Type 1 diabetes (T1D) is precipitated by the autoimmune destruction of the insulin-producing β cells in the pancreatic islets of Langerhans. Chemokines have been identified as major conductors of islet infiltration by autoaggressive leukocytes, including antigen-presenting cells and islet autoantigen-specific T cells. We have previously generated a road map of gene expression in the islet microenvironment during T1D in a mouse model and found that most of the chemokine axes are chronically upregulated during T1D. The XCL1/XCR1 chemokine axis is of particular interest, since XCR1 is exclusively expressed on conventional DCs type 1 (cDC1) that excel by their high capacity for T cell activation. Here, we demonstrate that cDC1-expressing XCR1 are present in and around the islets of patients with T1D and of individuals with islet autoantibody positivity. Furthermore, we show that XCL1 plays an important role in the attraction of highly potent DCs expressing XCR1 to the islets in an inducible mouse model for T1D. XCL1-deficient mice display a diminished infiltration of XCR1+ cDC1 and, subsequently, a reduced magnitude and activity of islet autoantigen-specific T cells, resulting in a profound decrease in T1D incidence. Interference with the XCL1/XCR1 chemokine axis might constitute a novel therapy for T1D.

OBJECTIVE:In autoimmune Type 1 Diabetes (T1D), aberrant immune activation promotes regulatory T cell (Treg) impairments thereby boosting progression of islet autoimmunity. Consequently, there is a progressive destruction of the insulin-producing beta cells in the pancreas. Controlling overshooting immune activation represents a relevant approach to allow for efficient Treg-targeting by broadening the window of opportunity to induce Tregs. METHODS:We investigated the effect of restricting pyrimidine de novo synthesis during islet autoimmunity and T1D by Dihydroorotate dehydrogenase (DHODH) inhibition using the next-generation DHODH inhibitor Vidofludimus calcium. We assessed Treg-inducing features of DHODH inhibition in T cells from ongoing murine islet autoimmunity and human T1D in vitro. To dissect the functional relevance of these observations, we tested the impact of DHODH inhibition on interfering with autoimmune activation and disease progression in pre-clinical models of T1D in vivo. RESULTS:We show that DHODH inhibition results in enhanced Treg induction in vitro especially during increased immune activation and reduced T cell proliferation. In addition, Vidofludimus calcium reduced T1D incidence in two mouse models. On the cellular level, treated mice showed reduced T cell activation accompanied by increased Treg frequencies. CONCLUSIONS:We demonstrate that restricting pyrimidine de novo synthesis by next-generation DHODH inhibition is a strategy to interfere with autoimmune activation while fostering Tregs.

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.

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.

Although type 1 diabetes (T1D) results from the autoimmune destruction of the insulin-producing β-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.

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 β 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 β-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.

Autoimmune liver diseases like autoimmune hepatitis, primary biliary cholangitis, primary sclerosing cholangitis, and IgG4-related cholangitis are chronic inflammatory diseases of the liver with an autoimmune background. The therapy of autoimmune hepatitis targets the autoreactive immune system and is largely dependent on the use of glucocorticoids and cytostatic drugs. In contrast, the treatment of cholestatic autoimmune liver diseases is restricted to the use of secondary or semi-synthetic bile acids, like ursodeoxycholic acid or obeticholic acid. Although the management of the disease using such drugs works well for the majority of patients, many individuals do not respond to standard therapy. In addition, chronic treatment with glucocorticoids results in well-known side effects. Further, the use of bile acids is a symptomatic therapy that has no direct immunomodulatory effect. Thus, there is still a lot of room for improvement. The use of animal models has facilitated to elucidate the pathogenesis of autoimmune liver diseases and many potential target structures for immunomodulatory therapies have been identified. In this review, we will focus on autoimmune hepatitis for which the first animal models have been established five decades ago, but still a precise treatment for autoimmune hepatitis, as obtainable for other autoimmune diseases such as rheumatoid arthritis or multiple sclerosis has yet to be introduced. Thus, the question arises if our animal models are too far from the patient reality and thus findings from the models cannot be reliably translated to the patient. Several factors might be involved in this discrepancy. There is first and foremost the genetic background and the inbred status of the animals that is different from human patients. Here the use of humanized animals, such as transgenic mice, might reduce some of the differences. However, there are other factors, such as housing conditions, nutrition, and the microbiome that might also play an important role. This review will predominantly focus on the current status of animal models for autoimmune hepatitis and the possible ways to overcome discrepancies between model and patient.

