Excessive visceral fat is an essential risk factor of metabolic dysfunction-associated steatotic liver disease (MASLD). Yet, the cellular and molecular mechanisms implicated in the correlation between visceral fat metabolism and hepatic steatosis remain poorly understood. Here we report post-expansive epididymal adipose tissue (EAT) atrophy and hepatic steatosis in mice fed a high-fat diet (HFD) primarily due to elevated adipocyte lipolysis in EAT. Mast cell (MC) accumulation in EAT represents a lipolysis-associated feature. Pharmacological stabilization of MCs suppresses EAT adipocyte lipolysis, and improves EAT atrophy and hepatic steatosis. MC-derived serotonin (5-HT) correlates with visceral adipose tissue (VAT) lipolysis in HFD-fed mice as well as in MASLD patients. Conditional deletion of 5-HT from MCs or its receptor HTR2b on adipocytes demonstrates that MC-derived 5-HT promotes adipocyte lipolysis, EAT atrophy, and hepatic steatosis by binding on adipocyte HTR2b. These results suggest that MCs and MC-derived 5-HT are potential therapeutic targets for obesity-associated MASLD. ### Competing Interest Statement The authors have declared no competing interest. the National Natural Science Foundation of China, 32070757, 32200630 the Fundamental Research Funds for the Central Universities, PA2025GDGP0026, JZ2024HGTB0242

Objective Immune checkpoint inhibitors (ICIs) have revolutionised cancer treatment and patients' survival. However, ICIs also cause severe immune-related adverse events, notably colitis, resulting in ICIs therapy discontinuation and tumour immunotherapy failure. This study investigates long myosin light chain kinase 1 (MLCK1), a known regulator of tight junction and gut permeability, to elucidate the mechanisms underlying ICI-mediated colitis and identify approaches to reduce this toxicity.Design This study employed an integrated approach, using clinical samples, in vivo models and in vitro organoid systems. Biopsies from patients with ICIs colitis were profiled using single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics. To recapitulate human ICIs colitis, we used a wild mouse microbiota (WildR) model, alongside various genetically modified and tumour-bearing models (including melanoma and MC38). Furthermore, mechanisms were investigated through organoid-immune cell co-cultures. Finally, surface plasmon resonance, microscale thermophoresis, full-spectrum flow cytometry, bulk RNA sequencing, immunostaining, ELISA and gut permeability assays were performed to comprehensively delineate the underlying molecular mechanism.Results Tight junction integrity was compromised in both human ICIs colitis and our WildR mouse model. We determined that this barrier dysfunction is driven by activation of the MLCK1-mediated leak pathway following ICI treatment. Using murine models, we identified tumour necrosis factor secreted by CD8+ and CD4+ T cells as an upstream regulator that induces colitis through this MLCK-dependent mechanism, as genetic deletion of MLCK preserved the tight junction structure and ameliorated the inflammation and ICIs colitis. Furthermore, a pharmacological screen identified the small molecule Epicatechin, which blocks MLCK1-FKBP8 interaction and inhibits the recruitment of MLCK1 to the perijunctional actomyosin ring and prevents the intestinal barrier loss. Finally, treatment with Epicatechin mitigated ICI-induced colitis without compromising the antitumour efficacy of the immunotherapy.Conclusions These findings suggest that MLCK1-dependent tight junction regulation is essential for ICIs colitis, positioning barrier restoration as a potential therapeutic strategy.

Intestinal stem cells (ISCs) reside in regionally variable niches that provide diverse microenvironmental cues such as tissue oxygen status, and morphogen signaling. Integration of these cues with ISC metabolism and fate remains poorly understood. Here, we show that cellular redox balance orchestrates niche factors with metabolic state to govern cell fate decisions. We demonstrate that hypoxia and Wnt signaling synergistically restrict the reactive oxygen species generating enzyme NADPH oxidase 1 (NOX1) regionally to the crypt base in the distal colon. NOX1 enables maintenance of an oxidative cell state that licenses cell cycle entry, altering the balance of asymmetric ISC self-renewal and lineage commitment. Mechanistically, cell redox state directs a self-reinforcing circuit that connects hypoxia inducible factor 1α-dependent signaling with post-translational regulation of the metabolic enzyme isocitrate dehydrogenase 1. Our studies show redox balance acts as a cellular rheostat that is central and causative for metabolic control of the ISC cell-cycle.

