Silencing of aryl hydrocarbon receptor repressor restrains Th17 cell immunity in autoimmune hepatitis.

Th17-cells play a key role in the pathogenesis of autoimmune hepatitis (AIH). Dysregulation of Th17-cells in AIH is linked to defective response to aryl-hydrocarbon-receptor (AhR) activation. AhR modulates adaptive immunity and is regulated by aryl-hydrocarbon-receptor-repressor (AHRR), which inhibits AhR transcriptional activity. In this study, we investigated whether defective Th17-cell response to AhR derives from aberrant AHRR regulation in AIH. Th17-cells, obtained from the peripheral blood of AIH patients (n = 30) and healthy controls (n = 30) were exposed to AhR endogenous ligands, and their response assessed in the absence or presence of AHRR silencing. Therapeutic effects of AHRR blockade were tested in a model of Concanavalin-A (Con-A)-induced liver injury in humanized mice. AHRR was markedly upregulated in AIH Th17-cells, following exposure to l-kynurenine, an AhR endogenous ligand. In patients, silencing of AHRR boosted Th17-cell response to l-kynurenine, as reflected by increased levels of CYP1A1, the main gene controlled by AhR; and decreased IL17A expression. Blockade of AHRR limited the differentiation of naïve CD4-cells into Th17 lymphocytes; and modulated Th17-cell metabolic profile by increasing the levels of uridine via ATP depletion or pyrimidine salvage. Treatment with 2'-deoxy-2'-fluoro-d-arabinonucleic acid (FANA) oligonucleotides to silence human AHRR in vivo, reduced ALT levels, attenuated lymphocyte infiltration on histology, and heightened frequencies of regulatory immune subsets in NOD/scid/gamma mice, reconstituted with human CD4 cells, and exposed to Con-A. In conclusion, blockade of AHRR in AIH restores Th17-cell response to AHR, and limits Th17-cell differentiation through generation of uridine. In vivo, silencing of AHRR attenuates liver damage in NOD/scid/gamma mice. Blockade of AHRR might therefore represent a novel therapeutic strategy to modulate effector Th17-cell immunity and restore homeostasis in AIH.

Endogenous DNase Activity in an Animal Model of Acute Liver Failure.

Deoxyribonucleases (DNases) cleave extracellular DNA (ecDNA) and are under intense research as interventions for diseases associated with high ecDNA, such as acute live injury. DNase I treatment decreases morbidity and mortality in this animal model. Endogenous DNase activity has high interindividual variability. In this study, we tested the hypothesis that high endogenous DNase activity is beneficial in an animal model of acute liver failure. DNase activity was measured in the plasma of adult male mice taken before i.p. injection of thioacetamide to induce acute liver failure. The survival of mice was monitored for 48 h. Mice were retrospectively divided into two groups based on the median DNase activity assessed using the gel-based single-radial enzyme diffusion assay. In acute liver failure, mice with a higher baseline DNase activity had lower mortality after 48 h (by 25%). Different protection of ecDNA against nucleases by vesicles or DNA-binding proteins could play a role and should be further evaluated. Similarly, the role of endogenous DNase activity should be analyzed in other disease models associated with high ecDNA.

Fecal Microbiota Transplantation as a Tool for Therapeutic Modulation of Non-gastrointestinal Disorders.

Fecal microbiota transplantation has been primarily investigated as a therapeutic tool for a number of gut disorders. Optimistic results from clinical studies on Clostridium difficile infection, inflammatory bowel disease and irritable bowel syndrome have stimulated the expansion of possible indications in which FMT might represent a game changing approach. Microbial dysbiosis was shown in a number of non-gastrointestinal disorders. Moreover, FMT was proven to be effective in therapy of numerous animal models of disease. However, only a proportion of these disorders have been addressed in clinical studies using FMT. These include obesity, non-alcoholic fatty liver disease, cardiovascular inflammation and neurological disorders such as autism, depression and Parkinson's disease. Results from preclinical and clinical studies also outlined possible molecular mechanisms that contribute to alleviation of the disease. These range from increasing the circulating levels of microbial metabolites (trimethylamine N-oxide, lipopolysaccharide, short chain fatty acids) to stimulation of the enteric nervous system. Several methodological shortcomings are still to be addressed; however, positive results of the clinical studies indicate that further investigation of FMT as a therapeutic tool for non-gastrointestinal disorders can be expected in upcoming years.

