve and Adx mice after 7 days of 25 mM ZnSO4 supplementation to the drinking water (N = 6 per group). C. Survival curves of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 50 μg (GRWT) or 12.5 μg (GRDim) TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 50 h after TNF injection. Results represent combined data of two experiments. D, E. Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of C57BL/6J naïve and Adx mice (D, N = 3–6), or GRWT and GRDim mice (E, N = 6–9). F. Agtpbp1 gene expression measured with RT–qPCR in the ileum of GRWT and GRDim mice treated for 7 days with 25 mM ZnSO4 in the drinking water (N = 3 per group). G. Survival curves of GRfl/fl and GRVillKO mice treated for 7 days with 25 mM ZnSO4 in the drinking water and challenged, i.p., with 35 μg TNF, solved in sterile PBS, per 20 g bodyweight. No deaths occurred later than 150 h after TNF injection. Results represent combined data of six experiments. H. Mouse Nr3c1 gene expression measured with RT–qPCR in the intestinal epithelial cells (IECs) of GRfl/fl and GRVillKO mice (N = 3–6). Hprt and Villin were used as housekeeping genes. I. Zinc levels in the serum after 7 days of 25 mM ZnSO4 supplementation to the drinking water of GRfl/fl and GRVillKO mice (N = 9 per group). J. Number of genes differentially expressed (up and down) by zinc (LFC > 0.8 and P < 0.05) measured by RNA sequencing in the ileum and liver of C57BL/6J mice treated with 25 mM ZnSO4 in the drinking water for 7 days (N = 3). Data information: For the survival curves, P-values were analyzed with a chi-square test. In (B, D, E, F, H, I), the data are shown as mean ± SD. P-values were analyzed with Student's t-test (unpaired, two-tailed) on the log-transformed data in (B, H) and with a two-way ANOVA followed by a Tukey multiple comparisons test in (D, E, F, I). Significant expression of genes in the RNA sequencing was assessed with a Wald test with negative binomial distribution in DESEQ2. Download figure Download PowerPoint We further confirmed that the complete lack of protection of zinc in Adx and GRDim mice is not reflected in a lack of zinc uptake by these mice, since blood zinc levels 1 week after 25 mM ZnSO4 were equally increased in these as in control mice (Fig 1D and E). Blood zinc levels fluctuate in mammals between 75 and 125 μg/dl (Reyes, 1996). In our studies, 1 week of treatment increases these levels to 200–300 μg/dl. Zinc is able to upregulate the mRNA expression of several genes (see further Fig 2) in the ileum of mice, and several key control genes, e.g., Agtpbp1, were found to be perfectly induced by zinc in GRDim as in GRWT mice, illustrating that zinc in these GRDim mice does have biological activities (Fig 1F). Figure 2. Transcriptional effects of ZnSO4 in the ileum and the role of antibiotics and germ-free aspects A–C. RNA sequencing on ileum samples of C57BL/6J mice treated with water or 25 mM ZnSO4 water for 7 days and then injected i.p. with 200 μg Dex (Rapidexon) solved in 200 μl PBS, or with PBS only. 2 h after injection, the ileum was isolated (N = 3 per group). Overlap between genes (A) upregulated or (B) downregulated by ZnSO4 (Zn) or Dex (LFC > 0.8, P < 0.05). (C) Scheme of numbers of genes upregulated in ZnSO4, Dex, or ZnSO4+Dex-treated mice (LFC > 0.8, P < 0.05). Significant expression of genes in the RNA sequencing was assessed with a Wald test with negative binomial distribution in DESEQ2. D. Regression curve plotting LFCs of genes induced by ZnSO4 in GRWT and GRDim mice, as measured by RNA-seq in ileum samples. All genes, induced in ileum by DEX in GRWT with LFC > 0.8, P < 0.05 are considered, while these genes with P < 0.05 in GRDim were considered. The slope of the correlation curve as well as Pearson correlation coefficient was calculated by GraphPad Prism. E. C57BL/6J mice received antibiotics (AB) in the drinking water for 3 weeks, followed by 1 week of 25 mM ZnSO4 in the drinking water. During this week, AB administration was continued by oral gavage. Mice were challenged i.v. with 12.5 μg TNF solved in sterile PBS and survival recorded. Combined data of two experiments. F. Zinc levels measured in serum after the antibiotics and ZnSO4 protocol (N = 5–8). G,H. Mice housed in germ-free (GF) or specific pathogen-free (SPF) conditions received 25 mM ZnSO4 in the drinking water for 1 week and were then challenged i.p. with 35 μg TNF, dissolved in sterile PBS, per 20 g bodyweight. Combined data of two experiments. (H) displays the blood zinc levels in SPF and GF mice treated for 1 week with water or ZnSO4 (N = 3/4 per group). I. Ileum Slurry (IS) was isolated from C57BL/6J mice put on 25 mM ZnSO4 or control water for 1 week, and 750 μl of this IS was injected i.p. in C57BL/6J GF mice. Data information: For the survival curves, P-values were analyzed with a chi-square test. In (F) and (H), data are shown as mean ± SD and P-values were analyz

