Congfeng Xu

IL-17 is one of the most potent and most actively investigated proinflammatory cytokines. In this study, we examined the effect of IL-17 on mesenchymal stem cells (MSCs) under the influence of inflammatory cytokines. Ironically, IL-17 dramatically enhanced the immunosuppressive effect of MSCs induced by IFNγ and TNFα, revealing a novel role of IL-17 in immunosuppression. Interestingly, we found that this action of IL-17 was dependent on the promoted expression of a key immune suppressive molecule, inducible nitric oxide synthase (iNOS), in MSCs. In a concanavalin A (ConA)-induced hepatitis mouse model, we found that IL-17 also enhanced the in vivo immunosuppressive effect of MSCs in an iNOS-dependent manner. Moreover, this promoting effect of IL-17 was found to be exerted through enhancing mRNA stability by modulating the protein level of ARE/poly(U)-binding/degradation factor 1 (AUF1), a well-known factor that promotes mRNA decay. In auf1(-/-) MSCs, IFNγ and TNFα could induce maximal immunosuppressive effect, both in vitro and in vivo, without the need for IL-17. Thus, our studies demonstrated that in the presence of MSCs, IL-17 promotes immunosuppression.

The NLRP3 inflammasome responds to various pathogen-derived factors and danger-associated molecules, mediating IL-1β maturation, therefore is involved in multiple inflammatory diseases. Curcumin has been shown to possess strong anti-inflammatory activity, but the underlying mechanism is not fully understood. Here, we sought to investigate the role and mechanism of curcumin on the inhibition of mature IL-1β production via the regulation of NLRP3 inflammasome.Curcumin dramatically inhibited the production of mature IL-1β in LPS-primed macrophages triggered by multiple NLRP3 inflammasome activators, and also reduced the level of cleaved caspase-1 as measured by western blot and ELISA. Curcumin prevented K(+) efflux, the common trigger for NLRP3 inflammasome activation, and attenuated lysosomes disruption and intracellular ROS formation as well. The inhibition of NLRP3 inflammasome by curcumin was in part mediated via the suppression of extracellular regulated protein kinases phosphorylation. Furthermore, administration of curcumin significantly reduced peritoneal IL-1β and HMGB-1 concentration induced by LPS and improved the survival of mice suffering from lethal endotoxic shock.Curcumin potently inhibits the activation of NLRP3 inflammasome which may contribute to its anti-inflammatory activity. Our finding offers a mechanistic basis for the therapeutic potential of curcumin in septic shock and other NLRP3 inflammasome-driven diseases.

Accumulation of hydrophobic bile acids in the liver contributes to cholestatic liver injury.Inflammation induced by excessive bile acids is believed to play a crucial role, however, the mechanisms of bile acids triggered inflammatory response remain unclear.Recent studies have highlighted the effect of NLRP3 inflammasome in mediating liver inflammation and fibrosis.In this study, we for the first time showed that chenodeoxycholic acid (CDCA), the major hydrophobic primary bile acid involved in cholestatic liver injury, could dose-dependently induce NLRP3 inflammasome activation and secretion of pro-inflammatory cytokine-IL-1β in macrophages by promoting ROS production and K + efflux.Mechanistically, CDCA triggered ROS formation in part through TGR5/EGFR downstream signaling, including protein kinase B, extracellular regulated protein kinases and c-Jun N-terminal kinase pathways.Meanwhile, CDCA also induced ATP release from macrophages which subsequently causes K + efflux via P2X7 receptor.Furthermore, in vivo inhibition of NLRP3 inflammasome with caspase-1 inhibitor dramatically decreased mature IL-1β level of liver tissue and ameliorated liver fibrosis in bile duct ligation (BDL) mouse model.In conclusion, excessive CDCA may represent an endogenous danger signal to activate NLRP3 inflammasome and initiate liver inflammation during cholestasis.Our finding offers a mechanistic basis to ameliorate cholestatic liver fibrosis by targeting inflammasome activation.

Mesenchymal stem cell (MSC)-based therapy is a promising approach to treat various inflammatory disorders including multiple sclerosis. However, the fate of MSCs in the inflammatory microenvironment is largely unknown. Experimental autoimmune encephalomyelitis (EAE) is a well-studied animal model of multiple sclerosis. We demonstrated that autophagy occurred in MSCs during their application for EAE treatment. Inflammatory cytokines, e.g., interferon gamma and tumor necrosis factor, induced autophagy in MSCs synergistically by inducing expression of BECN1/Beclin 1. Inhibition of autophagy by knockdown of Becn1 significantly improved the therapeutic effects of MSCs on EAE, which was mainly attributable to enhanced suppression upon activation and expansion of CD4(+) T cells. Mechanistically, inhibition of autophagy increased reactive oxygen species generation and mitogen-activated protein kinase 1/3 activation in MSCs, which were essential for PTGS2 (prostaglandin-endoperoxide synthase 2 [prostaglandin G/H synthase and cyclooxygenase]) and downstream prostaglandin E2 expression to exert immunoregulatory function. Furthermore, pharmacological treatment of MSCs to inhibit autophagy increased their immunosuppressive effects on T cell-mediated EAE. Our findings indicate that inflammatory microenvironment-induced autophagy downregulates the immunosuppressive function of MSCs. Therefore, modulation of autophagy in MSCs would provide a novel strategy to improve MSC-based immunotherapy.

Autophagy is an evolutionarily conserved biological process involved in an array of physiological and pathological events. Without proper control, autophagy contributes to various disorders, including cancer and autoimmune and inflammatory diseases. It is therefore of vital importance that autophagy is under careful balance. Thus, additional regulators undoubtedly deepen our understanding of the working network, and provide potential therapeutic targets for disorders. In this study, we found that RNF216 (ring finger protein 216), an E3 ubiquitin ligase, strongly inhibits autophagy in macrophages. Further exploration demonstrates that RNF216 interacts with BECN1, a key regulator in autophagy, and leads to ubiquitination of BECN1, thereby contributing to BECN1 degradation. RNF216 was involved in the ubiquitination of lysine 48 of BECN1 through direct interaction with the triad (2 RING fingers and a DRIL [double RING finger linked]) domain. We further showed that inhibition of autophagy through overexpression of RNF216 in alveolar macrophages promotes Listeria monocytogenes growth and distribution, while knockdown of RNF216 significantly inhibited these outcomes. These effects were confirmed in a mouse model of L. monocytogenes infection, suggesting that manipulating RNF216 expression could be a therapeutic approach. Thus, our study identifies a novel negative regulator of autophagy and suggests that RNF216 may be a target for treatment of inflammatory diseases.

High-fat diet (HFD) leads to systemic low-grade inflammation, which has been involved in the pathogenesis of diverse metabolic and inflammatory diseases. Colon is thought to be the first organ suffering from inflammation under HFD conditions due to the pro-inflammatory macrophages infiltration, however, the mechanisms concerning the induction of pro-inflammatory phenotype of colonic macrophages remains unclear. In this study, we show that HFD increased the percentage of gram-positive bacteria, especially genus Clostridium, and resulted in the significant increment of fecal deoxycholic acid (DCA), a gut microbial metabolite produced by bacteria mainly restricted to genus Clostridium. Notably, reducing gram-positive bacteria with vancomycin diminished fecal DCA and profoundly alleviated pro-inflammatory macrophage infiltration in colon, whereas DCA-supplemented feedings to vancomycin-treated mice provoked obvious pro-inflammatory macrophage infiltration and colonic inflammation. Meanwhile, intra-peritoneal administration of DCA also elicited considerable recruitment of macrophages with pro-inflammatory phenotype. Mechanistically, DCA dose-dependently promoted M1 macrophage polarization and pro-inflammatory cytokines production at least partially through toll-like receptor 2 (TLR2) transactivated by M2 muscarinic acetylcholine receptor (M2-mAchR)/Src pathway. In addition, M2-mAchR mediated increase of TLR2 transcription was mainly achieved via targeting AP-1 transcription factor. Moreover, NF-κB/ERK/JNK signalings downstream of TLR2 are involved in the DCA-induced macrophage polarization. In conclusion, our findings revealed that high level DCA induced by HFD may serve as an initiator to activate macrophages and drive colonic inflammation, thus offer a mechanistic basis that modulation of gut microbiota or intervening specific bile acid receptor signaling could be potential therapeutic approaches for HFD-related inflammatory diseases.

