Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain.

The function of somatic stem cells declines with age. Understanding the molecular underpinnings of this decline is key to counteract age-related disease. Here, we report a dramatic drop in the neural stem cells (NSCs) number in the aging murine brain. We find that this smaller stem cell reservoir is protected from full depletion by an increase in quiescence that makes old NSCs more resistant to regenerate the injured brain. Once activated, however, young and old NSCs show similar proliferation and differentiation capacity. Single-cell transcriptomics of NSCs indicate that aging changes NSCs minimally. In the aging brain, niche-derived inflammatory signals and the Wnt antagonist sFRP5 induce quiescence. Indeed, intervention to neutralize them increases activation of old NSCs during homeostasis and following injury. Our study identifies quiescence as a key feature of old NSCs imposed by the niche and uncovers ways to activate NSCs to repair the aging brain.

Blockade of neutrophil extracellular trap components ameliorates cholestatic liver disease in Mdr2 (Abcb4) knockout mice.

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.

Gene-expression profiling of laser-dissected islets and studies in deficient mice reveal chemokines as differential driving force of type 1 diabetes.

Although type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing ß-cells, its treatment is largely restricted to exogenous insulin administration. Only few therapies targeting the autoaggressive immune system have been introduced into clinical practice or are considered in clinical trials. Here, we provide a gene expression profile of the islet microenvironment obtained by laser-dissection microscopy in an inducible mouse model. Thereby, we have identified novel targets for immune intervention. Increased gene expression of most inflammatory proteins was apparent at day 10 after T1D induction and largely paralleled the observed degree of insulitis. We further focused on genes involved in leukocyte migration, including chemokines and their receptors. Besides the critical chemokine CXCL10, we found several other chemokines upregulated locally in temporary or chronic manner. Localization of the chemokine ligand/receptor pairs to the islet microenvironment has been confirmed by RNAscope. Interference with the CXCL16-CXCR6 and CX3CL1-CX3CR1 axes, but not the CCL5-CCR1/3/5 axis, resulted in reduced insulitis and lower T1D incidence. Further, we found that the receptors for the differentially expressed chemokines CXCL10, CXCL16 and CX3CL1 are distributed unevenly among islet autoantigen-specific T cells, which explains why the interference with just one chemokine axis cannot completely abrogate insulitis and T1D.

Combination treatment of a novel CXCR3 antagonist ACT-777991 with an anti-CD3 antibody synergistically increases persistent remission in experimental models of type 1 diabetes.

Treatment of patients with recent-onset type 1 diabetes with an anti-CD3 antibody leads to the transient stabilization of C-peptide levels in responder patients. Partial efficacy may be explained by the entry of islet-reactive T-cells spared by and/or regenerated after the anti-CD3 therapy. The CXCR3/CXCL10 axis has been proposed as a key player in the infiltration of autoreactive T cells into the pancreatic islets followed by the destruction of ß cells. Combining the blockade of this axis using ACT-777991, a novel small-molecule CXCR3 antagonist, with anti-CD3 treatment may prevent further infiltration and ß-cell damage and thus, preserve insulin production. The effect of anti-CD3 treatment on circulating T-cell subsets, including CXCR3 expression, in mice was evaluated by flow cytometry. Anti-CD3/ACT-777991 combination treatment was assessed in the virally induced RIP-LCMV-GP and NOD diabetes mouse models. Treatments started at disease onset. The effects on remission rate, blood glucose concentrations, insulitis, and plasma C-peptide were evaluated for the combination treatment and the respective monotherapies. Anti-CD3 treatment induced transient lymphopenia but spared circulating CXCR3+ T cells. Combination therapy in both mouse models synergistically and persistently reduced blood glucose concentrations, resulting in increased disease remission rates compared to each monotherapy. At the study end, mice in disease remission demonstrated reduced insulitis and detectable plasma C-peptide levels. When treatments were initiated in non-severely hyperglycemic NOD mice at diabetes onset, the combination treatment led to persistent disease remission in all mice. These results provide preclinical validation and rationale to investigate the combination of ACT-777991 with anti-CD3 for the treatment of patients with recent-onset diabetes.

