Cyr61 Alleviates Cholangitis by Inhibiting Cytotoxic Effects of CD8+ T Cells on Biliary Epithelial Cells.

OBJECTIVE: Primary biliary cholangitis (PBC) is a chronic progressive cholestatic liver disease. In recent years, researchers have found that cysteine-rich angiogenic inducer 61 (Cyr61, also known as CCN1) has a potential role in reducing portal inflammation in patients with PBC. This study aimed to explore the relationship between Cyr61 and PBC to provide new ideas and an experimental basis for the clinical treatment of PBC. METHODS: After induction of the overexpression of Cyr61 in a mouse model of PBC using recombinant adenovirus, hematoxylin and eosin staining and pathological scores were used to indicate intrahepatic inflammation and bile duct damage. Real-time PCR was used to detect changes in inflammation-related cytokines in the liver. To further study the mechanism, we assessed whether Cyr61 protects bile duct epithelial cells from cytotoxic effects. RESULTS: Serum and hepatic Cyr61 levels were increased in the murine model of PBC. Overexpression of Cyr61 alleviated hepatic inflammation and bile duct injury in vivo. Cyr61 inhibited the cytotoxic effects of CD8+ T cells by acting on biliary epithelial cells (BECs) in vitro. CONCLUSION: Our results provide novel insight into the pathogenesis of PBC and suggest that Cyr61 plays a dominant role in the cytotoxic effects on BECs in PBC. Consequently, therapeutic strategies targeting Cyr61 could be a potent therapy for PBC.

Mast Cells Regulate Ductular Reaction and Intestinal Inflammation in Cholestasis Through Farnesoid X Receptor Signaling.

BACKGROUND AND AIMS: Cholestasis is characterized by increased total bile acid (TBA) levels, which are regulated by farnesoid X receptor (FXR)/FGF15. Patients with primary sclerosing cholangitis (PSC) typically present with inflammatory bowel disease (IBD). Mast cells (MCs) (i) express FXR and (ii) infiltrate the liver during cholestasis promoting liver fibrosis. In bile-duct-ligated (BDL) MC-deficient mice (B6.Cg-KitW-sh /HNihrJaeBsmJ [KitW-sh ]), ductular reaction (DR) and liver fibrosis decrease compared with BDL wild type, and MC injection exacerbates liver damage in normal mice. APPROACH AND RESULTS: In this study, we demonstrated that MC-FXR regulates biliary FXR/FGF15, DR, and hepatic fibrosis and alters intestinal FXR/FGF15. We found increased MC number and biliary FXR expression in patients with liver injury compared with control. Histamine and FGF19 serum levels and small heterodimer partner expression increase in patients PSC and PSC-IBD compared with healthy controls. MC injection increased liver damage, DR, inflammation, biliary senescence/senescence-associated secretory phenotype (SASP), fibrosis, and histamine in KitW-sh mice. Inhibition of MC-FXR before injection reduced these parameters. BDL and KitW-sh mice injected with MCs displayed increased TBA content, biliary FXR/FGF15, and intestinal inflammation, which decreased in BDL KitW-sh and KitW-sh mice injected with MC-FXR. MCs increased ileal FXR/FGF15 expression in KitW-sh mice that was reduced following FXR inhibition. BDL and multidrug resistance 2/ATP-binding cassette family 2 member 4 knockout (Mdr2-/- ) mice, models of PSC, displayed increased intestinal MC infiltration and FXR/FGF15 expression. These were reduced following MC stabilization with cromolyn sodium in Mdr2-/- mice. In vitro, MC-FXR inhibition decreased biliary proliferation/SASP/FGF and hepatic stellate cell activation. CONCLUSIONS: Our studies demonstrate that MC-FXR plays a key role in liver damage and DR, including TBA regulation through alteration of intestinal and biliary FXR/FGF15 signaling.

Cholangiopathy After Severe COVID-19: Clinical Features and Prognostic Implications.

