Elevated apoptosis impairs epithelial cell turnover and shortens villi in TNF-driven intestinal inflammation

The intestinal epithelial monolayer, at the boundary between microbes and the host immune system, plays an important role in the development of inflammatory bowel disease (IBD), particularly as a target and producer of pro-inflammatory TNF. Chronic overexpression of TNF leads to IBD-like pathology over time, but the mechanisms driving early pathogenesis events are not clear. We studied the epithelial response to inflammation by combining mathematical models with in vivo experimental models resembling acute and chronic TNF-mediated injury. We found significant villus atrophy with increased epithelial cell death along the crypt-villus axis, most dramatically at the villus tips, in both acute and chronic inflammation. In the acute model, we observed overexpression of TNF receptor I in the villus tip rapidly after TNF injection and concurrent with elevated levels of intracellular TNF and rapid shedding at the tip. In the chronic model, sustained villus atrophy was accompanied by a reduction in absolute epithelial cell turnover. Mathematical modelling demonstrated that increased cell apoptosis on the villus body explains the reduction in epithelial cell turnover along the crypt-villus axis observed in chronic inflammation. Cell destruction in the villus was not accompanied by changes in proliferative cell number or division rate within the crypt. Epithelial morphology and immunological changes in the chronic setting suggest a repair response to cell damage although the villus length is not recovered. A better understanding of how this state is further destabilised and results in clinical pathology resembling IBD will help identify suitable pathways for therapeutic intervention

Defective ATG16L1-mediated removal of IRE1α drives Crohn's disease-like ileitis.

ATG16L1$^{T300A}$, 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 IRE1α. IRE1α accumulates in Paneth cells of Atg16l1$^{ΔIEC}$ mice, and humans homozygous for ATG16L1$^{T300A}$ exhibit a corresponding increase of IRE1α in intestinal epithelial crypts. In contrast to a protective role of the IRE1β isoform, hyperactivated IRE1α also drives a similar ileitis developing earlier in life in Atg16l1;Xbp1$^{ΔIEC}$ mice, in which ER stress is induced by deletion of the unfolded protein response transcription factor XBP1. The selective autophagy receptor optineurin interacts with IRE1α, and optineurin deficiency amplifies IRE1α levels during ER stress. Furthermore, although dysbiosis of the ileal microbiota is present in Atg16l1;Xbp1$^{ΔIEC}$ 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 IRE1α aggregates during ER stress at this site.This study was supported by the European Research Council under the European Community’s Seventh Framework Program (grant FP7/2007-2013)/ERC, agreement no. 260961 to A. Kaser and grant HORIZON2020/ERC, agreement no. 648889 to A. Kaser), the Wellcome Trust (Investigator Award 106260/Z/14/Z to A. Kaser and Principal Research Fellowship 2008/Z/16/Z to D. Ron), the Cambridge Biomedical Research Centre (A. Kaser), a Medical Research Council PhD for clinicians training fellowship (grant MR/N001893/1 to J. Bhattacharyya), fellowships from the European Crohn’s and Colitis Organization (M. Tschurtschenthaler and T.E. Adolph), the Research Training Group Genes, Environment, and Inflammation supported by the Deutsche Forschungsgemeinschaft (grant RTG 1743/1 to P. Rosenstiel), the SFB877 subproject B9 and CLVIII ExC 306 Inflammation at Interfaces (P. Rosenstiel), and the National Institutes of Health (grants DK044319, DK051362, DK053056, and DK088199 to the Harvard Digestive Diseases Center and grant DK0034854 to R.S. Blumberg)

Beneficial effects of Limosilactobacillus fermentum CECT 5716 administration to infants delivered by Cesarean Section

Cesarean section (CS) disrupts the natural microbiota colonization process in infants, which might compromise immune system maturation, leading to a higher risk of infections. We evaluated the effect of the probiotic Limosilactobacillus (L.) fermentum CECT 5716 on the incidence of gastrointestinal and respiratory infections in the CS infant subgroups (n = 173) of three randomized clinical trials in which this probiotic strain was demonstrated to be safe and effective for preventing infections. Therefore, the data for the CS infants were extracted to obtain the incidence rate ratio (IRR) and 95% CI for gastrointestinal and respiratory infections for each study and were then combined to obtain a pooled IRR and 95% CI using the generic inverse variance method. There was a significant reduction of 73% in the incidence of gastrointestinal infections in CS infants receiving L. fermentum CECT 5716 compared with those receiving the control formula [n = 173, IRR: 0.27 (0.13, 0.53), p = 0.0002]. Regarding respiratory infections, although pooled results showed a reduction of 14% in the probiotic group, the difference was not statistically significant [n = 173, IRR (95% CI): 0.86 (0.67, 1.11), p = 0.25]. In conclusion, the administration of L. fermentum CECT 5716 to CS-born infants protects them from gastrointestinal infections by reducing the risk by up to 73% in this population

Microbial Colonization in Adulthood Shapes the Intestinal Macrophage Compartment

Background and Aims: Contact with distinct microbiota early in life has been shown to educate the mucosal immune system, hence providing protection against immune-mediated diseases. However, the impact of early versus late colonization with regard to the development of the intestinal macrophage compartment has not been studied so far. Methods: Germ-free mice were colonized with specific-pathogen-free [SPF] microbiota at the age of 5 weeks. The ileal and colonic macrophage compartment were analysed by immunohistochemistry, flow cytometry, and RNA sequencing 1 and 5 weeks after colonization and in age-matched SPF mice, which had had contact with microbiota since birth. To evaluate the functional differences, dextran sulfate sodium [DSS]-induced colitis was induced, and barrier function analyses were undertaken. Results: Germ-free mice were characterized by an atrophied intestinal wall and a profoundly reduced number of ileal macrophages. Strikingly, morphological restoration of the intestine occurred within the first week after colonization. In contrast, ileal macrophages required 5 weeks for complete restoration, whereas colonic macrophages were numerically unaffected. However, following DSS exposure, the presence of microbiota was a prerequisite for colonic macrophage infiltration. One week after colonization, mild colonic inflammation was observed, paralleled by a reduced inflammatory response after DSS treatment, in comparison with SPF mice. This attenuated inflammation was paralleled by a lack of TNF alpha production of LPS-stimulated colonic macrophages from SPF and colonized mice, suggesting desensitization of colonized mice by the colonization itself. Conclusions: This study provides the first data indicating that after colonization of adult mice, the numeric, phenotypic, and functional restoration of the macrophage compartment requires the presence of intestinal microbiota and is time dependent

Circular synthesized CRISPR/Cas gRNAs for functional interrogations in the coding and noncoding genome

Current technologies used to generate CRISPR/Cas gene perturbation reagents are labor intense and require multiple ligation and cloning steps. Furthermore, increasing gRNA sequence diversity negatively affects gRNA distribution, leading to libraries of heterogeneous quality. Here, we present a rapid and cloning-free mutagenesis technology that can efficiently generate covalently-closed-circular-synthesized (3Cs) CRISPR/Cas gRNA reagents and that uncouples sequence diversity from sequence distribution. We demonstrate the fidelity and performance of 3Cs reagents by tailored targeting of all human deubiquitinating enzymes (DUBs) and identify their essentiality for cell fitness. To explore high-content screening, we aimed to generate the largest up-to-date gRNA library that can be used to interrogate the coding and noncoding human genome and simultaneously to identify genes, predicted promoter flanking regions, transcription factors and CTCF binding sites that are linked to doxorubicin resistance. Our 3Cs technology enables fast and robust generation of bias-free gene perturbation libraries with yet unmatched diversities and should be considered an alternative to established technologies