Gut dysbiosis promotes islet-autoimmunity by increasing T-cell attraction in islets via CXCL10 chemokine.

CXCL10 is an IFNγ-inducible chemokine implicated in the pathogenesis of type 1 diabetes. T-cells attracted to pancreatic islets produce IFNγ, but it is unclear what attracts the first IFNγ -producing T-cells in islets. Gut dysbiosis following administration of pathobionts induced CXCL10 expression in pancreatic islets of healthy non-diabetes-prone (C57BL/6) mice and depended on TLR4-signaling, and in non-obese diabetic (NOD) mice, gut dysbiosis induced also CXCR3 chemokine receptor in IGRP-reactive islet-specific T-cells in pancreatic lymph node. In amounts typical to low-grade endotoxemia, bacterial lipopolysaccharide induced CXCL10 production in isolated islets of wild type and RAG1 or IFNG-receptor-deficient but not type-I-IFN-receptor-deficient NOD mice, dissociating lipopolysaccharide-induced CXCL10 production from T-cells and IFNγ. Although mostly myeloid-cell dependent, also ß-cells showed activation of innate immune signaling pathways and Cxcl10 expression in response to lipopolysaccharide indicating their independent sensitivity to dysbiosis. Thus, CXCL10 induction in response to low levels of lipopolysaccharide may allow islet-specific T-cells imprinted in pancreatic lymph node to enter in healthy islets independently of IFN-g, and thus link gut dysbiosis to early islet-autoimmunity via dysbiosis-associated low-grade endotoxemia.

Effects of adenovirus-induced hepatocyte damage on chronic bile duct inflammation in a sclerosing cholangitis mouse model.

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.

Antibody Neutralization of CXCL10 in Vivo Is Dependent on Binding to Free and Not Endothelial-bound Chemokine: IMPLICATIONS FOR THE DESIGN OF A NEW GENERATION OF ANTI-CHEMOKINE THERAPEUTIC ANTIBODIES.

To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10-60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.

Junctional adhesion molecules JAM-B and JAM-C promote autoimmune-mediated liver fibrosis in mice.

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.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds.

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G- cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G-) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.

Non-alcoholic fatty liver disease (NAFLD) potentiates autoimmune hepatitis in the CYP2D6 mouse model.

Non-alcoholic fatty liver disease (NAFLD) and its more severe development non-alcoholic steatohepatitis (NASH) are increasing worldwide. In particular NASH, which is characterized by an active hepatic inflammation, has often severe consequences including progressive fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Here we investigated how metabolic liver injury is influencing the pathogenesis of autoimmune hepatitis (AIH). We used the CYP2D6 mouse model in which wild type C57BL/6 mice are infected with an Adenovirus expressing the major liver autoantigen cytochrome P450 2D6 (CYP2D6). Such mice display several features of human AIH, including interface hepatitis, formation of LKM-1 antibodies and CYP2D6-specific T cells, as well as hepatic fibrosis. NAFLD was induced with a high-fat diet (HFD). We found that pre-existing NAFLD potentiates the severity of AIH. Mice fed for 12 weeks with a HFD displayed increased cellular infiltration of the liver, enhanced hepatic fibrosis and elevated numbers of liver autoantigen-specific T cells. Our data suggest that a pre-existing metabolic liver injury constitutes an additional risk for the severity of an autoimmune condition of the liver, such as AIH.

Two separate mechanisms of enforced viral replication balance innate and adaptive immune activation.

The induction of innate and adaptive immunity is essential for controlling viral infections. Limited or overwhelming innate immunity can negatively impair the adaptive immune response. Therefore, balancing innate immunity separately from activating the adaptive immune response would result in a better antiviral immune response. Recently, we demonstrated that Usp18-dependent replication of virus in secondary lymphatic organs contributes to activation of the innate and adaptive immune responses. Whether specific mechanisms can balance innate and adaptive immunity separately remains unknown. In this study, using lymphocytic choriomeningitis virus (LCMV) and replication-deficient single-cycle LCMV vectors, we found that viral replication of the initial inoculum is essential for activating virus-specific CD8(+) T cells. In contrast, extracellular distribution of virus along the splenic conduits is necessary for inducing systemic levels of type I interferon (IFN-I). Although enforced virus replication is driven primarily by Usp18, B cell-derived lymphotoxin beta contributes to the extracellular distribution of virus along the splenic conduits. Therefore, lymphotoxin beta regulates IFN-I induction independently of CD8(+) T-cell activity. We found that two separate mechanisms act together in the spleen to guarantee amplification of virus during infection, thereby balancing the activation of the innate and adaptive immune system.

