ABSTRACT
Primary sclerosing cholangitis (PSC) is an (auto)immune-mediated cholestatic liver disease with a yet unclear etiology. Increasing evidence points to an involvement of neutrophils in chronic liver inflammation and cirrhosis but also liver repair. Here, we investigate the role of the neutrophil extracellular trap (NET) component myeloperoxidase (MPO) and the therapeutic potential of DNase I and of neutrophil elastase (NE) inhibitor GW311616A on disease outcome in the multidrug resistance 2 knockout (Mdr2-/-) mouse, a PSC animal model. Initially, we observed the recruitment of MPO expressing cells and the formation of NETs in liver biopsies of PSC patients and in Mdr2-/- livers. Furthermore, sera of Mdr2-/- mice contained perinuclear anti-neutrophil cytoplasmic antibody (p-ANCA)-like reactivity similar to PSC patient sera. Also, hepatic NE activity was significantly higher in Mdr2-/- mice than in wild type littermates. Flow cytometry analyses revealed that during disease development a highly active neutrophil subpopulation established specifically in the liver of Mdr2-/- mice. However, absence of their MPO activity, as in MPO-deficient Mdr2-/- mice, showed no effect on hepatobiliary disease severity. In contrast, clearance of extracellular DNA by DNase I reduced the frequency of liver-resident neutrophils, plasmacytoid dendritic cells (pDCs) and CD103+ conventional DCs and decreased cholangiocyte injury. Combination of DNase I with a pDC-depleting antibody was additionally hepatocyte-protective. Most importantly, GW311616A, an orally bioavailable inhibitor of human NE, attenuated hepatobiliary injury in a TNFα-dependent manner and damped hyperproliferation of biliary epithelial cells. Further, hepatic immigration and activity of CD11b+ DCs as well as the secretion of IFNγ by hepatic CD4 and CD8 T cells were reduced. Our findings delineate neutrophils as important participants in the immune cell crosstalk that drives cholestatic liver disease and identify NET components as potential therapeutic targets.
Subject(s)
ATP-Binding Cassette Sub-Family B Member 4 , Cholangitis, Sclerosing , Disease Models, Animal , Extracellular Traps , Mice, Knockout , Neutrophils , Animals , Extracellular Traps/immunology , Extracellular Traps/metabolism , Mice , Humans , Cholangitis, Sclerosing/immunology , Neutrophils/immunology , Neutrophils/metabolism , Cholestasis/immunology , Cholestasis/metabolism , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/deficiency , ATP Binding Cassette Transporter, Subfamily B/metabolism , Liver/pathology , Liver/immunology , Liver/metabolism , Peroxidase/metabolism , Peroxidase/immunology , Deoxyribonuclease I/metabolism , Leukocyte Elastase/metabolism , Leukocyte Elastase/antagonists & inhibitors , Male , FemaleABSTRACT
MHC class I (MHC I) expression in the host influences NK cells in a process termed education. The result of this education is reflected in the responsiveness of NK cells at the level of individual cells as well as in the repertoire of inhibitory MHC I-specific receptors at the NK cell system level. The presence of MHC I molecules in the host environment gives rise to a skewed receptor repertoire in spleen NK cells where subsets expressing few (one or two) inhibitory receptors are expanded whereas subsets with many (three or more) receptors are contracted. It is not known whether this MHC I-dependent skewing is imposed during development or after maturation of NK cells. In this study, we tested the hypothesis that the NK cell receptor repertoire is shaped already early during NK cell development in the bone marrow. We used mice with a repertoire imposed by a single MHC I allele, as well as a C57BL/6 mutant strain with exaggerated repertoire skewing, to investigate Ly49 receptor repertoires at different stages of NK cell differentiation. Our results show that NK cell inhibitory receptor repertoire skewing can indeed be observed in the bone marrow, even during the earliest developmental steps where Ly49 receptors are expressed. This may partly be accounted for by selective proliferation of certain NK cell subsets, but other mechanisms must also be involved. We propose a model for how repertoire skewing is established during a developmental phase in the bone marrow, based on sequential receptor expression as well as selective proliferation.
Subject(s)
Bone Marrow , NK Cell Lectin-Like Receptor Subfamily A , Animals , Antigens, Ly/metabolism , Bone Marrow/metabolism , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/metabolism , Killer Cells, Natural , Lectins, C-Type/metabolism , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A/genetics , NK Cell Lectin-Like Receptor Subfamily A/metabolism , Receptors, NK Cell Lectin-Like/metabolism , Receptors, Natural Killer Cell/metabolismABSTRACT
Systemic lupus erythematosus (SLE) is a multi-organ inflammatory disease with kidney inflammation, lupus nephritis (LN), being one of the most severe manifestations. Immune complex deposits, particularly in glomeruli, and T cells, B cells, and myeloid cells, mainly with extraglomerular localization, contribute to the inflammatory process. Natural killer (NK) cells have been suggested to play a role in autoimmune diseases, but have not been investigated in detail in renal lupus before. In this exploratory study, we performed the first characterization of NK cell number and distribution in LN kidney biopsies. Twelve SLE patients were analyzed in the active phase of disease and five patients following immunosuppressive therapy. CD56+ cells, corresponding to NK cells or NK-like T-cells, were identified in all patients; however, with reduced numbers in four out of five patients at follow-up. Furthermore, cells were present in the kidney interstitium and peri-glomerular areas, but only rarely in glomeruli. Fluorescent co-staining of CD56 or NKp46 and CD3 revealed the presence of both CD56+/NKp46+CD3-NK cells and CD56+/NKp46+CD3+NK-like T-cells. Compared to healthy kidney sections, one out of four LN patients showed increased numbers of NK cells. A correlation between CD56+ and NK cells with clinical parameters could not be observed, perhaps due to the small patient cohort. In conclusion, we have identified NK cells and NK-like T-cells in the LN kidney and performed the first detailed analysis of their localization during active and inactive diseases. Their role in LN pathogenesis is, however, unclear and deserves further studies.
