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Fatty liver disease or the buildup of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of fat and alcohol diet towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified 4 relevant protein-protein interaction network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover 4 additional CpG loci and validate that aging related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

A close interaction between gut immune responses and distant organ-specific autoimmunity including the CNS in multiple sclerosis has been established in recent years. This so-called gut–CNS axis can be shaped by dietary factors, either directly or via indirect modulation of the gut microbiome and its metabolites. Here, we report that dietary supplementation with conjugated linoleic acid, a mixture of linoleic acid isomers, ameliorates CNS autoimmunity in a spontaneous mouse model of multiple sclerosis, accompanied by an attenuation of intestinal barrier dysfunction and inflammation as well as an increase in intestinal myeloid-derived suppressor-like cells. Protective effects of dietary supplementation with conjugated linoleic acid were not abrogated upon microbiota eradication, indicating that the microbiome is dispensable for these conjugated linoleic acid-mediated effects. Instead, we observed a range of direct anti-inflammatory effects of conjugated linoleic acid on murine myeloid cells including an enhanced IL10 production and the capacity to suppress T-cell proliferation. Finally, in a human pilot study in patients with multiple sclerosis (n = 15, under first-line disease-modifying treatment), dietary conjugated linoleic acid-supplementation for 6 months significantly enhanced the anti-inflammatory profiles as well as functional signatures of circulating myeloid cells. Together, our results identify conjugated linoleic acid as a potent modulator of the gut–CNS axis by targeting myeloid cells in the intestine, which in turn control encephalitogenic T-cell responses., Fleck et al. report that dietary supplementation with conjugated linoleic acid elicits anti-inflammatory and suppressive myeloid cell responses in the intestine, resulting in amelioration of CNS autoimmunity in mice and a beneficial modulation of myeloid cell responses in patients with relapsing-remitting multiple sclerosis.

Background & Aims Loss-of-function variants in nucleotide-binding oligomerization domain-containing protein 2 (NOD2) impair the recognition of the bacterial cell wall component muramyl-dipeptide and are associated with an increased risk for developing Crohn’s disease. Likewise, exposure to antibiotics increases the individual risk for developing inflammatory bowel disease. Here, we studied the long-term impact of NOD2 on the ability of the gut bacterial and fungal microbiota to recover after antibiotic treatment. Methods Two cohorts of 20-week-old and 52-week-old wild-type (WT) C57BL/6J and NOD2 knockout (Nod2-KO) mice were treated with broad-spectrum antibiotics and fecal samples were collected to investigate temporal dynamics of the intestinal microbiota (bacteria and fungi) using 16S ribosomal RNA and internal transcribed spacer 1 sequencing. In addition, 2 sets of germ-free WT mice were colonized with either WT or Nod2-KO after antibiotic donor microbiota and the severity of intestinal inflammation was monitored in the colonized mice. Results Antibiotic exposure caused long-term shifts in the bacterial and fungal community composition. Genetic ablation of NOD2 was associated with delayed body weight gain after antibiotic treatment and an impaired recovery of the bacterial gut microbiota. Transfer of the postantibiotic fecal microbiota of Nod2-KO mice induced an intestinal inflammatory response in the colons of germ-free recipient mice compared with respective microbiota from WT controls based on histopathology and gene expression analyses. Conclusions Our data show that the bacterial sensor NOD2 contributes to intestinal microbial community composition after antibiotic treatment and may add to the explanation of how defects in the NOD2 signaling pathway are involved in the etiology of Crohn’s disease., Graphical abstract

Viruses and bacteriophages have a strong impact on intestinal barrier function and the composition and functional properties of commensal bacterial communities. To improve our understanding of the role of the enteric intestinal virome, we longitudinally characterized the virome in fecal samples from wild-type (WT) C57BL/6J and knock-out (KO) NOD2 mice in response to an antibiotic perturbation. Sequencing of viral-like-particles (VLPs) demonstrated both a high diversity and high inter-individual variation of the murine gut virome composed of eukaryotic viruses and bacteriophages. Antibiotics also had a significant impact on the gut murine virome causing a delayed resilience independent of genotype. However, compositional shifts in the virome and bacteriome were highly correlated, suggesting that the loss of specific phages may contribute to a dysregulation of the bacterial community composition. Bacteriophage species may be playing an important role in either upregulating or downregulating the bacterial community, and restoring a healthy virome may therefore be a central goal of microbiota-targeted therapies.

