Splenectomy induces biochemical remission and regeneration in experimental murine autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic immune-mediated inflammatory liver disease. It is known that AIH originates not from the spleen but from the liver itself. Nonetheless, most details of the etiology and pathophysiology are unknown. We induced experimental murine AIH (emAIH) in NOD/Ltj mice by single administration of a replication-deficient adenovirus and performed splenectomy during late-stage disease. Biochemical disease remission occurred, which was characterized by improvement in transaminase levels. The causes of this remission included a shift in the transcriptomic signature of serum proteins toward regeneration. At the cellular level, there was a marked decrease in activated CD8+ T cells and an increase in intrahepatic regulatory T cells (Tregs). Here, intrahepatic Treg numbers correlated with biochemical remission. Notably, an imbalance in the T-cell/B-cell ratio was observed, with a disproportionate increase in total B cells. In summary, intrahepatic increases in Tregs, biochemical remission, and regeneration could be induced by splenectomy in the late stage of emAIH.

The Detrimental Role of Regulatory T Cells in Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is induced by steatosis and metabolic inflammation. While involvement of the innate immune response has been shown, the role of the adaptive immune response in NASH remains controversial. Likewise, the role of regulatory T cells (Treg) in NASH remains unclear although initial clinical trials aim to target these regulatory responses. High-fat high-carbohydrate (HF-HC) diet feeding of NASH-resistant BALB/c mice as well as the corresponding recombination activating 1 (Rag)-deficient strain was used to induce NASH and to study the role of the adaptive immune response. HF-HC diet feeding induced strong activation of intrahepatic T cells in BALB/c mice, suggesting an antigen-driven effect. In contrast, the effects of the absence of the adaptive immune response was notable. NASH in BALB/c Rag1-/- mice was substantially worsened and accompanied by a sharp increase of M1-like macrophage numbers. Furthermore, we found an increase in intrahepatic Treg numbers in NASH, but either adoptive Treg transfer or anti-cluster of differentiation (CD)3 therapy unexpectedly increased steatosis and the alanine aminotransferase level without otherwise affecting NASH. Conclusion: Although intrahepatic T cells were activated and marginally clonally expanded in NASH, these effects were counterbalanced by increased Treg numbers. The ablation of adaptive immunity in murine NASH led to marked aggravation of NASH, suggesting that Tregs are not regulators of metabolic inflammation but rather enhance it.

Anti-CD20 Therapy Alters the Protein Signature in Experimental Murine AIH, but Not Exclusively towards Regeneration.

BACKGROUND: Autoimmune hepatitis (AIH) is a chronic autoimmune inflammatory disease that usually requires lifelong immunosuppression. Frequent recurrences after the discontinuation of therapy indicate that intrahepatic immune regulation is not restored by current treatments. Studies of other autoimmune diseases suggest that temporary depletion of B cells can improve disease progression in the long term. METHODS: We tested a single administration of anti-CD20 antibodies to reduce B cells and the amount of IgG to induce intrahepatic immune tolerance. We used our experimental murine AIH (emAIH) model and treated the mice with anti-CD20 during the late stage of the disease. RESULTS: After treatment, the mice showed the expected reductions in B cells and serum IgGs, but no improvements in pathology. However, all treated animals showed a highly altered serum protein expression pattern, which was a balance between inflammation and regeneration. CONCLUSIONS: In conclusion, anti-CD20 therapy did not produce clinically measurable results because it triggered inflammation, as well as regeneration, at the proteomic level. This finding suggests that anti-CD20 is ineffective as a sole treatment for AIH or emAIH.

Dulaglutide Alone and in Combination with Empagliflozin Attenuate Inflammatory Pathways and Microbiome Dysbiosis in a Non-Diabetic Mouse Model of NASH.

Dysregulation of glucose homeostasis plays a major role in the pathogenesis of non-alcoholic steatohepatitis (NASH) as it activates proinflammatory and profibrotic processes. Beneficial effects of antiglycemic treatments such as GLP-1 agonist or SGLT-2 inhibitor on NASH in patients with diabetes have already been investigated. However, their effect on NASH in a non-diabetic setting remains unclear. With this aim, we investigated the effect of long-acting GLP1-agonist dulaglutide and SGLT-2 inhibitor empagliflozin and their combination in a non-diabetic mouse model of NASH. C57BL/6 mice received a high-fat-high-fructose (HFHC) diet with a surplus of cholesterol for 16 weeks. After 12 weeks of diet, mice were treated with either dulaglutide, empagliflozin or their combination. Dulaglutide alone and in combination with empagliflozin led to significant weight loss, improved glucose homeostasis and diminished anti-inflammatory and anti-fibrotic pathways. Combination of dulaglutide and empagliflozin further decreased MoMFLy6CHigh and CD4+Foxp3+ T cells. No beneficial effects for treatment with empagliflozin alone could be shown. While no effect of dulaglutide or its combination with empaglifozin on hepatic steatosis was evident, these data demonstrate distinct anti-inflammatory effects of dulaglutide and their combination with empagliflozin in a non-diabetic background, which could have important implications for further treatment of NASH.