Immunopathogenic Mechanisms of Autoimmune Hepatitis: How Much Do We Know from Animal Models?

Autoimmune hepatitis (AIH) is characterized by a progressive destruction of the liver parenchyma and a chronic fibrosis. The current treatment of autoimmune hepatitis is still largely dependent on the administration of corticosteroids and cytostatic drugs. For a long time the development of novel therapeutic strategies has been hampered by a lack of understanding the basic immunopathogenic mechanisms of AIH and the absence of valid animal models. However, in the past decade, knowledge from clinical observations in AIH patients and the development of innovative animal models have led to a situation where critical factors driving the disease have been identified and alternative treatments are being evaluated. Here we will review the insight on the immunopathogenesis of AIH as gained from clinical observation and from animal models.

Upregulation of matrilin-2 expression in murine hepatic stellate cells during liver injury has no effect on fibrosis formation and resolution.

BACKGROUND & AIMS: Matrilins are a family of four oligomeric adaptor proteins whose functions in extracellular matrix assembly during pathophysiological events still need to be explored in more detail. Matrilin-2 is the largest family member and the only matrilin expressed in the naive liver. Several studies demonstrate that matrilin-2 interacts with collagen I, fibronectin or laminin-111-nidogen-1 complexes. All these matrix components get upregulated during hepatic scar tissue formation. Therefore, we tested whether matrilin-2 has an influence on the formation and/or the resolution of fibrotic tissue in the mouse liver. METHODS: Fibrosis was induced by infection with an adenovirus encoding cytochrome P450 2D6 (autoimmune liver damage) or by exposure to the hepatotoxin carbon tetrachloride. Fibrosis severity and matrilin-2 expression were assessed by immunohistochemistry. Hepatic stellate cells (HSCs) were isolated and analysed by immunocytochemistry and Transwell migration assays. RESULTS: Both autoimmune as well as chemically induced liver damage led to simultaneous upregulation of matrilin-2 and collagen I expression. Discontinuation of carbon tetrachloride exposure resulted in concomitant dissolution of both proteins. Activated HSCs were the source of de novo matrilin-2 expression. Comparing wild type and matrilin-2-deficient mice, no differences were detected in fibronectin and collagen I upregulation and resolution kinetics as well as amount or location of fibronectin and collagen I production and degradation. CONCLUSIONS: Our findings suggest that the absence of matrilin-2 has no effect on HSC activation and regression kinetics, synthetic activity, proliferative capacity, motility, or HSC apoptosis.

GAD autoantibody affinity in schoolchildren from the general population.

AIMS/HYPOTHESIS: Subtyping GAD autoantibody (GADA) responses using affinity measurement allows the identification of GADA-positive children with a family history of type 1 diabetes who are at risk of developing diabetes. Here, we asked whether GADA affinity is a useful marker to stratify the risk of type 1 diabetes in GADA-positive schoolchildren from the general population. METHODS: GADA affinity was measured by competitive binding experiments with [(125)I]-labelled and unlabelled human 65 kDa isoform of GAD (GAD65) in sera from 97 GADA-positive children identified in the Karlsburg Type 1 Diabetes Risk Study of a general schoolchild population in north-eastern Germany. GADA epitope specificity was determined using radiobinding assays with [(35)S]-labelled GAD65/67 kDa isoform of GAD (GAD67) chimeric proteins. RESULTS: GADA affinity was high, ≥ 10(10) l/mol, in 33 of 35 multiple islet autoantibody-positive children. In contrast, the affinity ranged widely among 62 single GADA-positive children (median 3.1 × 10(9) l/mol; range 5.6 × 10(6) to >4.0 × 10(11) l/mol; p < 0.0001). High-affinity GADA were associated with HLA-DRB1*03 (p = 0.02) and predominantly directed against the C-terminal and/or middle part of the GAD65 protein. At follow-up, the affinity remained relatively constant. Five of the single GADA-positive children developed additional islet autoantibodies and had high-affinity GADA. Twenty-six children progressed to type 1 diabetes; among them, 23 had GADA affinities of ≥ 10(10) l/mol before disease onset. CONCLUSIONS/INTERPRETATION: Schoolchildren from the general population may develop heterogeneous GADA responses, and a high affinity can identify those GADA-positive children who are more likely to progress to type 1 diabetes.

