Gut microbiota-mediated bile acid metabolism aggravates biliary injury after liver transplantation through mitochondrial apoptosis.

Ischemic-type biliary lesions (ITBL) are a major cause of graft loss and even mortality after liver transplantation (LT). The underlying cellular mechanisms for ITBL remain unclear. Gut microbiota has been found to be closely related to complications after LT. Here, using gut microbiome compositions, we found patients with ITBL had a higher abundance of bacteria associated with bile salt metabolism. These bacteria are reported to convert cholic acid (CA) into deoxycholic acid (DCA), consistent with our data that there were higher DCA concentrations and DCA/CA ratio in patients with ITBL than patients without ITBL. Using an in vitro model, human intrahepatic biliary epithelial cells (HIBEC) subjected to DCA showed a higher apoptosis rate, lower viability, and higher levels of cleaved-caspase3 than CA at the same concentration. DCA also changed the morphology of mitochondria and farnesoid X receptor (FXR) expression. Interestingly, DCA-induced apoptosis rate was significantly reduced in HIBEC when the FXR or BAX gene was knocked down, suggesting that DCA-induced apoptosis was dependent on FXR-mitochondrial pathway. Furthermore, increasing DCA/CA ratio in a bile acid-feeding mouse model resulted in cholangiocyte apoptosis and impaired liver function. The patients with ITBL also showed an increased proportion of TUNEL-positive biliary epithelial cells than those without ITBL. These suggest that changes in the gut microbiota following LT may enhance the conversion of CA to DCA, and may contribute to biliary damage via FXR-mitochondrial apoptosis pathway, providing new ideas for the early monitoring and treatment of ITBL.

FOS+ Macrophages Promote Chronic Rejection of Cardiac Transplantation.

OBJECTIVES: Chronic rejection remains the leading cause of progressive decline in graft function. Accumulating evidence indicates that macrophages participate in chronic rejection dependent on CD40-CD40L. The FOS family members are critical in inflammatory and immune responses. However, the mechanisms underlying the role of FOS family members in chronic rejection remain unclear. In this study, we aimed to elucidate the role and underlying mechanisms of FOS-positive macrophages regulated by CD40 that mediate chronic allograft rejection. MATERIALS AND METHODS: We downloaded publicly accessible chronic rejection kidney transplant single-cell sequencing datasets from the gene expression omnibus database. Differentially expressed genes between the CD40hi and CD40low macrophage chronic rejection groups were analyzed. We established a chronic rejection mouse model by using CTLA-4-Ig. We treated bone marrow-derived macrophages with an anti-CD40 antibody. We assessed expression of the FOS family by flow cytometry, real-time quantitative polymerase chain reaction, Western blotting, and immunofluorescence. We identified altered signaling pathways by using RNA sequencing analysis. We detected DNA specifically bound to transcription factors by using ChIP-sequencing, with detection of the degree of graft fibrosis and survival. RESULTS: FOS was highly expressed on CD40hi macrophages in patients with chronic transplantrejection. Mechanistically, we showed that CD40 activated NF-κB2 translocation into the nucleus to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant.We showed thatNF-κB2 regulated c-Fos and FosB expression by binding to the c-fos and fosb promoter regions. Inhibition of c-Fos/activator protein-1 decreased graft fibrosis and prolonged graft survival. CONCLUSIONS: CD40 may activate transcription factor NF-κB2 translocation into the nucleus of macrophages to upregulate c-Fos and FosB expression, thus promoting chronic rejection of cardiac transplant. Inhibition of c-Fos/activator protein-1 decreased grafts fibrosis and prolonged graft survival.

CBX4 suppresses CD8+ T cell antitumor immunity by reprogramming glycolytic metabolism.