Subject(s)
Lupus Erythematosus, Systemic , Lupus Nephritis , CD56 Antigen , Humans , Kidney/pathology , Killer Cells, Natural , Lupus Nephritis/drug therapy , Lupus Nephritis/pathologyABSTRACT
Anti-neutrophil cytoplasmic antibody (ANCA)- associated vasculitis (AAV) is a group of systemic autoimmune diseases characterized by inflammation of small- and medium-sized vessels. The three main types of AAV are granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA). A growing number of studies focus on natural killer (NK) cells in AAV. NK cells are innate lymphoid cells with important roles in anti-viral and anti-tumor defense, but their roles in the pathogenesis of autoimmunity is less well established. In this review, we will present a summary of what is known about the number, phenotype and function of NK cells in patients with AAV. We review the literature on NK cells in the circulation of AAV patients, studies on tissue resident NK cells and how the treatment affects NK cells.
Subject(s)
Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/etiology , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/metabolism , Antibodies, Antineutrophil Cytoplasmic/immunology , Autoimmunity , Killer Cells, Natural/immunology , Killer Cells, Natural/metabolism , Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/diagnosis , Antibody-Dependent Cell Cytotoxicity/immunology , Autoimmune Diseases/etiology , Autoimmune Diseases/metabolism , Autoimmune Diseases/pathology , Biomarkers , Cytokines/metabolism , Diagnosis, Differential , Disease Susceptibility , Humans , Organ Specificity/immunologyABSTRACT
BACKGROUND & AIMS: Four major autoimmune diseases target the liver. They develop because of bile duct destruction, leading to chronic cholestasis or result from hepatocyte damage like autoimmune hepatitis (AIH). Interestingly, some patients simultaneously show features of both cholangitis and AIH. Our goal was to mimic such concurrent characteristics in a mouse model that would help deciphering mechanisms possibly involved in an inflammatory crosstalk between cholestatic disease and hepatitis. METHODS: Mdr2-/- mice, which spontaneously develop sclerosing cholangitis because of accumulation of toxic bile salts, were infected with adenovirus (Ad) encoding human Cytochrome P4502D6 (hCYP2D6), the major target autoantigen in type-2 AIH, to trigger hepatocyte injury. Wild type FVB mice were controls. RESULTS: Resulting Ad-Mdr2-/- mice presented with cholangitis, fibrosis and cellular infiltrations that were higher than in Mdr2-/- or Ad-FVB mice. Increased levels of anti-neutrophil cytoplasmic antibodies but similar anti-hCYP2D6 antibody titres were detected in Ad-Mdr2-/- compared to Mdr2-/- and Ad-FVB mice respectively. IFNγ-expressing hCYP2D6-specific CD4 T cells declined, whereas hCYP2D6-specific CD8 T cells increased in Ad-Mdr2-/- compared to Ad-FVB mice. The overall T cell balance in Ad-Mdr2-/- mice was a combination of a type 17 T cell response typically found in Mdr2-/- mice with a type 1 dominated T cell response characteristic for Ad-FVB mice. Simultaneously, the type 2 T cell compartment was markedly reduced. CONCLUSIONS: Experimental hepatitis induction in a mouse with sclerosing cholangitis results in a disorder which represents not simply the sum of the individual characteristics but depicts a more complex entity which urges on further analysis.
