Soybean Diacylglycerol Regulates Lipid Metabolism in D-galactose induced Aging Rats by Altering Gut Microbiota and Gene Expression of Colonic Epithelial Cells

Lipid metabolism is closely related to the health of aging bodies, and its disorder often leads to cardiovascular diseases and chronic diseases. Dietary fat is one of the important sources...

Macrophages and Epithelial Cells Mutually Interact through NLRP3 to Clear Infection and Enhance the Gastrointestinal Barrier

Activation of the nod-like receptor protein 3 (NLRP3) leads to the release of the proinflammatory cytokine IL-1β, which then facilitates pathogen control by macrophages. The role of NLRPs in controlling infection of epithelial cells is not well understood. Our hypothesis was that activation of the NLRP3 inflammasome in colonic epithelial cells would promote macrophage-mediated epithelial recovery after infection with the pathogen Citrobacter rodentium. We devised a co-culture model using mouse colonic epithelial cells (CMT-93) and macrophages (J774A.1) during infection with C. rodentium. Inflammasome was activated using LPS and ATP and inhibited by YVAD. We assessed cytokine secretion (ELISA), macrophage recruitment and pathogen penetration (immunofluorescence), and epithelial barrier integrity (transepithelial electrical resistance). Macrophages were recruited to the apical membrane of epithelial cells, associated with tight junctions, promoted epithelial barrier recovery, and displaced C. rodentium. While NLRP3 was expressed in infected epithelial cells, IL-18 or IL-1β secretion remained unchanged. Supernatants from infected epithelial cells promoted infection clearance by macrophage; while this was inflammasome-independent, ATP significantly improved epithelial barrier recovery. The inflammasome appears to promote epithelial barrier function, independent of IL-18 and IL-1β secretion. Inflammasome activation in macrophages plays a dual role of promoting pathogen clearance and improving epithelial barrier integrity.

Lacticaseibacillus casei Strain T21 Attenuates Clostridioides difficile Infection in a Murine Model Through Reduction of Inflammation and Gut Dysbiosis With Decreased Toxin Lethality and Enhanced Mucin Production

Clostridioides difficile is a major cause of diarrhea in patients with antibiotic administration. Lacticaseibacillus casei T21, isolated from a human gastric biopsy, was tested in a murine C. difficile infection (CDI) model and colonic epithelial cells (Caco-2 and HT-29). Daily administration of L. casei T21 [1 × 108 colony forming units (CFU)/dose] for 4 days starting at 1 day before C. difficile challenge attenuated CDI as demonstrated by a reduction in mortality rate, weight loss, diarrhea, gut leakage, gut dysbiosis, intestinal pathology changes, and levels of pro-inflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α, macrophage inflammatory protein 2 (MIP-2), and keratinocyte chemoattractant (KC)] in the intestinal tissue and serum. Conditioned media from L. casei T21 exerted biological activities that fight against C. difficile as demonstrated in colonic epithelial cells by the following: (i) suppression of gene expression and production of IL-8, an important chemokine involved in C. difficile pathogenesis, (ii) reduction in the expression of SLC11A1 (solute carrier family 11 member 1) and HuR (human antigen R), important genes for the lethality of C. difficile toxin B, (iii) augmentation of intestinal integrity, and (iv) up-regulation of MUC2, a mucosal protective gene. These results supported the therapeutic potential of L. casei T21 for CDI and the need for further study on the intervention capabilities of CDI.

Ketogal Safety Profile in Human Primary Colonic Epithelial Cells and in Mice

In our previous studies, a ketorolac–galactose conjugate (ketogal) showed prolonged anti-inflammatory and analgesic activity, causing less gastric ulcerogenic effect and renal toxicity than its parent drug ketorolac. In order to demonstrate the safer profile of ketogal compared to ketorolac, histopathological changes in the small intestine and liver using three staining techniques before and after repeated oral administration in mice with ketorolac or an equimolecular dose of its galactosylated prodrug ketogal were assessed. Cytotoxicity and oxidative stress parameters were evaluated and compared in ketorolac- and ketogal-treated Human Primary Colonic Epithelial cells at different concentrations and incubation times. Evidence of mitochondrial oxidative stress was found after ketorolac treatment; this was attributable to altered mitochondrial membrane depolarization and oxidative stress parameters. No mitochondrial damage was observed after ketogal treatment. In ketorolac-treated mice, severe subepithelial vacuolation and erosion with inflammatory infiltrates and edematous area in the intestinal tissues were noted, as well as alterations in sinusoidal spaces and hepatocytes with foamy cytoplasm. In contrast, treatment with ketogal provided a significant improvement in the morphology of both organs. The prodrug clearly demonstrated a safer profile than its parent drug both in vitro and ex vivo, confirming that ketogal is a strategic alternative to ketorolac.

Loss of aryl hydrocarbon receptor suppresses the response of colonic epithelial cells to IL22 signaling by upregulating SOCS3

IL22 signaling plays an important role in maintaining gastrointestinal epithelial barrier function, cell proliferation and protection of intestinal stem cells from genotoxicants. Emerging studies indicate that the aryl hydrocarbon receptor (AhR), a ligand activated transcription factor, promotes production of IL22 in gut immune cells. However, it remains to be determined if AhR signaling can also affect the responsiveness of colonic epithelial cells to IL22. Here, we show that IL22 treatment induces the phosphorylation of STAT3, inhibits colonic organoid growth, and promotes colonic cell proliferation in vivo. Notably, intestinal cell specific AhR knockout (KO) reduces responsiveness to IL22 and compromises DNA damage response following exposure to carcinogen, in part due to the enhancement of SOCS3 expression. Deletion of SOCS3 increases levels of pSTAT3 in AhR KO organoids, and phenocopies the effects of IL22 treatment on wildtype (WT) organoid growth. In addition, pSTAT3 levels are inversely associated with increased AOM/DSS induced colon tumorigenesis in AhR KO mice. These findings indicate that AhR function is required for optimal IL22 signaling in colonic epithelial cells and provide rationale for targeting AhR as a means of reducing colon cancer risk.

Vitamin D Receptor Binding with DNA in Duodenal Crypt, Duodenal Villi, and Colonic Epithelial Cells of Mice

Vitamin D receptor (VDR) is a transcription factor that mediates calcium absorption by intestinal epithelial cells. Although calcium absorption is ca-nonically thought to occur only in the small intestine, recent studies have shown that VDR activity in the co-lon alone is sufficient to prevent calcium deficiency in mice. Here, we further investigate VDR activity in the colon. We assess VDR-DNA binding in mouse duodenal crypt, duodenal villi, and colonic epithelial cells using Chromatin Immunoprecipitation se-quencing (ChIP-seq). We find that most VDR-respon-sive elements are common to all intestinal epithelial cells, though some VDR-responsive elements are re-gionally-enriched and exhibit greater VDR-binding affinity in either duodenal epithelial cells or colonic epithelial cells. We also assess chromatin accessibil-ity in the same three cell types using Assay for Trans-posase-Accessible Chromatin sequencing (ATAC-seq). By integrating the VDR ChIP-seq and ATAC-seq data, we find that regionally-enriched VDR-re-sponsive elements exhibit greater chromatin acces-sibility in the region of their enrichment. Finally, we assess the transcription factor motifs present in VDR-responsive elements. We find that duodenum- and colon-enriched VDR-responsive elements exhibit different sets of transcription factor motifs other than VDR, suggesting that VDR may act together with dif-ferent partner transcription factors in the two re-gions. Our work is the first investigation of VDR-DNA binding in the colon and provides a basis for further investigations of VDR activity in the colon.