A westernized high-fat diet (HFD) is associated with the development of inflammatory bowel disease (IBD). High level fecal deoxycholic acid (DCA) caused by HFD contributes to the colonic inflammatory injury of IBD, however, the mechanism concerning the initiation of inflammatory response by DCA remains unclear. In this study, we sought to investigate the role and mechanism of DCA in the induction of inflammation via promoting NLRP3 inflammasome activation. Here we for the first time showed that DCA dose-dependently induced NLRP3 inflammasome activation and highly pro-inflammatory cytokine-IL-1β production in macrophages. Mechanistically, DCA triggered NLRP3 inflammasome activation by promoting cathepsin B release at least partially through sphingosine-1-phosphate receptor 2 (S1PR2). Colorectal instillation of DCA significantly increased mature IL-1β level in colonic tissue and exacerbated DSS-induced colitis, while in vivo blockage of NLRP3 inflammasome or macrophage depletion dramatically reduced the mature IL-1β production and ameliorated the aggravated inflammatory injury imposed by DCA. Thus, our findings show that high level fecal DCA may serve as an endogenous danger signal to activate NLRP3 inflammasome and contributes to HFD-related colonic inflammation. NLRP3 inflammasome may represent a new potential therapeutical target for treatment of IBD.

Toll-like receptors play important roles in regulating immunity against microbial infections. Toll-like receptor 8 (TLR8) belongs to a subfamily comprising TLR7, TLR8 and TLR9. Human TLR8 mediates anti-viral immunity by recognizing ssRNA viruses, and triggers potent anti-viral and antitumor immune responses upon ligation by synthetic small molecular weight ligands. Interestingly, distinct from human TLR8, mouse TLR8 was not responsive to ligand stimulation in the absence of polyT-oligodeoxynucleotides (polyT-ODN). The molecular basis for this distinct ligand recognition is still unclear. In the present study, we compared the activation of TLR8 from different species including mouse, rat, human, bovine, porcine, horse, sheep, and cat by ligand ligations. Only the TLR8s from the rodent species (i.e., mouse and rat TLR8s) failed to respond to ligand stimulation in the absence of polyT-ODN. Multiple sequence alignment analysis suggested that these two rodent TLR8s lack a five-amino-acid motif that is conserved in the non-rodent species with varied sequence. This small motif is located in an undefined region of the hTLR8 ectodomain, immediately following LRR-14. Deletion mutation analysis suggested that this motif is essential for the species-specific ligand recognition of hTLR8, whereas it is not required for self-dimerization and intracellular localization of this receptor.

Autophagy is one of the downstream effector mechanisms for elimination of intracellular microbes following activation of the Toll-like receptors (TLRs). Although the detailed molecular mechanism for this cellular process is still unclear, Beclin 1, a key molecule for autophagy, has been suggested to play a role. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the stability of signaling proteins. Herein, we show that Hsp90 forms a complex with Beclin 1 through an evolutionarily conserved domain to maintain the stability of Beclin 1. In monocytic cells, geldanamycin (GA), an Hsp90 inhibitor, effectively promoted proteasomal degradation of Beclin 1 in a concentration-dependent (EC(50) 100 nM) and time-dependent (t(50) 2 h) manner. In contrast, KNK437/Hsp inhibitor I had no effect. Hsp90 specifically interacted with Beclin 1 but not with other adapter proteins in the TLR signalsome. Treatment of cells with GA inhibited TLR3- and TLR4-mediated autophagy. In addition, S. typhimurium infection-induced autophagy was blocked by GA treatment. This further suggested a role of the Hsp90/Beclin 1 in controlling autophagy in response to microbial infections. Taken together, our data revealed that by maintaining the homeostasis of Beclin 1, Hsp90 plays a novel role in TLR-mediated autophagy.

Biological pretreatment can increase the methane production of anaerobic digestion. In this study, stover was pretreated via microbial consortium prior to anaerobic digestion; through 16S rRNA gene and 16S rRNA amplicon sequencing and metatranscriptomic analysis, and the effects of the pretreatment on the microbial community and critical factors of the increased methane production were studied. Microbial community structure was less affected by the pretreatment, which ensures the stable performance of anaerobic digestion. The methane production increased by 62.85% at the peak phase compared to the untreated stover. The activity of Methanosaeta increased from 2.0% to 10.1%, significantly enhancing the ability of the community to capture acetic acid and reduce CO2 to methane. The main contribution to the increase in methane production was a unique acetyl-CoA synthetase, which showed significant up-regulation (121.8%). This research demonstrated the importance of Methanosaeta and its unique metabolic pathways in anaerobic digestion utilizing a biological pretreatment.

The degradation of lignin is the main rate-limiting step in the bio-pulping of rice straw. A lignin-degrading bacterial consortium LDC, which can efficiently degrade lignin of reed, was screened in the early stage of our laboratory work. In present study, 7-day incubation of LDC can degrade rice straw lignin by 31.18% in mineral salt medium. The communities’ structure of different incubation phases varied greatly, in which high abundance (44.78%) of Anaerocolumna was first found. The expression levels of lignin degradation enzyme class II peroxidase (AA2), vanillyl alcohol oxidase (AA4) and 1,4-benzoquinone reductase (AA6) during peak phase (48 h) were significantly up-regulated than initial phase (24 h), increasing by 112%, 165% and 67%, respectively, and 42.86% AA2 was from Thaurea; 100% AA4 was from Clostridium; 62.5% AA6 was from Pseudomonas. These provide microbial resources and data support for the industrialization of rice straw bio-pulping.

We recently have proved that excessive fecal DCA caused by high-fat diet may serve as an endogenous danger-associated molecular pattern to activate NLRP3 inflammasome and thus contributes to the development of inflammatory bowel disease (IBD). Moreover, the effect of DCA on inflammasome activation is mainly mediated through bile acid receptor sphingosine-1-phosphate receptor 2 (S1PR2); however, the intermediate process remains unclear. Here, we sought to explore the detailed molecular mechanism involved and examine the effect of S1PR2 blockage in a colitis mouse model. In this study, we found that DCA could dose dependently upregulate S1PR2 expression. Meanwhile, DCA-induced NLRP3 inflammasome activation is at least partially achieved through stimulating extracellular regulated protein kinases (ERK) signaling pathway downstream of S1PR2 followed by promoting of lysosomal cathepsin B release. DCA enema significantly aggravated DSS-induced colitis in mice and S1PR2 inhibitor as well as inflammasome inhibition by cathepsin B antagonist substantially reducing the mature IL-1β production and alleviated colonic inflammation superimposed by DCA. Therefore, our findings suggest that S1PR2/ERK1/2/cathepsin B signaling plays a critical role in triggering inflammasome activation by DCA and S1PR2 may represent a new potential therapeutic target for the management of intestinal inflammation in individuals on a high-fat diet.

Tetraspanin CD82 suppresses cell migration, tumor invasion, and tumor metastasis. To determine the mechanism by which CD82 inhibits motility, most studies have focused on the cell surface CD82, which forms tetraspanin-enriched microdomains (TEMs) with other transmembrane proteins, such as integrins. In this study, we found that CD82 undergoes endocytosis and traffics to endosomes and lysosomes. To determine the endocytic mechanism of CD82, we demonstrated that dynamin and clathrin are not essential for CD82 internalization. Depletion or sequestration of sterol in the plasma membrane markedly inhibited the endocytosis of CD82. Despite the demand on Cdc42 activity, CD82 endocytosis is distinct from macropinocytosis and the documented dynamin-independent pinocytosis. As a TEM component, CD82 reorganizes TEMs and lipid rafts by redistributing cholesterol into these membrane microdomains. CD82-containing TEMs are characterized by the cholesterol-containing microdomains in the extreme light- and intermediate-density fractions. Moreover, the endocytosis of CD82 appears to alleviate CD82-mediated inhibition of cell migration. Taken together, our studies demonstrate that lipid-dependent endocytosis drives CD82 trafficking to late endosomes and lysosomes, and CD82 reorganizes TEMs and lipid rafts through redistribution of cholesterol.