INTRODUCTION: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 virus, is a predominantly respiratory tract infection with the capacity to affect multiple organ systems. Abnormal liver tests, mainly transaminase elevations, have been reported in hospitalized patients. We describe a syndrome of cholangiopathy in patients recovering from severe COVID-19 characterized by marked elevation in serum alkaline phosphatase (ALP) accompanied by evidence of bile duct injury on imaging. METHODS: We conducted a retrospective study of COVID-19 patients admitted to our institution from March 1, 2020, to August 15, 2020, on whom the hepatology service was consulted for abnormal liver tests. Bile duct injury was identified by abnormal liver tests with serum ALP > 3x upper limit of normal and abnormal findings on magnetic resonance cholangiopacreatography. Clinical, laboratory, radiological, and histological findings were recorded in a Research Electronic Data Capture database. RESULTS: Twelve patients were identified, 11 men and 1 woman, with a mean age of 58 years. Mean time from COVID-19 diagnosis to diagnosis of cholangiopathy was 118 days. Peak median serum alanine aminotransferase was 661 U/L and peak median serum ALP was 1855 U/L. Marked elevations of erythrocyte sedimentation rate, C-reactive protein, and D-dimers were common. Magnetic resonance cholangiopacreatography findings included beading of intrahepatic ducts (11/12, 92%), bile duct wall thickening with enhancement (7/12, 58%), and peribiliary diffusion high signal (10/12, 83%). Liver biopsy in 4 patients showed acute and/or chronic large duct obstruction without clear bile duct loss. Progressive biliary tract damage has been demonstrated radiographically. Five patients were referred for consideration of liver transplantation after experiencing persistent jaundice, hepatic insufficiency, and/or recurrent bacterial cholangitis. One patient underwent successful living donor liver transplantation. DISCUSSION: Cholangiopathy is a late complication of severe COVID-19 with the potential for progressive biliary injury and liver failure. Further studies are required to understand pathogenesis, natural history, and therapeutic interventions.

Atezolizumab-induced Sclerosing Cholangitis in a patient with lung cancer: A case report.

Atezolizumab is an immune checkpoint inhibitor that is a key drug in non-small-cell lung cancer treatment. However, it can cause immune-related adverse events, including liver injury. Several patterns of liver injury associated with immune checkpoint inhibitor therapy have been reported; however, not much is known about sclerosing cholangitis. We present here a case of lung adenocarcinoma with atezolizumab-induced secondary sclerosing cholangitis diagnosed using needle biopsy of the liver. A 77-year-old woman with lung adenocarcinoma, cT3N2M0, stage IIIA, was treated with concurrent chemoradiotherapy involving carboplatin and paclitaxel, which markedly reduced the tumor diameter. However, 5 months later, the lesion regrew, and she underwent 39 cycles of pemetrexed monotherapy. As pulmonary metastasis progressed, she was treated with atezolizumab. After 13 cycles of atezolizumab therapy, she complained of nausea. Laboratory tests showed elevated levels of the biliary tract and hepatic enzymes. Nevertheless, abdominal computed tomography and ultrasonography revealed no underlying related cause. Ultrasound-guided needle biopsy of the liver was performed, and histopathological analysis of biopsy samples showed features of sclerosing cholangitis. Further examinations were performed, and a diagnosis of atezolizumab-induced secondary sclerosing cholangitis without strictures and dilatations of the large bile ducts was established. Prednisolone was administered orally, after which the biliary tract and hepatic enzyme levels improved immediately. In patients presenting with a hepatic injury during immune checkpoint inhibitor therapy, clinicians should be aware of the possibility of immune checkpoint inhibitor-induced sclerosing cholangitis, even if the large bile ducts have no strictures and dilatations.

IL-17A/F enable cholangiocytes to restrict T cell-driven experimental cholangitis by upregulating PD-L1 expression.