Tumour-specific activation of a tumour-blood transport improves the diagnostic accuracy of blood tumour markers in mice.

BACKGROUND: The accuracy of blood-based early tumour recognition is compromised by signal production at non-tumoral sites, low amount of signal produced by small tumours, and variable tumour production. Here we examined whether tumour-specific enhancement of vascular permeability by the particular tumour homing peptide, iRGD, which carries dual function of binding to integrin receptors overexpressed in the tumour vasculature and is known to promote extravasation via neuropilin-1 receptor upon site-specific cleavage, might be useful to improve blood-based tumour detection by inducing a yet unrecognised vice versa tumour-to-blood transport. METHODS: To detect an iRGD-induced tumour-to-blood transport, we examined the effect of intravenously injected iRGD on blood levels of α-fetoprotein (AFP) and autotaxin in several mouse models of hepatocellular carcinoma (HCC) or in mice with chronic liver injury without HCC, and on prostate-specific antigen (PSA) levels in mice with prostate cancer. FINDINGS: Intravenously injected iRGD rapidly and robustly elevated the blood levels of AFP in several mouse models of HCC, but not in mice with chronic liver injury. The effect was primarily seen in mice with small tumours and normal basal blood AFP levels, was attenuated by an anti-neuropilin-1 antibody, and depended on the concentration gradient between tumour and blood. iRGD treatment was also able to increase blood levels of autotaxin in HCC mice, and of PSA in mice with prostate cancer. INTERPRETATION: We conclude that iRGD induces a tumour-to-blood transport in a tumour-specific fashion that has potential of improving diagnosis of early stage cancer. FUNDING: Deutsche Krebshilfe, DKTK, LOEWE-Frankfurt Cancer Institute.

Mechanism of autoimmune hepatic fibrogenesis induced by an adenovirus encoding the human liver autoantigen cytochrome P450 2D6.

Autoimmune hepatitis type 2 (AIH-2) is a severe autoimmune liver disease with unknown etiology. We recently developed the CYP2D6 mouse model for AIH-2, in which mice are challenged with an adenovirus (Ad-2D6) expressing human cytochrome P450 2D6 (hCYP2D6), the major autoantigen in AIH-2. Such mice develop chronic hepatitis with cellular infiltrations and generation of hCYP2D6-specific antibodies and T cells. Importantly, the CYP2D6 model represents the only model displaying chronic fibrosis allowing for a detailed investigation of the mechanisms of chronic autoimmune-mediated liver fibrogenesis. We found that hCYP2D6-dependent chronic activation of hepatic stellate cells (HSC) resulted in an increased extracellular matrix deposition and elevated expression of α-smooth muscle actin predominantly in and underneath the liver capsule. The route of Ad-2D6 infection dramatically influenced the activation and trafficking of inflammatory monocytes, NK cells and hCYP2D6-specific T cells. Intraperitoneal Ad-2D6 infection caused subcapsular fibrosis and persistent clustering of inflammatory monocytes. In contrast, intravenous infection caused an accumulation of hCYP2D6-specific CD4 T cells throughout the liver parenchyma and induced a strong NK cell response preventing chronic HSC activation and fibrosis. In summary, we found that the location of the initial site of inflammation and autoantigen expression caused a differential cellular trafficking and activation and thereby determined the outcome of AIH-2-like hepatic damage and fibrosis.

