Segmental patterning of microbiota and immune cells in the murine intestinal tract.

The intestine exhibits distinct characteristics along its length, with a substantial immune cell reservoir and diverse microbiota crucial for maintaining health. This study investigates how anatomical location and regional microbiota influence intestinal immune cell abundance. Using conventionally colonized and germ-free mice, segment-specific immune cell composition and microbial communities were assessed. Metagenomic sequencing analyzed microbiome variations, while flow cytometry and immunofluorescence examined immune cell composition. Microbiome composition varied significantly along the intestine, with diversity and abundance increasing from upper to lower segments. Immune cells showed distinct segment-specific patterning influenced by microbial colonization and localization. T cell subsets displayed varied dependence on microbiome presence and anatomical location. This study highlights locoregional differences in intestinal immune cell and microbiome composition, identifying immune subsets susceptible to microbiota presence. The findings provide context for understanding immune cell alterations in disease models.

PROTAC based STING degrader attenuates acute colitis by inhibiting macrophage M1 polarization and intestinal epithelial cells pyroptosis mediated by STING-NLRP3 axis.

Inflammatory bowel diseases (IBDs) are chronic, relapsing, and inflammatory disorders of the gastrointestinal tract characterized by abnormal immune responses. Recently, STING has emerged as a promising therapeutic target for various autoinflammatory diseases. However, few STING-selective small molecules have been investigated as novel strategies for IBD. In this study, we sought to examine the effects of PROTAC-based STING degrader SP23 on acute colitis and explore its underlying mechanism. SP23 treatment notably alleviates dextran sulfate sodium (DSS)-induced colitis. Pharmacological degradation of STING significantly reduced the production of inflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, and inhibited macrophage polarization towards the M1 type. Furthermore, SP23 administration decreased the loss of tight junction proteins, including ZO-1, occludin, and claudin-1, and downregulated STING and NLRP3 signaling pathways in intestinal inflammation. In vitro, STING activated NLRP3 inflammasome-mediated pyroptosis in intestinal epithelial cells, which could be abrogated by SP23 and STING siRNA intervention. In conclusion, these findings provide new evidence for STING as a novel therapeutic target for IBD, and reveal that hyperactivation of STING could exaggerate colitis by inducing NLRP3/Caspase-1/GSDMD axis mediated intestinal epithelial cells pyroptosis.

Unlocking the power of short-chain fatty acids in ameliorating intestinal mucosal immunity: a new porcine nutritional approach.

Short-chain fatty acids (SCFAs), a subset of organic fatty acids with carbon chains ranging from one to six atoms in length, encompass acetate, propionate, and butyrate. These compounds are the endproducts of dietary fiber fermentation, primarily catalyzed by the glycolysis and pentose phosphate pathways within the gut microbiota. SCFAs act as pivotal energy substrates and signaling molecules in the realm of animal nutrition, exerting a profound influence on the intestinal, immune system, and intestinal barrier functions. Specifically, they contibute to 60-70% of the total energy requirements in ruminants and 10-25% in monogastric animals. SCFAs have demonstrated the capability to effectively modulate intestinal pH, optimize the absorption of mineral elements, and impede pathogen invasion. Moreover, they enhance the expression of proteins associated with intestinal tight junctions and stimulate mucus production, thereby refining intestinal tissue morphology and preserving the integrity of the intestinal structure. Notably, SCFAs also exert anti-inflammatory properties, mitigating inflammation within the intestinal epithelium and strengthening the intestinal barrier's defensive capabilities. The present review endeavors to synthesize recent findings regarding the role of SCFAs as crucial signaling intermediaries between the metabolic activities of gut microbiota and the status of porcine cells. It also provides a comprehensive overview of the current literature on SCFAs' impact on immune responses within the porcine intestinal mucosa.

TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma.

Immune memory has been expanded to group 2 innate lymphoid cells (ILC2s), but the cellular and molecular bases remain incompletely understood. Based on house dust mite (HDM)-induced mice asthma models and human samples, we applied flow cytometry, parabiosis, in vivo imaging and adoptive transplantation to confirm the persistence, migration and function of CD45+lineage-CD90.2+NK1.1-NKp46-ST2-KLRG1+IL-17RB+ memory-like ILC2s (ml-ILC2s). Regulated by CCR9/CCL25 and S1P signaling, ml-ILC2s reside in the lamina propria of small intestines (siLP) in asthma remission, and subsequently move to airway upon re-encountering antigens or alarmins. Furthermore, ml-ILC2s possess properties of longevity, potential of rapid proliferation and producing IL-13, and display transcriptional characteristics with up-regulation of Tox and Tcf-7. ml-ILC2s transplantation restore the asthmatic changes abrogated by Tox and Tcf7 knockdown. Our data identify siLP ml-ILC2s as a memory-like subset, which promotes asthma relapse. Targeting TCF-1 and TOX might be promising for preventing asthma recurrence.

