Extracellular vesicles microRNA analysis in type 1 autoimmune pancreatitis: Increased expression of microRNA-21.

BACKGROUND: The molecular basis of type 1 autoimmune pancreatitis (AIP) remains unclear. Recent attention on the role of extracellular vesicles microRNA (EV miRNA) in immune homeostasis has prompted us to perform an extensive miRNA screening of serum-derived EV in AIP. METHODS: EV miRNA expression was analyzed using microarrays in AIP, chronic pancreatitis (CP), and healthy adult (HC) samples (n = 10 from each group). Differences in signals, > 3 or <1/3 times, represented significant differences in expression. Another cohort of AIP (n = 14), CP (n = 10), and HC (n = 10) samples of EV miRNA was analyzed using reverse-transcription polymerase chain reaction (RT-PCR). miRNA expression in pancreatic tissues was evaluated using in situ hybridization (ISH) in three additional subjects from each group. RESULTS: Signals of eight miRNAs (miR-659-3p, -27a-3p, -99a-5p, -21-5p, -205-5p, -100-5p, -29c-3p, and -125b-1-3p) were significantly higher, while those of two miRNAs (miR-4252 and -5004-5p) were significantly lower in AIP than in HC. EV miR-21-5p was significantly up-regulated in AIP than in HC (P = 0.035) and CP (P = 0.048). The number of miR-21-5p positive inflammatory cells was significantly elevated in AIP than in CP (P = 0.014). CONCLUSIONS: Circulating EVs exhibited altered miRNA expression patterns with elevated miR-21-5p in AIP when compared with those in HC and CP. miR-21-5p was highly expressed in pancreatic inflammatory cells in AIP. Our data suggests that miR-21-5p may be involved in the regulation of effector pathways in the pathophysiology of AIP, thus differentiating AIP from CP.

Pathogen infections and primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a multi-factorial disease caused by the interaction of both genetic predisposition and environmental triggers. Bacterial infection has been investigated most intensively, both epidemiologically and experimentally, as a prime environmental aetiology in PBC. The association of recurrent history of urinary tract infection (UTI) with PBC has been frequently confirmed by several large-scale, case-control studies, despite variation in geographic area or case-finding methods. Escherichia coli is a predominant pathogen in most cases with UTI. Animal studies and molecular mimicry analysis between the human and E. coli E2 subunit of the 2-oxo-acid dehydrogenase complexes demonstrated that E. coli infection is a key factor in breaking immunological tolerance against the mitochondria, resulting in the production of anti-mitochondrial autoantibodies (AMA), the disease-specific autoantibodies of PBC. Novosphingobium aromaticivorans, a ubiquitous xenobiotic-metabolizing bacterium, is another candidate which may be involved in the aetiology of PBC. Meanwhile, improved environmental hygiene and increased prevalence of PBC, especially in males, may argue against the aetiological role of bacterial infection in PBC. Multiple mechanisms can result in the loss of tolerance to mitochondrial autoantigens in PBC; nonetheless, bacterial infection is probably one of the dominant pathways, especially in female patients. Notably, there is a rising prevalence of male patients with PBC. With increasing exposure to environmental xenobiotics in both genders, studies directed towards identifying the environmental culprit with systematically designed case-control studies are much needed to further determine the environmental factors and role of bacterial infections in PBC.

The microbiome and autoimmunity: a paradigm from the gut-liver axis.

Microbial cells significantly outnumber human cells in the body, and the microbial flora at mucosal sites are shaped by environmental factors and, less intuitively, act on host immune responses, as demonstrated by experimental data in germ-free and gnotobiotic studies. Our understanding of this link stems from the established connection between infectious bacteria and immune tolerance breakdown, as observed in rheumatic fever triggered by Streptococci via molecular mimicry, epitope spread and bystander effects. The availability of high-throughput techniques has significantly advanced our capacity to sequence the microbiome and demonstrated variable degrees of dysbiosis in numerous autoimmune diseases, including rheumatoid arthritis, type 1 diabetes, multiple sclerosis and autoimmune liver disease. It remains unknown whether the observed differences are related to the disease pathogenesis or follow the therapeutic and inflammatory changes and are thus mere epiphenomena. In fact, there are only limited data on the molecular mechanisms linking the microbiota to autoimmunity, and microbial therapeutics is being investigated to prevent or halt autoimmune diseases. As a putative mechanism, it is of particular interest that the apoptosis of intestinal epithelial cells in response to microbial stimuli enables the presentation of self-antigens, giving rise to the differentiation of autoreactive Th17 cells and other T helper cells. This comprehensive review will illustrate the data demonstrating the crosstalk between intestinal microbiome and host innate and adaptive immunity, with an emphasis on how dysbiosis may influence systemic autoimmunity. In particular, a gut-liver axis involving the intestinal microbiome and hepatic autoimmunity is elucidated as a paradigm, considering its anatomic and physiological connections.