Abstract OBJECTIVES Activated Clotting Time (ACT) is commonly used to monitor anticoagulation during cardiac surgeries. Final-ACT values may be essential to predict postoperative bleeding and transfusions, although ideal values remain unknown. Our aim was to evaluate the utility of ACT as a predictor of postoperative bleeding and transfusion use. METHODS Retrospective study (722 patients) submitted to surgery between July 2018–October 2021. We compared patients with Final-ACT&amp;lt;basal-ACT and Final-ACT≥basal-ACT; and Final-ACT &amp;lt; 140s with ≥140 s. Continuous variables were analyzed with Wilcoxon Rank-Sum test; categorical variables using Chi-square or Fisher's exact test. A Linear Mixed Regression model was used to analyze bleeding in patients with Final-ACT &amp;lt; 140s and ≥140 s.Independent variables were analyzed with Binary Logistic Regression models to investigate their association with bleeding and transfusion. RESULTS Patients with Final-ACT ≥ 140s presented higher postoperative bleeding than Final-ACT &amp;lt; 140s at 12 h (P = 0.006) and 24 h (**P = 0.004). CPB time (OR 1.009,1.002–1.015,95% CI) and masculine sex (OR 2.842,1.721–4.821,95% CI) were significant predictors of bleeding. Patients with Final-ACT ≥ 140s had higher risk of UT (OR 1.81, 1.13–2.89, 95% CI; P = 0.0104), compared to Final-ACT &amp;lt; 140s. CPB time (OR 1.019,1.012–1.026,95% CI) and Final-ACT (OR 1.021,1.010–1.032,95% CI) were significant predictors of transfusion. Female sex was a predictor of UT, with a probability for use of 27.23% (21.84–33.39%,95% CI) in elective surgeries, and 60.38% (37.65–79.36%,95% CI) in urgent surgeries, higher than in males. CONCLUSIONS Final-ACT has a good predictive value for the use of transfusion. Final-ACT ≥ 140s correlate with higher risk of transfusion and increased bleeding. The risk of bleeding and transfusion is higher with longer periods of CPB. Males have a higher risk of bleeding, but females have a higher risk of transfusion.

Several classes of antibiotics have long been known for protective properties that cannot be explained through their direct antimicrobial effects. However, the molecular bases of these beneficial roles have been elusive. In this issue of the JCI, Mottis et al. report that tetracyclines induced disease tolerance against influenza virus infection, expanding their protection potential beyond resistance and disease tolerance against bacterial infections. The authors dissociated tetracycline's disease-resistance properties from its disease-tolerance properties by identifying potent tetracycline derivatives with minimal antimicrobial activity but increased capacity to induce an adaptive mitochondrial stress response that initiated disease tolerance mechanisms. These findings have potential clinical applications in viral infections.

Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here, we show that the extensively used anthracyclines Doxorubicin, Daunorubicin, and Epirubicin decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon-responsive genes in primary mouse ( Mus musculus ) macrophages. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA-binding sites. The anthracycline variants Aclarubicin, Doxorubicinone, and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

ABSTRACT Background Seroepidemiological studies provide estimates of population‐level immunity, prevalence/incidence of infections, and evaluation of vaccination programs. We assessed the seroprevalence of protective antibodies against influenza and evaluated the correlation of seroprevalence with the cumulative annual influenza incidence rate. Methods We conducted an annual repeated cross‐sectional seroepidemiological survey, during June–August, from 2014 to 2019, in Portugal. A total of 4326 sera from all age groups, sex, and regions was tested by hemagglutination inhibition assay. Seroprevalence and geometric mean titers (GMT) of protective antibodies against influenza were assessed by age group, sex, and vaccine status (65+ years old). The association between summer annual seroprevalence and the difference of influenza incidence rates between one season and the previous one was measured by Pearson correlation coefficient ( r ). Results Significant differences in seroprevalence of protective antibodies against influenza were observed in the population. Higher seroprevalence and GMT for A(H1N1)pdm09 and A(H3N2) were observed in children (5–14); influenza B seroprevalence in adults 65+ was 1.6–4.4 times than in children (0–4). Vaccinated participants (65+) showed significant higher seroprevalence/GMT for influenza. A strong negative and significant correlation was found between seroprevalence and ILI incidence rate for A(H1N1)pdm09 in children between 5 and 14 ( r = −0.84; 95% CI, −0.98 to −0.07); a weak negative correlation was observed for A(H3N2) and B/Yamagata ( r ≤ −0.1). Conclusions The study provides new insight into the anti‐influenza antibodies seroprevalence measured in summer on the ILI incidence rate in the next season and the need for adjusted preventive health care measures to prevent influenza infection and transmission.

Basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) are high-incidence, non-melanoma skin cancers (NMSCs). The success of immune-targeted therapies in advanced NMSCs led us to anticipate that NMSCs harbored significant populations of tumor-infiltrating lymphocytes with potential anti-tumor activity. The main aim of this study was to characterize T cells infiltrating NMSCs. Flow cytometry and immunohistochemistry were used to assess, respectively, the proportions and densities of T cell subpopulations in BCCs (n = 118), SCCs (n = 33), and normal skin (NS, n = 30). CD8+ T cells, CD4+ T cell subsets, namely, Th1, Th2, Th17, Th9, and regulatory T cells (Tregs), CD8+ and CD4+ memory T cells, and γδ T cells were compared between NMSCs and NS samples. Remarkably, both BCCs and SCCs featured a significantly higher Th1/Th2 ratio (~four-fold) and an enrichment for Th17 cells. NMSCs also showed a significant enrichment for IFN-γ-producing CD8+T cells, and a depletion of γδ T cells. Using immunohistochemistry, NMSCs featured denser T cell infiltrates (CD4+, CD8+, and Tregs) than NS. Overall, these data favor a Th1-predominant response in BCCs and SCCs, providing support for immune-based treatments in NMSCs. Th17-mediated inflammation may play a role in the progression of NMSCs and thus become a potential therapeutic target in NMSCs.

Background: We previously demonstrated that celastrol has significant anti-inflammatory and bone protective effects when administered via the intraperitoneal route. For further preclinical evaluation, an effective oral administration of celastrol is crucial. Here we aimed to study the therapeutic dose range for its oral administration. Methods: Celastrol (1-25 μg/g/day, N = 5/group) was administrated orally to female adjuvant-induced arthritis (AIA) rats after 8 days of disease induction for a period of 14 days. A group of healthy (N = 8) and arthritic (N = 15) gender- and age-matched Wistar rats was used as controls. During the treatment period, the inflammatory score, ankle perimeter, and body weight were measured. At the end of the treatment, the animals were sacrificed, blood was collected for clinical pathology, necropsy was performed with collection of internal organs for histopathological analysis, and paw samples were used for disease scoring. Results: Doses higher than 2.5 μg/g/day of celastrol reduced the inflammatory score and ankle swelling, preserved joint structure, halted bone destruction, and diminished the number of synovial CD68+ macrophages. Bone resorption and turnover were also reduced at 5 and 7.5 μg/g/day doses. However, the dose of 7.5 μg/g/day was associated with thymic and liver lesions, and higher doses showed severe toxicity. Conclusion: Oral administration of celastrol above 2.5 μg/g/day ameliorates arthritis. This data supports and gives relevant information for the development of a preclinical test of celastrol in the setting of a chronic model of arthritis since rheumatoid arthritis is a long-term disease.