Sirtuin 6 inhibits group 3 innate lymphoid cell function and gut immunity by suppressing IL-22 production.

Introduction: Group 3 innate lymphoid cells (ILC3s) are enriched in the intestinal mucosa and play important roles in host defense against infection and inflammatory diseases. Sirtuin 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD+)- dependent deacetylase and has been shown to control intestinal epithelial cell differentiation and survival. However, the role of SIRT6 in ILC3s remains unknown. Methods: To investigate the role of SIRT6 in gut ILC3s, we generated SIRT6 conditional knockout mice by crossing Rorccre and Sirt6flox/flox mice. Cell number and cytokine production was examined using flow cytometry. Citrobacter rodentium infection and dextran sodium sulfate-induced colitis models were used to determine the role of SIRT6 in gut defense. RT-qPCR, flow cytometry and immunohistochemistry were used to assess the intestinal inflammatory responses. Results: Here we show that SIRT6 inhibits IL-22 expression in intestinal ILC3s in a cell-intrinsic manner. Deletion of SIRT6 in ILC3s does not affect the cell numbers of total ILC3s and subsets, but results in increased IL-22 production. Furthermore, ablation of SIRT6 in ILC3s protects mice against Citrobacter rodentium infection and dextran sodium sulfate-induced colitis. Our results suggest that SIRT6 may play a role in ILC3 function by regulating gut immune responses against bacterial infection and inflammation. Discussion: Our finding provided insight into the relation of epigenetic regulators with IL-22 production and supplied a new perspective for a potential strategy against inflammatory bowel disease.

Neighbor's feedback helps macrophages learn tolerance in the gut.

Intestinal macrophages play a key role in regulating immune tolerance in the gut. In this issue of Immunity, Mertens et al. uncover a mechanism for the establishment of memory in macrophage tolerance in the gut involving a bistable metabolic switch in macrophages and an intercellular positive feedback between macrophages and intestinal epithelial cells (IECs).

Role of chemokine receptors in gastrointestinal mucosa.

Chemokine receptors are essential for the immune response in the oral and gut mucosa. The gastrointestinal mucosa is characterized by the presence of immune populations because it is susceptible to inflammatory and infectious diseases, necessitating immune surveillance. Chemokine receptors are expressed on immune cells and play a role in gastrointestinal tissue-homing, although other non-immune cells also express them for various biological functions. CCR9, CXCR3 and CXCR6 play an important role in the T cell response in inflammatory and neoplastic conditions of the gastrointestinal mucosa. However, CXCR6 could also be found in gastric cancer cells, highlighting the different roles of chemokine receptors in different pathologies. On the other hand, CCR4 and CCR8 are critical for Treg migration in gastrointestinal tissues, correlating with poor prognosis in mucosal cancers. Other chemokine receptors are also important in promoting myeloid infiltration with context-dependent roles. Further, CXCR4 and CXCR7 are also present in gastrointestinal tumor cells and are known to stimulate proliferation, migration, and invasion into other tissues, among other pro-tumorigenic functions. Determining the processes underlying mucosal immunity and creating tailored therapeutic approaches for gastrointestinal diseases requires an understanding of the complex interactions that occur between chemokine receptors and their ligands in these mucosal tissues.

Recipient tissue microenvironment determines developmental path of intestinal innate lymphoid progenitors.

Innate lymphoid cells (ILCs) are critical in maintaining tissue homeostasis, and during infection and inflammation. Here we identify, by using combinatorial reporter mice, a rare ILC progenitor (ILCP) population, resident to the small intestinal lamina propria (siLP) in adult mice. Transfer of siLP-ILCP into recipients generates group 1 ILCs (including ILC1 and NK cells), ILC2s and ILC3s within the intestinal microenvironment, but almost exclusively group 1 ILCs in the liver, lung and spleen. Single cell gene expression analysis and high dimensional spectral cytometry analysis of the siLP-ILCPs and ILC progeny indicate that the phenotype of the group 1 ILC progeny is also influenced by the tissue microenvironment. Thus, a local pool of siLP-ILCP can contribute to pan-ILC generation in the intestinal microenvironment but has more restricted potential in other tissues, with a greater propensity than bone marrow-derived ILCPs to favour ILC1 and ILC3 production. Therefore, ILCP potential is influenced by both tissue of origin and the microenvironment during development. This may provide additional flexibility during the tuning of immune reactions.