Cellular and Molecular Mechanisms of Autoimmune Hepatitis.

Autoimmune hepatitis is an uncommon idiopathic syndrome of immune-mediated destruction of hepatocytes, typically associated with autoantibodies. The disease etiology is incompletely understood but includes a clear association with human leukocyte antigen (HLA) variants and other non-HLA gene variants, female sex, and the environment. Pathologically, there is a CD4+ T cell-rich lymphocytic inflammatory infiltrate with variable hepatocyte necrosis and subsequent hepatic fibrosis. Attempts to understand pathogenesis are informed by several monogenetic syndromes that may include autoimmune liver injury, by several drug and environmental agents that have been identified as triggers in a minority of cases, by human studies that point toward a central role for CD4+ effector and regulatory T cells, and by animal models of the disease. Nonspecific immunosuppression is the current standard therapy. Further understanding of the disease's cellular and molecular mechanisms may assist in the design of better-targeted therapies, aid the limitation of adverse effects from therapy, and inform individualized risk assessment and prognostication.

The risk predictive values of UK-PBC and GLOBE scoring system in Chinese patients with primary biliary cholangitis: the additional effect of anti-gp210.

BACKGROUND: Adequate risk stratification is critical for the management of the patients with primary biliary cholangitis (PBC). The UK-PBC and GLOBE scoring systems for prognosis of PBC have been proposed recently, but have not been validated in Asian population. AIM: To validate the UK-PBC and GLOBE scoring systems in Chinese patients for prognosis of PBC. To clarify the role of anti-gp210 as a biomarker, and to investigate whether anti-gp210 could affect the prognostic values of UK-PBC and GLOBE scoring systems. METHODS: We retrospectively analysed 276 patients with PBC evaluated between September 2004 and May 2016, including 133 anti-gp210+ and 143 anti-gp210- patients. RESULTS: The 5-year adverse outcome-free survivals of anti-gp210+ vs. anti-gp210- patients were 70% and 85%, respectively (P = 0.005). Cirrhosis (P = 0.001), albumin level ≤40 g/L (P = 0.011) and platelet count ≤153 × 109 (P < 0.001) had a superimposition effect on anti-gp210 antibody as a risk factor. Furthermore, long-term prognoses were evaluated using the UK-PBC and GLOBE scores. For UK-PBC scoring system, the area under receiver operating characteristic curve (AUROC) was 0.924 for all patients with PBC (n = 223), 0.940 for anti-gp210+ patients (n = 110) and 0.888 for anti-gp210- patients (n = 113). For GLOBE scoring system, the area under receiver operating characteristic curve was 0.901 for all patients with PBC (n = 223), 0.924 for anti-gp210+ patients (n = 110) and 0.848 for anti-gp210- patients (n = 113). UK-PBC score >0.0578 (P < 0.001, HR: 32.736, 95% CI: 11.368-94.267) and GLOBE score <0.850 (P < 0.001, HR: 18.763, 95% CI: 7.968-44.180) were associated with poorer outcomes in the whole cohort. CONCLUSIONS: The UK-PBC and GLOBE scoring systems were good 5-year prognostic predictors in Chinese patients with PBC, especially in anti-gp210+ patients. As a biomarker, anti-gp210 antibody was associated with a more severe cholestatic manifestation and a worse long-term prognosis. The anti-gp210 antibody could be added to further optimise the UK-PBC and GLOBE scoring systems.

Endogenous interleukin-22 protects against inflammatory bowel disease but not autoimmune cholangitis in dominant negative form of transforming growth factor beta receptor type II mice.