IgE mediates type I hypersensitivity reaction and can be found in the mucosa of organs affected by allergy. Acute appendicitis (AA) is a common disease, but its etiology remains poorly understood. Here, we investigated IgE deposition in histological sections of AA samples to test the hypothesis that an allergic reaction may substantially contribute to the pathophysiology of AA.In a retrospective study, we assessed the presence of IgE in appendicular specimens of histologically confirmed appendicitis and in the control group, comprised of negative appendicitis and incidental appendectomies, using a monoclonal antibody against human IgE. Samples from 134 appendectomies were included: 38 phlegmonous and 27 gangrenous appendicitis from the study group and 52 incidental appendectomies and 17 negative appendicitis from the control group. The slides were visualized by light microscopy, and a standard procedure was used to manually count the positive IgE staining cells.IgE staining was present in the cells of all but 5 appendicular specimens. We found a significantly increased number of IgE-positive cells in phlegmonous AA (median = 28) when compared to incidental appendectomy (median = 17) (p = 0.005; p < 0.0001 when adjusted for age and gender). No difference was found for gangrenous appendicitis. Discussion. The presence of IgE supports the contribution of an allergic reaction for the pathophysiology of phlegmonous appendicitis. The reduced number of IgE staining cells in gangrenous appendicitis can be due to tissue destruction, or, as been claimed by others, gangrenous appendicitis is a distinct entity, with different etiology.In this study, phlegmonous appendicitis had the highest number of IgE-positive appendicular cells. These findings suggest that an allergic reaction can contribute to the pathophysiology of AA, opening a novel possibility for preventive measures in a disease that typically requires surgery.

The generation of diversity and plasticity of transcriptional programs are key components of effective vertebrate immune responses. The role of Alternative Splicing has been recognized, but it is underappreciated and poorly understood as a critical mechanism for the regulation and fine-tuning of physiological immune responses. Here we report the generation of loss-of-function phenotypes for a large collection of genes known or predicted to be involved in the splicing reaction and the identification of 19 novel regulators of IL-1β secretion in response to E. coli challenge of THP-1 cells. Twelve of these genes are required for IL-1β secretion, while seven are negative regulators of this process. Silencing of SFRS3 increased IL-1β secretion due to elevation of IL-1β and caspase-1 mRNA in addition to active caspase-1 levels. This study points to the relevance of splicing in the regulation of auto-inflammatory diseases.

Sleep related breathing disorders (SRBD) cause sleep fragmentation, intermittent hypoxia or a combination of both leading to homeostasis perturbations, including in the immune system. We investigated whether SRBD patients with or without intermittent hypoxia show substantial differences in perforin and granzyme-B positive peripheral blood lymphocytes. A total of 87 subjects were included and distributed as follows: 24 controls (C), 19 patients with respiratory effort related arousals due to increased upper airway resistance (UAR) without hypoxic events, 24 obese patients with obstructive sleep apnea (OSA) (oOSA), and 20 without obesity (noOSA). After polysomnographic recording, we analyzed in fasting blood samples routine hematologic and biochemical parameters and the percentage of lymphocytes containing the proteins perforin and granzyme-B (GrB). Kruskal-Wallis tests and a posteriori multiple comparisons were applied for statistical analysis of results. Perforin-positive γδ-cells revealed significant differences between groups (p = 0.017), especially between the Control group and the oOSA (p-value = 0.04); the remaining SRBD groups also showed differences from the control (C vs UAR: p = 0.08; C vs noOSA = 0.09), but they did not raise to statistical significance. There were no differences among the SRBD groups. Granzyme-B cells were decreased in SRBD patients, but the differences were not statistically significant. No additional statistical significant result was found in the other investigated lymphocyte subsets. Obstructive sleep-disordered breathing is associated with a decrease in perforin-positive CD3+γδ-T cells. Although this finding was detected in lean patients without intermittent hypoxia, the reduction was only statistically significant in obese patients with severe OSA. Because CD3+γδ-T cells play an important role in the control of tumor cells, our findings are directly relevant for the study of the association of OSA and cancer.

