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Background & Aims Tumor necrosis factor alpha (TNFα) is considered a major tissue damage-promoting effector in Crohn’s disease (CD) pathogenesis. Patient-derived intestinal organoid (enteroid) recapitulates the disease-specific characteristics of the intestinal epithelium. This study aimed to evaluate the intestinal epithelial responses to TNFα in enteroids derived from healthy controls and compare them with those of CD patient-derived enteroids. Methods Human enteroids derived from patients with CD and controls were treated with TNFα (30 ng/mL), and cell viability and gene expression patterns were evaluated. Results TNFα induced MLKL-mediated necroptotic cell death, which was more pronounced in CD patient-derived enteroids than in control enteroids. Immunohistochemistry and RNA sequencing revealed that treatment with TNFα caused expansion of the intestinal stem cell (ISC) populations. However, expanded ISC subpopulations differed in control and CD patient-derived enteroids, with LGR5+ active ISCs in control enteroids and reserve ISCs, such as BMI1+ cells, in CD patient-derived enteroids. In single-cell RNA sequencing, LGR5+ ISC-enriched cell cluster showed strong expression of TNFRSF1B (TNFR2) and cyclooxygenase-prostaglandin E2 (PGE2) activation. In TNFα-treated CD patient-derived enteroids, exogenous PGE2 (10 nmol/L) induced the expansion of the LGR5+ ISC population and improved organoid-forming efficiency, viability, and wound healing. Conclusions TNFα increases necroptosis of differentiated cells and induces the expansion of LGR5+ ISCs. In CD patient-derived enteroids, TNFα causes LGR5+ stem cell dysfunction (expansion failure), and exogenous PGE2 treatment restored the functions of LGR5+ stem cells. Therefore, PGE2 can be used to promote mucosal healing in patients with CD., Graphical abstract

Background & Aims Although nonalcoholic fatty liver disease (NAFLD) is closely associated with obesity, the role of adipose tissue in NAFLD is not well-understood. Because autophagy has been reported to be involved in the degradation of lipid droplets, we investigated the role of adipose tissue autophagy in the liver pathogenesis of NAFLD. Methods C57BL/6J mice and adipocyte-specific Atg7-knockout mice (Adipoq-Atg7 KO mice) were fed a high-fat diet (HFD). Results HFD feeding for up to 4 months increased both inguinal and epididymal white adipose tissue (iWAT and eWAT, respectively; the former represents subcutaneous fat, and the latter represents visceral fat) in mice. After HFD feeding, autophagy flux in both types of white adipose tissue was increased, and the levels of Rubicon, a negative autophagy regulator, were decreased, suggesting autophagy promotion. Adipoq-Atg7 KO mice exhibited suppressed autophagy in both iWAT and eWAT. Adipocyte-specific Atg7 KO enhanced HFD-induced iWAT hypertrophy. On the other hand, eWAT levels in Adipoq-Atg7 KO mice were increased after 1 month of HFD feeding but decreased after 4 months of HFD feeding compared with those in wild-type controls. Cleaved caspase 3 and JNK pathway protein expression in eWAT was increased without cytokine elevation in Adipoq-Atg7 KO mice fed an HFD compared with wild-type mice fed an HFD. Adipocyte-specific Atg7 KO decreased serum free fatty acid levels and ameliorated HFD-induced steatosis, liver inflammation, and fibrosis. Conclusions Autophagy was enhanced in the white adipose tissues of mice fed an HFD. Autophagy inhibition in white adipose tissues ameliorated the liver pathology of NAFLD via adipose-liver crosstalk., Graphical abstract

Background & Aims: CD36 has immunometabolic actions and is abundant in the small intestine on epithelial, endothelial, and immune cells. We examined the role of CD36 in gut homeostasis by using mice null for CD36 (CD36KO) and with CD36 deletion specific to enterocytes (Ent-CD36KO) or endothelial cells (EC-CD36KO). Methods: Intestinal morphology was evaluated by using immunohistochemistry and electron microscopy. Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors, and cyclooxygenase-2. Barrier integrity was assessed from circulating lipopolysaccharide and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized by using two-photon microscopy. Results: The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix accumulation with increased expression of extracellular matrix proteins, evidence of neutrophil infiltration, inflammation, and compromised barrier function. Electron microscopy showed shortened desmosomes with decreased desmocollin 2 expression. Systemically, leukocytosis and neutrophilia were present together with 80% reduction of anti-inflammatory Ly6Clow monocytes. Bone marrow transplants supported the primary contribution of non-hematopoietic cells to the inflammatory phenotype. Specific deletion of endothelial but not of enterocyte CD36 reproduced many of the gut phenotypes of germline CD36KO mice including fibronectin deposition, increased interleukin 6, neutrophil infiltration, desmosome shortening, and impaired epithelial barrier function. Conclusions: CD36 loss results in chronic neutrophil infiltration of the gut, impairs barrier integrity, and systemically causes subclinical inflammation. Endothelial cell CD36 deletion reproduces the major intestinal phenotypes. The findings suggest an important role of the endothelium in etiology of gut inflammation and loss of epithelial barrier integrity. Keywords: Neutrophils, Endothelium, Fibronectin, Collagen

Background & Aims Mitochondria exist in a constantly remodelling network, and excessive fragmentation can be pathophysiological. Mitochondrial dysfunction can accompany enteric inflammation, but any contribution of altered mitochondrial dynamics (ie, fission/fusion) to gut inflammation is unknown. We hypothesized that perturbed mitochondrial dynamics would contribute to colitis. Methods Quantitative polymerase chain reaction for markers of mitochondrial fission and fusion was applied to tissue from dextran sodium sulfate (DSS)-treated mice. An inhibitor of mitochondrial fission, P110 (prevents dynamin related protein [Drp]-1 binding to mitochondrial fission 1 protein [Fis1]) was tested in the DSS and di-nitrobenzene sulfonic acid (DNBS) models of murine colitis, and the impact of DSS ± P110 on intestinal epithelial and macrophage mitochondria was assessed in vitro. Results Analysis of colonic tissue from mice with DSS-colitis revealed increased mRNA for molecules associated with mitochondrial fission (ie, Drp1, Fis1) and fusion (optic atrophy factor 1) and increased phospho-Drp1 compared with control. Systemic delivery of P110 in prophylactic or treatment regimens reduced the severity of DSS- or DNBS-colitis and the subsequent hyperalgesia in DNBS-mice. Application of DSS to epithelial cells or macrophages caused mitochondrial fragmentation. DSS-evoked perturbation of epithelial cell energetics and mitochondrial fragmentation, but not cell death, were ameliorated by in vitro co-treatment with P110. Conclusions We speculate that the anti-colitic effect of systemic delivery of the anti-fission drug, P110, works at least partially by maintaining enterocyte and macrophage mitochondrial networks. Perturbed mitochondrial dynamics can be a feature of intestinal inflammation, the suppression of which is a potential novel therapeutic direction in inflammatory bowel disease., Graphical abstract