ning Guo

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.

Intrahepatic Expression of Programmed Death-1 and its Ligands in Patients with HBV-Related Acute-on-Chronic Liver Failure

Hepatitis B virus (HBV) infection is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of understanding of pathogenesis as well as a lack of effective treatments. Signals from the inhibitory receptor programmed death-1 (PD-1) have been demonstrated to be involved in regulating the pathogenesis of infectious diseases. However, the expression of PD-1 and its ligands in HBV-ACLF patients has yet to be evaluated. In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in liver biopsies from HBV-ACLF as well as chronic hepatitis B (CHB) patients were analyzed by immunohistochemistry. The results showed that all three molecules were observed in the HBV-ACLF samples and their levels were significantly higher than they were in CHB. Immunofluorescence double-staining showed that PD-1 was found on CD3+, CD8+ T cells, CD56+ NK cells, CD68+ macrophages, CK-18+ epithelial cells, and CD16+ monocytes. The PD-L1 expression was observed on all cell types detected and the PD-L2 was chiefly on CK-18+ epithelial cells and CD31+ endothelial cells. Interestingly, high levels of virus-induced procoagulant molecule fibrinogen-like protein 2 (FGL2) were observed in liver sections from HBV-ACLF, and PD-L1 and PD-L2 expression was also observed on FGL2+ cells in these patients. Our combined results suggest that the expression of PD-L1 and PD-L2 may be biomarkers to identify and diagnose ACLF, and a clear understanding of their functional roles should further elucidate the pathogenesis of this disease.

The characteristic expression of B7-associated proteins in Langerhans cell sarcoma.

Langerhans cell sarcoma (LCS) is a rare malignancy derived from dendritic cells of the epidermis that is characterized by cytological atypia, frequent mitoses, and aggressive clinical behavior. Cancer-associated B7 molecules including B7-H1, B7-DC, B7-H3 and B7-H4 are thought to be involved in the immunoescape of cancer cells and to function as prognostic markers. However, the expression and distribution of these molecules in LCS have not been described. Here we report that all of these molecules were observed in LCS sample sections by immunohistochemistry analysis. At the cellular level, they were found on the cell membrane and in the cytoplasm. Fluorescence dual staining indicated that B7-H1, B7-H3 and B7-H4 were principally associated with Langerin(+) tumor cells. More interestingly, B7-H1, B7-H3 and B7-H4 were co-expressed on the same tumor cells. Z39Ig, the novel B7-related protein, was also found in the LCS sample sections. Fluorescence dual staining showed that Z39Ig was restricted on CD68(+) macrophages. Our results suggest that B7-H1, B7-H3 and B7-H4 may be potential biomarkers to identify LCS, and a clear understanding of their functional roles may further elucidate the pathogenesis of this carcinoma and potentially contribute to the development of novel immunotherapeutic strategies.

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.

Intracellular B7-H4 Suppresses Bile Duct Epithelial Cell Apoptosis in Human Primary Biliary Cirrhosis

The expression and function of B7-H4, a recently identified co-inhibitory molecule of the B7 superfamily, in the pathogenesis of primary biliary cirrhosis (PBC) is still unclear. Here the expression of B7-H4 in sections from PBC patients (n = 16) was examined by immunohistochemistry and it was detected in primary bile duct epithelial cells (BECs) which were isolated from PBC patients by flow cytometry (FACs). Moreover, we also analyzed BECs-associated B7-H4 function through knock-down of its expression via RNA interference (RNAi) in vitro. Immunohistochemistry and FACs evidenced that the expression of B7-H4 was restricted in the cytoplasm of BECs from PBC patients, while it was completely absent in normal liver tissues. The cytoplasmic B7-H4 gene was cloned, and sequenced analysis showed it was encoded by the same gene to the membrane B7-H4. Interesting, silencing B7-H4 by specific RNAi resulted in enhanced FasL expression and BEC apoptosis. Conversely, interruption of Fas\FasL interaction with using FasL blocking antibodies (clone 4H9) reversed cell apoptosis. Our results suggested that the intracellular B7-H4 appears to prevent Fas/FasL-mediated BEC apoptosis during the progression of PBC, and indicates B7-H4 is a possible target for therapeutic intervention of this disease.

Expression and anatomical distribution of TIM-containing molecules in Langerhans cell sarcoma.

Signals from the T cell immunoglobulin and mucin-domain (TIM)-containing molecules have been demonstrated to be involved in regulating the progress of carcinoma. However, the expression and anatomical distribution of TIMs in Langerhans cell sarcoma (LCS), which is a rare malignancy derived from dendritic cells of the epidermis, has yet to be determined. In this study, the expression of TIM-1, TIM-3 and TIM-4 in LCS samples were detected by immunohistochemistry. Our results showed that these three molecules were found in LCS sections. At the cellular level, these molecules were found on the cell membrane and in the cytoplasm. Immunofluorescence double-staining demonstrated that these TIMs were co-expressed with Langerin, a potential biomarker for detecting LCS. In addition, TIM-1 was also expressed on CD68+ macrophages and CK-18+ epithelial cells, while TIM-3 and TIM-4 were expressed on all cell types investigated, including CD3+T cells, CD68+ macrophages, CD11c+ dendritic cells, CD16+ NK Cells, CD31+ endothelial cells and CK-18+ epithelial cells. Interestingly, TIMs were also co-expressed with some members of the B7 superfamily, including B7-H1, B7-H3 and B7-H4 on sarcoma cells. Our results clearly showed the characteristic expression and anatomical distribution of TIMs in LCS, and a clear understanding of their functional roles may further elucidate the pathogenesis of this carcinoma and potentially contribute to the development of novel immunotherapeutic strategies.