Effective CD 8 + T ‐cell responses against tumor or microbial antigens that are not directly expressed in antigen‐presenting cells ( APC s) depend on the cross‐presentation of these antigens on MHC c lass I in APC s. To identify signaling molecules that regulate cross‐presentation, we used lentiviral‐based RNA interference to test the roles of hundreds of kinases and phosphatases in this process. Our study uncovered eight previously unknown genes, consisting of one positive and seven negative regulators of antigen cross‐presentation. Depletion of Acvr1c, a type I receptor for TGF ‐β family of signaling molecules, led to an increase in CD 80 and CD 86 co‐stimulator surface expression and secreted IL ‐12 in mouse bone marrow‐derived DC s, as well as antigen‐specific T ‐cell proliferation.

Sepsis is a life-threatening condition that arises as a systemic inflammatory response syndrome to an infection. Its uncontrolled progression can in frequent cases lead to multiple organ failure, which is still associated with high mortality rates. Modern antibiotics made clear that the infection is only an initiating, and not always necessary, event of this syndrome as many patients with sepsis die despite effective eradication of the inciting pathogen. This observation critically contributed to a paradigm shift that focused the pathogenesis of sepsis on the host and not on the pathogen. However, therapeutic strategies based on the inhibition of proinflammatory critical mediators of sepsis or immunostimulation have so far failed to improve sepsis outcome and, therefore, this condition urgently needs transformative therapeutic ideas and strategies. Here we argue that the induction of tolerance, a defence strategy that minimises the impact of an infection on organ function without directly affecting the pathogen burden, is perhaps the missing but essential element to add to the current components of sepsis care and treatment.

Abstract While antibiotics are intended to specifically target bacteria, most are known to affect host cell physiology. In addition, some antibiotic classes are reported as immunosuppressive, for reasons that remain unclear. Here we show that linezolid, a ribosomal-targeting antibiotic (RAbo), effectively blocked the course of a T cell-mediated autoimmune disease. Linezolid and other RAbos were strong inhibitors of Th17 effector function in vitro , showing that this effect was independent of their antibiotic activity. Perturbing mitochondrial translation in differentiating T cells, either with RAbos or through the inhibition of mitochondrial elongation factor G1 (mEF-G1) progressively compromises the integrity of the electron transport chain (ETC). Ultimately, this leads to loss of mitochondrial metabolism and cytokine production in differentiating Th cells. In accordance, mice lacking Gfm1 in T cells are protected from EAE, demonstrating that this pathway plays a key role in maintaining T cell function and pathogenicity.

Summary Synergy of resistance and disease tolerance mechanisms is necessary for an effective immune response leading to survival and return to homeostasis when an organism is challenged by infection. Antibiotics are used for their resistance enhancement capabilities by decreasing pathogen load, but several classes have long been known to have beneficial effects that cannot be explained strictly on the basis of their capacity to control the infectious agent. Here we report that tetracycline antibiotics, a class of ribosome-targeting drugs, robustly protects against sepsis by inducing disease tolerance, independently from their direct antibiotic properties. Mechanistically, we find that mitochondrial inhibition of protein synthesis perturbs the electron transfer chain and leads to improved damage repair in the lung and fatty acid oxidation and glucocorticoid sensitivity in the liver. Using a partial and acute deletion of CRIF1 in the liver, a critical mitoribosomal component for protein synthesis, we find that mice are protected against bacterial sepsis, an observation which is phenocopied by the transient inhibition of complex I of ETC by phenformin. Together, we demonstrate that ribosome-targeting antibiotics are beneficial beyond their antibacterial activity and that mitochondrial protein synthesis inhibition leading to ETC perturbation is a novel mechanism for the induction of disease tolerance.