Macrophages and Gut Barrier Function: Guardians of Gastrointestinal Health in Post-Inflammatory and Post-Infection Responses.

The gut barrier is essential for protection against pathogens and maintaining homeostasis. Macrophages are key players in the immune system, are indispensable for intestinal health, and contribute to immune defense and repair mechanisms. Understanding the multifaceted roles of macrophages can provide critical insights into maintaining and restoring gastrointestinal (GI) health. This review explores the essential role of macrophages in maintaining the gut barrier function and their contribution to post-inflammatory and post-infectious responses in the gut. Macrophages significantly contribute to gut barrier integrity through epithelial repair, immune modulation, and interactions with gut microbiota. They demonstrate active plasticity by switching phenotypes to resolve inflammation, facilitate tissue repair, and regulate microbial populations following an infection or inflammation. In addition, tissue-resident (M2) and infiltration (M1) macrophages convert to each other in gut problems such as IBS and IBD via major signaling pathways mediated by NF-κB, JAK/STAT, PI3K/AKT, MAPK, Toll-like receptors, and specific microRNAs such as miR-155, miR-29, miR-146a, and miR-199, which may be good targets for new therapeutic approaches. Future research should focus on elucidating the detailed molecular mechanisms and developing personalized therapeutic approaches to fully harness the potential of macrophages to maintain and restore intestinal permeability and gut health.

A mouse protozoan boosts antigen-specific mucosal IgA responses in a specific lipid metabolism- and signaling-dependent manner.

IgA antibodies play an important role in mucosal immunity. However, there is still no effective way to consistently boost mucosal IgA responses, and the factors influencing these responses are not fully understood. We observed that colonization with the murine intestinal symbiotic protozoan Tritrichomonas musculis (T.mu) boosted antigen-specific mucosal IgA responses in wild-type C57BL/6 mice. This enhancement was attributed to the accumulation of free arachidonic acid (ARA) in the intestinal lumen, which served as a signal to stimulate the production of antigen-specific mucosal IgA. When ARA was prevented from undergoing its downstream metabolic transformation using the 5-lipoxygenase inhibitor zileuton or by blocking its downstream biological signaling through genetic deletion of the Leukotriene B4 receptor 1 (Blt1), the T.mu-mediated enhancement of antigen-specific mucosal IgA production was suppressed. Moreover, both T.mu transfer and dietary supplementation of ARA augmented the efficacy of an oral vaccine against Salmonella infection, with this effect being dependent on Blt1. Our findings elucidate a tripartite circuit linking nutrients from the diet or intestinal microbiota, host lipid metabolism, and the mucosal humoral immune response.

Moderate Aerobic Exercise Induces Homeostatic IgA Generation in Senile Mice.

A T-cell-independent (TI) pathway activated by microbiota results in the generation of low-affinity homeostatic IgA with a critical role in intestinal homeostasis. Moderate aerobic exercise (MAE) provides a beneficial impact on intestinal immunity, but the action of MAE on TI-IgA generation under senescence conditions is unknown. This study aimed to determine the effects of long-term MAE on TI-IgA production in young (3 month old) BALB/c mice exercised until adulthood (6 months) or aging (24 months). Lamina propria (LP) from the small intestine was obtained to determine B cell and plasma cell sub-populations by flow cytometry and molecular factors related to class switch recombination [Thymic Stromal Lymphopoietin (TSLP), A Proliferation-Inducing Ligand (APRIL), B Cell Activating Factor (BAFF), inducible nitric oxide synthase (iNOS), and retinal dehydrogenase (RDH)] and the synthesis of IgA [α-chain, interleukin (IL)-6, IL-21, and Growth Factor-ß (TGF-ß)]; and epithelial cells evaluated IgA transitosis [polymeric immunoglobulin receptor (pIgR), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-4] by the RT-qPCR technique. The results were compared with data obtained from sedentary age-matched mice. Statistical analysis was computed with ANOVA, and p < 0.05 was considered to be a statistically significant difference. Under senescence conditions, MAE promoted the B cell and IgA+ B cells and APRIL, which may improve the intestinal response and ameliorate the inflammatory environment associated presumably with the downmodulation of pro-inflammatory mediators involved in the upmodulation of pIgR expression. Data suggested that MAE improved IgA and downmodulate the cytokine pro-inflammatory expression favoring homeostatic conditions in aging.

Human organoids with an autologous tissue-resident immune compartment.

The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.

Protective effect of dihydromyricetin on intestinal epithelium in weaned pigs upon enterotoxigenic Escherichia coli challenge.