BACKGROUND & AIMS: IL-17A-producing T cells are present in autoimmune cholestatic liver diseases; however, little is known about the contribution of IL-17 to periductal immune responses. Herein, we investigated the role of IL-17 produced by antigen-specific CD8+ T cells in a mouse model of cholangitis and in vitro in human cholangiocyte organoids. METHODS: K14-OVAp mice express a major histocompatibility complex I-restricted ovalbumin (OVA) peptide sequence (SIINFEKL) on cholangiocytes. Cholangitis was induced by the adoptive transfer of transgenic OVA-specific ovalbumin transgene (OT)-1 CD8+ T cells that either had OT-1wt or lacked IL-17A/F (OT-1IL17ko). The response of mouse and human cholangiocytes/organoids to IL-17A was assessed in vitro. RESULTS: Transfer of OVA-specific OT-1IL17ko cells significantly aggravated periductal inflammation in K14-OVAp recipient mice compared with transfer of OT-1wt T cells. OT-1IL17ko T cells were highly activated in the liver and displayed increased cytotoxicity and proliferation. IL-17A/F produced by transferred OT-1wt CD8+ T cells induced upregulation of the inhibitory molecule programmed cell death ligand 1 (PD-L1) on cholangiocytes, restricting cholangitis by limiting cytotoxicity and proliferation of transferred cells. In contrast, OT-1IL17ko T cells failed to induce PD-L1 on cholangiocytes, resulting in uncontrolled expansion of cytotoxic CD8+ T cells and aggravated cholangitis. Blockade of PD-L1 after transfer of OT-1wt T cells with anti-PD-L1 antibody also resulted in aggravated cholangitis. Using human cholangiocyte organoids, we were able to confirm that IL-17A induces PD-L1 expression in cholangiocytes. CONCLUSIONS: We demonstrate that by upregulating PD-L1 on cholangiocytes, IL-17 has an important role in restricting cholangitis and protecting against CD8+ T cell-mediated inflammatory bile duct injury. Caution should be exercised when targeting IL-17 for the treatment of cholangitis. LAY SUMMARY: IL-17 is assumed to be a driver of inflammation in several autoimmune diseases, such as psoriasis. IL-17 is also present in inflammatory diseases of the bile duct, but its role in these conditions is not clear, as the effects of IL-17 depend on the context of its expression. Herein, we investigated the role of IL-17 in an experimental autoimmune cholangitis mouse model, and we identified an important protective effect of IL-17 on cholangiocytes, enabling them to downregulate bile duct inflammation via checkpoint inhibitor PD-L1.

Inflammation Drives MicroRNAs to Limit Hepatocyte Bile Acid Transport in Murine Biliary Atresia.

BACKGROUND: Biliary atresia (BA) is an inflammatory pediatric cholangiopathy with only surgical means for treatment. Many contributors to bile acid synthesis and transport have previously been reported to be downregulated in patients with BA; yet, the driving factors of the abnormal bile acid synthesis and transport in regard to BA have not been previously studied. MATERIALS AND METHODS: Wild type or Ig-α-/- mice were injected with salt solution (control) or rotavirus on day of life 0, and analyses were performed on day of life 14. The mRNA levels of bile acid transporters/nuclear receptors and liver microRNAs (miRNAs) were compared between groups. A mouse hepatocyte cell line was used to examine the effects of innate cytokines on miRNA levels and bile acid transporter/nuclear receptor expression and miRNAs on bile acid transporter/nuclear receptor expression. RESULTS: BA mice had significantly increased mRNA expression of innate cytokines and miRNAs known to bind bile acid transporters/nuclear receptors (miRNAs -22-5p, -34a-5p, and -222-3p) and decreased mRNA expression of bile acid transporters and nuclear receptors. In vitro, TNF-α and IL-1ß decreased BSEP and CYP7A1 while increasing miRNA-34a-5p and miRNA 222-3p. LXR, SHP, CYP7A1, NTCP, and MRP2 were decreased by miRNA-34a-5p, whereas miRNA-222-3p decreased NTCP and MRP4. TNF-α and IL-1ß increased expression of miRNAs 34a-5p and 222-3p and these miRNAs then decrease expression of multiple bile acid transporters and nuclear receptors. CONCLUSIONS: Loss of bile acid transporters increases hepatotoxicity via bile acid retention. Therapeutic agents that increase bile acid transport or nuclear receptor functioning should be investigated in BA.