The intestinal epithelium is a rapidly regenerating tissue dependent on resident stem cell self-renewal and differentiation. The reactive oxygen species (ROS) generating enzyme NADPH oxidase 1 (NOX1) is spatially restricted to the crypt base epithelium in the distal colon. Using NOX1-deletion, along with cell-specific redox reporter mice, lineage tracing, single-cell transcriptomics, cysteine proteomics, and cell metabolic studies, we investigated the role of redox balance in colonic stem cell function. We show that distal colonic stem cells uniquely require NOX1-generated ROS to maintain a relatively oxidized state that promotes stem cell self-renewal. Mechanistically, this occurs through a cellular circuit that connects hypoxia inducible factor 1 (HIF1α)-dependent signaling to ROS regulation of isocitrate dehydrogenase 1 (IDH1), to maintain efficient cell cycle entry and cell fate determination in the relatively hypoxic distal colonic niche environment. Our studies therefore provide a basis for understanding regeneration dynamics and disease propensity in the distal large intestine. ### Competing Interest Statement J.O.M. reports compensation for consulting services with Tessel Biosciences and Radera Biotherapeutics.

Commensal bacteria of the Bacteroidetes phylum are the primary producers of sphingolipids in the gut lumen. These lipids serve dual roles as bacterial virulence factors and regulators of the host mucosal immune system, including regulatory T cells and invariant natural killer T cells (iNKT). Sphingolipid composition is significantly altered in fecal samples of patients with inflammatory bowel disease (IBD). However, the specific mechanisms by which bacterial sphingolipids modulate mucosal homeostasis and regulate intestinal inflammation remain unclear. In this study, we investigated the impact of bacterial sphingolipids on intestinal inflammation by mono-colonizing mice with Bacteroides fragilis strains that either express or lack sphingolipids during DSS-induced colitis. We discovered that B. fragilis sphingolipids exacerbate intestinal inflammation. Mice mono-colonized with B. fragilis lacking sphingolipids exhibited less severe DSS-induced colitis. This amelioration of colitis was associated with increased production of interleukin-22 (IL-22) by innate lymphoid cell type 3 (ILC3). Consistent with the inhibitory effect of sphingolipids on IL-22 production, mice colonized with B. fragilis lacking sphingolipids showed enhanced epithelial STAT3 activity, intestinal cell proliferation, and antimicrobial peptide production following DSS treatment compared to those colonized with B. fragilis producing sphingolipids. Additionally, colitis severity in mice colonized with B. fragilis lacking sphingolipids was exacerbated upon IL-22 blockade. Furthermore, our study reveals that bacterial sphingolipids restrict epithelial IL-18 production following DSS treatment and interfere with IL-22 production by a subset of ILC3 cells expressing both the interleukin-18 receptor (IL-18R) and major histocompatibility complex class II (MHC II). These findings indicate that B. fragilis -derived sphingolipids exacerbate mucosal inflammation by impeding epithelial IL-18 expression, resulting in compromised production of IL-22 by ILC3 cells. Highlights B. fragilis -derived sphingolipids exacerbate DSS-induced colitis in mono-colonized C57BL/6 mice. B. fragilis -derived sphingolipids constrain ILC3-derived IL-22, leading to reduced colonic epithelial cell proliferation and compromised barrier function. B. fragilis -derived sphingolipids restrict epithelial NLRC4 inflammasome activation and IL-18 secretion. B. fragilis -derived sphingolipids modulate IL-22 production by IL18R + MHC II + ILC3s.

Canonically, the complement system is known for its rapid response to remove microbes in the bloodstream. However, relatively little is known about a functioning complement system on intestinal mucosal surfaces. Herein, we report the local synthesis of complement component 3 (C3) in the gut, primarily by stromal cells. C3 is expressed upon commensal colonization and is regulated by the composition of the microbiota in healthy humans and mice, leading to an individual host's specific luminal C3 levels. The absence of membrane attack complex (MAC) components in the gut ensures that C3 deposition does not result in the lysis of commensals. Pathogen infection triggers the immune system to recruit neutrophils to the infection site for pathogen clearance. Basal C3 levels directly correlate with protection against enteric infection. Our study reveals the gut complement system as an innate immune mechanism acting as a vigilant sentinel that combats pathogens and spares commensals.