Rationale: CD8+ T cells undergo a series of metabolic reprogramming processes during their activation and proliferation, including increased glycolysis, decreased aerobic oxidation of sugars, increased amino acid metabolism and increased protein synthesis. However, it is still unclear what factors regulate these metabolic reprogramming processes in CD8+ T cells in the tumor immune microenvironment. Methods: T cell chromobox protein 4 (CBX4) knock-out mice models were used to determine the role of CBX4 in CD8+ T cells on the tumor immune microenvironment and tumor progression. Flow cytometry, Cut-Tag qPCR, Chip-seq, immunoprecipitation, metabolite detection, lentivirus infection and adoptive T cells transfer were performed to explore the underlying mechanisms of CBX4 knock-out in promoting CD8+ T cell activation and inhibiting tumor growth. Results: We found that CBX4 expression was induced in tumor-infiltrating CD8+ T cells and inhibited CD8+ T cell function by regulating glucose metabolism in tumor tissue. Mechanistically, CBX4 increases the expression of the metabolism-associated molecule aldolase B (Aldob) through sumoylation of trans-acting transcription factor 1 (SP1) and Krüppel-like factor 3 (KLF3). In addition, Aldob inhibits glycolysis and ATP synthesis in T cells by reducing the phosphorylation of the serine/threonine protein kinase (Akt) and ultimately suppresses CD8+ T cell function. Significantly, knocking out CBX4 may improve the efficacy of anti-PD-1 therapy by enhancing the function of CD8+ T cells in the tumor microenvironment. Conclusion: CBX4 is involved in CD8+ T cell metabolic reprogramming and functional persistence in tumor tissues, and serves as an inhibitor in CD8+ T cells' glycolysis and effector function.

A prognostic model based on CLEC6A predicts clinical outcome of breast cancer patients.

CLEC6A, (C-type lectin domain family 6, member A), plays a prominent role in regulating innate immunity and adaptive immunity. CLEC6A has shown great potential as a target for cancer immunotherapy. This study aims to explore the prognostic value of CLEC6A, and analyze the relationship associated with the common hematological parameters in breast cancer patients. We performed a retrospective analysis on 183 breast cancer patients data in hospital information system from January 2013 to December 2015. The expression of CLEC6A was recorded via semiquantitative immunohistochemistry in breast cancer. The association between expression of CLEC6A and relative parameters were performed by Chi-square test and Fisher's exact test. Kaplan-Meier assay and Log-rank test were performed to evaluate the survival time. The Cox proportional hazards regression analysis was applied to identify prognostic factors. Nomograms were conducted to predict 1-, 3-, and 5-year disease free survival (DFS) and overall survival (OS) for breast cancer, which could be a good reference in clinical practice. The nomogram model was estimated by calibration curve analysis for its function of discrimination. The accuracy and benefit of the nomogram model were appraised by comparing it to only CLEC6A via decision curve analysis (DCA). The prediction accuracy of CLEC6A was also determined by time-dependent receiver operating characteristics (TDROC) curves, and the area under the curve (AUC) for different survival time. There were 94 cases in the CLEC6A low-expression group and 89 cases in CLEC6A high-expression group. Compared to CLEC6A low-expression group, the CLEC6A high-expression group had better survival (DFS: 56.95 vs. 70.81 months, P = 0.0078 and OS: 67.98 vs. 79.05 months, P = 0.0089). The CLEC6A was a potential prognostic factor in multivariate analysis (DFS: P = 0.023, hazard ratio (HR): 0.454, 95 % confidence interval (CI): 0.229-0.898; OS: P = 0.020, HR: 0.504, 95 %CI: 0.284-0.897). The nomogram in accordance with these potential prognostic factors was constructed to predict survival and the calibration curve analysis had indicated that the predicted line was well-matched with reference line in 1-, 3-, and 5-year DFS and OS category. The 1-, 3-, and 5-year DCA curves have revealed that nomogram model yielded larger net benefits than CLEC6A alone. Finally, the TDROC curve indicated that CLEC6A could better predict 1-year DFS and OS than others. Furthermore, we combined these potential independent prognostic factors to analyze the relationship among these hematologic index and oxidative stress indicators, and indicated that higher CLEC6A level, higher CO2 level or low CHOL level or high HDL-CHO level would have survived longer and better prognosis. In breast cancer, high expression of CLEC6A can independently predict better survival. Our nomogram consisted of CLEC6A and other indicators has good predictive performance and can facilitate clinical decision-making.