Association of Chronic Opisthorchis Infestation and Microbiota Alteration on Tumorigenesis in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a common hepatobiliary cancer in East and Southeast Asia. The data of microbiota contribution in CCA are still unclear. Current available reports have demonstrated that an Opisthorchis viverrini (OV) infection leads to dysbiosis in the bile duct. An increase in the commensal bacteria Helicobacter spp. in OV-infected CCA patients is associated with bile duct inflammation, severity of bile duct fibrosis, and cholangiocyte proliferation. In addition, secondary bile acids, major microbial metabolites, can mediate cholangiocyte inflammation and proliferation in the liver. A range of samples from CCA patients (stool, bile, and tumor) showed different degrees of dysbiosis. The evidence from these samples suggests that OV infection is associated with alterations in microbiota and could potentially have a role in CCA. In this comprehensive review, reports from in vitro, in vivo, and clinical studies that demonstrate possible links between OV infection, microbiota, and CCA pathogenesis are summarized and discussed. Understanding these associations may pave ways for novel potential adjunct intervention in gut microbiota in CCA patients.

Increased p16INK4a-expressing senescent bile ductular cells are associated with inadequate response to ursodeoxycholic acid in primary biliary cholangitis.

BACKGROUND & AIMS: Senescent biliary epithelial cells (BECs) may be involved in the pathophysiology of primary biliary cholangitis (PBC) by secreting senescence-associated secretory phenotypes. We examined an association of the extent of cellular senescence in BECs with clinicopathological features including response to ursodeoxycholic acid (UDCA) and a possibility of senolytic therapy in PBC. METHODS: The expression of senescent markers (p21WAF1/Cip1, p16INK4a) and B-cell lymphoma-extra large (Bcl-xL), a key regulator of senescent cell anti-apoptotic pathway, was immunohistochemically examined in livers from patients with PBC (n = 145) and 103 control livers. Senolytic effect of Bcl-xL inhibitors (A-1331852 and Navitoclax) was examined in senescent murine BECs. RESULTS: Senescent BECs were increased in small bile ducts in PBC, compared with control livers (p < 0.01). Senescent BECs were increased in ductular reactions in PBC, stage 3-4, compared with PBC, stage 1-2 and control livers (p < 0.01). The extent of senescent BECs in bile ductules was significantly correlated with stage and hepatitis activity (p < 0.01) and the expression of p16INK4a in bile ductules was significantly correlated to inadequate response to UDCA in PBC (p < 0.01). Double immunofluorescence revealed an increased expression of Bcl-xL in p16INK4a-positive senescent BECs in PBC. Bcl-xL inhibitors selectively induced apoptosis in senescent murine BECs (p < 0.01). CONCLUSION: The extent of senescent BECs in small bile ducts and bile ductules was closely related to stage and activity of PBC and the increased expression of p16 INK4a in bile ductules was correlated with inadequate response to UDCA.

Emperipolesis mediated by CD8+ T cells correlates with biliary epithelia cell injury in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is an autoimmune disease characterized by chronic destruction of the bile ducts. A major unanswered question regarding the pathogenesis of PBC is the precise mechanisms of small bile duct injury. Emperipolesis is one of cell-in-cell structures that is a potential histological hallmark associated with chronic hepatitis B. This study aimed to clarify the pathogenesis and characteristics of emperipolesis in PBC liver injury. Sixty-six PBC patients, diagnosed by liver biopsy combined with laboratory test, were divided into early-stage PBC (stages I and II, n = 39) and late-stage PBC (stages III and IV, n = 27). Emperipolesis was measured in liver sections stained with haematoxylin-eosin. The expressions of CK19, CD3, CD4, CD8, CD20, Ki67 and apoptosis of BECs were evaluated by immunohistochemistry or immunofluorescence double labelling. Emperipolesis was observed in 62.1% of patients with PBC, and BECs were predominantly host cells. The number of infiltrating CD3+ and CD8+ T cells correlated with the advancement of emperipolesis (R2  = 0.318, P < .001; R2  = 0.060, P < .05). The cell numbers of TUNEL-positive BECs and double staining for CK19 and Ki67 showed a significant positive correlation with emperipolesis degree (R2  = 0.236, P < .001; R2  = 0.267, P < .001). We conclude that emperipolesis mediated by CD8+ T cells appears to be relevant to apoptosis of BEC and thus may aggravate the further injury of interlobular bile ducts.