Dihydromyricetin (DMY), a natural flavonoid compound, are believed to prevent inflammatory response, dealing with pathogens and repairing the intestinal barrier. The objective of this study was to investigate whether DMY supplementation could attenuate intestinal damage in the context of enterotoxigenic Escherichia coli K88 (ETEC F4+) infection. After weaning, different litters of pigs were randomly assigned to one of the following treatments: (1) non-challenged control (CON, fed with basal diet); (2) ETEC-challenged control (ECON, fed with basal diet); and (3) ETEC challenge + DMY treatment (EDMY, fed with basal diet plus 300 mg kg-1 DMY). We observed a significant reduction in fecal Escherichia coli shedding and diarrhea incidence, but an increase in ADG in pigs of EDMY group compared to the pigs of ECON group. Relative to the pigs of ECON group, dietary DMY treatment decreased (P < 0.05) concentrations of the serum D-xylose, D-lactate and diamine oxidase (DAO), but increased the abundance of zonula occludens-1 (ZO-1) in the jejunum of pigs. In addition, DMY also decreased (P < 0.05) the number of S-phase cells and the percentage of total apoptotic epithelial cells of jejunal epithelium in pigs of the EDMY group compared to the pigs of the ECON group. Furthermore, DMY decreased the mRNA expression levels of critical immune-associated genes TLR4, NFκB, Caspase3, Caspase9, IL-1ß, IL-6, TNF-α and the protein p-NFκB and p-IκBα expressions of intestinal epithelium in pigs of the EDMY group compared to the pigs of the ECON group. Compared to the ECON group, DMY elevated (P < 0.05) the expression levels of ß-defensins PBD1, PBD2, PBD3, PBD129, as well as the abundance of secreted IgA in intestinal mucosae of the EDMY group. Thus, our results indicate that DMY may relieve intestinal integrity damage due to Escherichia coli F4.

PLGA microspheres carrying EMSCs-CM for the effective treatment of murine ulcerative colitis.

Ectodermal mesenchymal stem cells-derived conditioned medium (EMSCs-CM) has been reported to protect against ulcerative colitis (UC) in mice, but its underlying mechanism in alleviating UC need to be further elucidated. Here, it is reported that EMSCs-CM could attenuate pro-inflammatory response of LPS-induced IEC-6 cells and regulate the polarization of macrophages towards anti-inflammatory type in vitro. Furthermore, PLGA microspheres prepared by the double emulsion method were constructed for oral delivery of EMSCs-CM (EMSCs-CM-PLGA), which are beneficial for colon-targeted adhesion of EMSCs-CM to the damaged colon mucosa. The results showed that orally-administered of EMSCs-CM-PLGA microspheres reduced inflammatory cells infiltration and maintained the intestinal mucosal barrier. Further investigation found that EMSCs-CM-PLGA microspheres treatment gradually inhibited the activation of NF-κB pathway to regulate M1/M2 polarization balance in colon tissue macrophages, thereby alleviating DSS-induced UC. These results of this study will provide a theoretical basis for clinical application of EMSCs-CM in UC repair.

UCP2 knockout exacerbates sepsis-induced intestinal injury by promoting NLRP3-mediated pyroptosis.

Sepsis-induced intestinal injury is a common complication that increases the morbidity and mortality associated with sepsis. UCP2, a mitochondrial membrane protein, is involved in numerous cellular processes, including metabolism, inflammation, and pyroptosis. According to our previous studies, UCP2 expression increases in septic intestinal tissue. However, its function in intestinal damage is not known. This work investigated UCP2's role in intestinal injury caused by sepsis. A sepsis mouse model was established in wild-type and UCP2-knockout (UCP2-KO) animals using cecal ligation and puncture (CLP). MCC950, an NLRP3 inflammasome inhibitor, was injected intraperitoneally 3 h before CLP surgery. Overall, significantly higher levels of UCP2 were observed in the intestines of septic mice. UCP2-KO mice subjected to CLP exhibited exacerbated intestinal damage, characterized by enhanced mucosal erosion, inflammatory cell infiltration, and increased intestinal permeability. Furthermore, UCP2 knockout significantly increased oxidative stress, inflammation, and pyroptosis in the CLP mouse intestines. Interestingly, MCC950 not only inhibited pyroptosis but also reversed inflammation, oxidative stress as well as damage to intestinal tissues as a result of UCP2 knockout. Our results highlighted the protective functions of UCP2 in sepsis-associated intestinal injury through modulation of inflammation and oxidative stress via NLRP3 inflammasome-induced pyroptosis.

ASB3 expression aggravates inflammatory bowel disease by targeting TRAF6 protein stability and affecting the intestinal microbiota.