Adenylyl cyclases (AC) are important regulators of airway smooth muscle function, because ␤-adrenergic receptor (AR) agonists stimulate AC activity and increase airway diameter.We assessed expression of AC isoforms in human bronchial smooth muscle cells (hBSMC).Reverse transcriptase-polymerase chain reaction and immunoblot analyses detected expression of AC2, AC4, and AC6.Forskolin-stimulated AC activity in membranes from hBSMC displayed Ca 2ϩ -inhibited and G ␤␥stimulated AC activity, consistent with expression of AC6, AC2, and AC4.Isoproterenol-stimulated AC activity was inhibited by Ca 2ϩ but unaltered by G ␤␥ , whereas butaprost-stimulated AC activity was stimulated by G ␤␥ but unaffected by Ca 2ϩ addition.Using sucrose density centrifugation to isolate lipid raft fractions, we found that only AC6 localized in lipid raft fractions, whereas AC2 and AC4 localized in nonraft fractions.Immuno-isolation of caveolae using caveolin-1 antibodies yielded Ca 2ϩinhibited AC activity (consistent with AC6 expression), whereas the nonprecipitated material displayed G ␤␥ -stimulated AC activity (consistent with expression of AC2 and/or AC4).Overexpression of AC6 enhanced cAMP production in response to isoproterenol and beraprost but did not increase responses to prostaglandin E 2 or butaprost.␤ 2 AR, but not prostanoid EP 2 or EP 4 receptors, colocalized with AC5/6 in lipid raft fractions.Thus, particular G protein-coupled receptors couple to discreet AC isoforms based, in part, on their colocalization in membrane microdomains.These different cAMP signaling compartments in airway smooth muscle cells are responsive to different hormones and neurotransmitters and can be regulated by different coincident signals such as Ca 2ϩ and G ␤␥ .

Hepatitis B virus (HBV) infection brings a huge challenge for medical health practitioners. It has been reported that invaded HBV escapes autophagic degradation through inhibiting lysosome maturation following enhanced autophagy formation, which putatively contributes to HBV replication and infection. However, the underlying mechanism by which HBV escapes from autophagic degradation remains elusive. In this study, we monitored the autophagic process using HepG2 cells and mice without or with transient HBV DNA plasmid transfection (pHepG2) or stable HBV infection (HepG2.2.15 cells) in vitro and in vivo. The results of Western blot, transmission electron microscopy and confocal microscopy, confirmed that HBV induced autophagy, while the fusion of autophagosomes with lysosomes was arrested. Furthermore, Rab7, a small GTPase that functions as a molecular switch responsible for the autophagosome-lysosome fusion, was inhibited, suggesting a potential mechanism for HBV-induced inhibition of autophagic degradation. In conclusion, our study proposes a potential mechanism for how HBV escapes autophagic degradation, which might be a novel therapeutic target for controlling HBV infection.

Originally identified as an E3 ligase regulating toll-like receptor (TLR) signaling, ring finger protein 216 (RNF216) also plays an essential role in autophagy, which is fundamental to cellular homeostasis.Autophagy dysfunction leads to an array of pathological events, including tumor formation.In this study, we found that RNF216 was upregulated in human colorectal cancer (CRC) tissues and cell lines, and was associated with progression of CRC.RNF216 promoted CRC cell proliferation and migration in vitro and in vivo, largely by enhancing proteasomal degradation of BECN1, a key autophagy regulator and tumor suppressor.RNF216 restricted CRC cell autophagy through BECN1 inhibition under nutritional starvation conditions.RNF216 knockdown increased the autophagy, limiting CRC cell proliferation and migration.Moreover, BECN1 knockdown or autophagy inhibition restored proliferation and migration of RNF216-knockdown CRC cells.Collectively, our results suggested that RNF216 promoted CRC cell proliferation and migration by negatively regulating BECN1-dependent autophagy.This makes RNF216 as a potential biomarker and novel therapeutic target for inhibiting CRC development and progression.

It is significant to understand corn stover (CS) in anaerobic digestion (AD) under high organic loadings. A semi-continuous mesophilic (37 ± 1 °C) CS AD was conducted in this study with increasing loadings. The initial total solids (TS) gradually increased with 1% gradient at every 10 days from 8% to 15% until the system was acidified. Adding different ratios of cattle manure (CM) (20%, 30% and 40% (v/v)) to rescue this system back to a stable operation was adopted. The diversity of bacteria and archaea was analyzed by 16S rRNA gene sequencing technology. The results showed that when loading TS content was increased to 15%, AD system was acidized with pH value of 5.13. 30% of CM was the optimal ratio to recover biogas production. High abundance (31.07%) of Bathyarchaeota was first found in AD system. Acidification of high loading CS AD can be highly correlating with bacterial community, specially Clostridium and Caproiciproducens.

Inefficient electron transfer is a key bottleneck for anaerobic digestion (AD), which often leads to system instability. Here, we propose for the first time a new strategy to solve the problem by adding surface electron-polarized biochar to enhance methanogenesis by providing electrons to methanogens through surface electron-poor/rich regions. Pyrolysis conversion of pigeon manure to conductive materials (CM) containing electron-poor/rich regions is realized. Different levels of CM (2.5, 5 and 7.5 g/L) are used to enhance methane production from pig manure and rice straw co-digestion systems. It is found that the addition of 5 g/L CM results in the highest cumulative CH4 production (187.4 ± 8.0 mL/g VS substrate), 35.4% higher (P < 0.05) than the control (0 g/L). Methanogenic archaea are enriched. Synergistic metabolic mechanisms between the functional microorganism bin.CM52.160 (cellulose degradation) and bin.CM03.101 (methane production) are revealed by the metagenomic binning strategy. A large number of potentially unculturable electroactive microorganisms have been identified. This work provides important insights into the construction of micro-electric fields on material surfaces and offers a practical approach to improving AD performance.

Background and Purpose Nicotinic ACh receptors containing the α7 sub‐unit (α7‐nAChRs) suppress inflammation through a wide range of pathways in immune cells. These receptors are thus potentially involved in a number of inflammatory diseases. However, the detailed mechanisms underlying the anti‐inflammatory effects of α7‐nAChRs remain to be described. Experimental Approach Anti‐inflammatory effects of α7‐nAChR agonists were assessed in both murine macrophages (RAW 264.7) and bone marrow‐derived macrophages (BMDM), stimulated with LPS, using immunoblotting, RT‐PCR and luciferase reporter assays. The role of adenylyl cyclase‐6 in the degradation of Toll‐like receptor 4 (TLR4) following endocytosis, was explored via overexpression and knockdown. A mouse model of chronic obstructive pulmonary disease (COPD) induced by porcine pancreatic elastase was used to confirm key findings. Results Anti‐inflammatory effects of α7‐nAChRs were largely dependent on adenylyl cyclase‐6 activation, as knockdown of adenylyl cyclase‐6 considerably reduced the effects of α7‐nAChR agonists while adenylyl cyclase‐6 overexpression promoted them. We found that α7‐nAChRs and adenylyl cyclase‐6 are co‐localized in lipid rafts of macrophages and directly interact. Activation of adenylyl cyclase‐6 led to increased degradation of TLR4. Administration of the α7‐nAChR agonist PNU‐282987 attenuated pathological and inflammatory end points in a mouse model of COPD. Conclusion and Implications The α7‐nAChRs inhibit inflammation through activating adenylyl cyclase‐6 and promoting degradation of TLR4. The use of α7‐nAChR agonists may represent a novel therapeutic approach for treating COPD and possibly other inflammatory diseases.

Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.

IL-17 participates in the development of many autoimmune diseases by promoting the expression of some chemokines. Chemokine C-C motif ligand 2 (CCL2) is an important factor at the infiltration of mononuclear cells in the myocardial tissue of viral myocarditis (VMC). It was found that IL-17 could aggravate myocardial injury by upregulating CCL2. But the underlying mechanism involved in CCL2 secretion induced by IL-17 in cardiac myocytes remains unclear. This study investigated the role of transcription factor AP-1 in IL-17 induced CCL2 expression. The results showed that IL-17 mediated the activation of Act1, TRAF6, p38MAPK and c-Jun/AP-1 not Wnt or PI3K signalling pathway to upregulate CCL2 expression in cardiac myocytes. After blocking Act1/TRAF6/p38MAPK cascade and interfering AP-1 with Curcumin or c-Jun siRNA, CCL2 expression induced by IL-17 was significantly attenuated at both mRNA and protein levels. Furthermore, the phosphorylation of c-Jun was suppressed when cardiac myocytes were treated with Act1 siRNA, TRAF6 siRNA, SB203580 (p38MAPK inhibitor) or SP600125 (JNK inhibitor) in cardiac myocytes. In conclusion, IL-17 could stimulate the expression of CCL2 in cardiac myocytes via Act1/TRAF6/p38MAPK-dependent AP-1 activation, which may provide a new target for the diagnosis and treatment of VMC.

