Timon E. Adolph

Paneth cells as a site of origin for intestinal inflammation

The recognition of autophagy related 16-like 1 (ATG16L1) as a genetic risk factor has exposed the critical role of autophagy in Crohn’s disease. Homozygosity for the highly prevalent ATG16L1 risk allele, or murine hypomorphic (HM) activity, causes Paneth cell dysfunction. As Atg16l1HM mice do not develop spontaneous intestinal inflammation, the mechanism(s) by which ATG16L1 contributes to disease remains obscure. Deletion of the unfolded protein response (UPR) transcription factor X-box binding protein-1 (Xbp1) in intestinal epithelial cells, the human orthologue of which harbours rare inflammatory bowel disease risk variants, results in endoplasmic reticulum (ER) stress, Paneth cell impairment and spontaneous enteritis. Unresolved ER stress is a common feature of inflammatory bowel disease epithelium, and several genetic risk factors of Crohn’s disease affect Paneth cells. Here we show that impairment in either UPR (Xbp1ΔIEC) or autophagy function (Atg16l1ΔIEC or Atg7ΔIEC) in intestinal epithelial cells results in each other’s compensatory engagement, and severe spontaneous Crohn’s-disease-like transmural ileitis if both mechanisms are compromised. Xbp1ΔIEC mice show autophagosome formation in hypomorphic Paneth cells, which is linked to ER stress via protein kinase RNA-like endoplasmic reticulum kinase (PERK), elongation initiation factor 2α (eIF2α) and activating transcription factor 4 (ATF4). Ileitis is dependent on commensal microbiota and derives from increased intestinal epithelial cell death, inositol requiring enzyme 1α (IRE1α)-regulated NF-κB activation and tumour-necrosis factor signalling, which are synergistically increased when autophagy is deficient. ATG16L1 restrains IRE1α activity, and augmentation of autophagy in intestinal epithelial cells ameliorates ER stress-induced intestinal inflammation and eases NF-κB overactivation and intestinal epithelial cell death. ER stress, autophagy induction and spontaneous ileitis emerge from Paneth-cell-specific deletion of Xbp1. Genetically and environmentally controlled UPR function within Paneth cells may therefore set the threshold for the development of intestinal inflammation upon hypomorphic ATG16L1 function and implicate ileal Crohn’s disease as a specific disorder of Paneth cells.

Crohn's disease: NOD2, autophagy and ER stress converge

Polymorphisms in NOD2, encoding an intracellular pattern recognition receptor, contribute the largest fraction of genetic risk for Crohns disease among the >40 risk loci identified so far. Autophagy plays a prominent role in the innate immune response towards intracellular bacteria. The discovery of the autophagy genes ATG16L1 and IRGM as risk factors for Crohns disease turned autophagy into the spotlight in inflammatory bowel disease (IBD). Remarkably, NOD2 has recently been identified as a potent autophagy inducer. A physical interaction of NOD2 and ATG16L1 appears to be required for autophagic clearance of intracellular pathogens. Moreover, Crohns disease-associated NOD2 and ATG16L1 variants exhibit a defect in the induction of an autophagic response and hence predict autophagy as a key converging mechanism that leads to Crohns disease. Another pathway that is closely intertwined with autophagy and mutually cross-regulated is the unfolded protein response (UPR), which is induced by endoplasmic reticulum (ER) stress. Genes involved in the UPR (XBP1, ORMDL3) have also been genetically associated with Crohns disease and ulcerative colitis. Moreover, the intestinal epithelium at the interface between host and microbe appears particularly affected by IBD-associated hypomorphic function of autophagy and the UPR. The functional convergence of main genetic risk factors for IBD on these innate immune pathways has hence important implications for the hosts interaction with the microbiota. Moreover, the genetic convergence on these molecular mechanisms may open novel therapeutic options for IBD that deserve further exploration.

ER stress transcription factor Xbp1 suppresses intestinal tumorigenesis and directs intestinal stem cells

X-box–binding protein 1 suppresses tumor formation in the gut by regulating Ire1α and Stat3-mediated regenerative responses in the epithelium as a consequence of ER stress.

The unfolded protein response and gastrointestinal disease

As the inner lining of the gastrointestinal tract, the intestinal epithelium serves an essential role in innate immune function at the interface between the host and microbiota. Given the unique environmental challenges and thus physiologic secretory functions of this surface, it is exquisitely sensitive to perturbations that affect its capacity to resolve endoplasmic reticulum (ER) stress. Genetic deletion of factors involved in the unfolded protein response (UPR), which functions in the resolution of ER stress that arises from misfolded proteins, result in spontaneous intestinal inflammation closely mimicking human inflammatory bowel disease (IBD). This is demonstrated by observations wherein deletion of genes such as Xbp1 and Agr2 profoundly affects the intestinal epithelium and results in spontaneous intestinal inflammation. Moreover, both genes, along with others (e.g., ORDML3) represent genetic risk factors for human IBD, both Crohns disease and ulcerative colitis. Here, we review the current mechanistic understanding for how unresolved ER stress can lead to intestinal inflammation and highlight the findings that implicate ER stress as a genetically affected biological pathway in IBD. We further discuss environmental and microbial factors that might impact on the epitheliums capacity to resolve ER stress and which may constitute exogenous factors that may precipitate disease in genetically susceptible individuals.