Extracellular DNA Correlates with Intestinal Inflammation in Chemically Induced Colitis in Mice.

Circulating extracellular DNA (ecDNA) is known to worsen the outcome of many diseases. ecDNA released from neutrophils during infection or inflammation is present in the form of neutrophil extracellular traps (NETs). It has been shown that higher ecDNA concentration occurs in a number of inflammatory diseases including inflammatory bowel disease (IBD). Enzymes such as peptidyl arginine deiminases (PADs) are crucial for NET formation. We sought to describe the dynamics of ecDNA concentrations and fragmentation, along with NETosis during a mouse model of chemically induced colitis. Plasma ecDNA concentration was highest on day seven of dextran sulfate sodium (DSS) intake and the increase was time-dependent. This increase correlated with the percentage of cells undergoing NETosis and other markers of disease activity. Relative proportion of nuclear ecDNA increased towards more severe colitis; however, absolute amount decreased. In colon explant medium, the highest concentration of ecDNA was on day three of DSS consumption. Early administration of PAD4 inhibitors did not alleviate disease activity, but lowered the ecDNA concentration. These results uncover the biological characteristics of ecDNA in IBD and support the role of ecDNA in intestinal inflammation. The therapeutic intervention aimed at NETs and/or nuclear ecDNA has yet to be fully investigated.

Altered aryl-hydrocarbon-receptor signalling affects regulatory and effector cell immunity in autoimmune hepatitis.

BACKGROUND & AIMS: In autoimmune hepatitis (AIH), the imbalance between regulatory T cells (Tregs) and T-helper type 17 (Th17) cells has been linked to low levels of CD39, an ectoenzyme that hydrolyses ATP, ultimately generating immunosuppressive adenosine. Upregulation of CD39 results from activation of aryl hydrocarbon receptor (AHR), which mediates toxin responses to modulate T-cell immunity. In this study, we investigated whether altered AHR signalling underlies defective CD39 expression and function in AIH Tregs and Th17 cells, therefore contributing to regulatory/effector cell imbalance. METHODS: Tregs and Th17 cells, obtained from the peripheral blood of 49 patients with AIH and 21 healthy individuals (HI), were tested for response to endogenous and exogenous AHR ligands. RESULTS: When compared to those of HI, AIH-derived Tregs and Th17 cells displayed impaired responses to AHR activation, reflected by impaired upregulation of CD39, delayed increase in ectoenzymatic activity, and defective Treg suppressive function. These impairments resulted, at least in part, from heightened levels of AHRR and Erα in Tregs and high HIF-1α in Th17 cells, and were reverted upon molecular blockade. Importantly, in AIH-derived Tregs, the binding affinity of AHR was higher for Erα than ARNT. CONCLUSIONS: In AIH, high levels of AHRR and HIF-1α inhibit AHR signalling in Tregs and Th17 cells. AHR non-canonical binding to Erα further amplifies the lack of effective CD39 upregulation. Blockade of these inhibitory and/or non-canonical activation pathways represents a potential therapeutic approach to restore CD39 and immunohomeostasis in AIH. LAY SUMMARY: In patients with autoimmune hepatitis, the imbalance between regulatory T cells and T helper type-17 cells is linked to dysfunction of the aryl hydrocarbon receptor pathway, resulting from aberrant inhibition or non-canonical activation. These alterations impair Treg- and Th17 cell-induced upregulation of CD39, an ectoenzyme key to immunoregulation. Blockade of excessive inhibition or non-canonical activation of the aryl hydrocarbon receptor pathway might represent a novel therapeutic strategy to control inflammation while restoring immune balance in autoimmune hepatitis.

