Chengying Yang

Intrahepatic PD-1/PD-L1 up-regulation closely correlates with inflammation and virus replication in patients with chronic HBV infection.

Chronic hepatitis B was characterized by fluctuant immune response to infected hepatocytes resulting in hepatic inflammation and virus persistence. Recently, Programmed Death-1 (PD-1) and its ligand PD-L1 have been demonstrated to play an essential role in balancing antiviral immunity and inflammation in the livers of acute hepatitis B patients, significantly influencing disease outcome. PD-1 up-regulation in peripheral T cells is associated with immune dysfunction in chronic hepatitis B patients. However, the effect of PD-1/PD-L1 on hepatic damage and chronic infective status is still unknown in patients with chronic HBV infection. Here, we report up-regulation of PD-1 and PD-L1 in liver biopsies from 32 chronic HBV patients compared to 4 healthy donors. PD-1/PD-L1 up-regulation was significantly associated with hepatic inflammation and ALT elevation. Moreover, appropriate up-regulation but not overexpression of PD-L1 in the active phase of chronic hepatitis B as well as lower expression of PD-L1 in the inactive phase in liver residential antigen presenting cells (including Kupffer cells and sinusoidal endothelial cells) may contribute to viral inhibition. Our data suggest that the intrahepatic interaction of PD-1 and PD-L1 might play an important role in balancing the immune response to HBV and immune-mediated liver damage in chronic HBV infection.

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization.

Sublytic C5b‐9 has been described as a pro‐inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen‐specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b‐9 in DC function has not been described. In this report, we use in vitro assembled functional C5b‐9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b‐9. This was demonstrated by up‐regulation of CD83, HLA‐antigens and costimulatory molecules, including CD80, D86, B7‐H1, B7‐H3, B7‐H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)‐12 and tumor necrosis factor‐α was increased while the capacity for antigen uptake (FITC‐Dextran and Lucifer Yellow) was reduced in C5b‐9‐treated DC. Mixed lymphocyte reactions indicated that C5b‐9‐activated DC acted as stimulators that significantly promoted CD4+ T cell activation and elicited production of cytokines, including interferon‐γ and IL‐2. Interestingly, C5b‐9‐treated DC also orient CD4+CD45RA+ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b‐9, suggesting that C5b‐9 bridges innate and acquired immunity by inducing DC maturation.

Programmed Death (PD)-1-Deficient Mice Are Extremely Sensitive to Murine Hepatitis Virus Strain-3 (MHV-3) Infection

The inhibitory receptor programmed death-1 (PD-1) has the capacity to maintain peripheral tolerance and limit immunopathological damage; however, its precise role in fulminant viral hepatitis (FH) has yet to be described. Here, we investigated the functional mechanisms of PD-1 as related to FH pathogenesis induced by the murine hepatitis virus strain-3 (MHV-3). High levels of PD-1-positive CD4+, CD8+ T cells, NK cells and macrophages were observed in liver, spleen, lymph node and thymus tissues following MHV-3 infection. PD-1-deficient mice exhibited significantly higher expression of the effector molecule which initiates fibrinogen deposition, fibrinogen-like protein 2 (FGL2), than did their wild-type (WT) littermates. As a result, more severe tissue damage was produced and mortality rates were higher. Fluorescence double-staining revealed that FGL2 and PD-1 were not co-expressed on the same cells, while quantitative RT-PCR demonstrated that higher levels of IFN-γ and TNF-α mRNA transcription occurred in PD-1-deficient mice in response to MHV-3 infection. Conversely, in vivo blockade of IFN-γ and TNF-α led to efficient inhibition of FGL2 expression, greatly attenuated the development of tissue lesions, and ultimately reduced mortality. Thus, the up-regulation of FGL2 in PD-1-deficient mice was determined to be mediated by IFN-γ and TNF-α. Taken together, our results suggest that PD-1 signaling plays an essential role in decreasing the immunopathological damage induced by MHV-3 and that manipulation of this signal might be a useful strategy for FH immunotherapy.

Induced B7-H1 expression on human renal tubular epithelial cells by the sublytic terminal complement complex C5b-9.