IgG4-Related Diseases in the Gastrointestinal Tract: Clinical Presentation, Diagnosis and Treatment Challenges.

BACKGROUND: IgG4-related diseases are a rare but an important entity. Due to the variable clinical presentation, this multiorgan disease was attributed to single-organ systems for many years. Also, it often remains a challenge to differentiate between IgG4-related diseases and malignancies. The pathogenesis seems to be a mixture of Th1- and Th2- immune responses, whereas the role of the non-pathogenic IgG4 antibodies is still unclear. Histopathological characteristics are a lymphoplasmacellular infiltrate with IgG4+ plasma cells, a storiform fibrosis and an obliterative phlebitis. This can lead to the functional destruction of every organ affected. In most cases, glucocorticoid treatment leads to remission and is used as maintenance therapy as well. Immune modulatory therapies are employed in case of steroid resistance. However, a majority of patients achieve remission without any therapy. SUMMARY: In this study, we review the current state-of-the-art regarding pathophysiology, diagnostics, organ manifestation and therapeutic approaches. Key Messages: While the diagnosis of IgG4-related diseases is still challenging, there have been significant improvements in diagnostic as well as in therapeutic approaches. This is partially due to a better understanding of the pathophysiology of the disease but also due to improved imaging modalities and novel, more targeted therapies.

Periductal Lymphocytic Infiltrate at Bilary Atresia Diagnosis Increases the Risk of Addition to the Transplant Waitlist.

PURPOSE: Biliary atresia (BA) is the main indication for pediatric liver transplantation. The aim of this study is to correlate aspects of histological examinations of diagnostic hepatic biopsies for BA with the patients' clinical progression and successful addition to the liver transplant waitlist. METHODS: This was a retrospective study of all 108 BA cases treated at the Federal University of São Paulo (1998-2015). Demographic and clinical data were correlated with histological findings. A logistic regression was used for outcome analysis, while the Kaplan-Meier method was applied for survival analysis. RESULTS: There were 108 patients with BA, 68.5% of whom underwent Kasai surgery. Patients added to the transplant waitlist tended to undergo Kasai surgery at a later time (P = .035). Periductal lymphocytic infiltrate was correlated with the addition to the transplant waitlist, with an odds ratio of 3.92 (P = .033). Patients who developed ascites after surgery were more frequently added to the transplant waitlist (P = .05). CONCLUSION: Patients added to the transplant waitlist underwent Kasai surgery later than other patients. Periductal lymphocytic infiltrate in the diagnostic hepatic biopsy and ascites after Kasai surgery were associated with an increased likelihood of addition to the transplant waitlist.

Gut microbiota translocation promotes autoimmune cholangitis.

