Intestinal Epithelial Damage Research Articles

Citrulline and long-term mortality in patients with cardiovascular disease.

Cardiovascular disease (CVD) is associated with intestinal barrier dysfunction and increased intestinal permeability. Increased intestinal permeability to gut microbial metabolites may accelerate the progression of CVD. Plasma citrulline levels are a marker of functional enterocyte mass, and reduced citrulline levels indicate intestinal epithelial damage. Citrulline was reported as a useful prognostic marker in critically ill patients. However, data are lacking on the association of citrulline with long-term mortality in patients with CVD and with the levels of trimethylamine N-oxide (TMAO), a microbiota-derived metabolite which has been implicated in the pathogenesis of CVD. To assess the effect of citrulline levels, a marker of intestinal barrier disruption, on long-term mortality in patients with CVD. Moreover, the relationship between the concentrations of 2 biomarkers - citrulline and TMAO - was assessed. Serum citrulline levels were retrospectively assessed in 1036 consecutive patients with CVD (median age: 62 years; 61% men) hospitalized between 2013 and 2015. Associations of citrulline levels with 5-year mortality rates as well as anthropometric and biochemical parameters were evaluated for the entire study group and in subgroups of patients with acute coronary syndrome (ACS), chronic coronary syndrome, chronic heart failure (chronic HF), and atrial fibrillation (AF). Correlations between serum citrulline and TMAO levels were assessed. The median citrulline level in the study population was 22.5 μM (interquartile range (IQR): 17.8-27.9). Citrulline levels were not associated with 5-year mortality in patients with CVD (hazard ratio (HR) = 0.99; 95% confidence interval (95% CI): 0.97-1.00; p = 0.49). Median citrulline levels differed significantly between deceased patients and survivors at 5 years in patients with ACS (p = 0.025). There were no significant correlations between citrulline and TMAO levels (Kendall's tau = 0.027). Decreasing citrulline levels do not predict long-term mortality of hospitalized patients with CVD. Moreover, they are not associated with the serum levels of TMAO in these patients.

1260-P: Gut Microbiome and Metabolic Activity in Type 1 Diabetes—An Analysis Based on the Presence of GADA

Background: Type 1 diabetes (T1D) progression is affected by the level of circulating glutamic acid decarboxylase antibody (GADA) , a main manifestation of islet autoimmunity. The gut microbiome and serum metabolites may contribute to islet autoimmunity in patients with T1D. Methods: We used radiobinding assay to measure GADA titers and identify the GADA+ ones (n=49) in 1T1D patients. The gut microbiome was assessed by 16S rRNA gene sequencing, and the serum metabolites were analysed with GC/MS. Results: T1D patients with and without GADA exhibited compositional and functional changes in the gut microbiome. Profiles in patients with GADA were distinct, with a significant increase in the abundances of Alistipes, Ruminococcus, Prevotella, Dialister, and marked depletion of Bacteroides, Bifidobacterium, Roseburia. For the untargeted serum metabolites, compared with those of GADA-, there were 54 significantly different metabolites with tryptophan metabolism: phenylalanine, and tyrosine and tryptophan biosynthesis decreased in patients with GADA. In addition, patients with GADA had elevated concentrations of Indole-3-acetic-acid, a marker of intestinal inflammation and epithelial damage. Co-occurrence network analysis revealed that gut microbiome disturbances affected tryptophan metabolism, suggesting that disturbed gut microbiome may mediate T1D immunotypes. Tryptophan metabolism may mediate inflammation in the pathogenesis and pathophysiology of T1D. Conclusion: These findings suggest that metabolites produced by the patients with GADA gut microbiota could cause damage to intestinal and systemic homeostasis and could be targeted for preventing the development of T1D. Disclosure T.Yue: None. X.Zheng: None. Z.Liu: None. Y.Ding: None. J.W.Wei: None. W.Xu: None. J.Weng: None. S.Luo: None. Funding the National Key r&D Program (2017YFC1309600) the Anhui Provincial Natural science foundation (006212943003)

SARS-CoV-2 induces barrier damage and inflammatory responses in the human iPSC-derived intestinal epithelium