Long-term anaerobic co-digestion of swine manure (SM) and corn stover (CS) was conducted using semi-continuously loaded digesters under mesophilic conditions. A preliminary test was first conducted to test the effects of loading rates, and results indicated the 3 g-VS L−1 d−1 was the optimal loading rate. Based on the preliminary results, a verification replicated test was conducted with 3 g-VS L−1 d−1 loading rate and different SM/CS ratios (1:1, 2:1 and 1:2). Results showed that a SM/CS ratio of 2/1 was optimal, based on maximum observed methane-VSdes generation and carbon conversion efficiency (72.56 ± 3.40 mL g−1 and 40.59%, respectively). Amplicon sequencing analysis suggested that microbial diversity was increased with CS loading. Amino-acid-degrading bacteria were abundant in the treatment groups. Archaea Methanoculleus could enhance biogas and methane productions.

Abstract Proper intracellular localization of TLRs is essential for their signaling and biological function. Endocytosis constitutes a key step in protein turnover, as well as maintenance of TLR localization in plasma membrane and intracellular compartments, and thus provides important regulating points to their signaling. In this study, we demonstrate that adenylyl cyclase (AC) activation attenuates TLR4 signaling in a murine macrophage cell line (RAW 264.7) and bone marrow–derived macrophages when stimulated with LPS. We further show that the AC6 isoform plays a key role in negative regulation of TLR4 signaling by promoting protein degradation. TLR4 is normally endocytosed through the clathrin-mediated pathway, but concomitant AC6 activation shifts it to lipid raft-mediated endocytosis, which accelerates degradation of TLR4 and suppresses downstream signaling. Our studies unveil a new mechanism of negative regulation of TLR4 signaling through AC6-mediated endocytosis, which might provide a novel therapeutic approach for limiting inflammatory and autoimmune diseases.

Synthetic CpG-oligodeoxynucleotides (CpG-ODN) are potent adjuvants that accelerate and boost antigen-specific immune responses. Toll-like receptor 9 (TLR9) is the cellular receptor for these CpG-ODN. Previous studies have shown species-specific activation of mouse TLR9 (mTLR9) and human TLR9 (hTLR9) by their optimized CpG-ODN. The interaction between rabbit TLR9 (rabTLR9) and CpG-ODN, however, has not been previously investigated. Here, we cloned and characterized rabTLR9 and comparatively investigated the activation of the rabbit, mouse, and human TLR9 by CpG-ODN. The complete open reading frame of rabTLR9 encodes 1028 amino acid residues, which share 70.6% and 75.5% of the identities of mTLR9 and hTLR9, respectively. Rabbit TLR9 is preferentially expressed in immune cells rich tissues, and is localized in intracellular vesicles. While mTLR9 and hTLR9 displayed species-specific recognition of their optimized CpG-ODN, rabbit TLR9 was activated by these CpG-ODN without any preference. This result suggests that rabTLR9 has a broader ligand-recognition profile than mouse and human TLR9.

Toll-like receptors 7 (TLR7) and 8 (TLR8) recognize viral single-stranded RNA and small molecular weight agonists to activate anti-viral immune responses. TLR8s from different species have distinct ligand recognitions. For example, human TLR8 is responsive to ligand stimulation, but mouse and rat TLR8 are activated by small molecular weight agonists only in the presence of polyT-oligodeoxynucleotides. TLR7 and TLR8 have been reported to be absent and pseudogenized, respectively, in rabbit (Oryctolagus cuniculus). In this study, we detected the expression of rabbit (rab)TLR8 in immune-cell-associated tissues. Cell proliferation and cytokine expressions in rabbit splenocytes were induced by the TLR7/8 ligand but not by the TLR7 ligands, suggesting that rabTLR8 is functional but rabTLR7 is not. In rabbits, CL075, a TLR7/8 ligand, activated an antigen-specific antibody response, although one not as potent as aluminum salt or Freund's adjuvant. Nevertheless, CL075, alone or in combination with aluminum salt, generates fewer adverse effects than Freund's adjuvant at the injection sites. To further investigate the activation of rabTLR8, we cloned its cDNA. In cell-based assay, this rabTLR8 is activated by TLR7/8 ligand but not activated by TLR7 ligand. Upon stimulation the rabTLR8 had a lower activation compared to the activation of TLR8 from other species, except the mouse and rat TLR8s. Using different deletion and human-rabbit chimeric TLR8 expressing constructs, we showed that an extra peptide in the undefined region results in reduced activity of rabTLR8. These results provide a molecular basis for the mild activities of TLR7/8 ligands in rabbits, and suggest TLR7/8 agonists may provide safer immune stimuli in rabbits than in other non-rodent species.

Background: ECM proteins are instrumental for angiogenesis, which plays momentous roles during development and repair in various organs, including post cardiac insult. After a screening based on an open access RNA-seq database, we identified Nephronectin (NPNT), an extracellular protein, might be involved in cardiac repair post myocardial infarction (MI). However, the specific impact of nephronectin during cardiac repair in MI remains elusive. Methods and Results: In the present study, we established a system overexpressing NPNT locally in mouse heart by utilizing a recombinant adeno-associated virus. One-to-four weeks post MI induction, we observed improved cardiac function, limited infarct size, alleviated cardiac fibrosis, with promoted angiogenesis in infarct border zone in NPNT overexpressed mice. And NPNT treatment enhanced human umbilical vascular endothelial cell (HUVEC) migration and tube formation, putatively through advocating phosphorylation of EGFR/JAK2/STAT3. The migration and capillary-like tube formation events could be readily revoked by EGFR or STAT3 inhibition. Notably, phosphorylation of EGFR, JAK2 and STAT3 were markedly upregulated in AAV2/9-cTnT-NPNT-treated mice with MI. Conclusions: Our study thus identifies the beneficial effects of NPNT on angiogenesis and cardiac repair post MI by enhancing the EGFR/JAK2/STAT3 signaling pathway, implying the potential therapeutic application of NPNT on myocardial dysfunction post MI.

Objective: To investigate the effect of compound Xuanju capsule on T lymphocyte subsets in patients with unexplained recurrent abortion. Methods: The effect of compound Xuanju capsule for the treatment of unexplained recurrent abortion wasinvestigated in a randomized, single blind and clinical trial with control of prednisone and lymphocyte immunotherapy. 211 patients were randomly divided into three groups which were given compound Xuanju capsule, prednisone, and lymphocyte immunotherapy respectively. The main outcome measures were T lymphocyte subsets (T lymphocyte ratio, Th cell ratio, Ts cell ratio, Th/Ts cell ratio, NK cell ratio) whichwas detected in the three groups before and after treatment (at the end of one course of treatment) and the secondary outcome measure was TCM syndrome grade andpregnancy outcome. Results: (1) T lymphocyte subsets: Compared with beforetreatment, the Total T cell ratio showed no significant difference (P&gt;0.05) in the threegroups. The Th cell ratio was lower (P&lt;0.01) in the three groups and the decrease of Thcell ratio in compound Xuanju capsule group and prednisone group was moresignificant than that in the immunotherapy group (P &lt; 0.01). The Ts cell ratio was higher in compound Xuanju capsule group and prednisone group (P &lt; 0.05). The Th/Ts cellratio was lower in the three groups (P &lt; 0.05) and the decrease of Th/Ts cell ratio incompound Xuanju capsule group and prednisone group was more significant than thatin the immunotherapy group (P &lt; 0.05). Total proportion of NK was lower (P&lt;0.001) in the compound Xuanju capsule group and prednisone group. The Total NK cell ratioshowed no significant difference (P &gt; 0.05) in the immunotherapy group (P &gt; 0.05). (2)Pregnancy outcome: Pregnancy outcomes were significantly improved but there wasno significant difference (P &gt; 0.05) in the three groups. (3) TCM syndrome grade:Compared with before treatment, the TCM syndrome grade was lower (P&lt;0.05) in the Copyright © 2024 Cognizant Communication Corp.Acupuncture &amp; Electro-therapeutics Research E-pubthree groups and the decrease of TCM syndrome grade in compound Xuanju capsulegroup was more significant than that in the prednisone group and immunotherapy group(P &lt; 0.01). Conclusion: Compared with before treatment, the Th cell ratio, Th/Ts cellratio and Total NK cell ratio was lower and the Ts cell ratio was higher in compoundXuanju capsule group. Pregnancy outcomes of patients were significantly improved.Compound Xuanju capsule is superior to prednisone and lymphocyte immunotherapywhen it comes to TCM syndrome grade reduction. These results suggest that CompoundXuanju Capsule can regulate the immune function of unexplained recurrentspontaneous abortion (URSA) patients, suppress abnormal immune response, and playa certain role in the treatment of recurrent abortion with unknown causes

A large amount of agricultural waste causes global environmental pollution. Biogas production by microbial pretreatment is an important way to utilize agricultural waste resources. In this study, Sporocytophaga CG-1 (A, cellulolytic strain) was co-cultured with Bacillus Subtilis HP-1 (B, non-cellulolytic strain). We investigate the synergistic effect of rice straw degradation by the combination, and analyze the effect of pretreatment of rice straw on methanogenic capacity of anaerobic digestion. The results showed that weight loss rate of filter paper of co-culture combination is 53.38%, which is 29.37% higher than that of A. The synergistic effect of B on A can promote its degradation of cellulose. The cumulative methane production rate of the co-culture combination was the highest (93.04 mL/g VS substrate), which was significantly higher than that of A, B and the control group (82.38, 67.28 and 67.70 mL/g VS substrate). Auxiliary bacteria can improve cellulose degradation rate by promoting secondary product metabolism. These provide data support for the application of co-culture strategies in the field of anaerobic digestion.