The urinary bladder is markedly enlarged in the type 1 diabetes mellitus model of streptozotocin-injected rats, which may contribute to the frequent diabetic uropathy. Much less data exists for models of type 2 diabetes. Diabetic polyuria has been proposed as the pathophysiological mechanism behind bladder enlargement. Therefore, we explored such a relationship across nine distinct rodent models of diabetes including seven models of type 2 diabetes/obesity by collecting data on bladder weight and blood glucose from 16 studies with 2-8 arms each; some studies included arms with various diets and/or pharmacological treatments. Data were analysed for bladder enlargement and for correlations between bladder weight on the one and glucose levels on the other hand. Our data confirm major bladder enlargement in streptozotocin rats and minor if any enlargement in fructose-fed rats, db/db mice and mice on a high-fat diet; enlargement was present in some of five not reported previously models. Bladder weight was correlated with blood glucose as a proxy for diabetic polyuria within some but not other models, but correlations were moderate to weak except for RIP-LCMV mice (r(2) of pooled data from all studies 0.0621). Insulin levels also failed to correlate to a meaningful extent. Various diets and medications (elafibranor, empagliflozin, linagliptin, semaglutide) had heterogeneous effects on bladder weight that often did not match their effects on glucose levels. We conclude that the presence and extent of bladder enlargement vary markedly across diabetes models, particularly type 2 diabetes models; our data do not support the idea that bladder enlargement is primarily driven by glucose levels/glucosuria.

The urinary bladder is markedly enlarged in the type 1 diabetes mellitus model of streptozotocin (STZ)-injected rats, but much less data exist for models of type 2 diabetes (T2DM). Diabetic polyuria has been proposed to explain bladder enlargement. We have collected data on bladder weight and blood glucose from 16 studies representing 9 distinct rodent diabetes (7 T2DM) and obesity models; some included arms with diets and/or pharmacological treatments. Data were analyzed for bladder enlargement and for correlations between bladder weight on the one and glucose levels on the other hand. Our data confirm major bladder enlargements in STZ rats, minor if any enlargement in fructose-fed rats, db/db mice and mice on a high-fat diet. For the first time we report bladder weight data on 5 other models with presence and degree of bladder enlargement being heterogeneous across models. Bladder weight was correlated with plasma glucose in some but not other models, but correlations were moderate to weak except for RIP-LMCV mice. We conclude that the presence and extent of bladder enlargement varies markedly across diabetes models, particularly TD2M models; our data do not support the idea that bladder enlargement is primarily driven by glucose levels/glucosuria. ### Competing Interest Statement Authors TRC, RE and AK are former employees of Sanofi-Aventis. Other than that, the authors do not report conflicts of interest. * AMLN : Amylin liver NASH diet BBW : bladder/body weight BW : bladder weight CI : confidence interval HFD : high-fat diet IRS2 : insulin receptor substrate 2 RIP-LCMV : rat insulin promotor lymphocytic choriomeningitis virus SGLT2 : sodium-glucose transporter 2 STZ : streptozotocin T1DM : type 1 diabetes mellitus T2DM : type 2 diabetes mellitus ZSF1 : Zucker diabetic fatty/spontaneously hypertensive