Splenectomy Prior to Experimental Induction of Autoimmune Hepatitis Promotes More Severe Hepatic Inflammation, Production of IL-17 and Apoptosis.

Autoimmune hepatitis (AIH) is detected at a late stage in the course of the disease. Therefore, induction and etiology are largely unclear. It is controversial if the induction of autoimmunity occurs in the liver or in the spleen. In our experimental murine AIH model, the induction of autoimmunity did not occur in the spleen. Instead, a protective role of the spleen could be more likely. Therefore, we splenectomized mice followed by induction of experimental murine AIH. Splenectomized mice presented more severe portal inflammation. Furthermore, these mice had more IL-17, IL-23 receptor (IL-23R) and caspase 3 (casp3) and a decreased amount of erythropoietin in serum, while intrahepatic T cell compartments were unaffected. These results indicate that the spleen is not necessary for induction of AIH, and splenectomy disrupts the ability to immune regulate the intensity of hepatic inflammation, production of IL-17 and apoptosis.

Investigation of an outbreak caused by an unknown infectious pathogen in rural Zola.

This case study was written as part of a fellowship in biosafety and biosecurity organised by the German Biosecurity Programme, namely the Global Partnership Initiated Biosecurity Academia for Controlling Health Threats (GIBACHT). Among other objectives, the fellowship focuses on equipping participants with the skills of developing their own country-specific case studies with focus on biosafety- and biosecurity-related scenarios. Upon completion of the underlying case study, participants should be able to identify some existing gaps with regards to early detection and investigation of outbreaks, describe the key steps in outbreak investigation, explain the role of communication and coordination among the various stakeholders in outbreak investigations and analyse epidemiological data obtained during outbreak investigations. They should also be able to suggest appropriate control and prevention measures for specific disease outbreaks with focus on foodborne outbreaks and to distinguish between biosafety and biosecurity concepts.

Autoimmune hepatitis induction can occur in the liver.

The priming of T cells in the liver is widely accepted. Nonetheless, it is controversial whether immune activation in autoimmune hepatitis (AIH) occurs in the liver or in the spleen. To address this issue, we splenectomized mice and induced experimental murine AIH (emAIH) with an adenovirus (Ad)-expressing formiminotransferase cyclodeaminase (FTCD). Post-splenectomy, the experimental mice developed emAIH to a higher extent than the control mice. In addition, splenectomized mice harboured more intrahepatic B cells and a disproportionately small number of regulatory T cells (Tregs) within a reduced T cell population at the site of inflammation. These results imply that the spleen is not the site of AIH induction. In contrast, the spleen seems to have a protective function since the pathological score was more severe in splenectomized animals. These findings have important implications for the aetiology of AIH.

Regulatory T cells engineered with a novel insulin-specific chimeric antigen receptor as a candidate immunotherapy for type 1 diabetes.

Adoptive immunotherapy with ex vivo expanded, polyspecific regulatory T cells (Tregs) is a promising treatment for graft-versus-host disease. Animal transplantation models used by us and others have demonstrated that the adoptive transfer of allospecific Tregs offers greater protection from graft rejection than that of polyclonal Tregs. This finding is in contrast to those of autoimmune models, where adoptive transfer of polyspecific Tregs had very limited effects, while antigen-specific Tregs were promising. However, antigen-specific Tregs in autoimmunity cannot be isolated in sufficient numbers. Chimeric antigen receptors (CARs) can modify T cells and redirect their specificity toward needed antigens and are currently clinically used in leukemia patients. A major benefit of CAR technology is its "off-the-shelf" usability in a translational setting in contrast to major histocompatibility complex (MHC)-restricted T cell receptors. We used CAR technology to redirect T cell specificity toward insulin and redirect T effector cells (Teffs) to Tregs by Foxp3 transduction. Our data demonstrate that our converted, insulin-specific CAR Tregs (cTregs) were functional stable, suppressive and long-lived in vivo. This is a proof of concept for both redirection of T cell specificity and conversion of Teffs to cTregs.