To explore the role and mechanism of Notch signaling and ERK1/2 pathway in the inhibitory effect of sacubitril/valsartan on the proliferation of vascular smooth muscle cells (VSMCs).Human aortic vascular smooth muscle cells (HA-VSMCs) were cultured in vitro. The proliferating VSMCs were divided into three groups as control group, Ang II group and Ang II + sacubitril/valsartan group. Cell proliferation and migration were detected by CCK8 and scratch test respectively. The mRNA and protein expression of PCNA, MMP-9, Notch1 and Jagged-1 were detected by qRT-PCR and Western blot respectively. The p-ERK1/2 expression was detected by Western blot.Compared with the control group, proliferation and migration of VSMCs and the expression of PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 was increased in Ang II group. Sacubitril/valsartan significantly reduced the proliferation and migration. Additionally, pretreatment with sacubitril/valsartan reduced the PCNA, MMP-9, Notch1, Jagged-1 and p-ERK1/2 expression.

Appropriate fibrosis is required to prevent subsequent adverse remodeling and heart failure post myocardial infarction (MI), and cardiac fibroblasts (CFs) play a critical role during the process. Carbonic anhydrase 3 (CAR3) is an important mediator in multiple biological processes besides its CO2 hydration activity; however, the role and underlying mechanism of CAR3 on cardiac repair post MI injury remains unknown. Here, we found that CAR3 expression was up-regulated in cardiac tissue in infarct area at the reparative phase of MI, with a peak at 7 days post MI. The upregulation was detected mainly on fibroblast instead of cardiomyocyte, and primary cardiac fibroblasts treated with TGF-β1 recaptured our observation. While CAR3 deficiency leads to weakened collagen density, enlarged infarct size and aggravated cardiac dysfunction post-MI. In fibroblast, we observed that CAR3 deficiency restrains collagen synthesis, cell migration and gel contraction of cardiac fibroblasts, whereas overexpression of CAR3 in CFs improves wound healing and cardiac fibroblast activation. Mechanistically, CAR3 stabilizes Smad7 protein via modulating its acetylation, which dampens phosphorylation of Smad2 and Smad3, thus inhibiting fibroblast transformation. In contrast, inhibition of Smad7 acetylation with C646 blunts CAR3 deficiency induced suppression of fibroblast activation and impaired cardiac healing. Our data demonstrate a protective role of CAR3 in cardiac wound repair post MI via promoting fibroblasts activation through Smad7-TGF-β/Smad2/3 signaling pathway.

A large amount of agricultural waste causes global environmental pollution. Biogas production by microbial pretreatment is an important way to utilize agricultural waste resources. In this study, Sporocytophaga CG-1 (A, cellulolytic strain) was co-cultured with Bacillus clausii HP-1 (B, non-cellulolytic strain) to analyze the effect of pretreatment of rice straw on methanogenic capacity of anaerobic digestion (AD). The results showed that weight loss rate of filter paper of co-culture combination is 53.38%, which is 29.37% higher than that of A. The synergistic effect of B on A can promote its degradation of cellulose. The cumulative methane production rate of the co-culture combination was the highest (93.04 mL/g VS substrate), which was significantly higher than that of A, B and the control group (82.38, 67.28 and 67.70 mL/g VS substrate). Auxiliary bacteria can improve cellulose degradation rate by promoting secondary product metabolism. These results provide data support for the application of co-culture strategies in the field of anaerobic digestion practices.

Background The peroxisome proliferator-activated receptor (PPAR) activation has generally been shown to have anti-inflammatory effects and dendritic cells (DCs) are the most efficient antigen presenting cells that play an active role in the development of atherosclerosis. The effects of PPARγ on DCs maturation and immune function remain unknown now and we, therefore, studied the influence of PPARγ agonist ciglitazone on the maturation and immune function of DCs. Methods Human monocytes were purified and immature DCs derived; ciglitazone (25 μmol/L) was added to the medium for 24 hours; ox-LDL (50 μg/ml) was then added to the medium for another 24 hours. The immunophenotypic expressions (CD1a, CD40, CD86, and HLA-DR) were analyzed by FACS and endocytosis function by FITC-dextran and the cytokines secretions of culture supernatants (IL-12,IL-10,TNFα, and IL-2) were measured with ELISA. Results Ciglitazone reduced ox-LDL induced immunophenotypic expressions of DCs (CD40, CD1a, and HLA-DR). Ox-LDL inhibited the endocytosis of DCs, which was prevented by ciglitazone; ciglitazone attenuated ox-LDL induced cytokine secretions of DCs (IL-12, 116 ± 29 versus 34 ± 3 pg/ml*; IL-10, 49 ± 1 versus 28 ± 9 pg/ml*; TNFα, 46 ± 16 versus 24 ± 8pg/ml*, *P < 0.05 compared with ox-LDL, respectively). Conclusion Our study suggested that one of the anti-inflammatory mechanisms of PPAR-γ agonist ciglitazone was mediated by inhibiting the ox-LDL induced maturation and immune function of DCs.

Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifested as fatigable muscle weakness, which is typically caused by pathogenic autoantibodies against postsynaptic CHRN/AChR (cholinergic receptor nicotinic) in the endplate of skeletal muscle. Our previous studies have identified CA3 (carbonic anhydrase 3) as a specific protein insufficient in skeletal muscle from myasthenia gravis patients. In this study, we investigated the underlying mechanism of how CA3 insufficiency might contribute to myasthenia gravis. Using an experimental autoimmune myasthenia gravis animal model and the skeletal muscle cell C2C12, we find that inhibition of CAR3 (the mouse homolog of CA3) promotes CHRN internalization via a lipid raft-mediated pathway, leading to accelerated degradation of postsynaptic CHRN. Activation of CAR3 reduces CHRN degradation by suppressing receptor endocytosis. CAR3 exerts this effect by suppressing chaperone-assisted selective autophagy via interaction with BAG3 (BCL2-associated athanogene 3) and by dampening endoplasmic reticulum stress. Collectively, our study illustrates that skeletal muscle cell CAR3 is critical for CHRN homeostasis in the neuromuscular junction, and its deficiency leads to accelerated degradation of CHRN and development of myasthenia gravis, potentially revealing a novel therapeutic approach for this disorder.

Giant-cell tumor (GCT) of the bone is an invasiveness and high recurrent bone tumor that is considered borderline or potentially malignant. To explore the molecular mechanism leading to bone destruction and identify novel targets for treatment, we conducted silencing of miR-223 and miR-19a in stromal giant cells and identified TWIST and Runx2 as their target genes. We investigated the impact of these microRNAs and their target genes on stromal giant cells that promote the differentiation of monocyte/macrophages into osteoclast cells and recruitment to the bone microenvironment, which in turn enhances the bone destruction capacity of GCT. MiR-223 and miR-19a were found to regulate the expression of TWIST and Runx2, influence the RANKL-RANK pathway and the expression of MCP-1, and finally regulate the pathophysiological process of osteolytic bone destruction. Our results indicate that re-expression of miR-223 and miR-19a induces an inhibitory effect on the bone destruction capacity of GCT, suggesting that re-expression of miR-223 and miR-19a can be a novel strategy for the treatment of GCT.