Interleukin-22 in acute-on-chronic liver failure: A matter of ineffective levels, receptor dysregulation or defective signalling?Journal of HepatologyVol. 73Issue 4PreviewXiang et al. published interesting insights about the role of interleukin (IL)-22 in acute-on-chronic liver failure (ACLF).1 The authors elegantly analysed the effects of IL-22 in a mouse model of ACLF after chronic carbon tetrachloride (CCl4)-treatment (0.2 ml/kg, 2×/week, i.p.) and acute precipitating injury using 2 injections of 0.4 ml/kg CCl4 in combination with an i.p. challenge with Klebsiella pneumonia (K.P.), mimicking a bacterial infection. The model seems to induce a shift in the hepatic IL-22 signalling from the pro-regenerative IL-6/STAT3 towards the anti-regenerative IFNγ/STAT1 pathway paralleled by suppression of liver regeneration. Full-Text PDF Open AccessInterleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in miceJournal of HepatologyVol. 72Issue 4PreviewAcute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition. Full-Text PDF We greatly appreciate Schwarzkopf et al.'s comments on our work. The first question raised relates to the role of endogenous interleukin (IL)-22 and IL-22 binding protein (IL-22BP) in acute-on-chronic liver failure (ACLF). Serum levels of IL-22 are elevated in patients with ACLF[1]Schwarzkopf K. Ruschenbaum S. Barat S. Cai C. Mucke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar and in mouse models of ACLF induced by infection of adenovirus-CYP4502D6 plus injection of cecal slurry (CS) or chronic CCl4 plus 2 binges of ethanol.[2]Schwarzkopf K. Eberle L. Uschner F. Klein S. Schierwagen R. Muecke M. et al.Interleukin-22 (IL-22) in acute-on-chronic liver failure: a matter of ineffective levels, receptor dysregulation or defective signaling?.J Hepatol. 2020; 72: 736-745PubMed Google Scholar Actually, we also found that serum IL-22 levels were mildly but significantly elevated in our ACLF model, which was illustrated in Fig. S6B in our paper.[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar The role of this endogenous IL-22 elevation in ACLF, which was not the focus of our study, remains obscure, although endogenous IL-22 has been shown to protect against acute liver injury[4]Zenewicz L.A. Yancopoulos G.D. Valenzuela D.M. Murphy A.J. Karow M. Flavell R.A. Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation.Immunity. 2007; 27: 647-659Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar and bacterial infection in mice.[5]Zheng M. Horne W. McAleer J.P. Pociask D. Eddens T. Good M. et al.Therapeutic role of interleukin 22 in experimental intra-abdominal Klebsiella pneumoniae infection in mice.Infect Immun. 2016; 84: 782-789Crossref PubMed Scopus (26) Google Scholar One enigma is that humans have a thousand-fold higher circulating IL-22BP than mice (~40,000–70,000 pg/ml in humans vs. ~40 pg/ml in mice)[1]Schwarzkopf K. Ruschenbaum S. Barat S. Cai C. Mucke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar,[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar; the reason for this huge difference is not clear. Although IL-22BP has been shown to inhibit IL-22 signaling, the degree to which IL-22BP inhibits IL-22 signaling remains unclear, and the exact functions of IL-22BP still remain obscure. It is plausible that endogenous IL-22 may have less significant functions in humans than in mice because of much higher IL-22BP levels in humans; however, this speculation needs to be confirmed. Nevertheless, we agree with the hypothesis by Schwarzkopf et al. that the ineffective hepatoprotection by endogenous IL-22 in patients with ACLF may be due to high levels of IL-22BP.[1]Schwarzkopf K. Ruschenbaum S. Barat S. Cai C. Mucke M.M. Fitting D. et al.IL-22 and IL-22-binding protein are associated with development of and mortality from acute-on-chronic liver failure.Hepatol Commun. 2019; 3: 392-405Crossref PubMed Scopus (25) Google Scholar,[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar However, the pharmacological treatment with IL-22Fc, which was the focus of our study, caused a several thousand-fold increase in serum IL-22 (~700,000 pg/ml after IL-22Fc vs. ~200 pg/ml before IL-22Fc) and great biological responses in healthy volunteers[6]Tang K.Y. Lickliter J. Huang Z.H. Xian Z.S. Chen H.Y. Huang C. et al.Safety, pharmacokinetics, and biomarkers of F-652, a recombinant human interleukin-22 dimer, in healthy subjects.Cell Mol Immunol. 2019; 16: 473-482Crossref PubMed Scopus (45) Google Scholar,[7]Rothenberg M.E. Wang Y. Lekkerkerker A. Danilenko D.M. Maciuca R. Erickson R. et al.Randomized phase I healthy volunteer study of UTTR1147A (IL-22Fc): a potential therapy for epithelial injury.Clin Pharmacol Ther. 2019; 105: 177-189Crossref PubMed Scopus (32) Google Scholar and in patients with alcoholic hepatitis.[8]Arab J.P. Sehrawat T.S. Simonetto D.A. Verma V.K. Feng D. Tang T. et al.An open-label, dose-escalation study to assess the safety and efficacy of IL-22 agonist F-652 in patients with alcohol-associated hepatitis.Hepatology. 2019; https://doi.org/10.1002/hep.31046Crossref PubMed Scopus (67) Google Scholar IL-22Fc is a recombinant fusion protein consisting of 2 human IL-22 molecules linked to an immunoglobulin constant region (IgG-Fc) and has a long half-life in vivo.[6]Tang K.Y. Lickliter J. Huang Z.H. Xian Z.S. Chen H.Y. Huang C. et al.Safety, pharmacokinetics, and biomarkers of F-652, a recombinant human interleukin-22 dimer, in healthy subjects.Cell Mol Immunol. 