Macrophages show high plasticity and play a vital role in the progression of metabolic dysfunction-associated steatohepatitis (MASH). X-box binding protein 1 (XBP1), a key sensor of the unfolded protein response, can modulate macrophage-mediated pro-inflammatory responses in the pathogenesis of MASH. However, how XBP1 influences macrophage plasticity and promotes MASH progression remains unclear. Herein, we formulated an Xbp1 siRNA delivery system based on folic acid modified D-α-tocopheryl polyethylene glycol 1000 succinate nanoparticles (FT@XBP1) to explore the precise role of macrophage-specific Xbp1 deficiency in the progression of MASH. FT@XBP1 was specifically internalized into hepatic macrophages and subsequently inhibited the expression of spliced XBP1 both in vitro and in vivo. It promoted M1-phenotype macrophage repolarization to M2 macrophages, reduced the release of pro-inflammatory factors, and alleviated hepatic steatosis, liver injury, and fibrosis in mice with fat-, fructose- and cholesterol-rich diet-induced MASH. Mechanistically, FT@XBP1 promoted macrophage polarization toward the M2 phenotype and enhanced the release of exosomes that could inhibit the activation of hepatic stellate cells. A promising macrophage-targeted siRNA delivery system was revealed to pave a promising strategy in the treatment of MASH.

The loss of IL-10R function leads to severe early onset colitis and, in murine models, is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed, STAT1-/- mice exhibit defects in colonic macrophage accumulation after Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNγR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both wild-type and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R inhibits the generation of cell extrinsic signals that promote the accumulation of immature macrophages. These results define the essential mechanisms controlling the inflammatory macrophage accumulation in inflammatory bowel diseases.

The human gut microbiome constantly converts natural products derived from the host and diet into numerous bioactive metabolites 1 – 3 . Dietary fats are essential micronutrients that undergo lipolysis to release free fatty acids (FAs) for absorption in the small intestine 4 . Gut commensal bacteria modify some unsaturated FAs—for example, linoleic acid (LA)—into various intestinal FA isomers that regulate host metabolism and have anticarcinogenic properties 5 . However, little is known about how this diet–microorganism FA isomerization network affects the mucosal immune system of the host. Here we report that both dietary factors and microbial factors influence the level of gut LA isomers (conjugated LAs (CLAs)) and that CLAs in turn modulate a distinct population of CD4 + intraepithelial lymphocytes (IELs) that express CD8αα in the small intestine. Genetic abolition of FA isomerization pathways in individual gut symbionts significantly decreases the number of CD4 + CD8αα + IELs in gnotobiotic mice. Restoration of CLAs increases CD4 + CD8αα + IEL levels in the presence of the transcription factor hepatocyte nuclear factor 4γ (HNF4γ). Mechanistically, HNF4γ facilitates CD4 + CD8αα + IEL development by modulating interleukin-18 signalling. In mice, specific deletion of HNF4γ in T cells leads to early mortality from infection by intestinal pathogens. Our data reveal a new role for bacterial FA metabolic pathways in the control of host intraepithelial immunological homeostasis by modulating the relative number of CD4 + T cells that were CD4 + CD8αα + .

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Canonically, complement is a serum-based host defense system that protects against systemic microbial invasion. Little is known about the production and function of complement components on mucosal surfaces. Here we show gut complement component 3 (C3), central to complement function, is regulated by the composition of the microbiota in healthy humans and mice, leading to host-specific gut C3 levels. Stromal cells in intestinal lymphoid follicles (LFs) are the predominant source of intestinal C3. During enteric infection with Citrobacter rodentium or enterohemorrhagic Escherichia coli, luminal C3 levels increase significantly and are required for protection. C. rodentium is remarkably more invasive to the gut epithelium of C3-deficient mice than of wild-type mice. In the gut, C3-mediated phagocytosis of C. rodentium functions to clear pathogens. Our study reveals that variations in gut microbiota determine individuals’ intestinal mucosal C3 levels, dominantly produced by LF stromal cells, which directly correlate with protection against enteric infection. Highlights Gut complement component 3 (C3) is induced by the microbiome in healthy humans and mice at a microbiota-specific level. Gut stromal cells located in intestinal lymphoid follicles are a major source of luminal C3 During enteric infections with Citrobacter rodentium or enterohemorrhagic Escherichia coli, gut luminal C3 levels increase and are required for protection. C. rodentium is significantly more invasive of the gut epithelium in C3-deficient mice when compared to WT mice. In the gut, C3-mediated opsonophagocytosis of C. rodentium functions to clear pathogens.

Primary immunodeficiency may present with treatment-refractory enteropathy. We present two patients with celiac/celiac-like disease diagnosed in early childhood and refractory to the gluten-free diet. One patient had features of multi-system autoimmunity, whereas the other had celiac-like disease as an isolated clinical finding. Both patients underwent genetic testing given disease refractoriness and were ultimately diagnosed with cytotoxic T lymphocyte antigen 4 (CTLA4) haploinsufficiency. They are both now in complete clinical and endoscopic remission on abatacept. CTLA4 haploinsufficiency has incomplete penetrance and significant phenotypic heterogeneity but should be considered in the differential diagnosis of refractory celiac/celiac-like disease, as treatment implications are significant.