Myasthenia gravis (MG) is considered as an autoimmune disease mainly mediated by antibodies against acetylcholine receptor. In recent years, other targets related to MG have been the subject of interest. Our previous research found that protein P25 was lower in muscles of MG patients using two-dimensional electrophoresis. In present study, anti-serum to P25 was prepared, immunohistochemistry and ATPase staining revealed that P25 was a muscle specific cytosolic protein and was mainly distributed in type I muscle fibers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and precise molecular weight derived from mass spectrometer identified P25 as carbonic anhydrase III (CA III). Some members of CA family are related to autoimmune diseases and CA III is recently reported to be involved in rheumatoid arthritis. The results of immunoblot in this report showed that the level of CA III is specifically insufficient in the skeletal muscle of MG patients. The possible roles that CA III play in MG need further elucidation.

Lignocellulose is widely found in the nature. The highly efficient degradation of lignocellulose requires synergistic interactions of varieties of microorganisms. The mechanism of synergistic interaction relationship is not entirely clear because it needs multitudinous microorganisms to participate in the process of lignocellulose degradation. With the development of microbial molecular biology and omics technology, some new methods will be provided for the research on the mechanism of microbial synergistic degradation of lignocellulose. Our previous research found that the bacterial composite microbial system shows strong degradation ability of lignocellulose at 50 °C. The consortium is composed of cultured and uncultured bacteria, but the former has no degradation ability. Metagenomics and metatranscriptomics show that the expression levels of some genes related to lignocellulosic degradation change significantly. It is possible to explain the microbiological and enzymatic mechanisms of lignocellulosic degradation by microorganisms through omics in the future. The research progress of lignocellulose microbial degradation is reviewed from the aspects of enzyme, pure culture strain, and microbial consortium. The current situation and application prospect of omics technology in analyzing the function mechanism of microbial consortium are also introduced, to provide reference for exploring synergistic interactions of lignocellulose microbial degradation.木质纤维素广泛存在于自然界中，因结构复杂，其高效降解需要多种微生物的协同互作，由于参与木质纤维素降解的微生物种类繁多，其协同降解机理尚不完全明确。随着微生物分子生物学和组学技术的快速发展，将为微生物协同降解木质纤维素机制的研究提供新的方法和思路。笔者前期研究发现，细菌复合菌系在50 ℃下表现出强大的木质纤维素降解能力，菌系由可分离培养和暂时不可分离培养细菌组成，但是可分离培养细菌没有降解能力。通过宏基因组和宏转录组研究表明，与木质纤维素降解相关的某些基因表达量发生显著变化，通过组学方法有可能更加深入解释微生物协同降解木质纤维素的微生物学和酶学机理。文中从酶、纯培养菌株和复合菌群三个方面综述了木质纤维素微生物降解研究进展，着重介绍了组学技术在解析复合菌群作用机理方面的现状和应用前景，以期为探索微生物群落协同降解木质纤维素的机理提供借鉴。.

High-fat diet (HFD) is closely associated with the increased prevalence of inflammatory bowel disease (IBD). Excessive gut microbial metabolite deoxycholic acid (DCA) caused by HFD plays significant roles in eliciting intestinal inflammation, however, the mechanism underlining the induction of inflammatory response by DCA has not been fully elucidated. The purpose of this study was to investigate the role of DCA in the triggering of inflammation via affecting CD4+ T cell differentiation.Murine CD4+T cells were cultured under Th1, Th2 or Th17-polarizing conditions treated with or without different dosage of DCA, and flowcytometry was conducted to detect the effect of DCA on CD4+ T cell differentiation. Alteration of gene expression in CD4+ T cells upon DCA treatment was determined by RNA-sequencing and qRT-PCR. Bioinformatic analysis, cholesterol metabolic profiling, ChIP assay and immuno-fluorescent staining were further applied to explore the DCA-regulated pathway that involved in CD4+T cell differentiation. The results showed that DCA could dose-dependently promote the differentiation of CD4+ T cell into Th17 linage with pathogenic signature. Mechanistically, DCA stimulated the expression of cholesterol biosynthetic enzymes CYP51 and led to the increased generation of endogenous RORγt agonists, including zymosterol and desmosterol, therefore facilitating Th17 differentiation. Up-regulation of CYP51 by DCA was largely mediated via targeting transcription factor SREBP2 and at least partially through bile acid receptor TGR5. In addition, DCA-supplemented diet significantly increased intestinal Th17 cell infiltration and exacerbated TNBS-induced colitis. Administration of cholestyramine to eliminate fecal bile acid obviously alleviated colonic inflammation accompanied by decreased Th17 cells in HFD-fed mice.Our data establish a link between DCA-induced cholesterol biosynthesis in immune cells and gut inflammation. Modulation of bile acid level or targeting cholesterol metabolic pathway may be potential therapeutic measurements for HFD-related colitis.

After binding by acetylcholine released from a motor neuron, a nicotinic acetylcholine receptor at the neuromuscular junction produces a localized end-plate potential, which leads to muscle contraction. Improper turnover and renewal of acetylcholine receptors contributes to the pathogenesis of myasthenia gravis. In the present study, we demonstrate that endoplasmic reticulum (ER) stress contributes to acetylcholine receptor degradation in C2C12 myocytes. We further show that ER stress promotes acetylcholine receptor endocytosis and lysosomal degradation, which was dampened by blocking endocytosis or treating with lysosome inhibitor. Knockdown of ER stress proteins inhibited acetylcholine receptor endocytosis and degradation, while rescue assay restored its endocytosis and degradation, confirming the effects of ER stress on promoting endocytosis-mediated degradation of junction acetylcholine receptors. Thus, our studies identify ER stress as a factor promoting acetylcholine receptor degradation through accelerating endocytosis in muscle cells. Blocking ER stress and/or endocytosis might provide a novel therapeutic approach for myasthenia gravis.

For the sustainable reutilization of poultry feces (PF) to reduce environmental pollution, we present a novel approach for converting PF into a highly effective catalyst, consisting of trace copper (Cu) and sulfur (S) linked with ordered graphitized carbon (CS/CPF) for wastewater purification. Raman and EPR results verified that the disorderly organic matters in PF are transformed into orderly graphene structures that complexed with Cu to form large numbers of electron-poor/rich microregions on CS/CPF surface. The electrons from electron-rich organic pollutants can be directly captured by dissolved oxygen (DO) to produce abundant reactive oxygen species due to the enhanced electron polarization via the construction of Cu-S-C bond bridge on CS/CPF surface, which greatly enhance the removal efficiency of pollutants. CS/CPF achieves 100% removal for 2,4-dichlorophenoxyacetic acid (2,4-D) in just 10 min after adding trace peroxymonosulfate (PMS), keeping efficient catalytic activity after continuous reactions for 240 h. This strategy offers a practical and sustainable solution for the efficient resource recovery of poultry feces.

Sepsis-induced myocardiopathy, characterized by innate immune cells infiltration and proinflammatory cytokines release, may lead to perfusion failure or even life-threatening cardiogenic shock. Macrophages-mediated inflammation has been shown to contribute to sepsis-induced myocardiopathy. In the current study, we introduced two photoactivated adenylyl cyclases (PACs), Beggiatoa sp. PAC (bPAC) and Beggiatoa sp. IS2 PAC (biPAC) into macrophages by transfection to detect the effects of light-induced regulation of macrophage pro-inflammatory response and LPS-induced sepsis-induced myocardiopathy. By this method, we uncovered that blue light-induced bPAC or biPAC activation considerably inhibited the production of pro-inflammatory cytokines IL-1 and TNF-α, both at mRNA and protein levels. Further, we assembled a GelMA-Macrophages-LED system, which consists of GelMA-a type of light crosslink hydrogel, gene modulated macrophages and wireless LED device, to allow light to regulate cardiac inflammation in situ with murine models of LPS-induced sepsis. Our results showed significant inhibition of leukocytes infiltration, especially macrophages and neutrophils, suppression of pro-inflammatory cytokines release, and alleviation of sepsis-induced cardiac dysfunction. Thus, our study may represent an emerging means to treat sepsis-induced myocardiopathy and other cardiovascular diseases by photo-activated regulating macrophage function.