2019; 16: 473-482Crossref PubMed Scopus (45) Google Scholar,[7]Rothenberg M.E. Wang Y. Lekkerkerker A. Danilenko D.M. Maciuca R. Erickson R. et al.Randomized phase I healthy volunteer study of UTTR1147A (IL-22Fc): a potential therapy for epithelial injury.Clin Pharmacol Ther. 2019; 105: 177-189Crossref PubMed Scopus (32) Google Scholar Such high levels of IL-22Fc can probably overcome the inhibitory function of IL-22BP and ameliorate ACLF by activating the pro-regenerative STAT3 signaling in hepatocytes. Schwarzkopf et al. proposed a mouse model of ACLF induced by infection of adenovirus-CYP4502D6 plus injection of CS.[2]Schwarzkopf K. Eberle L. Uschner F. Klein S. Schierwagen R. Muecke M. et al.Interleukin-22 (IL-22) in acute-on-chronic liver failure: a matter of ineffective levels, receptor dysregulation or defective signaling?.J Hepatol. 2020; 72: 736-745PubMed Google Scholar We agree that CS is a good approach for inducing ACLF in mice due to induction of polymicrobial infection. Actually, we attempted to develop a CS-induced ACLF model as well. We prepared the CS according to a recently published CS preparation protocol[9]Starr M.E. Steele A.M. Saito M. Hacker B.J. Evers B.M. Saito H. A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis.PLoS One. 2014; 9: e115705Crossref PubMed Scopus (82) Google Scholar and injected a high dose of 200 μl/mouse as suggested by this protocol.[9]Starr M.E. Steele A.M. Saito M. Hacker B.J. Evers B.M. Saito H. A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis.PLoS One. 2014; 9: e115705Crossref PubMed Scopus (82) Google Scholar Although 40% mortality was reported in young mice after injection of this dose in this protocol,[9]Starr M.E. Steele A.M. Saito M. Hacker B.J. Evers B.M. Saito H. A new cecal slurry preparation protocol with improved long-term reproducibility for animal models of sepsis.PLoS One. 2014; 9: e115705Crossref PubMed Scopus (82) Google Scholar we failed to observe mortality in mice by injecting this CS dose although the blood cultures showed positive results (Fig. 1). We believe that this CS approach is highly dependent on the conditions of animal facilities and thus hard to control. For example, mice in our animal facility at the NIAAA are provided with water of pH ~7, whereas mice at the Jackson laboratory are given acidic water (pH ~2). This difference could greatly impact gut bacteria and the outcome of the CS injection experiment. In addition, we also performed cecal ligation and puncture (CLP) surgery to induce multi-bacterial infections in our ACLF model and found that CLP caused a similar pathological process and mortality like Klebsiella pneumonia (K.P.) injection.[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar However, in the CLP model, it is hard to accurately control the spillage amount of cecal contents into the peritoneal cavity, and the surgical procedure and environment of the animal facility significantly influence the outcome of the model. In contrast, a single K.P. injection is easy to handle and the dose of K.P. can be controlled precisely.[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Therefore, we prefer a single K.P. injection to introduce bacterial infection in the ACLF model with chronic plus acute CCl4 injection.[3]Xiang X. Feng D. Hwang S. Ren T. Wang X. Trojnar E. et al.Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.J Hepatol. 2020; 72: 736-745Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Lastly, establishment of ACLF in mice by combination of alcohol binges and chronic CCl4 treatment, as shared by Schwarzkopf et al., is of great interest to us at the NIAAA. We have not tested alcohol binges in our ACLF model, but it will be exciting to further explore this model and investigate the mechanism underlying the precipitating effect of alcohol binge in the course of alcohol-associated ACLF development. Again, we thank Schwarzkopf et al. for raising several stimulating questions. Because IL-22 has many beneficial functions in the liver and has shown promise in a phase IIb trial in patients with alcoholic hepatitis,[8]Arab J.P. Sehrawat T.S. Simonetto D.A. Verma V.K. Feng D. Tang T. et al.An open-label, dose-escalation study to assess the safety and efficacy of IL-22 agonist F-652 in patients with alcohol-associated hepatitis.Hepatology. 2019; https://doi.org/10.1002/hep.31046Crossref PubMed Scopus (67) Google Scholar it is worth exploring IL-22 therapy in ACLF and other of liver diseases, such as non-alcoholic steatohepatitis.[10]Hwang S. He Y. Xiang X. Seo W. Kim S.J. Ma J. et al.Interleukin-22 ameliorates neutrophil-driven nonalcoholic steatohepatitis through multiple targets.Hepatology. 2019; https://doi.org/10.1002/hep.31031Crossref Scopus (61) Google Scholar Further studies are needed to clarify the role of endogenous IL-22 and IL-22BP in the pathogenesis of liver diseases. This work was supported by the intramural program of NIAAA, NIH, United States (B.G.). XX performed the experiment for the Fig. 1 and wrote the paper; SH wrote and edited the paper; BG supervised the whole project, wrote and edited the paper. The authors declare no competing interests. Please refer to the accompanying ICMJE disclosure forms for further details. Download .pdf (.24 MB) Help with pdf files disclosures.pdf

Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acid-labile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS-Hz-Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS-Hz-Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders.

Cytotoxic T lymphocyte (CTL) activation contributes to liver damage during sepsis, but the mechanisms involved are largely unknown. Understanding the underlying principle will permit interference with CTL activation and thus, provide a new therapeutic option. Methods: To elucidate the mechanism leading to CTL activation we used the Hepa1-6 cell line in vitro and the mouse model of in vivo polymicrobial sepsis, following cecal-ligation and -puncture (CLP) in wildtype, myeloid specific NOX-2, global NOX2 and NOX4 knockout mice, and their survival as a final readout. In this in vivo setting, we also determined hepatic mRNA and protein expression as well as clinical parameters of liver damage - aspartate - and alanine amino-transaminases. Hepatocyte specific overexpression of PD-L1 was achieved in vivo by adenoviral infection and transposon-based gene transfer using hydrodynamic injection. Results: We observed downregulation of PD-L1 on hepatocytes in the murine sepsis model. Adenoviral and transposon-based gene transfer to restore PD-L1 expression, significantly improved survival and reduced the release of liver damage, as PD-L1 is a co-receptor that negatively regulates T cell function. Similar protection was observed during pharmacological intervention using recombinant PD-L1-Fc. N-acetylcysteine blocked the downregulation of PD-L1 suggesting the involvement of reactive oxygen species. This was confirmed in vivo, as we observed significant upregulation of PD-L1 expression in NOX4 knockout mice, following sham operation, whereas its expression in global as well as myeloid lineage NOX2 knockout mice was comparable to that in the wild type animals. PD-L1 expression remained high following CLP only in total NOX2 knockouts, resulting in significantly reduced release of liver damage markers. Conclusion: These results suggest that, contrary to common assumption, maintaining PD-L1 expression on hepatocytes improves liver damage and survival of mice during sepsis. We conclude that administering recombinant PD-L1 or inhibiting NOX2 activity might offer a new therapeutic option in sepsis.