Loss of IL-10R function leads to severe early onset colitis and in murine models is associated with the accumulation of immature inflammatory colonic macrophages. We have shown that IL-10R-deficient colonic macrophages exhibit increased STAT1-dependent gene expression, suggesting that IL-10R-mediated inhibition of STAT1 signaling in newly recruited colonic macrophages might interfere with the development of an inflammatory phenotype. Indeed Stat1 -/- mice exhibit defects in colonic macrophage accumulation following Helicobacter hepaticus infection and IL-10R blockade, and this was phenocopied in mice lacking IFNGR, an inducer of STAT1 activation. Radiation chimeras demonstrated that reduced accumulation of STAT1-deficient macrophages was based on a cell-intrinsic defect. Unexpectedly, mixed radiation chimeras generated with both WT and IL-10R-deficient bone marrow indicated that rather than directly interfering with STAT1 function, IL-10R prevents the generation of a cell extrinsic signal that promotes the accumulation of immature macrophages. These results define essential mechanisms controlling inflammatory macrophage accumulation in inflammatory bowel diseases. Summary Intrinsic STAT1-function drives the accumulation of macrophages within the colon following the loss of IL-10R signaling. IL-10R prevents this STAT1-dependent process through a non-cell autonomous mechanism.

Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.

The gut microbiome is altered in patients with inflammatory bowel disease, yet how these alterations contribute to intestinal inflammation is poorly understood. Murine models have demonstrated the importance of the microbiome in colitis since colitis fails to develop in many genetically susceptible animal models when re-derived into germ-free environments. We have previously shown that Wiskott-Aldrich syndrome protein (WASP)-deficient mice (Was−/−) develop spontaneous colitis, similar to human patients with loss-of-function mutations in WAS. Furthermore, we showed that the development of colitis in Was−/− mice is Helicobacter dependent. Here, we utilized a reductionist model coupled with multi-omics approaches to study the role of host-microbe interactions in intestinal inflammation. Was−/− mice colonized with both altered Schaedler flora (ASF) and Helicobacter developed colitis, while those colonized with either ASF or Helicobacter alone did not. In Was−/− mice, Helicobacter relative abundance was positively correlated with fecal lipocalin-2 (LCN2), a marker of intestinal inflammation. In contrast, WT mice colonized with ASF and Helicobacter were free of inflammation and strikingly, Helicobacter relative abundance was negatively correlated with LCN2. In Was−/− colons, bacteria breach the mucus layer, and the mucosal relative abundance of ASF457 Mucispirillum schaedleri was positively correlated with fecal LCN2. Meta-transcriptomic analyses revealed that ASF457 had higher expression of genes predicted to enhance fitness and immunogenicity in Was−/− compared to WT mice. In contrast, ASF519 Parabacteroides goldsteinii’s relative abundance was negatively correlated with LCN2 in Was−/− mice, and transcriptional analyses showed lower expression of genes predicted to facilitate stress adaptation by ASF519 in Was−/−compared to WT mice. These studies indicate that the effect of a microbe on the immune system can be context dependent, with the same bacteria eliciting a tolerogenic response under homeostatic conditions but promoting inflammation in immune-dysregulated hosts. Furthermore, in inflamed environments, some bacteria up-regulate genes that enhance their fitness and immunogenicity, while other bacteria are less able to adapt and decrease in abundance. These findings highlight the importance of studying host-microbe interactions in different contexts and considering how the transcriptional profile and fitness of bacteria may change in different hosts when developing microbiota-based therapeutics.

大肠杆菌在生物学领域的研究中,可作为食物维持秀丽隐杆线虫的生长发育;但是,不同大肠杆菌营养成分的差异,以及对线虫发育等的影响还不明确.文章对实验室可用作线虫食物的4种大肠杆菌品系OP50、MG1655、HT115、HB101进行能量营养成分分析,并且研究了4种大肠杆菌对线虫的进食速率、发育速率、寿命、生殖能力的影响.结果发现:4种大肠杆菌的碳水化合物质量、能量及营养物质总能量从高到低依次为MG1655、HT115、HB101、OP50;蛋白质的质量、能量相似;脂肪的质量、能量HB101远大于其他3种大肠杆菌,OP50与HT115相似,都大于MG1655;4种大肠杆菌对线虫的影响在进食速率和寿命上没有明显差异;在发育速率上,食用OP50的线虫大于食用MG1655和HB101的线虫;在生殖能力上,食用HT115的线虫大于食用HB101的线虫.结果表明,除了HB101含有较高的脂肪水平导致线虫的发育速率加快、生殖能力降低外,其他营养物质对线虫的发育没有显著影响.