Background and aims T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is mainly expressed by immune cells and plays an immunomodulatory role in cardiovascular disease. However, the prognostic value of Tim-3 in acute decompensated heart failure (ADHF) is unclear. This study aimed to investigate the expression profile of Tim-3 on CD4 + and CD8 + T cells in patients with ADHF and its impact on their prognosis. Methods In this prospective study, 84 patients who were hospitalized with ADHF and 83 patients without heart failure were enrolled. Main clinical data were collected during patient visits. The Tim-3 expression on CD4 + and CD8 + T cells in peripheral blood samples was assayed by flow cytometry. Long-term prognosis of the patients with ADHF was evaluated by major adverse cardiac and cerebrovascular events (MACCE) over a 12-month follow-up period. Results We found that the Tim-3 expression on CD4 + T cells [2.08% (1.15–2.67%) vs. 0.88% (0.56–1.39%), p &amp;lt; 0.001] and CD8 + T cells [3.81% (2.24–6.03%) vs. 1.36% (0.76–3.00%), p &amp;lt; 0.001] in ADHF group were significantly increased vs. the non-ADHF group. Logistic analysis revealed that high levels of Tim-3 expressed on CD4 + and CD8 + T cells were independent risk factors of ADHF (OR: 2.76; 95% CI: 1.34–5.65, p = 0.006; OR: 2.58; 95% CI: 1.26–5.31, p = 0.010, respectively). ROC curve analysis showed that the high level of Tim-3 on CD4 + or CD8 + T cells as a biomarker has predictive performance for ADHF (AUC: 0.75; 95% CI: 0.68–0.83; AUC: 0.78, 95% CI: 0.71–0.85, respectively). During a median follow-up of 12 months, the Cox regression analysis revealed that higher Tim-3 on CD4 + and CD8 + T cells were strongly associated with increased risks of MACCE within 12 months after ADHF (HR: 2.613; 95% CI: 1.11–6.13, p = 0.027; HR: 2.762, 95% CI: 1.15–6.63, p = 0.023; respectively). Conclusion Our research indicated that the expression level of Tim-3 on CD4 + and CD8 + T cells, elevated in patients with ADHF, was an independent predictor of MACCE within 12 months after ADHF. It suggests a potential immunoregulatory role of Tim-3 signaling system in the mechanism of ADHF.

Autophagy plays essential roles in diverse biological events, range from cellular homeostasis and proliferation to aging, immune response, and contributes to various disorders if it go awry. Because of the paramount importance of autophagy, it is therefore of vital significance that any autophagy is carefully balanced. Autophagy involves the sequestration of cytoplasmic cargo inside double-membrane vesicles (autophagosomes) and fused with lysosome (autolysosome), where the captured material is degraded. The initiation and formation of autophagosomes involves multiple steps through multiple protein complexes. Beclin 1 is a key regulatory protein in Phosphoinositide 3-kinase (PI3K) complex during the early steps. The initiation of phagophore formation is regulated by a protein complex comprising Vps15, Vps34, Beclin 1, and other regulatory proteins, such as Bcl-2 and Rubicon. Recent studies have shown the important role of Beclin 1 in Toll-like receptor (TLR) 4-mediated autophagy. Here we demonstrated that Triad3A, an E3 ubiquitin protein ligase, specifically interacted with Beclin 1 but not with other adapter proteins in PI3K complex in the murine macrophage cell line RAW264.7 and in bone marrow-derived macrophages (BMDM) stimulated with lipopolysaccharide (LPS). We further showed that Triad3A accelerated Beclin 1 degradation through ubiquitination, thus inhibition of autophagy. In addition, S. typhimurium infection-induced autophagy was enhanced if Triad3A was knockdown using specific siRNA in BMDM, and contributed to the restriction of bacteria growth. This further suggested a role of the Triad3A in controlling autophagy in response to microbial infections. Taken together, our study have unveiled a new mechanism of the regulation of autophagy through ubiquitination of Beclin 1 by Triad3A, which represent a novel pathway of innate immunity link to autophagy. Triad3A-meidiated regulation might provide a novel therapeutic approach for controlling infectious diseases.

To inhibit the expression of CVB3 VP1 protein and the replication of CVB3 with synthesized siRNAs.According to the sequence and secondary structure of CVB3 VP1 protein, four pieces of siRNAs were designed following the requirement from Journal of Nature Cell Biology were synthesized in Shanghai GeneChem Company. Then they were transfected into HeLa cells by liposome (Lipofectamine 2000), but the non-transfected cells and non-specific siRNAs were taken as control. 48 hours later, the patho-morphous changes were observed, virus titer changes were examined by TCID50, CVB3-VP1 protein expression were detected by immunofluorescence with FITC dyeing, and CVB3-RNA level was tested by semi-quantitative RT-PCR.Two pieces of the four specific synthesized siRNAs (VP1-1 and VP1-2) were found to have obvious inhibitory effect on CVB3 replication and VP1 protein expression were reduced greatly. Besides, the changes of pathological cells were obviously mitigated.Specific siRNAs can effectively inhibit the expression of CVB3 VP1 protein and the replication of CVB3 in HeLa cells.

Although some certain infectious pathogens could be detected in the patients with coronary artery disease, the roles of these infectious factors in the development of coronary artery diseases remain largely unknown. Since the number of infectious pathogens has been argued to be relative to the coronary artery diseases, we therefore examined whether there is a link between the number of infections and the incidence of in-stent restenosis after stent implantation.One hundred and eighty-one patients were enrolled in this study. Infectious pathogens including serum anti-Chlymydia pneumoniae, cytomegalovirus, Helico pylori, human herpes simplex virus-1, human herpes simplex virus-2 antibodies and hepatitis B virus antigen were measured in all patients before coronary stent implantation. Coronary angiography was performed before, immediately after and 6 months after stent implantation.Restenosis rate 6 months post stent implantation was similar in patients with low pathogen burden (< 3 pathogens, 33.3%) to those with high pathogen burden (> or = 3 pathogens, 29.1%).Previous infections with Chlymydia pneumoniae, cytomegalovirus, Helico pylori, human herpes simplex virus-1, human herpes simplex virus-2 and hepatitis B virus do not contribute to the incidence of restenosis after stent implantation.

Aim To induce Coxsackie virus B type 3 (CVB3)-specific immune response by using a DNA vaccine containing CVB3-VP1 and to observe its protection against CVB3 challenge. Methods The gene coding for VP1 was obtained by RT-PCR and then was cloned into plasmid pcDNA3 to construct pcDNA3-VP1. In-vitro expression of VP1 was performed by transfection of pcDNA3-VP1 into Hela cells. Expressed product was detected by ELISA. BALB/c mice were immunized intramuscularly with 50 microg DNA three times, and challenged by 5xLD(50) CVB3 four weeks after the last immunization. Results pcDNA3-VP1 had been constructed and the expression product was detected in the culture supernatant of Hela cells 24 hours after transfection. CVB3-specific IgM and IgG elicited in the mice immunized with pcDNA3-VP1 were significantly higher than those in the control mice immunized with pcDNA3. Specific proliferation of the splenic lymphocytes and activity of CVB3-specific CTLs from the pcDNA3-VP1 immunized mice were much stronger than those in the controls. pcDNA3-VP1 could protect 33.3% mice from lethal CVB3 challenge, while control mice only survived 6.7 days. Infiltration of inflammatory cells or unusual proliferation of connective tissue, indicating ongoing myocarditis or fibrosis, were not found in pcDNA3-VP1 immunized mice, but did exist in control mice. Conclusion Intramuscular immunization with pcDNA3-VP1 may be a promising approach against CVB3 infection.

Abstract Myasthenia gravis is an autoimmune disorder of the neuromuscular junction manifestated as fatigable muscle weakness. The disorder typically caused by pathogenic autoantibodies against the postsynaptic acetylcholine receptor in the endplate of skeletal muscle. Our previous studies have identified carbonic anhydrase III as a specific protein insufficient in skeletal muscle from myasthenia gravis patient. In this study, we determined to investigate the underlying mechanism how carbonic anhydrase III insufficiency contributes to myasthenia gravis. Based on experimental autoimmune myasthenia gravis animal model and myocyte C2C12 cells, we find that inhibition of carbonic anhydrase III promotes acetylcholine receptor, which is through lipid raft-mediated pathway, leading to accelerated degradation of surface acetylcholine receptor. While activation of carbonic anhydrase III negatively regulates acetylcholine receptor degradation, through suppressing the endocytosis of surface acetylcholine receptor, thus contributes to attenuation of myasthenia gravis. Further study demonstrates that carbonic anhydrase III regulates endocytosis of AChR through actin polymerization and Src phosphorylation. Collectively, our study illustrates that carbonic anhydrase III in skeletal muscle cells is critical for acetylcholine receptor homeostasis in neuromuscular junction, while carbonic anhydrase III deficiency induces accelerated degradation of acetylcholine receptor, leading to myasthenia gravis. This novel mechanism highlights the essential role of carbonic anhydrase III in regualtion acetylcholine receptor endocytosis, providing a potential therapeutic approaches for myasthenia gravis.