E3 ubiquitin ligase (E3) plays a vital role in regulating inflammatory responses by mediating ubiquitination. Previous studies have shown that ankyrin repeat and SOCS box-containing protein 3 (ASB3) is involved in immunomodulatory functions associated with cancer. However, the impact of ASB3 on the dynamic interplay of microbiota and inflammatory responses in inflammatory bowel disease (IBD) is unclear. Here, we systematically identify the E3 ligase ASB3 as a facilitative regulator in the development and progression of IBD. We observed that ASB3 exhibited significant upregulation in the lesions of patients with IBD. ASB3-/- mice are resistant to dextran sodium sulfate-induced colitis. IκBα phosphorylation levels and production of proinflammatory factors IL-1ß, IL-6, and TNF-α were reduced in the colonic tissues of ASB3-/- mice compared to WT mice. This colitis-resistant phenotype was suppressed after coprophagic microbial transfer and reversed after combined antibiotics removed the gut commensal microbiome. Mechanistically, ASB3 specifically catalyzes K48-linked polyubiquitination of TRAF6 in intestinal epithelial cells. In contrast, in ASB3-deficient organoids, the integrity of the TRAF6 protein is shielded, consequently decelerating the onset of intestinal inflammation. ASB3 is associated with dysregulation of the colitis microbiota and promotes proinflammatory factors' production by disrupting TRAF6 stability. Strategies to limit the protein level of ASB3 in intestinal epithelial cells may help in the treatment of colitis. IMPORTANCE: Ubiquitination is a key process that controls protein stability. We determined the ubiquitination of TRAF6 by ASB3 in intestinal epithelial cells during colonic inflammation. Inflammatory bowel disease patients exhibit upregulated ASB3 expression at focal sites, supporting the involvement of degradation of TRAF6, which promotes TLR-Myd88/TRIF-independent NF-κB aberrant activation and intestinal microbiota imbalance. Sustained inflammatory signaling in intestinal epithelial cells and dysregulated protective probiotic immune responses mediated by ASB3 collectively contribute to the exacerbation of inflammatory bowel disease. These findings provide insights into the pathogenesis of inflammatory bowel disease and suggest a novel mechanism by which ASB3 increases the risk of colitis. Our results suggest that future inhibition of ASB3 in intestinal epithelial cells may be a novel clinical strategy.

Polysaccharide from Areca catechu L. inflorescence enhances the intestinal mucosal immunity to maintain immune homeostasis.

Being the first line of defense, intestinal mucosal immunity serves as in maintaining immune homeostasis among organisms. This study investigated the impact of the areca inflorescence polysaccharide (AFP) on intestinal mucosal immunity and elucidated the mechanisms responsible for the immunomodulatory effects of AFP. The immunosuppression mouse model was established using the cyclophosphamide. The intestinal mucosal status was evaluated based on the intestinal integrity, chemical and mucosal immune barriers, and intestinal flora. According to the findings, AFP enhances intestinal integrity by up-regulating the expression of tight junction proteins and reinforcing the chemical barrier through increased mucin-2, ß-defensins, and SIgA expression and secretion. Furthermore, AFP restores the mucosal immune barrier by regulating immune cells within Peyer's patches and lamina propria. AFP also reverses the intestinal flora balance and regulates its metabolism. Additionally, AFP effectively modulates the immune response in the spleen and peripheral blood. Together, these results indicated that AFP repairs mucosal damage and restores mucosal immunity, thereby preserving the immune homeostasis of organisms.

EGCG drives gut microbial remodeling-induced epithelial GPR43 activation to lessen Th1 polarization in colitis.

Modulation of immune microenvironment is critical for inflammatory bowel disease (IBD) intervention. Epigallocatechin gallate (EGCG), as a natural low toxicity product, has shown promise in treating IBD. However, whether and how EGCG regulates the intestinal microenvironment is not fully understood. Here we report that EGCG lessens colitis by orchestrating Th1 polarization and self-amplification in a novel manner that required multilevel-regulated intestinal microecosystem. Mechanistically, EGCG activates GPR43 on IEC to inhibit Th1 polarization dependently of short chain fatty acid (SCFA)-producing gut microbiota. Inhibition of GPR43 activity weakens the protective effects of EGCG on colitis development. Moreover, we confirm that fecal SCFAs and/or intestinal GPR43 are limited in patients with colitis and are correlated with Th1 cell number. Taken together, our study reveals an intestinal microenvironment-dependent immunoregulatory effects of EGCG in treating IBD and provides insight into mechanisms of EGCG-based novel immunotherapeutic strategies for IBD.

A spatial expression atlas of the adult human proximal small intestine.

The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.