The Sequential Organ Failure Assessment (SOFA) score is a predictor of mortality in ICU patients. Although it is widely used and has been validated as a reliable and independent predictor of mortality and morbidity in cardiac ICU, few studies correlate early postoperative SOFA with long-term survival.Retrospective observational cohort study.Tertiary academic cardiac surgery ICU.One-thousand three-hundred seventy-nine patients submitted to cardiac surgery.SOFA 24 hours, SOFA 48 hours, mean, and highest SOFA scores were correlated with survival at 12 and 24 months. Wilcoxon tests were used to analyze differences in variables. Multivariate logistic regressions and likelihood ratio test were used to access the predictive modeling. Receiver operating characteristic curves were used to assess accuracy of the variables in separating survivor from nonsurvivors.Lower SOFA scores have better survival rates at 12 and 24 months. Highest SOFA and SOFA at 48 hours showed to be better predictors of outcome and to have higher accuracy in distinguishing survivors from nonsurvivors than initial SOFA and mean SOFA. A decreasing score during the first 48 hours had mortality rates of 4.9%, while an unchanged or increased score was associated with a mortality rate of 5.7%.SOFA score in the ICU after cardiac surgery correlated with survival at 12 and 24 months. Patients with lower SOFA scores had higher survival rates. Differences in survival at 12 months were better correlated with the absolute value at 48 hours than with its variation. SOFA score may be useful to predict long-term outcomes and to stratify patients with higher probability of mortality.

&lt;div&gt;Abstract&lt;p&gt;During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer &lt;i&gt;in vivo&lt;/i&gt; validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context. &lt;i&gt;Cancer Res; 72(19); 4920–30. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

&lt;p&gt;PDF file - 77K, Metastasis formation after i.v. injection and in vitro growth of 4T1; Metastasis formation by 4T1 after i.v. injection is decreased (A), whereas growth of 4T1 tumors in vitro is not affected (B) by Rab27a inhibition&lt;/p&gt;

&lt;p&gt;PDF file - 158K, Involvement of the immune system in differential growth of TS/A, 4T1, and their sh27a-expressing counterparts; A-Examples of FACS stainings and cytospin analysis of CD11b, CD11c, Ly6C and Ly6G in spleens of tumor-bearing mice. B, C- Growth of Scr- and sh27a-4T1 in Rag2-/- or CD4+ T cell-depleted hosts&lt;/p&gt;

&lt;p&gt;PDF file - 113K&lt;/p&gt;

&lt;p&gt;PDF file - 71K, Most shRNA to Rab27a or Rab27b affect the expression level of the other gene. Expression level of Rab27a and Rab27b in cells expressing 5 different shRNA to either Rab27a or Rab27b&lt;/p&gt;

&lt;p&gt;PDF file - 109K, Complete list of secreted proteins analyzed by antibody microarrays. PDF (109KB) Source File (PDF) 186KB A- Arbitrary expression level of the proteins expressed above background by 4T1 or TS/A. B-D- Scheme of the 4 antibody microarray membranes and complete list of the proteins analysed&lt;/p&gt;

&lt;p&gt;PDF file - 158K, Involvement of the immune system in differential growth of TS/A, 4T1, and their sh27a-expressing counterparts; A-Examples of FACS stainings and cytospin analysis of CD11b, CD11c, Ly6C and Ly6G in spleens of tumor-bearing mice. B, C- Growth of Scr- and sh27a-4T1 in Rag2-/- or CD4+ T cell-depleted hosts&lt;/p&gt;

&lt;p&gt;PDF file - 109K, Complete list of secreted proteins analyzed by antibody microarrays. PDF (109KB) Source File (PDF) 186KB A- Arbitrary expression level of the proteins expressed above background by 4T1 or TS/A. B-D- Scheme of the 4 antibody microarray membranes and complete list of the proteins analysed&lt;/p&gt;

&lt;p&gt;PDF file - 71K, Most shRNA to Rab27a or Rab27b affect the expression level of the other gene. Expression level of Rab27a and Rab27b in cells expressing 5 different shRNA to either Rab27a or Rab27b&lt;/p&gt;

&lt;p&gt;PDF file - 77K, Metastasis formation after i.v. injection and in vitro growth of 4T1; Metastasis formation by 4T1 after i.v. injection is decreased (A), whereas growth of 4T1 tumors in vitro is not affected (B) by Rab27a inhibition&lt;/p&gt;