Subject(s)
Cholangitis, Sclerosing/complications , Hepatitis, Autoimmune/complications , Liver/pathology , Adenoviridae , Animals , Cholangitis, Sclerosing/pathology , Cytochrome P-450 CYP2D6/immunology , Disease Models, Animal , Female , Hepatitis, Autoimmune/pathology , Hepatocytes/immunology , Mice , T-Lymphocytes/physiologyABSTRACT
Fibrosis remains a serious health concern in patients with chronic liver disease. We recently reported that chemically induced chronic murine liver injury triggers increased expression of junctional adhesion molecules (JAMs) JAM-B and JAM-C by endothelial cells and de novo synthesis of JAM-C by hepatic stellate cells (HSCs). Here, we demonstrate that biopsies of patients suffering from primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) or autoimmune hepatitis (AIH) display elevated levels of JAM-C on portal fibroblasts (PFs), HSCs, endothelial cells and cholangiocytes, whereas smooth muscle cells expressed JAM-C constitutively. Therefore, localization and function of JAM-B and JAM-C were investigated in three mouse models of autoimmune-driven liver inflammation. A PBC-like disease was induced by immunization with 2-octynoic acid-BSA conjugate, which resulted in the upregulation of both JAMs in fibrotic portal triads. Analysis of a murine model of PSC revealed a role of JAM-C in PF cell-cell adhesion and contractility. In mice suffering from AIH, endothelial cells increased JAM-B level and HSCs and capsular fibroblasts became JAM-C-positive. Most importantly, AIH-mediated liver fibrosis was reduced in JAM-B-/- mice or when JAM-C was blocked by soluble recombinant JAM-C. Interestingly, loss of JAM-B/JAM-C function had no effect on leukocyte infiltration, suggesting that the well-documented function of JAMs in leukocyte recruitment to inflamed tissue was not effective in the tested chronic models. This might be different in patients and may even be complicated by the fact that human leukocytes express JAM-C. Our findings delineate JAM-C as a mediator of myofibroblast-operated contraction of the liver capsule, intrahepatic vasoconstriction and bile duct stricture. Due to its potential to interact heterophilically with endothelial JAM-B, JAM-C supports also HSC/PF mural cell function. Together, these properties allow JAM-B and JAM-C to actively participate in vascular remodeling associated with liver/biliary fibrosis and suggest them as valuable targets for anti-fibrosis therapies.
Subject(s)
Cell Adhesion Molecules/metabolism , Cholangitis, Sclerosing/metabolism , Endothelial Cells/metabolism , Hepatitis, Autoimmune/metabolism , Immunoglobulins/metabolism , Inflammation/metabolism , Liver Cirrhosis, Biliary/metabolism , Liver/pathology , Myocytes, Smooth Muscle/metabolism , Myofibroblasts/metabolism , Animals , Cell Adhesion , Cell Adhesion Molecules/genetics , Cells, Cultured , Disease Models, Animal , Fatty Acids, Monounsaturated/immunology , Female , Fibrosis , Humans , Immunoglobulins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Vascular Remodeling , VasoconstrictionABSTRACT
Ceramides are known to be key players in intracellular signaling and are involved in apoptosis, cell senescence, proliferation, cell growth and differentiation. They are synthesized by ceramide synthases (CerS). So far, six different mammalian CerS (CerS1-6) have been described. Recently, we demonstrated that human breast cancer tissue displays increased activity of CerS2, 4, and 6, together with enhanced generation of their products, ceramides C(16:0), C(24:0), and C(24:1). Moreover, these increases were significantly associated with tumor dignity. To clarify the impact of this observation, we manipulated cellular ceramide levels by overexpressing ceramide synthases 2, 4 or 6 in MCF-7 (breast cancer) and HCT-116 (colon cancer) cells, respectively. Overexpression of ceramide synthases 4 and 6 elevated generation of short chain ceramides C(16:0), C(18:0) and C(20:0), while overexpression of ceramide synthase 2 had no effect on ceramide production in vivo, presumably due to limited substrate availability, because external addition of very long chain acyl-CoAs resulted in a significant upregulation of very long chain ceramides. We also demonstrated that upregulation of CerS4 and 6 led to the inhibition of cell proliferation and induction of apoptosis, whereas upregulation of CerS2 increased cell proliferation. On the basis of our data, we propose that a disequilibrium between ceramides of various chain length is crucial for cancer progression, while normal cells require an equilibrium between very long and long chain ceramides for normal physiology.
Subject(s)
Breast Neoplasms/pathology , Ceramides/chemistry , Ceramides/metabolism , Colonic Neoplasms/pathology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Cell Survival , Ceramides/biosynthesis , Gene Expression Regulation, Neoplastic , Humans , Intracellular Space/metabolism , Oxidoreductases/genetics , Oxidoreductases/metabolism , TransfectionABSTRACT
Ceramide synthases (CerSs) are key enzymes in the biosynthesis of ceramides and display a group of at least six different isoenzymes (CerS1-6). Ceramides itself are bioactive molecules. Ceramides with different N-acyl side chains (C(14:0)-Cer - C(26:0)-Cer) possess distinct roles in cell signaling. Therefore, the selective inhibition of specific CerSs which are responsible for the formation of a specific ceramide holds promise for a number of new clinical treatment strategies, e.g., cancer. Here, we identified four of hitherto unknown functional inhibitors of CerSs derived from the FTY720 (Fingolimod) lead structure and showed their inhibitory effectiveness by two in vitro CerS activity assays. Additionally, we tested the substances in two cell lines (HCT-116 and HeLa) with different ceramide patterns. In summary, the in vitro activity assays revealed out that ST1058 and ST1074 preferentially inhibit CerS2 and CerS4, while ST1072 inhibits most potently CerS4 and CerS6. Importantly, ST1060 inhibits predominately CerS2. First structure-activity relationships and the potential biological impact of these compounds are discussed.