During chronic inflammation, interleukin (IL)-22 expression is up-regulated in both CD4 and CD8 T cells, exerting a protective role in infections. However, in autoimmunity, IL-22 appears to have either a protective or a pathogenic role in a variety of murine models of autoimmunity and, by extrapolation, in humans. It is not clear whether IL-22 itself mediates inflammation or is a by-product of inflammation. We have taken advantage of the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice that develop both inflammatory bowel disease and autoimmune cholangitis and studied the role and the biological function of IL-22 by generating IL-22(-/-) dnTGF-ßRII mice. Our data suggest that the influence of IL-22 on autoimmunity is determined in part by the local microenvironment. In particular, IL-22 deficiency exacerbates tissue injury in inflammatory bowel disease, but has no influence on either the hepatocytes or cholangiocytes in the same model. These data take on particular significance in the previously defined effects of IL-17A, IL-12p40 and IL-23p19 deficiency and emphasize that, in colitis, there is a dominant role of IL-23/T helper type 17 (Th17) signalling. Furthermore, the levels of IL-22 are IL-23-dependent. The use of cytokine therapy in patients with autoimmune disease has significant potential, but must take into account the overlapping and often promiscuous effects that can theoretically exacerbate inflammation.

Serum microRNAs as novel biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

The diagnosis of primary sclerosing cholangitis (PSC) is difficult due to the lack of sensitive and specific biomarkers, as is the early diagnosis of cholangiocarcinoma (CC), a complication of PSC. The aim of this study was to identify specific serum miRNAs as diagnostic biomarkers for PSC and CC. The levels of 667 miRNAs were evaluated in 90 human serum samples (30 PSC, 30 CC and 30 control subjects) to identify disease-associated candidate miRNAs (discovery phase). The deregulated miRNAs were validated in an independent cohort of 140 samples [40 PSC, 40 CC, 20 primary biliary cirrhosis (PBC) and 40 controls]. Receiver operating characteristic (ROC) curves were established and only miRNAs with an area under the curve (AUC) > 0·70 were considered useful as biomarkers. In the discovery phase we identified the following: 21 miRNAs expressed differentially in PSC, 33 in CC and 26 in both in comparison to control subjects as well as 24 miRNAs expressed differentially between PSC and CC. After the validation phase, miR-200c was found to be expressed differentially in PSC versus controls, whereas miR-483-5p and miR-194 showed deregulated expression in CC compared with controls. We also demonstrate a difference in the expression of miR-222 and miR-483-5p in CC versus PSC. Combination of these specific miRNAs further improved the specificity and accuracy of diagnosis. This study provides a basis for the use of miRNAs as biomarkers for the diagnosis of PSC and CC.

The cumulative effects of known susceptibility variants to predict primary biliary cirrhosis risk.

Multiple genetic variants influence the risk for development of primary biliary cirrhosis (PBC). To explore the cumulative effects of known susceptibility loci on risk, we utilized a weighted genetic risk score (wGRS) to evaluate whether genetic information can predict susceptibility. The wGRS was created using 26 known susceptibility loci and investigated in 1840 UK PBC and 5164 controls. Our data indicate that the wGRS was significantly different between PBC and controls (P=1.61E-142). Moreover, we assessed predictive performance of wGRS on disease status by calculating the area under the receiver operator characteristic curve. The area under curve for the purely genetic model was 0.72 and for gender plus genetic model was 0.82, with confidence limits substantially above random predictions. The risk of PBC using logistic regression was estimated after dividing individuals into quartiles. Individuals in the highest disclosed risk group demonstrated a substantially increased risk for PBC compared with the lowest risk group (odds ratio: 9.3, P=1.91E-084). Finally, we validated our findings in an analysis of an Italian PBC cohort. Our data suggested that the wGRS, utilizing genetic variants, was significantly associated with increased risk for PBC with consistent discriminant ability. Our study is a first step toward risk prediction for PBC.

Immunological potential of cytotoxic T lymphocyte antigen 4 immunoglobulin in murine autoimmune cholangitis.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) immunoglobulin (Ig) is an important regulator of T cell activation and a fusion protein directed at CD80 and CD86; it blocks co-stimulatory signalling and T cell activation. We have taken advantage of a murine model of human primary biliary cirrhosis (PBC), mice expressing a transforming growth factor (TGF)-ß receptor II dominant negative (dnTGF-ßRII) transgene to address the potential therapeutic efficacy of CTLA-4 Ig. To mimic patients with PBC at different stages or duration of disease, we treated mice with either CTLA-4 Ig or control IgG three times weekly from 3 to 12 or 24 weeks of age, or from 12 to 24 weeks of age. CTLA-4 Ig treatment from 3 weeks of age significantly reduced liver inflammation to 12 weeks of age. Treatment initiated at 12 weeks of age also ameliorated the autoimmune cholangitis at 24 weeks of age. However, in mice treated at 3 weeks of age, suppression of liver inflammation was not sustained and colitis was aggravated when treatment was extended to 24 weeks of age. Our data indicate that, in dnTGF-ßRII mice, CTLA-4 Ig treatment has short-term beneficial effects on autoimmune cholangitis, but the effect varies according to duration of treatment and the time in which therapy was initiated. Further dissection of the events that lead to the reduction in therapeutic effectiveness of CTLA-4 Ig will be critical to determining whether such efforts can be applied to human PBC.

Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg ) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8(+) T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-ßRII) mice to recombination-activating gene (Rag)1(-/-) recipients. We then used this robust established adoptive transfer system and co-transferred CD8(+) T cells from dnTGF-ßRII mice with either C57BL/6 or dnTGF-ßRII forkhead box protein 3 (FoxP3(+) ) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF-ßRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF-ßRII Treg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in Treg from C57BL/6 compared to dnTGF-ßRII mice. Our data reflect the therapeutic potential of wild-type CD4(+) FoxP3(+) Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

Innate immunity drives xenobiotic-induced murine autoimmune cholangitis.

Although primary biliary cirrhosis (PBC) is considered a model autoimmune disease, it has not responded therapeutically to traditional immunosuppressive agents. In addition, PBC may recur following liver transplantation, despite the absence of major histocompatibility complex (MHC) matching, in sharp contrast to the well-known paradigm of MHC restriction. We have suggested previously that invariant natural killer T (iNK T) cells are critical to the initiation of PBC. In this study we have taken advantage of our ability to induce autoimmune cholangitis with 2-octynoic acid, a common component of cosmetics, conjugated to bovine serum albumin (2-OA-BSA), and studied the natural history of pathology in mice genetically deleted for CD4 or CD8 following immunization with 2-OA-BSA in the presence or absence of α-galactosylceramide (α-GalCer). In particular, we address whether autoimmune cholangitis can be induced in the absence of traditional CD4 and CD8 responses. We report herein that CD4 and CD8 knock-out mice immunized with 2-OA-BSA/PBS or 2-OA-BSA/α-GalCer develop anti-mitochondrial antibodies (AMAs), portal infiltrates and fibrosis. Indeed, our data suggest that the innate immunity is critical for immunopathology and that the pathology is exacerbated in the presence of α-GalCer. In conclusion, these data provide not only an explanation for the recurrence of PBC following liver transplantation in the absence of MHC compatibility, but also suggest that effective therapies for PBC must include blocking of both innate and adaptive pathways.

Complex regional pain syndrome: a comprehensive and critical review.

Complex regional pain syndrome (CRPS) is a term used to describe a variety of disorders characterized by spontaneous or stimulus-induced pain that is disproportional to the inciting event and accompanied by a myriad of autonomic and motor disturbances in highly variable combinations. There are no standards which can be applied to the diagnosis and would fulfill definitions of evidence-based medicine. Indeed, there are almost as many diagnostic criteria as there are names to this disorder. The umbrella term CRPS has been subdivided into type I and type II. CRPS I is intended to encompass reflex sympathetic dystrophy and similar disorders without a nerve injury; while CRPS II occurs after damage to a peripheral nerve. There are numerous etiological pathophysiological events that have been incriminated in development of CRPS, including inflammation, autoimmune responses, abnormal cytokine production, sympathetic-sensory disorders, altered blood flow and central cortical reorganization. However, the number of studies that have included appropriate controls and have sufficient numbers of patients to allow statistical analysis with appropriate power calculations is vanishingly small. This has led to over-diagnosis and often excessive pharmacotherapy and even unnecessary surgical interventions. In this review we provide a detailed critical overview of not only the history of CRPS, but also the epidemiology, the clinical features, the pathophysiological studies, the proposed criteria, the therapy and, in particular, an emphasis that future research should apply more rigorous standards to allow a better understanding of CRPS, i.e. what it is, if it is, and when it is.

Escherichia coli infection induces autoimmune cholangitis and anti-mitochondrial antibodies in non-obese diabetic (NOD).B6 (Idd10/Idd18) mice.