&lt;p&gt;PDF file - 113K&lt;/p&gt;

&lt;div&gt;Abstract&lt;p&gt;During progression from single cancer cells to a tumor mass and metastases, tumor cells send signals that can subvert their tissue microenvironment. These signals involve soluble molecules and various extracellular vesicles, including a particular type termed exosomes. The specific roles of exosomes secreted in the tumor microenvironment, however, is unclear. The small GTPases RAB27A and RAB27B regulate exocytosis of multivesicular endosomes, which lead to exosome secretion, in human HeLa cells. Here, we used mouse models to show that Rab27a blockade in mammary carcinoma cells decreased secretion of exosomes characterized by endocytic markers, but also of matrix metalloproteinase 9, which is not associated with exosomes. Rab27a blockade resulted in decreased primary tumor growth and lung dissemination of a metastatic carcinoma (4T1), but not of a nonmetastatic carcinoma (TS/A). Local growth of 4T1 tumors required mobilization of a population of neutrophil immune cells induced by Rab27a-dependent secretion of exosomes together with a specific combination of cytokines and/or metalloproteinases. Our findings offer &lt;i&gt;in vivo&lt;/i&gt; validation of the concept that exosome secretion can exert key pathophysiologic roles during tumor formation and progression, but they also highlight the idiosyncratic character of the tumor context. &lt;i&gt;Cancer Res; 72(19); 4920–30. ©2012 AACR&lt;/i&gt;.&lt;/p&gt;&lt;/div&gt;

The family of thioester-containing proteins (TEPs) appeared early in evolution: members of this family have been found in such diverse organisms as nematodes, insects, molluscs, fish, birds, and mammals (1). They are characterized by homologous sequence features, including a unique intrachain β-cysteinyl-γ-glutamyl thioester, and a propensity for multiple conformationally sensitive binding interactions (2). The presence of the highly reactive thioester bond renders the molecules unstable at elevated temperature and results in their autocatalytic fragmentation at the thioester site (3,4). Moreover, when exposed, the thioester bond is readily hydrolyzed by water. To avoid precocious inactivation, the thioester in the native protein is protected by a shielded environment (5,6). Proteolytic cleavage exposes a previously hidden thioester bond, which mediates covalent attachment through transacylation (7). The reactivity associated with the thioester is one of the defining features of this protein family. Another important feature is the propensity for diverse conformationally sensitive interactions with other molecules. This includes covalent attachment to activating self and nonself surfaces (complement factors), covalent or noncovalent crosslinking to the attacking proteases [α2-macroglobulins (α2Ms)], interactions with receptors (complement factors and α2Ms), and binding of cleavage-generated products to corresponding receptors (anaphylatoxins of complement factors). In addition, α2Ms bind cytokines and growth factors and regulate their clearance and activity (8,9).

Abstract Sepsis is a life-threatening organ dysfunction condition caused by a dysregulated host response to an infection. Here we report that the circulating levels of growth-differentiation factor-15 (GDF15) are strongly increased in septic shock patients and correlate with mortality. In mice, we find that peptidoglycan is a potent ligand that signals through the TLR2-Myd88 axis for the secretion of GDF15 and that Gdf15 -deficient animals are protected against abdominal sepsis due to increased chemokine CXC ligand 5 (CXCL5)-mediated recruitment of neutrophils into the peritoneum leading to better local bacterial control. Our results identify GDF15 as a potential target to improve sepsis treatment. Its inhibition should increase neutrophil recruitment to the site of infection and consequently lead to better pathogen control and clearance.