The co-inhibitory molecule B7-H1 has been broadly detectable on human inflammatory renal tubular epithelial cells (TECs) and is proposed to limit tubular damage through down-regulation of tubulointerstitial infiltration T cell activation. Nevertheless, factors that initiate B7-H1 expression on TECs remain unclarified. The terminal complement complex C5b-9, which deposits diffusely on tubules and glomerules of diseased kidneys, is now recognized as a mediator that triggers cellular activation rather than inducing cell death. Whether the up-regulation of B7-H1 on tubules is also induced by C5b-9 is uncertain. Here, after assembling functional sublytic C5b-9 on the membranes of TECs based on purified complement components, we found that B7-H1 gene transcription and protein synthesis was enhanced by C5b-9. Promoter constructs in a luciferase assay, site-directed mutagenesis and laser scanning confocal microscopy assay (LSCM) revealed that the transcription factor NF-kappaB is primarily responsible for C5b-9-mediated B7-H1 expression. To further detect the physiologic function of B7-H1, triggering B7-H1 with its agonist mAb (clone 5H1) profoundly enhanced Fas expression on C5b-9-treated TECs and thus induced TEC apoptosis. Interestingly, pretreatment of TECs with Fas blocking antibodies prevented this effect. Our results propose that C5b-9 regulates tubular pathogenesis in glomerulonephritis or other renal autoimmune diseases, possibly through enhances cell apoptosis mediated by B7-H1 signals, in addition to it directly promotes tubular damage.

Down-regulation of Z39Ig on macrophages by IFN-γ in patients with chronic HBV infection.

Co-inhibitory signals from the B7 superfamily have been demonstrated to induce T cell dysfunction in chronic HBV infection (CHB). However, the expression and function of Z39Ig, a new inhibitor of the B7 superfamily, is still unclear in CHB. Here immunohistochemical staining showed that Z39Ig was restricted to macrophages and that its level was decreased significantly in CHB patients compared to healthy controls. Moreover, reduced Z39Ig expression was positively correlated with plasma HBV load but was inversely related to serum alanine aminotransaminase levels. Further, Z39Ig mRNA had a negative relation to IFN-gamma in vivo, and IFN-gamma also down-regulated Z39Ig expression on monocyte-derived macrophages (MDMs) in a time- and dose-dependent manner in vitro. Interestingly, Z39Ig expression on MDMs was restored when IFN-gamma neutralizing antibodies were added to the T cell/MDM co-culture system, indicating that the IFN-gamma derived from activated-T cells may contribute to the reduction of Z39Ig in the CHB environment. Our results suggest that T cells can opposite T cell hyporesponsiveness through dampening Z39Ig inhibitory signals from macrophages and thus maintain their anti-viral function in CHB. Therefore, decreasing Z39Ig signals from macrophages could contribute to CHB clinical therapy.

The NLRP3 Inflammasome and IL-1β Accelerate Immunologically Mediated Pathology in Experimental Viral Fulminant Hepatitis.

Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1β expression in the serum and liver. Whereas, the viral infection in IL-1β receptor-I deficient (IL-1R1 -/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45+Gr-1high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47 phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1β secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1β induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.

Upregulated BclGL expression enhances apoptosis of peripheral blood CD4 + T lymphocytes in patients with systemic lupus erythematosus

Increased lymphocyte apoptosis has been suggested to contribute to the development of systemic lupus erythematosus (SLE), but the critical factors involved in the apoptotic pathways are still unknown. By long serial analysis of gene expression (LongSAGE) profiles and microarray analyses, a novel apoptosis-related gene BclG(L) expression was found significantly increased in peripheral blood CD4+ T cells of SLE patients, which was correlated with the enhanced CD4+ T cells apoptosis, anti-nuclear antibody (ANA) titer and proteinuria. In vitro, BclG(L) expression could be specially upregulated by SLE serum stimulation and positively correlated with induced CD4+ T cell apoptosis. Enforcing BclG(L) overexpression by lentivirus could directly enhance CD4+ T cell apoptosis, but these apoptosis-inducing effects could be partially inhibited by knockdown of BclG(L) expression. Collectively, these results indicate that increased BclG(L) expression may contribute to the aberrant CD4+ T cell apoptosis which causes an inappropriate immune response and impaired homeostasis in SLE.