We studied cytochrome c oxidase (COX) expression patterns in nuclear and mtDNA gene defects. Using quantitative immunocytochemical assay for COX, heteroplasmic staining was seen in MELAS patients with mtDNA mutations but similar staining variability was seen in control cell lines and nuclear gene defects. All fibroblast lines showed a wide variability in cell-to-cell COX I staining intensity. All 8 patient fibroblast lines had reduced COX staining on immunocytochemistry. In 6 lines reduced protein amount was seen on Western blotting and 7 had low COX activity. This study demonstrates that nuclear gene defects can produce a heteroplasmic appearance on immunocytochemistry.

Bacterial and viral DNA are potent stimuli to immune cells. Synthetic CpG-oligodeoxynucleotides (CpG-ODN) mimic the stimulatory effect of these microbial DNA in activation of immune cells. The activity of a CpG-ODN is determined by its sequence context including its length, the number of CpG motifs, and the spacing, position, and surrounding bases of these motifs. Toll-like receptor 9 (TLR9) is the cellular receptor for these CpG-ODN. Previous studies have shown different ligand recognition profiles of mouse TLR9 and human TLR9. Rabbits are commonly used for production of antibodies. Although CpG-ODN have been shown to be a safety and effective adjuvant in boosting antibody production in rabbits, the interaction between CpG-ODN have not yet investigated. In the present study, we cloned and characterized rabbit TLR9 cDNA. We further established cell-based activations with this rabbit TLR9 cDNA, and cDNAs for mouse and human TLR9 to comparatively investigate activation of these TLR9s. The complete open reading frame of rabTLR9 encodes 1028 amino acid residues, which share 70.6% and 75.5% of the identities of mTLR9 and hTLR9, respectively. Similiar to that of human TLR9, rabbit TLR9 is preferentially expressed in immune cells riched tissues, and is localized in intracellular vesicles. While mTLR9 and hTLR9 displayed species-specific recognition of their optimized CpG-ODN, rabbit TLR9 was activated by these CpG-ODN without any preference. The results indicated a different ligand recognition profiles of these TLR9 from these species.

Abstract Proper intracellular localization of Toll-like receptors is essential for their signaling and biological functions. Endocytosis constitutes a key step in protein turnover as well as maintenance of localization, and thus provides important points to regulate their signaling. Here we demonstrated that adenylyl cyclase activation attenuated membrane Toll-like receptor 4-dependent NF-κB signaling and IRF activation in the murine macrophage cell line RAW264.7 and in bone marrow-derived macrophages stimulated with lipopolysaccharide. We further showed that the adenylyl cyclase 6 isoform distributed in lipid raft-enriched domains played key roles in the negative regulation of Toll-like receptor 4 signaling by promoting its degradation. Toll-like receptor 4 is normally endocytosed through the clathrin-mediated pathway, which is a critical step in signal transduction, while adenylyl cyclase 6 activation initiated its colocalization with Toll-like receptor 4 and their interaction, and shifted clathrin-mediated endocytosis of Toll-like receptor 4 to lipid raft-mediated endocytosis, leading to accelerated degradation of Toll-like receptor 4 and consequently suppressed downstream signaling. Our studies have unveiled a new mechanism of negative regulation of Toll-like receptor 4 signaling through adenylyl cyclase 6-mediated endocytosis. Adenylyl cyclase 6-specific activation might provide a novel therapeutic approach for limiting inflammatory and autoimmune diseases.

Purpose To explore the influence of oxidized low-density lipoprotein (oxLDL) on the immune function of monocyte derived dendritic cells (MDCs). Methods Monocytes were purified (over 98%) using anti-CD14 microbeads,after 5 day's culture with DCs Cellgro medium containing rhGM-CSF (100 ng/mL) and rhIL-4 (20 ng/mL),immature MDCs were derived,50 μg/mL LDL and oxLDL were added to the medium for 48 hours,PBS and LPS (500 μg/mL) were as the negative and positive control;FACS was used to investigate the immunophenotypic expression (CD1a,CD40,CD86,and HLA-DR),FITC-Dextran was used to analyze the endocytosis function,allogeneic mixed T lymphocytes reaction and supernatants'cytokine measurements were used for immune functional assays. Results Compared with LDL and PBS,oxLDL treated MDCs up-regulated CD86 and CD1a expression,decreased the endocytosis and promoted allogeneic T cells proliferation.Furthermore,the cytokines (IL-12,IL-10 and TNFα) secretion of oxLDL group were increased (P0.05). Conclusions oxLDL can promote the maturation of DCs and augments their capacity to stimulate T-cell proliferation and cytokine secretion.These effects of oxLDL on DCs may contribute to the immunopathogenesis of atherosclerotic lesions.

Objectives To investigate the relationship between Chlamydia pneumoniae(CP) infection,inflammation and coronary atherosclerosis, and to evaluate their potential roles in restenosis.Methods Five hundred and fifty five subjects were divided into coronary artery atherosclerosis patients and non-coronary artery atherosclerosis controls according to coronary angiography. Their baseline characteristics were carefully recorded. The serum CRP and CP-specific antibody IgA were measured by ELISA.Of the subjects,89 patients who had stent implantation were followed up for six months. Quantitative coronary angiography was used to assess the rate of lumen loss. Results Positive rate and titer of CP IgA in patients with coronary atherosclerosis were higher than those in control group .Serum CRP level was also higher in coronary atherosclerotic group than in control group, and increased with increasing extent of the lesions. There was no significant difference in rate of neointima proliferation after stent placement between the seropositive group and seronegative group.But CRP increased in patients with neointima proliferation.Conclusions Previous infection with CP was associated with coronary artery atherosclerosis, but it could not predict the restenosis after stent placement. However, serum CRP played an important role in the process of both atherosclerosis and restenosis.

Background and purpose: Alpha 7 nicotinic acetylcholine receptors (CHRNA7) suppress inflammation through diverse pathways in immune cells, so is potentially involved in a number of inflammatory diseases.However, the detailed mechanisms underlying CHRNA7's anti-inflammatory effects remain elusive.Experimental approach: The anti-inflammatory effects of CHRNA7 agonists in both murine macrophages (RAW 264.7) and bone marrow-derived macrophages (BMDM) stimulated with LPS were examined.The role of adenylyl cyclase 6 (AC6) in Toll-like Receptor 4 (TLR4) degradation was explored via overexpression

Objective To study the relationship between CMV infection and atherogenesis and the putative mechanism.Method To detect CMV IgG of subject serum with ELISA and the existence of CMV IE gene in the atherosclerotic plaque by PCR. The expression of chemokines in endothelial cells after infection with CMV was also studied.Results The positive rate of CMV IgG was significantly higher in athe- rosclerosis group than that in non-atherosclerosis group (82.2% vs 61.0%, P=0.02); HCMV-specific gene in atherosclerotic plaque was much more frequently detected than that in normal vascular tissue(13/15 vs 2/7, P=0.01). CMV infection can up-regulate the expression of chemokines in endothelial cells ECV-304.Conclusion CMV infection was involved in the pathogenesis of atherosclerosis, which might be me- diated by up-regulated expression of chemokines in ECV-304 after CMV infection.

To evaluate the relationship between infection burden and coronary atherosclerosis and the plaque feature.One hundred and eighty two patients underwent coronary angiography in Zhongshan Hospital from 2002 - 2003. Atherosclerosis and vulnerable plaque were determined by intravascular ultrasound (IVUS). Seropositivity of cytomegalovirus, helicobacter pylori, chlamydia pneumonia, hepatitis B virus, EB virus, CoxB virus, influenza A virus, influenza B virus and mycobacterium tuberculosis were determined by ELISA. The serum hs-CRP was detected by Dade Behring prospect (Immuno-nehelomitery). Patients were divided into three groups according to the pathogen burden: group A, n <or= 3, group B, n = 4 - 5 and group C, n >or= 6.The pathogen burden was independent of the C-reactive protein level. Increasing pathogen burden was significantly associated with increasing atherosclerosis risk, the prevalence of atherosclerosis was 44.4%, 70.6% and 76.7% in group A, B and C. The risk associated with elevated pathogen burden was much higher when CRP was also elevated (> 5.0 mg/L) (43.8%, 70.0%, 70.8%) vs (45.5%, 63.7%, 96.8%). The positively of vulnerable plaque increased significantly when the pathogen burden was high (n > 5) (33.3%, 32.4% and 51.7% P < 0.05).Our data suggested that infection burden was associated with prevalence of coronary atherosclerosis, and it was particularly important when C-reactive protein was elevated. The high level infection burden could predict vulnerable plaque.