Molecular mimicry rather than identity breaks T-cell tolerance in the CYP2D6 mouse model for human autoimmune hepatitis.

In our novel mouse model for autoimmune hepatitis (AIH), wildtype FVB mice infected with an Adenovirus (Ad) expressing the major AIH autoantigen human cytochrome P450 2D6 (hCYP2D6) show persistent histological and immunological features associated with AIH, including the generation of anti-hCYP2D6 antibodies with an epitope specificity identical to LKM-1 autoantibodies in AIH-patients. Since FVB mice do not express hCYP2D6, the immune response was directed against mouse CYP (mCYP) homologues. Additional expression of hCYP2D6 in transgenic mice resulted in amelioration of the liver disease. In the present study we used the CYP2D6 model to assess why tolerance breakdown and induction of autoimmune liver disease is more efficient if the triggering antigen is similar but not identical to the target autoantigen. We found that in contrast to the specificity and magnitude of anti-hCYP2D6 antibody responses, T-cell responses differ profoundly between wildtype and transgenic mice. Detailed T-cell epitope mapping studies show a robust, antigen-specific T-cell reactivity in FVB mice largely directed against one CD4 and three CD8 epitopes, activating a total of approximately 1% CD4 and 10% CD8 T-cells, respectively, while infected hCYP2D6 mice generated almost no hCYP2D6-specific T-cells. The frequency of hCYP2D6-specific T-cells was approximately 3-fold higher in the liver compared with the spleen. Amino acid sequence comparison revealed that the immunodominant epitopes were located in hCYP2D6-segments of intermediate homology between hCYP2D6 and its mCYP homologues. Our data indicate that self/non-self molecular mimicry, rather than molecular identity, is a prerequisite for breaking T-cell tolerance in the liver.

Distinct immune surveillance in primary biliary cholangitis and primary sclerosing cholangitis is linked with discrete cholangiocarcinoma risk.

Primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are 2 major liver autoimmune diseases. PBC is common in women and primarily affects intrahepatic small bile duct epithelial cells, known as cholangiocytes. In contrast, PSC is dominant in men and primarily affects medium and big intrahepatic and extrahepatic bile duct epithelial cells. Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes, and its incidence is increasing worldwide in both men and women. Numerous retrospective and clinical studies have suggested that PBC patients rarely develop CCA compared to PSC patients. CCA is accountable for the higher deaths in PSC patients due to ineffective therapies and our inability to diagnose the disease at an early stage. Therefore, it is paramount to understand the differences in immune surveillance mechanisms that render PBC patients more resistant while PSC patients are susceptible to CCA development. Here, we review several potential mechanisms contributing to differences in the susceptibility to CCA in PBC versus PSC patients.

A comparison of urinary bladder weight in male and female mice across five models of diabetes and obesity.

Introduction: Diabetes often leads to lower urinary tract dysfunction. The most frequently assessed parameter of urinary bladder dysfunction in animal models of diabetes is an enlargement of the bladder, which is consistently observed in type 1 and less consistently in type 2 diabetes. The vast majority of studies on bladder weight in animal models of diabetes and obesity has been performed in males, and no studies have directly compared this outcome parameter between sexes. Methods: Therefore, we have compared bladder weight and bladder/body weight ratio in five mouse models of obesity and diabetes (RIP-LCMV, db/db, ob/ob (two studies), insulin receptor substrate 2 (IRS2) knock-out mice and mice on a high-fat diet; pre-specified secondary analysis of a previously reported study). Results: In a pooled analysis of the control groups of all studies, females exhibited slightly lower glucose levels, lower body weight, and lower bladder weight, but bladder/body weight ratio was similar in both sexes (0.957 vs. 0.986 mg/g, mean difference 0.029 [-0.06; 0.118]). Among the six diabetic/obese groups, bladder/body weight ratio was similar in both sexes in three but smaller in female mice in three other groups. The mRNA expression of a panel of genes implied in the pathophysiology of bladder enlargement and/or fibrosis and inflammation did not differ systematically between sexes. Conclusions: We conclude that sex differences in diabetes/obesity-associated bladder enlargement may be model dependent.