Type I interferon signalling in the intestinal epithelium affects Paneth cells, microbial ecology and epithelial regeneration

Objective Intestinal epithelial cells (IECs) at the internal/external interface orchestrate the mucosal immune response. Paneth cells secrete antimicrobial peptides and inflammatory mediators, protect from pathogens and shape the commensal microbiota. Prompted by the genetic association of the locus harbouring the type I interferon (IFN) receptor (IFNAR1) with Crohns disease, and a transcriptional signature for type I IFN signalling in Paneth cells, we studied the function of IFNAR1 in IECs. Design Type I IFN signalling was studied in mice with conditional deletion of Ifnar1 in IECs. Phenotype was characterised at baseline, and gut microbiota composition was assessed by 16S rDNA ribotyping. The role of IFNAR1 was also investigated in experimental colitis induced by dextran sodium sulfate (DSS) and colitis-associated cancer induced by DSS in conjunction with azoxymethane (AOM). Results Ifnar1−/−(IEC) mice displayed expansion of Paneth cell numbers and epithelial hyperproliferation compared with Ifnar1-sufficient littermates. While Ifnar1−/−(IEC) mice did not exhibit spontaneous inflammation or increased severity in DSS colitis compared with Ifnar1+/+(IEC) mice, they exhibited an increased tumour burden in the AOM/DSS model. Both hyperproliferation and tumour promotion were dependent on the microbial flora, as the differences between genotypes were marked upon separately housing mice, but disappeared when Ifnar1−/−(IEC) and Ifnar1+/+(IEC) mice were co-housed. Accordingly, ribotyping revealed marked differences between Ifnar1−/−(IEC) and Ifnar1+/+(IEC) mice that where diminished upon co-housing. Conclusions IFNAR1 in IECs, and Paneth cells in particular, contributes to the regulation of the host–microbiota relationship, with consequences for intestinal regeneration and colitis-associated tumour formation.

Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis

ATG16L1T300A, a major risk polymorphism in Crohn’s disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1&agr;. IRE1&agr; accumulates in Paneth cells of Atg16l1&Dgr;IEC mice, and humans homozygous for ATG16L1T300A exhibit a corresponding increase of IRE1&agr; in intestinal epithelial crypts. In contrast to a protective role of the IRE1&bgr; isoform, hyperactivated IRE1&agr; also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1&Dgr;IEC mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1&agr;, and optineurin deficiency amplifies IRE1&agr; levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1&Dgr;IEC mice as predicted from impaired Paneth cell antimicrobial function, such structural alteration of the microbiota does not trigger ileitis but, rather, aggravates dextran sodium sulfate–induced colitis. Hence, we conclude that defective autophagy in IECs may predispose to CD ileitis via impaired clearance of IRE1&agr; aggregates during ER stress at this site.

Lipocalin 2 Protects from Inflammation and Tumorigenesis Associated with Gut Microbiota Alterations

High mucosal and fecal concentrations of the antimicrobial siderophore-binding peptide Lipocalin-2 (Lcn2) are observed in inflammatory bowel disease. However, Lcn2 function in chronic intestinal inflammation remains unclear. Here, we demonstrate that Lcn2 protects from early-onset colitis and spontaneous emergence of right-sided colonic tumors resulting from IL-10 deficiency. Exacerbated inflammation in Lcn2(-/-)/Il10(-/-) mice is driven by IL-6, which also controls tumorigenesis. Lcn2(-/-)/Il10(-/-) mice exhibit profound alterations in gut microbial composition, which contributes to inflammation and tumorigenesis, as demonstrated by the transmissibility of the phenotype and protection conferred by antibiotics. Specifically, facultative pathogenic Alistipes spp. utilize enterobactin as iron source, bloom in Lcn2(-/-)/Il10(-/-) mice, and are sufficient to induce colitis and right-sided tumors when transferred into Il10(-/-) mice. Our results demonstrate that Lcn2 protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota.

Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease

Objective Alcoholic liver disease (ALD) is a global health problem with limited therapeutic options. Intestinal barrier integrity and the microbiota modulate susceptibility to ALD. Akkermansia muciniphila, a Gram-negative intestinal commensal, promotes barrier function partly by enhancing mucus production. The aim of this study was to investigate microbial alterations in ALD and to define the impact of A. muciniphila administration on the course of ALD. Design The intestinal microbiota was analysed in an unbiased approach by 16S ribosomal DNA (rDNA) sequencing in a Lieber-DeCarli ALD mouse model, and faecal A. muciniphila abundance was determined in a cohort of patients with alcoholic steatohepatitis (ASH). The impact of A. muciniphila on the development of experimental acute and chronic ALD was determined in a preventive and therapeutic setting, and intestinal barrier integrity was analysed. Results Patients with ASH exhibited a decreased abundance of faecal A. muciniphila when compared with healthy controls that indirectly correlated with hepatic disease severity. Ethanol feeding of wild-type mice resulted in a prominent decline in A. muciniphila abundance. Ethanol-induced intestinal A. muciniphila depletion could be restored by oral A. muciniphila supplementation. Furthermore, A. muciniphila administration when performed in a preventive setting decreased hepatic injury, steatosis and neutrophil infiltration. A. muciniphila also protected against ethanol-induced gut leakiness, enhanced mucus thickness and tight-junction expression. In already established ALD, A. muciniphila used therapeutically ameliorated hepatic injury and neutrophil infiltration. Conclusion Ethanol exposure diminishes intestinal A. muciniphila abundance in both mice and humans and can be recovered in experimental ALD by oral supplementation. A. muciniphila promotes intestinal barrier integrity and ameliorates experimental ALD. Our data suggest that patients with ALD might benefit from A. muciniphila supplementation.

Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease

BACKGROUND & AIMS Alcoholic steatohepatitis (ASH) is characterised by neutrophil infiltration that contributes to hepatic injury and disease. Lipocalin-2 (LCN2) was originally identified as siderophore binding peptide in neutrophils, which exerted tissue protective effects in several disease models. Here we investigate the role of LCN2 in the pathogenesis of alcohol-induced liver injury. METHODS We compared hepatic LCN2 expression in ASH patients, alcoholic cirrhosis patients without evidence of ASH and patients with non-alcoholic fatty liver disease (NAFLD; i.e. simple steatosis). To mechanistically dissect LCN2 function in alcohol-induced liver injury, we subjected wild-type (WT) and Lcn2-deficient (Lcn2(-/-)) mice to the Lieber-DeCarli diet containing 5% ethanol (EtOH) or isocaloric maltose. Adoptive transfer experiments were performed to track neutrophil migration. Furthermore, we tested the effect of antibody-mediated LCN2 neutralisation in an acute model of ethanol-induced hepatic injury. RESULTS Patients with ASH exhibited increased hepatic LCN2 immunoreactivity compared to patients with alcoholic cirrhosis or simple steatosis, which mainly localised to neutrophils. Similarly, ethanol-fed mice exhibited increased LCN2 expression that mainly localised to leukocytes and especially neutrophils. Lcn2(-/-) mice were protected from alcoholic liver disease (ALD) as demonstrated by reduced neutrophil infiltration, liver injury and hepatic steatosis compared to WT controls. Adoptive transfers revealed that neutrophil-derived LCN2 critically determines hepatic neutrophil immigration and persistence during chronic alcohol exposure. Antibody-mediated neutralisation of LCN2 protected from hepatic injury and neutrophilic infiltration after acute alcohol challenge. CONCLUSIONS LCN2 drives ethanol-induced neutrophilic inflammation and propagates the development of ALD. Despite a critical role for LCN2 in immunity and infection, pharmacological neutralisation of LCN2 might be of promise in ALD.

Influence of the human intestinal microbiome on obesity and metabolic dysfunction.

Purpose of review Recent studies have suggested that there may be a strong link between the gut microbiota, energy extraction and body metabolism. Recent findings Evidence is accumulating that the intestinal microbiota, in addition to other major factors such as diet and host genetics, contributes to obesity, metabolic dysfunction and diabetes. Both preclinical experimental and human studies have shown that obesity and metabolic dysfunction are characterized by a profound dysbiosis. Several human metagenome-wide association studies have demonstrated highly significant correlations of certain members of intestinal microbiota with obesity and type 2 diabetes. In addition dietary factors that substantially affect microbial composition, microbiota disruption, and the consequence of early-life antibiotic use, may contribute to childhood obesity and metabolic dysfunction. Further evidence for an association between microbiota and metabolic dysfunction has been derived from studies in pregnancy demonstrating that major gut microbial shifts occur during pregnancy thereby affecting host metabolism. In particular, the high rate of obesity following caesarean section could be partially explained by functional alterations in the intestinal microbiota. Summary Obesity and associated metabolic dysfunction emerge from disturbed interactions between the intestinal microbiota, dietary changes and host immune functions. A better understanding of this relationship might lead to better therapies for human metabolic and inflammatory diseases in the future.