LRP11 promotes stem-like T cells via MAPK13-mediated TCF1 phosphorylation, enhancing anti-PD1 immunotherapy.

BACKGROUND: Tumor-infiltrating T cells enter an exhausted or dysfunctional state, which limits antitumor immunity. Among exhausted T cells, a subset of cells with features of progenitor or stem-like cells has been identified as TCF1+ CD8+ T cells that respond to immunotherapy. In contrast to the finding that TCF1 controls epigenetic and transcriptional reprogramming in tumor-infiltrating stem-like T cells, little is known about the regulation of TCF1. Emerging data show that elevated body mass index is associated with outcomes of immunotherapy. However, the mechanism has not been clarified. METHODS: We investigated the proliferation of splenic lymphocytes or CD8+ T cells induced by CD3/CD28 stimulation in vitro. We evaluated the effects of low-density lipoprotein (LDL) and LRP11 inhibitors, as well as MAPK13 inhibitors. Additionally, we used shRNA technology to validate the roles of LRP11 and MAPK13. In an in vivo setting, we employed male C57BL/6J injected with B16 cells or MC38 cells to build a tumor model to assess the effects of LDL and LRP11 inhibitors, LRP11 activators, MAPK13 inhibitors on tumor growth. Flow cytometry was used to measure cell proportions and activation status. Molecular interactions and TCF1 status were examined using Western blotting. Moreover, we employed RNA sequencing to investigate the effects of LDL stimulation and MAPK13 inhibition in CD8+ T cells. RESULTS: By using a tumor-bearing mouse model, we found that LDL-induced tumor-infiltrating TCF1+PD1+CD8+ T cells. Using a cell-based chimeric receptor screening system, we showed that LRP11 interacted with LDL and activated TCF1. LRP11 activation enhanced TCF1+PD1+CD8+ T-cell-mediated antitumor immunity, consistent with LRP11 blocking impaired T-cell function. Mechanistically, LRP11 activation induces MAPK13 activation. Then, MAPK13 phosphorylates TCF1, leading to increase of stem-like T cells. CONCLUSIONS: LRP11-MAPK13-TCF1 enhanced antitumor immunity and induced tumor-infiltrating stem-like T cells.

The Role of Immune Cells in the Pathogenesis of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown etiology with limited treatment options. The role of the immune system in IPF has received increasing attention. Uncontrolled immune responses drive the onset and progression of IPF. This article provides an overview of the role of innate immune cells (including macrophages, neutrophils, mast cells, eosinophils, dendritic cells, nature killer cells, nature kill cells and γδ T cells) and adaptive immune cells (including Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, cytotoxic T cells, B lymphocytes and Treg cells) in IPF. In addition, we review the current status of pharmacological treatments for IPF and new developments in immunotherapy. A deeper comprehension of the immune system's function in IPF may contribute to the development of targeted immunomodulatory therapies that can alter the course of the disease.

Targeting Alpha-Ketoglutarate Disruption Overcomes Immunoevasion and Improves PD-1 Blockade Immunotherapy in Renal Cell Carcinoma.

The Warburg effect-related metabolic dysfunction of the tricarboxylic acid (TCA) cycle has emerged as a hallmark of various solid tumors, particularly renal cell carcinoma (RCC). RCC is characterized by high immune infiltration and thus recommended for immunotherapeutic interventions at an advanced stage in clinical guidelines. Nevertheless, limited benefits of immunotherapy have prompted investigations into underlying mechanisms, leading to the proposal of metabolic dysregulation-induced immunoevasion as a crucial contributor. In this study, a significant decrease is found in the abundance of alpha-ketoglutarate (αKG), a crucial intermediate metabolite in the TCA cycle, which is correlated with higher grades and a worse prognosis in clinical RCC samples. Elevated levels of αKG promote major histocompatibility complex-I (MHC-I) antigen processing and presentation, as well as the expression of ß2-microglobulin (B2M). While αKG modulates broad-spectrum demethylation activities of histone, the transcriptional upregulation of B2M is dependent on the demethylation of H3K4me1 in its promoter region. Furthermore, the combination of αKG supplementation and PD-1 blockade leads to improved therapeutic efficacy and prolongs survival in murine models when compared to monotherapy. Overall, the findings elucidate the mechanisms of immune evasion in anti-tumor immunotherapies and suggest a potential combinatorial treatment strategy in RCC.