Gut microbiota and bacterial translocation have been implicated as significant contributors to mucosal immune responses and tolerance; alteration of microbial molecules, termed pathogen-associated molecular patterns (PAMP) and bacterial translocation are associated with immune pathology. However, the mechanisms by which dysregulated gut microbiota promotes autoimmunity is unclear. We have taken advantage of a well-characterized murine model of primary biliary cholangitis, dnTGFßRII mice, and an additional unique construct, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice coined dnTGFßRIITLR2-/- mice to investigate the influences of gut microbiota on autoimmune cholangitis. Firstly, we report that dnTGFßRII mice manifest altered composition of gut microbiota and that alteration of this gut microbiota by administration of antibiotics significantly alleviates T-cell-mediated infiltration and bile duct damage. Second, toll-like receptor 2 (TLR2)-deficient dnTGFßRII mice demonstrate significant exacerbation of autoimmune cholangitis when their epithelial barrier integrity was disrupted. Further, TLR2-deficiency mediates downregulated expression of tight junction-associated protein ZO-1 leading to increased gut permeability and bacterial translocation from gut to liver; use of antibiotics reduces microbiota translocation to liver and also decreases biliary pathology. In conclusion, our data demonstrates the important role of gut microbiota and bacterial translocation in the pathogenesis of murine autoimmune cholangitis.

Autoimmune sclerosing cholangitis: Evidence and open questions.

Juvenile sclerosing cholangitis is a rare chronic hepatobiliary disorder characterized by inflammation of the intra- and/or extrahepatic bile ducts, bile duct dilatation, narrowing and obliteration, and, histologically, by inflammatory bile duct damage leading to periductular fibrosis. The diagnosis is based on endoscopic retrograde cholangiopancreatography or magnetic resonance cholangiopancreatography. In children, it may be associated to a variety of systemic and hepatic conditions: thus, the term "primary" sclerosing cholangitis should be reserved for the rare cases without a known cause. Small duct disease is diagnosed in the presence of histological features diagnostic of sclerosing cholangitis and normal cholangiography. Autoimmune sclerosing cholangitis (ASC) is a form of sclerosing cholangitis with strong autoimmune features overlapping with those of autoimmune hepatitis (AIH). It is a well-recognized nosological entity in paediatrics, where it accounts for the majority of sclerosing cholangitis cases. It is as prevalent as AIH in children, is equally frequent in males and females, half of the patients have concomitant inflammatory bowel disease, virtually all patients have raised immunoglobulin G levels and positive anti-nuclear and/or anti-smooth muscle antibodies. Half of the ASC patients respond well to standard immunosuppressive treatment for AIH with the addition of ursodeoxycholic acid, but the transplant rate is higher than in AIH, and post-transplant recurrence is frequent. A number of open questions remain: are ASC and AIH distinct entities or different manifestations of the same condition? What is the role of histology? Is small duct disease a specific entity? What is the relationship between ASC and adult primary sclerosing cholangitis? What is the role of inflammatory bowel disease? In addition, validated diagnostic criteria for ASC are needed.

Primary sclerosing cholangitis leads to dysfunction and loss of MAIT cells.

Primary sclerosing cholangitis (PSC) is a severe chronic liver disease of the small and large bile ducts. The pathogenesis is unknown but a strong immune cell component has been suggested. Mucosal-associated invariant T (MAIT) cells are abundant in human liver and localize around bile ducts. Yet, the role of MAIT cells in PSC remains unclear. Here, we performed a detailed characterization of MAIT cells in circulation and assessed their presence in bile ducts of PSC patients as well as non-PSC controls. We observed a dramatic reduction in MAIT cell levels in PSC patients. High-dimensional phenotypical analysis using stochastic neighbor embedding revealed the MAIT cells to be activated, a phenotype shared by the investigated disease control groups. In line with the noted phenotypic alterations, MAIT cell function was reduced in response to Escherichia coli and to cytokine stimulation in PSC patients as compared to healthy controls. Using a novel sampling approach of human bile ducts, we found MAIT cells to be specifically enriched within bile ducts. Finally, distinct from the dramatic decline observed in circulation, PSC-patients had retained levels of MAIT cells within bile ducts. Altogether, our results provide a detailed insight into how the human MAIT cell compartment is affected in PSC.

Mesenchymal stem cells alleviate experimental autoimmune cholangitis through immunosuppression and cytoprotective function mediated by galectin-9.