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.

Fibrogenesis is a progressive scarring event resulting from disrupted regular wound healing due to repeated tissue injury and can end in organ failure, like in liver cirrhosis. The protagonists in this process, either liver-resident cells or patrolling leukocytes attracted to the site of tissue damage, interact with each other by soluble factors but also by direct cell-cell contact mediated by cell adhesion molecules. Since cell adhesion molecules also support binding to the extracellular matrix, they represent excellent biosensors, which allow cells to modulate their behavior based on changes in the surrounding microenvironment. In this review, we focus on selectins, cadherins, integrins and members of the immunoglobulin superfamily of adhesion molecules as well as some non-classical cell adhesion molecules in the context of hepatic fibrosis. We describe their liver-specific contributions to leukocyte recruitment, cell differentiation and survival, matrix remodeling or angiogenesis and touch on their suitability as targets in antifibrotic therapies.

Autoimmune hepatitis (AIH), primary biliary cholangitis (PBC), and primary sclerosing cholangitis (PSC), are serious autoimmune liver diseases that are characterized by a progressive destruction of the liver parenchyma and/or the hepatic bile ducts and the development of chronic fibrosis. Left untreated autoimmune liver diseases are often life-threatening and patients require a liver transplantation to survive. Thus, an early and reliable diagnosis is paramount for the initiation of a proper therapy with immunosuppressive and/or anticholelithic drugs. Besides the analysis of liver biopsies and serum markers indicating liver damage, the screening for specific autoantibodies is an indispensable tool for the diagnosis of autoimmune liver diseases. Such liver autoantigen-specific antibodies might be involved in the disease pathogenesis and their epitope specificity may give some insight into the etiology of the disease. Here, we will mainly focus on the generation and specificity of autoantibodies in AIH patients. In addition, we will review data from animal models that aim toward a better understanding of the origins and pathogenicity of such autoantibodies.

Many animal models for autoimmune hepatitis (AIH) have been described in the past. Most models had to deal with the relative immunosuppressive environment of the liver. Therefore, some models used a combination of several triggering factors often on a susceptible background to generate an aggressive immune response that targets the liver. In addition, in order to be able to track the immune response the models used specific model autoantigens as targets that are either not present or have not been identified as a natural autoantigen in AIH patients. Thereby the feasibility of such models is somewhat questionable. Although many historic approaches included challenges of experimental animals with liver homogenates it was only in the last decade that natural occurring liver autoantigens have been used in animal models. This article reflects on the requirements for breaking liver tolerance and on how an ideal experimental model for AIH would look like. In addition, it discusses historic as well as recent animal models in the context of feasibility of induction, similarity of the clinical outcome to human AIH, and gain of knowledge for possible future therapies.

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.

Type 1 diabetes (T1D) results from the autoimmune destruction of insulin-producing β-cells in the pancreas. Thereby, the chemokine CXC-motif ligand 10 (CXCL10) plays an important role in the recruitment of autoaggressive lymphocytes to the islets of Langerhans. Transplantation of isolated islets as a promising therapy for T1D has been hampered by early graft rejection. Here, we investigated the influence of CXCL10 on the autoimmune destruction of islet isografts using RIP-LCMV mice expressing a lymphocytic choriomeningitis virus (LCMV) protein in the β-cells. RIP-LCMV islets express CXCL10 after isolation and maintain CXCL10 production after engraftment. Thus, we isolated islets from either normal or CXCL10-deficient RIP-LCMV mice and transferred them under the kidney capsule of diabetic RIP-LCMV mice. We found that the autoimmune destruction of CXCL10-deficient islet isografts was significantly reduced. The autoimmune destruction was also diminished in mice administered with an anti-CXCL10 antibody. The persistent protection from autoimmune destruction was paralleled by an increase in FoxP3+ regulatory T cells within the cellular infiltrates around the islet isografts. Consequently, CXCL10 might influence the cellular composition locally in the islet graft, thereby playing a role in the autoimmune destruction. CXCL10 might therefore constitute a potential therapeutic target to prolong islet graft survival.