Activation of immune signals during organ transplantation.

The activation of host's innate and adaptive immune systems can lead to acute and chronic graft rejection, which seriously impacts graft survival. Thus, it is particularly significant to clarify the immune signals, which are critical to the initiation and maintenance of rejection generated after transplantation. The initiation of response to graft is dependent on sensing of danger and stranger molecules. The ischemia and reperfusion of grafts lead to cell stress or death, followed by releasing a variety of damage-associated molecular patterns (DAMPs), which are recognized by pattern recognition receptors (PRRs) of host immune cells to activate intracellular immune signals and induce sterile inflammation. In addition to DAMPs, the graft exposed to 'non-self' antigens (stranger molecules) are recognized by the host immune system, stimulating a more intense immune response and further aggravating the graft damage. The polymorphism of MHC genes between different individuals is the key for host or donor immune cells to identify heterologous 'non-self' components in allogeneic and xenogeneic organ transplantation. The recognition of 'non-self' antigen by immune cells mediates the activation of immune signals between donor and host, resulting in adaptive memory immunity and innate trained immunity to the graft, which poses a challenge to the long-term survival of the graft. This review focuses on innate and adaptive immune cells receptor recognition of damage-associated molecular patterns, alloantigens and xenoantigens, which is described as danger model and stranger model. In this review, we also discuss the innate trained immunity in organ transplantation.

USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

BACKGROUND: During organ transplantation, pharmacologic drugs targeting T cell activation signal to inhibit T cell-mediated allo-rejection are insufficient and not durable to suppress chronic rejection. Recent advances highlight an exhausted or dysfunctional status of T cells, which favor transplant acceptance. METHODS: The models of MHC-mismatched (BALB/c to C57BL/6 or USP25 KO mice) heterotopic heart transplantation and skin transplantation were utilized to evaluate the regulatory effects of ubiquitin-specific protease 25(USP25) deficiency in vivo. The consequences of USP25 deficiency on murine T-cell proliferation, activation, cytokine secretion, mixed lymphocyte reaction (MLR) and energy metabolism were investigated in vitro. The signaling pathway of T cells in knock out mice was detected by Western blotting and Co-IP. RESULTS: We found T cells were dysfunctional inUSP25KO mice. Due to T cell dysfunction, skin and heart graft had a longer survival. In these dysfunctional T cells, mitochondria number and cristae condensation were decreased. Impaired mitochondrial mass and function favored to allo-graft acceptance. Furthermore, USP25 interacted with ATP5A and ATP5B to promote their stability. CONCLUSIONS: Our data suggest that USP25 is a potential target to induce T cell dysfunction and allo-graft tolerance. And USP25 mediated mitochondrial homeostasis may contribute to reverse T cell exhaustion or dysfunction in tumor and chronic infection.

Immune Checkpoint and Other Receptor-Ligand Pairs Modulating Macrophages in Cancer: Present and Prospects.

Immunotherapy, especially immune checkpoint blocking, has become the primary anti-tumor treatment in recent years. However, the current immune checkpoint inhibitor (ICI) therapy is far from satisfactory. Macrophages are a key component of anti-tumor immunity as they are a common immune cell subset in tumor tissues and act as a link between innate and adaptive immunity. Hence, understanding the regulation of macrophage activation in tumor tissues by receptor-ligand interaction will provide promising macrophage-targeting strategies to complement current adaptive immunity-based immunotherapy and traditional anti-tumor treatment. This review aims to offer a systematic summary of the current advances in number, structure, expression, biological function, and interplay of immune checkpoint and other receptor-ligand between macrophages and tumor cells.