The interaction between the mammalian host and its resident gut microbiota is known to license adaptive immune responses. Nutritional constituents strongly influence composition and functional properties of the intestinal microbial communities. Here, we report that omission of a single essential amino acid - tryptophan – from the diet abrogates CNS autoimmunity in a mouse model of multiple sclerosis. Dietary tryptophan restriction results in impaired encephalitogenic T cell responses and is accompanied by a mild intestinal inflammatory response and a profound phenotypic shift of gut microbiota. Protective effects of dietary tryptophan restriction are abrogated in germ-free mice, but are independent of canonical host sensors of intracellular tryptophan metabolites. We conclude that dietary tryptophan restriction alters metabolic properties of gut microbiota, which in turn have an impact on encephalitogenic T cell responses. This link between gut microbiota, dietary tryptophan and adaptive immunity may help to develop therapeutic strategies for protection from autoimmune neuroinflammation., Food intake shapes intestinal microbiome composition, which in turn shapes adaptive immune responses. Here the authors show that dietary tryptophan restriction (DTR) protects mice from subsequent autoimmune neuropathology challenge by altering intestinal microbiota, highlighting the potential of diet-regulated microbiota to prevent immune pathology.

The involvement of the IL-23/IL23R pathway is well known in the disease pathogenesis of sarcoidosis and other inflammatory diseases. To date, the pathogenic mechanism of IL-23 is most notably described on CD4+ Th17 lymphocytes. However, the function of the IL23R on myeloid cells in sarcoidosis is poorly understood. Thus, the aim of the study is to investigate the role of the IL23R on myeloid cell in pulmonary granuloma formation. Methods: We generated IL23RLysMCre mice lacking the IL23R gene in myeloid cells. The importance of IL23R in myeloid cells for the development of sarcoidosis was studied in a mouse model of inflammatory lung granuloma formation through embolization of PPD from Mycobacterium bovis-coated Sepharose beads into previously PPD-immunized mice. In addition the function of IL23R on myeloid cells was studied in LPS or IFNγ stimulated BMDMs and BMDCs. The mRNA and protein expression levels of relevant cytokines were analyzed by RT-PCR (TaqMan) and ELISA. The composition of immune cells in BALF was quantified by flow cytometry and alteration in granuloma sizes were observed by H&E stained lung sections. Results: Mycobacterium Ag-elicted pulmonary granulomas tend to be smaller in IL23RLysMCre mice and NF-κB dependent Th1 cytokines in the murine lungs are reduced compared to wildtype mice. In line, we observed that IL23R-deficient bone marrow-derived macrophages show a reduced production of Th1 cytokines after LPS stimulation. Conclusion: We here for the first time demonstrate a role for IL23R on myeloid cells in pulmonary inflammation and granuloma formation. Our findings provide essential insights in the pathogenesis of inflammatory lung diseases like sarcoidosis, which might be useful for the development of novel therapeutics targeting distinct immunological pathways like IL-23/IL23R.

Summary Epigenetic modifications (e.g. DNA methylation) in NAFLD and their contribution to disease progression and extrahepatic complications are poorly explored. Here, we use an integrated epigenome and transcriptome analysis of mouse NAFLD hepatocytes and identify alterations in glyoxylate metabolism, a pathway relevant in kidney damage via oxalate release—a harmful waste product and kidney stone-promoting factor. Downregulation and hypermethylation of alanine-glyoxylate aminotransferase (Agxt), which detoxifies glyoxylate, preventing excessive oxalate accumulation, is accompanied by increased oxalate formation after metabolism of the precursor hydroxyproline. Viral-mediated Agxt transfer or inhibiting hydroxyproline catabolism rescues excessive oxalate release. In human steatotic hepatocytes, AGXT is also downregulated and hypermethylated, and in NAFLD adolescents, steatosis severity correlates with urinary oxalate excretion. Thus, this work identifies a reduced capacity of the steatotic liver to detoxify glyoxylate, triggering elevated oxalate, and provides a mechanistic explanation for the increased risk of kidney stones and chronic kidney disease in NAFLD patients.