BACKGROUND: Mesenchymal stem cells (MSCs) play an anti-inflammatory role by secreting certain bioactive molecules to exert their therapeutic effects for disease treatment. However, the underlying mechanism of MSCs in chronic autoimmune liver diseases-primary biliary cholangitis (PBC), for example-remains to be elucidated. METHODS: Human umbilical cord-derived MSCs (UC-MSCs) were injected intravenously into 2-octynoic acid coupled to bovine serum albumin (2OA-BSA)-induced autoimmune cholangitis mice. Serum levels of biomarkers and autoantibodies, histologic changes in the liver, diverse CD4+ T-cell subsets in different tissues, and chemokine activities were analyzed. Moreover, we investigated galectin-9 (Gal-9) expression and its function in UC-MSCs. RESULTS: In this study, UC-MSC transplantation (UC-MSCT) significantly ameliorated liver inflammation, primarily by diminishing T helper 1 (Th1) and Th17 responses as well as modifying liver chemokine activities in experimental autoimmune cholangitis mice. Mechanistically, UC-MSCs significantly repressed the proliferation of CD4+ T cells and suppressed the differentiation of Th1 and Th17 cells, which was likely dependent on Gal-9. Furthermore, the signal transducer and activator of transcription (STAT) and c-Jun N-terminal kinase (JNK) signaling pathways were involved in the production of Gal-9 in UC-MSCs. CONCLUSIONS: These results suggest that Gal-9 contributes significantly to UC-MSC-mediated therapeutic effects and improve our understanding of the immunomodulatory mechanisms of MSCs in the treatment of PBC.

Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b+F4/80- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Cholestasis induced liver pathology results in dysfunctional immune responses after arenavirus infection.

Immune responses are critical for defense against pathogens. However, prolonged viral infection can result in defective T cell immunity, leading to chronic viral infection. We studied immune activation in response to arenavirus infection during cholestasis using bile duct ligation (BDL). We monitored T cell responses, virus load and liver pathology markers after infection with lymphocytic choriomeningitis virus (LCMV). BDL mice failed to induce protective anti-viral immunity against LCMV and consequently exhibited chronic viral infection. BDL mice exhibited reduced anti-viral T cell immunity as well as reduced type 1 interferon production early after LCMV infection. Consistently, the presence of serum from BDL mice reduced the responsiveness of dendritic cell (DC) and T cell cultures when compared to Sham controls. Following fractionation and mass spectrometry analyses of sera, we identified several serum factors to be upregulated following BDL including bilirubin, bile acids, 78 kDa Glucose regulated protein (GRP78) and liver enzymes. Bilirubin and GRP78 were capable of inhibiting DC and T cell activation. In this work, we demonstrate that liver damage mediated by cholestasis results in defective immune induction following arenavirus infection.

Anti-inflammatory consequences of bile acid accumulation in virus-infected bile duct ligated mice.

Cholestatic patients exhibiting high bile acid serum levels were reported to be more susceptible to bacterial and viral infections. Animal studies in bile duct ligated (BDL) mice suggest that cholestasis leads to an aggravation of hepatic bacterial infections. We have investigated the impact of cholestasis on mouse cytomegalovirus (MCMV)-induced immune responses and viral replication. While MCMV did not aggravate BDL-induced liver damage, BDL markedly reduced MCMV-triggered chemokine expression and immune cell recruitment to the liver. MCMV-infected BDL mice showed diminished trafficking of Ly6C+/F4/80+ myeloid cells and NK1.1+ NK cells to the liver compared to MCMV infected control mice. Moreover, virus-driven expression of CCL7, CCL12, CXCL9 and CXCL10 was clearly impaired in BDL- compared to sham-operated mice. Furthermore, production of the anti-inflammatory cytokine IL-10 was massively augmented in infected BDL mice. In contrast, intra- and extrahepatic virus replication was unaltered in BDL-MCMV mice when compared to sham-MCMV mice. Cholestasis in the BDL model severely impaired pathogen-induced chemokine expression in the liver affecting CCR2- and CXCR3-dependent cell trafficking. Cholestasis resulted in reduced recruitment of inflammatory monocytes and NK cells to the liver.