PIRs mediate innate myeloid cell memory to nonself MHC molecules.

Immunological memory specific to previously encountered antigens is a cardinal feature of adaptive lymphoid cells. However, it is unknown whether innate myeloid cells retain memory of prior antigenic stimulation and respond to it more vigorously on subsequent encounters. In this work, we show that murine monocytes and macrophages acquire memory specific to major histocompatibility complex I (MHC-I) antigens, and we identify A-type paired immunoglobulin-like receptors (PIR-As) as the MHC-I receptors necessary for the memory response. We demonstrate that deleting PIR-A in the recipient or blocking PIR-A binding to donor MHC-I molecules blocks memory and attenuates kidney and heart allograft rejection. Thus, innate myeloid cells acquire alloantigen-specific memory that can be targeted to improve transplant outcomes.

OX40 Costimulation Inhibits Foxp3 Expression and Treg Induction via BATF3-Dependent and Independent Mechanisms.

Naive CD4+ T cells can be converted to Foxp3+ T regulatory cells (Tregs) in the periphery (iTregs), where induction of Foxp3 gene expression is central to Treg differentiation. OX40 signaling is known to inhibit Foxp3 expression and Treg induction, but the underlying mechanisms remain poorly defined. Here, we found that OX40 costimulation activates two distinct molecular pathways to suppress Foxp3 expression in freshly activated naive CD4+ T cells. Specifically, OX40 upregulates BATF3 and BATF, which produce a closed chromatin configuration to repress Foxp3 expression in a Sirt1/7-dependent manner. Moreover, OX40 can also activate the AKT-mTOR pathway, especially in the absence of BATF3 and BATF, to inhibit Foxp3 induction, and this is mediated by phosphorylation and nuclear exclusion of the transcription factor Foxo1. Taken together, our results provide key mechanistic insights into how OX40 inhibits Foxp3 expression and Treg induction in the periphery.

Oncofetal gene SALL4 reactivation by hepatitis B virus counteracts miR-200c in PD-L1-induced T cell exhaustion.

A chronic viral or tumor microenvironment can push T cells to exhaustion by promoting coinhibitory ligand expression. However, how host factors control coinhibitory ligand expression and whether viral infection breaks this control during tumor progress is unknown. Here we show a close negative correlation between SALL4 or PD-L1 and miR-200c in tumors from 98 patients with HBV-related hepatocellular carcinoma. SALL4 or PD-L1 expression correlates negatively with miR-200c expression, and patients with lower levels of SALL4 or PD-L1 and higher miR-200c survive longer. Moreover, over-expression of miR-200c antagonizes HBV-mediated PD-L1 expression by targeting 3'-UTR of CD274 (encoding PD-L1) directly, and reverses antiviral CD8+ T cell exhaustion. MiR-200c transcription is inhibited by oncofetal protein SALL4, which is re-expressed through HBV-induced STAT3 activation in adulthood. We propose that an HBV-pSTAT3-SALL4-miR-200c axis regulates PD-L1. Therapeutic strategies to influence this axis might reverse virus-induced immune exhaustion.

Pim-3 enhances melanoma cell migration and invasion by promoting STAT3 phosphorylation.

Melanoma is the deadliest form of commonly encountered skin cancer, and has fast propagating and highly invasive characteristics. Pim-3, a highly expressed oncogene in melanoma, is a highly conserved serine/threonine kinase with various biological activities, such as proliferation-accelerating and anti-apoptosis effects on cancer progression. However, whether Pim-3 regulates melanoma metastasis has not been determined. Here, we constructed a Pim-3-silencing short hairpin RNA (sh-Pim-3), a TLR7-stimulating ssRNA and a dual-function vector containing a sh-Pim-3 and a ssRNA, and transfected them into the B16F10 melanoma cell line to investigate the effects of Pim-3 on migration and invasion in melanoma. We found that sh-Pim-3 inhibited B16F10 cell migration and invasion in vitro. In a tumor-bearing mouse model, sh-Pim-3 significantly downregulated pulmonary metastasis of B16F10 melanoma cell in vivo. Mechanistically, sh-Pim-3 inhibited metastasis by regulating the expression of genes related to epithelial-mesenchymal transition (EMT). Further study revealed that by promoting the phosphorylation of STAT3 (signal transducer and activator of transcription 3), Pim-3 induced the expression of Slug, Snail, and ZEB1, which enhanced EMT-related changes and induced melanoma migration and invasion. Our study suggests that Pim-3 is a potential effective target for melanoma therapy.