VSIG4 expression on macrophages facilitates lung cancer development

Tumor-associated macrophages are a prominent component of lung cancer stroma and contribute to tumor progression. The protein V-set and Ig domain-containing 4 (VSIG4), a novel B7 family-related macrophage protein that has the capacity to inhibit T-cell activation, has a potential role in the development of lung cancer. In this study, 10 human non-small-cell lung cancer specimens were collected and immunohistochemically analyzed for VSIG4 expression. Results showed massive VSIG4+ cell infiltration throughout the samples. Immunofluorescent double staining showed that VSIG4 was present on CD68+ macrophages, but absent from CD3+ T cells, CD31+ endothelial cells, and CK-18+ epithelial cells. Moreover, VSIG4 was coexpressed on B7-H1+ and B7-H3+ cells in these tumor specimens. Transfection of the VSIG4 gene into 293FT cells demonstrated that the VSIG4 signal could inhibit cocultured CD4+ and CD8+ T-cell proliferation and cytokine (IL-2 and IFN-γ) production in vitro. Interestingly, in a murine tumor model induced by Lewis lung carcinoma cell line, we found that tumors grown in VSIG4-deficient (VSIG4−/−) mice were significantly smaller than those found in wild-type littermates. All of these results demonstrate that macrophage-associated VSIG4 is an activator that facilitates lung carcinoma development. Specific targeting of VSIG4 may prove to be a novel, efficacious strategy for the treatment of this carcinoma.

Expression of B and T lymphocyte attenuator (BTLA) in macrophages contributes to the fulminant hepatitis caused by murine hepatitis virus strain-3

Objectives Fulminant viral hepatitis (FH) remains a serious clinical problem for which the underlying pathogenesis remains unclear. The B and T lymphocyte attenuator (BTLA) is an immunoglobulin-domain-containing protein that has the capacity to maintain peripheral tolerance and limit immunopathological damage during immune responses. However, its precise role in FH has yet to be investigated. Design BTLA-deficient (BTLA−/−) mice and their wild-type littermates were infected with murine hepatitis virus strain-3 (MHV-3), and the levels of tissue damage, cell apoptosis, serum liver enzymes, fibrinogen-like protein 2 (FGL2) and cytokine production were measured and compared. Survival rate was studied after MHV-3 infection with or without adoptive transferring macrophages. Results FGL2 production, liver and spleen damage, and mortality were significantly reduced in BTLA−/− mice infected with MHV-3. This effect is due to rapid, TRAIL (TNF-related apoptosis-inducing ligand)-dependent apoptosis of MHV-3-infected macrophages in BTLA−/− mice. The early loss of macrophages resulted in reduced pathogenic tumour necrosis factor α (TNFα) and FGL2 levels and lower viral titres. The importance of TNFα in MHV-3-induced pathology was demonstrated by increased mortality in TNFα-treated MHV-3-infected BTLA−/− mice, whereas TNFα−/− mice were resistant to the infection. Moreover, adoptively transferring macrophages to BTLA−/− mice caused sensitisation, whereas blocking BTLA protected wild-type mice from virus-induced FH mortality. Conclusions BTLA promotes the pathogenesis of virus-induced FH by enhancing macrophage viability and function. Targeting BTLA may be a novel strategy for the treatment of FH.

Stimulation of B7-H1 in Hepatocarcinoma Cells by Hepatitis B virus X Antigen

The cross-talk between the hepatitis B virus X protein (HBx) and B7-H1 in hepatocarcinoma (HCC) is unclear. This study analyzed the potential relationships between HBx and B7-H1 in hepatocarcinogenesis. One of human HCC cell lines, HepG2 cells, was transfected to stably express HBx protein (HBx+-HepG2). The transcription of B7-H1 mRNA was increased significantly in these cells compared to cells transfected with control vector (HBx--HepG2), as confirmed by a comparative genome-wide microarray analysis (Capitalbio) and real time quantitative PCR (qPCR). Flow cytometry and western-blot further demonstrated that B7-H1 protein synthesis was enhanced in HBx+-HepG2 cells. Site-directed mutagenesis of promoter constructs revealed that the transcription factor (NF)-κB binding site between 128 and 137 bp upstream of B7-H1 gene transcriptional start site is primarily responsible for HBx‐mediated B7-H1 expression. Co-culture experiments with HBx+-HepG2/T cells showed that the number of apoptotic T cells increased profoundly, and this effect could be partially prevented when a neutralizing mAb against B7-H1 was added to the culture, demonstrating that B7-H1 signaling can promote T cell apoptosis. Our results suggest that the expression of B7-H1 in hepatocarcimona cells can be initiated by HBx antigen, thus inducing T cell apoptosis and finally potentially facilitates the genesis of HCC.