Abstract Anthracyclines are among the most used and effective anticancer drugs. Their activity has been attributed to DNA double-strand breaks resulting from topoisomerase II poisoning and to eviction of histones from select sites in the genome. Here we show that the extensively used anthracyclines Doxorubicin, Daunorubicin and Epirubicin, decrease the transcription of nuclear factor kappa B (NF-κB)-dependent gene targets, but not interferon responsive genes. Using an NMR-based structural approach, we demonstrate that anthracyclines disturb the complexes formed between the NF-κB subunit RelA and its DNA binding sites. The variant anthracyclines Aclarubicin, Doxorubicinone and the newly developed Dimethyl-doxorubicin, which share anticancer properties with the other anthracyclines but do not induce DNA damage, also suppressed inflammation, thus uncoupling DNA damage from the effects on inflammation. These findings have implications for anticancer therapy and for the development of novel anti-inflammatory drugs with limited side effects for life-threatening conditions such as sepsis.

This dataset was used in the analysis which composes the GitHub repository: https://github.com/andrebolerbarros/Chora_etal_2022 The files presented here correspond to: <em>gene_counts.tab:</em> the table for the gene counts produced by the alignment of fastq files using STAR; <em>sampleTable.csv:</em> the treatment information for each sample produced.

This dataset was used in the analysis which composes the GitHub repository: https://github.com/andrebolerbarros/Chora_etal_2022 The files presented here correspond to: <em>gene_counts.tab:</em> the table for the gene counts produced by the alignment of fastq files using STAR; <em>sampleTable.csv:</em> the treatment information for each sample produced.

Abstract Background: The Hypoxia Altitude Simulation Test (HAST) is the Gold Standard to evaluate hypoxia in response to altitude and to decide on in-flight requirements for oxygen supplementation. Several equations are available to predict PaO 2 in altitude (PaO 2alt ), but it remains unclear whether their predictive value is equivalent. We aimed to compare the results obtained by the available methods in a population of cystic fibrosis (CF) adults. Methods: Eighty-eight adults (58 healthy controls and 30 CF patients) performed a spirometry followed by an HAST. HAST results were compared with the predicted PaO 2alt made by five equations: 1 st : PaO 2alt = 0,410 x PaO 2ground + 1,7652; 2 nd : PaO 2alt = 0,519 x PaO 2ground + 11,855 x FEV 1 (L) − 1,760; 3 rd : PaO 2alt = 0,453 x PaO 2ground + 0,386 x FEV 1 (%) + 2,44; 4 th : PaO 2alt = 0,88 + 0,68 x PaO 2ground ; 5 th : PaO 2alt = PaO 2ground − 26,6. Results: None of the controls required in-flight oxygen neither by HAST or by the five predictive equations. Eleven CF-patients had PaO 2alt &lt; 50 mmHg, accessed by HAST. The positive predictive value was 50% (1 st ), 87.5% (2 nd and 3 rd ), 77.78% (4 th ) and 58.33% (5 th ). Areas under the curve were 78.95% (1 st ), 84.69% (2 nd ), 88.04% (3 rd ) and 78.95% (4 th and 5 th ). FEV 1 and PaO 2ground were correlated with HAST results. Conclusions: The 3rd equation gave the best predictions in comparison with results obtained by HAST. However, because the individual differences found were substantial for all equations, we still recommend performing a HAST whenever possible to confidently access in-flight hypoxia and the need for oxygen.

Abstract Acute appendicitis is the most frequent surgical abdominal emergency, but its etiology remains poorly understood. Histological examination of the appendix, following its removal due to acute appendicitis, consistently shows features in common with bronchial asthma, suggesting an allergic reaction as a candidate etiologic factor. Here we propose the concept of appendicular lavage and used it to study the levels of the Th2 cytokines IL-4, IL-5 and IL-9 in patients with a clinical diagnosis of acute appendicitis. The study group included 20 patients with a histological diagnosis of phlegmonous appendicitis, 13 patients with gangrenous appendicitis, and a control group of 8 patients with clinical diagnosis of appendicitis but with normal histology. Cytokines levels were higher in acute appendicitis. The difference was more pronounced when comparing phlegmonous appendicitis with non-pathological appendix (p=0.01) for IL-4 (48,3 vs 21,3 pg/mL), IL-5 (29,2 vs 8,0 pg/mL) and IL-9 (34,1 vs 16,6 pg/mL). This Th2 cytokine profile is compatible with the hypothesis of allergy as an etiologic factor for acute appendicitis and may have important implications for the diagnosis, prevention and treatment of this condition.