Guidance of super-enhancers in regulation of IL-9 induction and airway inflammation.

Th9 cells are prominently featured in allergic lung inflammation, but the mechanism that regulates IL-9 induction in T helper cells remains poorly defined. Here we demonstrate that formation of super-enhancers (SEs) is critical in robust induction of IL-9 and that assembly of the Il9 SEs in Th cells requires OX40-triggered chromatin acetylation. Mechanistically, we found that OX40 costimulation induces RelB expression, which recruits the histone acetyltransferase p300 to the Il9 locus to catalyze H3K27 acetylation. This allows binding of the SE factor Brd4 to organize assembly of the SE complex, which in turn drives robust IL-9 expression and Th9 cell induction. Thus, Th9 cells are strongly induced upon OX40 stimulation, and disruption of SEs abolished Th9 cell induction in vitro and inhibited Th9 cell-mediated allergic airway inflammation in vivo. Together, our data suggest that formation of SEs is essential in IL-9 expression and Th9 cell induction. These findings may have important clinical implications.

Macrophage subpopulations and their impact on chronic allograft rejection versus graft acceptance in a mouse heart transplant model.

Macrophages infiltrating the allografts are heterogeneous, consisting of proinflammatory (M1 cells) as well as antiinflammatory and fibrogenic phenotypes (M2 cells); they affect transplantation outcomes via diverse mechanisms. Here we found that macrophage polarization into M1 and M2 subsets was critically dependent on tumor necrosis factor receptor-associated factor 6 (TRAF6) and mammalian target of rapamycin (mTOR), respectively. In a heart transplant model we showed that macrophage-specific deletion of TRAF6 (LysMCre Traf6 fl/fl ) or mTOR (LysMCre Mtorfl/fl ) did not affect acute allograft rejection. However, treatment of LysMCre Mtorfl/fl recipients with CTLA4-Ig induced long-term allograft survival (>100 days) without histological signs of chronic rejection, whereas the similarly treated LysMCre Traf6 fl/fl recipients developed severe transplant vasculopathy (chronic rejection). The presentation of chronic rejection in CTLA4-Ig-treated LysMCre Traf6 fl/fl mice was similar to that of CTLA4-Ig-treated wild-type B6 recipients. Mechanistically, we found that the graft-infiltrating macrophages in LysMCre Mtorfl/fl recipients expressed high levels of PD-L1, and that PD-L1 blockade readily induced rejection of otherwise survival grafts in the LysMCre Mtorfl/fl recipients. Our findings demonstrate that targeting mTOR-dependent M2 cells is critical for preventing chronic allograft rejection, and that graft survival under such conditions is dependent on the PD-1/PD-L1 coinhibitory pathway.

TNF superfamily receptor OX40 triggers invariant NKT cell pyroptosis and liver injury.

Tissue-resident immune cells play a key role in local and systemic immune responses. The liver, in particular, hosts a large number of invariant natural killer T (iNKT) cells, which are involved in diverse immune responses. However, the mechanisms that regulate survival and homeostasis of liver iNKT cells are poorly defined. Here we have found that liver iNKT cells constitutively express the costimulatory TNF superfamily receptor OX40 and that OX40 stimulation results in massive pyroptotic death of iNKT cells, characterized by the release of potent proinflammatory cytokines that induce liver injury. This OX40/NKT pyroptosis pathway also plays a key role in concanavalin A-induced murine hepatitis. Mechanistically, we demonstrated that liver iNKT cells express high levels of caspase 1 and that OX40 stimulation activates caspase 1 via TNF receptor-associated factor 6-mediated recruitment of the paracaspase MALT1. We also found that activation of caspase 1 in iNKT cells results in processing of pro-IL-1ß to mature IL-1ß as well as cleavage of the pyroptotic protein gasdermin D, which generates a membrane pore-forming fragment to produce pyroptotic cell death. Thus, our study has identified OX40 as a death receptor for iNKT cells and uncovered a molecular mechanism of pyroptotic cell death. These findings may have important clinical implications in the development of OX40-directed therapies.