Nuclear and Mitochondrial Circulating Cell-Free DNA Is Increased in Patients With Inflammatory Bowel Disease in Clinical Remission.

Background: The role of cell-free DNA (cfDNA) in the pathogenesis of inflammatory bowel disease (IBD) has been recently suggested. The aim of this study was to analyze circulating cfDNA and deoxyribonuclease (DNase) activity in IBD patients in clinical remission. Materials and Methods: Plasma and serum were obtained from 72 patients with Crohn's disease and 28 patients with ulcerative colitis. Total cfDNA, nuclear DNA (ncDNA), mitochondrial DNA (mtDNA) and DNase activity were measured. Results: IBD patients showed higher levels of both ncDNA and mtDNA compared to healthy controls. Concentration of ncDNA was higher in males compared to females, including patients and healthy controls. However, unlike males higher amount of ncDNA was found in female IBD patients compared to healthy controls. DNase activity was significantly lower in male IBD patients compared with healthy controls. In addition, there was a negative correlation between DNase activity and ncDNA levels in male IBD patients. Conclusions: Herein we present increased amount of circulating ncDNA and mtDNA in IBD patients in clinical remission. Thus, unlike total cfDNA, circulating ncDNA and mtDNA might not represent the optimal biomarkers of disease activity. This is also the first report on sex difference in circulating ncDNA levels, possibly associated with lower DNase activity in males.

Endogenous antisense RNA curbs CD39 expression in Crohn's disease.

CD39 is an ectonucleotidase that initiates conversion of extracellular nucleotides into immunosuppressive adenosine. CD39 is expressed by regulatory T (Treg)-cells, where it mediates immunosuppression, and by a subset of T-helper (Th) 17-cells, where it limits pathogenicity. CD39 is regulated via single-nucleotide-polymorphisms and upon activation of aryl-hydrocarbon-receptor and oxygen-mediated pathways. Here we report a mechanism of CD39 regulation that relies on the presence of an endogenous antisense RNA, transcribed from the 3'-end of the human CD39/ENTPD1 gene. CD39-specific antisense is increased in Treg and Th17-cells of Crohn's disease patients over controls. It largely localizes in the cell nucleus and regulates CD39 by interacting with nucleolin and heterogeneous-nuclear-ribonucleoprotein-A1. Antisense silencing results in CD39 upregulation in vitro and amelioration of disease activity in a trinitro-benzene-sulfonic-acid model of colitis in humanized NOD/scid/gamma mice. Inhibition/blockade of antisense might represent a therapeutic strategy to restore CD39 along with immunohomeostasis in Crohn's disease.

Reverse phenotype transfer via fecal microbial transplantation in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by a disbalance in the composition of intestinal microbiota. It is not clear whether such dysbiosis is a cause or a consequence of a disease state. Fecal microbiota transplantation (FMT) from a healthy donor to a patient or diseased animal is a valuable tool for targeted modification of microbiome leading to therapeutic response. Positive effect has been shown in therapy of a number of gastrointestinal as well as non-gastrointestinal diseases. In addition, FMT has been successfully used to transfer the diseased phenotype form a donor with the disease to a healthy recipient. However, targeted modification of the microbiome before the onset of colitis has not been shown previously. Based on our preliminary results, we propose the hypothesis of so called reverse phenotype transfer in IBD. This term describes the phenomenon, in which the transplantation of gut microbiota from a donor more sensitive to IBD to a healthy recipient leads to resistance of the recipient to IBD and vice versa. Mice that received FMT from donors with severe colitis have shown improved colitis score compared with mice that received FMT from donors more resistant to development of colitis. Such reverse phenotype transfer has broad implications, especially in terms of preventive medicine. However, detailed mechanisms need to be elucidated to conclude the validity of the phenomenon.