Hepatic T Cell Tolerance Induction in An Inflammatory Environment.

For the development of autoimmune hepatitis (AIH), genetic predisposition and environmental triggers are of major importance. Although experimental AIH can be induced in genetically susceptible mice, the low precursor frequency of autoreactive T cells hampers a deeper analysis of liver-specific T cells. Here, we established a system where the model antigen hemagglutinin (HA) is expressed exclusively in hepatocytes of Rosa26-HA mice following administration of a replication deficient adenovirus expressing Cre recombinase (Ad-Cre). Under these conditions, hepatocytes mimic the generation of altered-self neoantigens. To follow autoreactive T cells during AIH, we adoptively transferred HA--specific Cl4-TCR and 6.5-TCR T cells into Ad-Cre infected -Rosa26-HA mice. Alternatively, Rosa26-HA mice have been crossed with TCR transgenic mice that were infected with Ad-Cre to break hepatic tolerance and induce the expression of the HA antigen as a hepatic self-antigen. Surprisingly, neither adoptive transfer nor a very high precursor frequency of autoreactive T cells was able to break tolerance in the context of adenoviral infection. The low proliferation of the antigen experienced autoreactive T cells despite the presence of the autoantigen and inflammation points to anergy as a potential tolerance mechanism. This model underscores the crucial importance of genetic susceptibility to break tolerance against hepatic autoantigens.

The influence of genetic predisposition and autoimmune hepatitis inducing antigens in disease development.

Autoimmune hepatitis (AIH) is defined as a chronic liver inflammation with loss of tolerance against hepatocytes. The etiology and pathophysiology of AIH are still poorly understood because reliable animal models are limited. Therefore, we recently introduced a model of experimental murine AIH by a self-limited adenoviral infection with the AIH type 2 antigen formiminotransferase cyclodeaminase (FTCD). We could demonstrate that break of humoral tolerance towards liver specific autoantigens like FTCD and cytochrome P450 2D6 (CYP2D6) is not dependent on the genetic background. However, the development of AIH in autoantibody positive animals is determined by genetic background genes. We could also show that the break of humoral tolerance is necessary but not sufficient for the development of AIH. In contrast the break of tolerance against the ubiquitously expressed nuclear antigens (ANAs) is strictly dependent on genetic predisposition. Priming with the UGA suppressor tRNA-associated protein (soluble liver antigen; SLA) is a strong inducer of ANA reactivity, but not sufficient to cause AIH development thereby questioning the importance of anti-SLA immune response as an important driver in AIH. Monogenetic mutations such as Aire-deficiency can cause AIH in otherwise genetically resistant strains. CONCLUSION: The results have important implications for our understanding of the pathophysiology of AIH development and for the interpretation of humoral antibody responses in AIH.

Autoimmune hepatitis in a murine autoimmune polyendocrine syndrome type 1 model is directed against multiple autoantigens.

UNLABELLED: Autoimmune polyendocrine syndrome type 1 (APS-1) is caused by mutations of the autoimmune regulator (AIRE) gene. Mouse studies have shown that this results in defective negative selection of T cells and defective early seeding of peripheral organs with regulatory T cells (Tregs). Aire deficiency in humans and mice manifests as spontaneous autoimmunity against multiple organs, and 20% of patients develop an autoimmune hepatitis (AIH). To study AIH in APS-1, we generated a murine model of human AIH on a BALB/c mouse background, in which Aire is truncated at exon 2. A subgroup of 24% of mice is affected by AIH, characterized by lymphoplasmacytic and periportal hepatic infiltrates, autoantibodies, elevated aminotransferases, and a chronic and progressive course of disease. Disease manifestation was dependent on specific Aire mutations and the genetic background of the mice. Though intrahepatic Treg numbers were increased and hyperproliferative, the intrahepatic CD4/CD8 ratio was decreased. The targets of the adaptive autoimmune response were polyspecific and not focussed on essential autoantigens, as described for other APS-1-related autoimmune diseases. The AIH could be treated with prednisolone or adoptive transfer of polyspecific Tregs. CONCLUSION: Development of AIH in APS-1 is dependent on specific Aire mutations and genetic background genes. Autoimmune response is polyspecific and can be controlled by steroids or transfer with Tregs. This might enable new treatment options for patients with AIH.