Several epidemiological studies have demonstrated that patients with primary biliary cirrhosis (PBC) have a higher incidence of urinary tract infections (UTI) and there is significant homology of the immunodominant mitochondrial autoantigen, the E2 component of the pyruvate dehydrogenase complex (PDC-E2), between mammals and bacteria. Previous work has demonstrated that non-obese diabetic (NOD).B6 Idd10/Idd18 infected with Novosphingobium aromaticivorans developed liver lesions similar to human PBC. It was postulated that the biliary disease was dependent upon the presence of the unique N. aro glycosphingolipids in activating natural killer T (NK T) cells. To address this issue, we infected NOD.B6 Idd10/Idd18 mice with either Escherichia coli, N. aro or use of a phosphate-buffered saline (PBS) vehicle control and serially followed animals for the appearance of liver pathology and anti-mitochondrial autoantibodies (AMA). Of striking importance, the biliary disease of E. coli-infected mice was more severe than N. Aro-infected mice and the titre of AMA was higher in E. coli-infected mice. Furthermore, the immunopathology did not correlate with the ability of bacterial extracts to produce antigen-dependent activation of NK T cells. Our data suggest that the unique glycosphingolipids of N. aro are not required for the development of autoimmune cholangitis. Importantly, the data highlight the clinical significance of E. coli infection in a genetically susceptible host, and we suggest that the appearance of autoimmune cholangitis is dependent upon molecular mimicry. These data highlight that breach of tolerance to PDC-E2 is probably the first event in the natural history of PBC in genetically susceptible hosts.

Anti-CD4 treatment inhibits autoimmunity in scurfy mice through the attenuation of co-stimulatory signals.

A major concept in autoimmunity is that disruption of Foxp3(+) regulatory T cells (Tregs) predisposes to breach of tolerance. This is exemplified by the Foxp3-linked disorder termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) which affects newborn children. There has been considerable clinical interest in the role of non-depleting anti-CD4 antibodies as a means of upregulating the function of Foxp3(+) Tregs in order to control detrimental inflammatory responses such as transplant rejection. However, according to the paradigm of a Treg-dependent mechanism of action, the effectiveness of anti-CD4 antibodies as a therapy for human autoimmune diseases is unclear considering that Treg function might be intrinsically impaired. Specifically, anti-CD4 therapy is expected to fail in patients suffering from the IPEX syndrome due to the lack of functional Foxp3(+) Tregs. Taking advantage of natural Foxp3 mutant scurfy (sf) mice closely resembling the IPEX syndrome, and genetically engineered mice depleted of Foxp3(+) Tregs, we report here that anti-CD4 treatment induces tolerance independent of Foxp3(+) Tregs. This so far undefined mechanism is dependent on the recessive non-infectious tolerization of autoreactive T cells. Treg-independent tolerance alone is powerful enough to suppress both the onset and severity of autoimmunity and reduces clinically relevant autoantibody levels and liver fibrosis. Mechanistically, tolerance induction requires the concomitant activation of autoreactive T cells and is associated with the down-regulation of the co-stimulatory TNF-receptor superfamily members OX40 and CD30 sustaining CD4(+) T cell survival. In the light of ongoing clinical trials, our results highlight an unexpected potency of anti-CD4 antibodies for the treatment of autoimmune diseases. Particularly, CD4 blockade might represent a novel therapeutic option for the human IPEX syndrome.

Anti-CD40 ligand monoclonal antibody delays the progression of murine autoimmune cholangitis.

While there have been significant advances in our understanding of the autoimmune responses and the molecular nature of the target autoantigens in primary biliary cirrhosis (PBC), unfortunately these data have yet to be translated into new therapeutic agents. We have taken advantage of a unique murine model of autoimmune cholangitis in which mice expressing a dominant negative form of transforming growth factor ß receptor II (dnTGFßRII), under the control of the CD4 promoter, develop an intense autoimmune cholangitis associated with serological features similar to human PBC. CD40-CD40 ligand (CD40L) is a major receptor-ligand pair that provides key signals between cells of the adaptive immune system, prompting us to determine the therapeutic potential of treating autoimmune cholangitis with anti-CD40L antibody (anti-CD40L; MR-1). Four-week-old dnTGFßRII mice were injected intraperitoneally with either anti-CD40L or control immunoglobulin (Ig)G at days 0, 2, 4 and 7 and then weekly until 12 or 24 weeks of age and monitored for the progress of serological and histological features of PBC, including rigorous definition of liver cellular infiltrates and cytokine production. Administration of anti-CD40L reduced liver inflammation significantly to 12 weeks of age. In addition, anti-CD40L initially lowered the levels of anti-mitochondrial autoantibodies (AMA), but these reductions were not sustained. These data indicate that anti-CD40L delays autoimmune cholangitis, but the effect wanes over time. Further dissection of the mechanisms involved, and defining the events that lead to the reduction in therapeutic effectiveness will be critical to determining whether such efforts can be applied to PBC.