The Expression and Anatomical Distribution of BTLA and Its Ligand HVEM in Rheumatoid Synovium

Co-inhibitory signaling from B and T lymphocyte attenuator (BTLA) can suppress lymphocyte activation and maintain peripheral tolerance. However, the expression and anatomical distribution of BTLA and its ligand, herpesvirus entry mediator (HVEM), in rheumatoid arthritis (RA) synovium have not been reported. In this study, we analyzed the expression of HVEM and BTLA in RA synovium by immunohistochemistry, and our results showed that both factors were observed in all four cases of RA samples. At the cellular level, both HVEM and BTLA were found on the cell membrane and in the cytoplasm. Fluorescence dual staining demonstrated that HVEM was chiefly on CD3+ T cells, CD68+ macrophages, and to a lesser extent was found on CD31+ endothelial cells. Similarly, the expression of BTLA was observed on infiltrated CD3+ T cells and CD68+ macrophages. The co-expression of HVEM and BTLA with some members of the B7 family in these sections was also analyzed, and the results showed that HVEM antigen was also found on B7-H3+ capillaries, while it was absent on B7-H1+, B7-DC+, B7-H4+, and Z39Ig+ cells. Interestingly, BTLA was observed on B7-H1+, B7-H4+, and HVEM+ cells in the synovium. The characteristic expression and distribution of BTLA/HVEM in the synovium indicated that their signaling probably affects the pathogenesis of RA, and a clear understanding of their functional roles may further elucidate the pathogenesis of this disease.

The Intrahepatic Expression and Distribution of BTLA and its Ligand HVEM in patients with HBV-related acute-on-chronic liver failure

ObjectiveIt has been demonstrated that signals from the inhibitory receptor B and T lymphocyte attenuator (BTLA) are involved in regulating the pathogenesis of infectious diseases. However, the expression and anatomical distribution of BTLA and its ligand, the herpes virus entry mediator (HVEM), have not yet been determined in cases of HBV-related acute-on-chronic liver failure (HBV-ACLF) patients.MethodsIn this study, the expression of BTLA and HVEM in liver tissues from HBV-ACLF, chronic hepatitis B (CHB) patients and healthy individuals was analyzed by immunohistochemistry.ResultsThe results of this analysis demonstrated that both molecules were observed in the HBV-ACLF samples and that their expression was chiefly in the infiltrating inflammatory cells and the damaged bile ducts. However, they were absent in liver sections from CHB patients and healthy controls. Immunofluorescence double-staining indicated that BTLA was found on CK-18+ epithelial cells, CD31+ endothelial cells, CD68+ macrophages, CD56+ NK cells, CD16+ monocytes, CD3+ , CD8+ T cells, and Foxp3+ regulatory T cells (Treg). By contrast, HVEM expression was restricted to CK18+ epithelial cells and CD68+ macrophages. Moreover, the expression of several members of the B7 superfamily, including PD-L1, PD-L2, B7-H3 and B7-H4, was also detected in these liver tissues, and these proteins were co-expressed with HVEM. Interestingly, the expression of fibrinogen-like protein 2 (FGL2), a virus-induced procoagulant molecule, was also found in liver sections from HBV-ACLF, this molecule also co-expresses with BTLA and HVEM.ConclusionsThese results suggest that BTLA-HVEM signaling is likely to affect the pathogenesis of HBV-ACLF, a clear understanding of the functional roles of these proteins should further elucidate the disease process.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/8080806838149123

The development of endometrial hyperplasia in aged PD-1-deficient female mice.

BackgroundProgrammed death-1 (PD-1, Pdcd1)-deficient mice develop different types of autoimmune diseases depending on the mouse strain but its role in uterus development has not been reported.MethodsIn this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in uterine tissues from aged WT mice in a 129svEv-Brd background was analyzed by immunohistochemistry and the uterine morphology between WT and PD-1-/- mice was compared by hematoxylin and eosin staining.ResultsThe aged PD-1-/- female mice in a 129svEv-Brd rather than Balb/c background develop endometrial hyperplasia. H&E staining showed an increase in the number of glands, neovascularization and an extremely large luminal cavity in aged PD-1-/- uteri. Immunohistochemical assay showed that the expression of PD-1 was observed in glandular/luminal epithelium and cells infiltrating the stroma. Fluorescent double staining demonstrated that PD-1 expresses on CD68+ macrophages, CD3+ T cells, CD16+ monocytes, CD56+ NK cells and CK-18+ epithelial cells, respectively. Additionally, PD-1 co-expresses with vascular endothelial growth factor (VEGF), and PD-1 deficiency resulted in an accumulation of glandular/luminal epithelium derived VEGF, which accelerates the expression of the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), and thus potentially lead to epithelial proliferation in aged PD-1-/- uteri.ConclusionsThese findings showed that PD-1 deficiency augments luminal epithelial cell proliferation probably through induced VEGF secretion, suggesting PD-1 plays an important role in controlling the growth and differentiation of the uterine epithelium.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5809067461223905