HBV inhibits LPS-induced NLRP3 inflammasome activation and IL-1ß production via suppressing the NF-κB pathway and ROS production.

BACKGROUND & AIMS: Hepatitis B virus (HBV) has developed strategies to evade immune responses. However, the mechanisms involved remain unclear. The NLRP3 inflammasome plays crucial roles in antiviral host defense and its downstream factor IL-1ß has been shown to inhibit HBV infection in vivo. This study aims to assess whether HBV can affect the NLRP3 inflammasome signaling pathways and shed light on the underlying mechanisms HBV utilizes to evade host innate immune responses. METHODS: HBV inhibition of the lipopolysaccharide (LPS)-induced NLRP3 inflammasome activation was evaluated by Western blot, quantitative RT-PCR, flow cytometry and immunofluorescence. RESULTS: Kupffer cells expressed significantly more NLRP3 and IL-1ß after LPS stimulation; whereas, chronic HBV infection suppressed LPS-induced NLRP3 and pro-IL-1ß expression as well as IL-1ß maturation. This inhibitory activity is mediated by HBeAg, and is involved in the inhibition of NF-κB signal pathway and reactive oxygen species (ROS) production. The inhibitory effect of HBeAg was confirmed in patients with chronic hepatitis B (CHB) and hepatocellular carcinoma by comparing the levels of IL-1ß and NLRP3-related proteins in para-carcinoma tissues from HBeAg-positive or negative patients. Moreover, chronic HBV infection increases the susceptibility of mice to S. typhimurium infection, possibly via inhibiting the NLRP3 inflammasome activation and IL-1ß production. CONCLUSIONS: HBeAg inhibits LPS-induced NLRP3 inflammasome activation and IL-1ß production via suppressing NF-κB pathway and ROS production. This finding provides a novel mechanism for HBV-mediated suppression of innate immune responses, and identifies new therapeutic targets for chronic HBV infection and related diseases. LAY SUMMARY: HBeAg suppresses LPS-induced NLRP3 inflammasome activation and IL-1ß production in two ways, one is to repress NLRP3 and pro-IL-1ß expression via inhibiting NF-κB phosphorylation, and the other is to repress caspase-1 activation and IL-1ß maturation via inhibiting ROS production. This effect contributes to the HBV persistence and immune tolerance.

HBV suppresses expression of MICA/B on hepatoma cells through up-regulation of transcription factors GATA2 and GATA3 to escape from NK cell surveillance.

Decreased expression of NKG2D ligands on HBV-infected human hepatoma cells impairs NK cells lysis. However, which components of HBV exert this effect and the precise mechanisms need to be further investigated. In the present study, we observed that the HBx and HBc genes significantly down-regulated MICA expression. Through analysis with the chromatin immunoprecipitation assay, we found that HBV infection promotes the expression of transcription factors GATA-2 and GATA-3, which specifically suppressed MICA/B expression by directly binding to the promoter region of MICA/B. HBx protein, acting as a co-regulator, forms a tripolymer with GATA2 and GATA3, thus promotes the GATA-2 or GATA-3-mediated of MICA/B suppression. HBc protein inhibits MICA/B expression via directly binding to the CpG island in the MICA/B promoter. Thus, our study identified the novel role of transcription factors GATA-2 and GATA-3 in suppressing MICA/B expression and clarified the mechanisms of HBx and HBc in downregulation of MICA/B expression. These findings provide novel mechanisms for the contribution of HBV to hepatoma cells escape from NK cell surveillance.