Characteristics of splenic CD8+ T cell exhaustion in patients with hepatitis C.

There is increasing interest in the role of T cell exhaustion and it is well known that the natural history of chronic hepatitis C virus infection (HCV) is modulated by CD8(+) T cell immunobiology. There are many pathways that alter the presence of exhaustive T cells and, in particular, they are functionally impaired by inhibitory receptors, such as programmed death-1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3). We obtained spleen, liver and peripheral blood (before and after splenectomy) lymphoid cells from 25 patients with HCV-related cirrhosis undergoing liver transplantation for end-stage disease or splenectomy for portal hypertension. In all samples we performed an extensive phenotypic study of exhaustion markers [PD-1, Tim-3, interferon (IFN)-γ) and their ligands (PD-L1, PD-L2, galectin-9] in CD8(+) T cell subpopulations (both total and HCV-specific) and in antigen-presenting cells (APC; monocytes and dendritic cells). In the spleen, total and HCV-specific CD8(+) T cells demonstrated enhanced markers of exhaustion, predominantly in the effector memory subpopulation. Similarly, splenic APC over-expressed inhibitory receptor ligands when compared to peripheral blood. Finally, when peripheral blood CD8(+) T cells were compared before and after splenectomy, markers of exhaustion were reduced in splenic CD8(+) T cells and APC. Our data in HCV-related cirrhosis suggest that CD8(+) T cells in the spleen manifest a significantly higher exhaustion compared to peripheral blood and may thus contribute to the failure to control HCV. Counteracting this process may contribute to inducing an effective immune response to HCV.

Human intrahepatic biliary epithelial cells engulf blebs from their apoptotic peers.

The phagocytic clearance of apoptotic cells is critical for tissue homeostasis; a number of non-professional phagocytic cells, including epithelial cells, can both take up and process apoptotic bodies, including the release of anti-inflammatory mediators. These observations are particularly important in the case of human intrahepatic biliary cells (HiBEC), because such cells are themselves a target of destruction in primary biliary cirrhosis, the human autoimmune disease. To address the apoptotic ability of HiBECs, we have focused on their ability to phagocytize apoptotic blebs from autologous HiBECs. In this study we report that HiBEC cells demonstrate phagocytic function from autologous HiBEC peers accompanied by up-regulation of the chemokines CCL2 [monocyte chemotactic protein-1 (MCP-1)] and CXCL8 [interleukin (IL)-8]. In particular, HiBEC cells express the phagocytosis-related receptor phosphatidylserine receptors (PSR), implying that HiBECs function through the 'eat-me' signal phosphatidylserine expressed by apoptotic cells. Indeed, although HiBEC cells acquire antigen-presenting cell (APC) function, they do not change the expression of classic APC function surface markers after engulfment of blebs, both with and without the presence of Toll-like receptor (TLR) stimulation. These results are important not only for understanding of the normal physiological function of HiBECs, but also explain the inflammatory potential and reduced clearance of HiBEC cells following the inflammatory cascade in primary biliary cirrhosis.

Pathway-based analysis of primary biliary cirrhosis genome-wide association studies.

Genome-wide association studies (GWAS) have successfully identified several loci associated with primary biliary cirrhosis (PBC) risk. Pathway analysis complements conventional GWAS analysis. We applied the recently developed linear combination test for pathways to datasets drawn from independent PBC GWAS in Italian and Canadian subjects. Of the Kyoto Encyclopedia of Genes and Genomes and BioCarta pathways tested, 25 pathways in the Italian dataset (449 cases, 940 controls) and 26 pathways in the Canadian dataset (530 cases, 398 controls) were associated with PBC susceptibility (P<0.05). After correcting for multiple comparisons, only the eight most significant pathways in the Italian dataset had FDR <0.25 with tumor necrosis factor/stress-related signaling emerging as the top pathway (P=7.38 × 10⁻4, FDR=0.18). Two pathways, phosphatidylinositol signaling and hedgehog signaling, were replicated in both datasets (P<0.05), and subjected to two additional complementary pathway tests. Both pathway signals remained significant in the Italian dataset on modified gene set enrichment analysis (P<0.05). In both GWAS, variants nominally associated with PBC were significantly overrepresented in the phosphatidylinositol pathway (Fisher exact P<0.05). These results point to established and novel pathway-level associations with inherited predisposition to PBC that, on further independent replication and functional validation, may provide fresh insights into PBC etiology.