The disintegrin metalloprotease ADAM17 has a critical role in intestinal inflammation and regeneration in mice, as illustrated by the dramatically increased susceptibility of ADAM17 hypomorphic (ADAM17ex/ex) mice to dextran sulfate sodium (DSS)-induced colitis. Similarly, necroptosis has been implicated in inflammatory responses in the intestine. In this study, we have investigated the contribution of necroptosis to ADAM17-regulated intestinal inflammation in vivo by crossing ADAM17ex/ex mice with mice that lack the necroptotic core protein RIPK3. Despite the loss of RIPK3, ADAM17ex/ex/RIPK3-/- mice showed the same increased susceptibility as ADAM17ex/ex mice in both acute and chronic models of DSS-induced colitis. Mice of both genotypes revealed comparable results with regard to weight loss, disease activity index and colitis-associated changes of inner organs. Histopathological analyses confirmed similar tissue destruction, loss of barrier integrity, immune cell infiltration, and cell death; serum analyses revealed similar levels of the pro-inflammatory cytokine KC. Resolving these unexpected findings, ADAM17ex/ex mice did not show phosphorylation of RIPK3 and its necroptotic interaction partner MLKL during DSS-induced colitis, although both proteins were clearly expressed. Consistent with these findings, murine embryonic fibroblasts derived from ADAM17ex/ex mice were protected from tumor necrosis factor (TNF)-induced necroptosis and failed to show phosphorylation of MLKL and RIPK3 after induction of necroptosis by TNF, revealing a novel, undescribed role of the protease ADAM17 in necroptosis.

Endoplasmic reticulum (ER) stress and autophagy are tightly controlled cellular processes, which are responsible for maintaining protein homeostasis in a cell. Impairment of the interlinking pathways have been implicated in a number of human diseases, prominently in inflammatory bowel disease, where genetic variants in several independent autophagy and ER stress related loci have been associated to increased disease risk. Autophagy is a selective quality control process, which governs the integrity of the cell by removal of aged organelles and proteins via the lysosome, but recently has been shown to actively license the outcome of other signaling pathways by guiding the proteolytic removal of signaling protein complexes (adaptophagy). In this review, we summarize our knowledge on regulated proteolytic events involved in ER stress responses and autophagy, their interplay and potential regulatory effects with a particular focus on intestinal inflammation. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1 beta by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1 beta generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-Delta LysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1 beta-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

Dysregulated autophagy and ER stress are involved in the etiology of human IBD. Aden et al. show that loss of ATG16L1 function renders intestinal epithelial cells vulnerable to IL-22–induced ER stress and necroptosis via STING signaling, which induces ileal inflammation in vivo., A coding variant of the inflammatory bowel disease (IBD) risk gene ATG16L1 has been associated with defective autophagy and deregulation of endoplasmic reticulum (ER) function. IL-22 is a barrier protective cytokine by inducing regeneration and antimicrobial responses in the intestinal mucosa. We show that ATG16L1 critically orchestrates IL-22 signaling in the intestinal epithelium. IL-22 stimulation physiologically leads to transient ER stress and subsequent activation of STING-dependent type I interferon (IFN-I) signaling, which is augmented in Atg16l1ΔIEC intestinal organoids. IFN-I signals amplify epithelial TNF production downstream of IL-22 and contribute to necroptotic cell death. In vivo, IL-22 treatment in Atg16l1ΔIEC and Atg16l1ΔIEC/Xbp1ΔIEC mice potentiates endogenous ileal inflammation and causes widespread necroptotic epithelial cell death. Therapeutic blockade of IFN-I signaling ameliorates IL-22–induced ileal inflammation in Atg16l1ΔIEC mice. Our data demonstrate an unexpected role of ATG16L1 in coordinating the outcome of IL-22 signaling in the intestinal epithelium., Graphical Abstract

Summary The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.

A plethora of functional and genetic studies have suggested a key role for the IL-23 pathway in chronic intestinal inflammation. Currently, pathogenic actions of IL-23 have been ascribed to specific effects on immune cells. Herein, we unveil a protective role of IL-23R signaling. Mice deficient in IL-23R expression in intestinal epithelial cells (Il23R(ΔIEC)) have reduced Reg3b expression, show a disturbed colonic microflora with an expansion of flagellated bacteria, and succumb to DSS colitis. Surprisingly, Il23R(ΔIEC) mice show impaired mucosal IL-22 induction in response to IL-23. αThy-1 treatment significantly deteriorates colitis in Il23R(ΔIEC) animals, which can be rescued by IL-22 application. Importantly, exogenous Reg3b administration rescues DSS-treated Il23R(ΔIEC) mice by recruiting neutrophils as IL-22-producing cells, thereby restoring mucosal IL-22 levels. The study identifies a critical barrier-protective immune pathway that originates from, and is orchestrated by, IL-23R signaling in intestinal epithelial cells.