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly spread and led to global health crises. COVID-19 causes well-known respiratory failure and gastrointestinal symptoms, such as diarrhea, nausea, and vomiting. Thus, human gastrointestinal cell models are urgently needed for COVID-19 research; however, it is difficult to obtain primary human intestinal cells. In this study, we examined whether human induced pluripotent stem cell (iPSC)-derived small intestinal epithelial cells (iPSC-SIECs) could be used as a SARS-CoV-2 infection model. We observed that iPSC-SIECs, such as absorptive and Paneth cells, were infected with SARS-CoV-2, and remdesivir treatment decreased intracellular SARS-CoV-2 replication in iPSC-SIECs. SARS-CoV-2 infection decreased expression levels of tight junction markers, ZO-3 and CLDN1, and transepithelial electrical resistance (TEER), which evaluates the integrity of tight junction dynamics. In addition, SARS-CoV-2 infection increased expression levels of proinflammatory genes, which are elevated in patients with COVID-19. These findings suggest iPSC-SIECs as a useful in vitro model for elucidating COVID-19 pathology and drug development.

Expression of a BTB-ZF transcription factor controls the function of an innate-like Tregs essential for intestinal homeostasis

Abstract The BTB-ZF genes are a family of 49 transcription factors that control the lineage specification and development of key effector functions of different subsets of lymphocytes. Using a single-cell BTB-ZF transcription factor expression analysis, we identified a unique subset of T regulatory cells (Tregs). These Tregs are genetically and phenotypically distinct from the larger conventional Treg population. For instance, analogous to natural killer (NKT) cells they have an activated phenotype (CD62Llo, CD44hi) and constitutively express IL-10. Interestingly, these characteristics are acquired early during T cell development. The defining transcription factor is induced during thymic development, binds the IL-10 promoter, and enables T cells to rapidly produce the cytokine. The subset of Tregs is enriched in the intestine and expands during acute colitis. The deletion of the defining transcription factor in T cells results in intestinal epithelial layer damage causing loss of the barrier function making mice highly vulnerable to severe disease and death from induced colitis. Moreover, in the absence of the transcription factor in T cells, normal intestinal macrophage responses are disrupted most likely due to decreased production of IL-10 by the Tregs. Therefore, by profiling BTB-ZF transcription factor expression, we have identified a distinct subset of T cells with potent immunosuppressive abilities that are essential for the health of the intestine. Numerous aspects of these Tregs are parallel to NKT cells, indicating an existence of “naïve” Tregs that have innate-like effector functions acquired independently from the differentiation in the periphery. Supported by NIH/NIAID R01 AI122757-01 Careers in Immunology Fellowships – 2021

Disordered Expression of Tight Junction Proteins Is Involved in the Mo-induced Intestinal Microenvironment Dysbiosis in Sheep.

To evaluate the molybdenum (Mo)-induced changes of intestinal morphology and the relationship of intestinal tight junction (TJ) proteins expression and intestinal barrier function, a total of 20 healthy sheep were randomly divided into five groups of four: 0, 5, 10, 20, and 50mg/kg BW/day Na2MoO4·2H2O were administrated in five groups named control group, Mo 5 group, Mo 10 group, Mo 20 group, and Mo 50 group, respectively. After 28days of Mo treatment, the duodenum, the jejunum, and the ileum tissue were collected. The histopathology and the developmental parameters were evaluated by hematoxylin-eosin staining. The intestinal epithelial cell DNA damage was detected by TdT-mediated dUTP nick end labeling assay. The intestinal glycoprotein and the goblet cells were analyzed by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining and PAS staining, respectively. TJ proteins were determined by immunofluorescence technology. Results showed that excessive Mo significantly decreased the small intestinal villus height (VH), crypt depth (CD), VH/CD, and mucosal thickness (P < 0.05 or P < 0.01) while induced the damage of DNA in small intestinal epithelial cells. Moreover, excessive Mo injured intestinal barrier function by decreasing the percent of glycoprotein distribution area (P < 0.05) and the relative density of intestinal goblet cells (P < 0.05). Mo treatment induced decreased (P < 0.01) expression of Zonula Occludens-1, Occludin, and Claudin-1. In conclusion, excessive Mo interfered with the expression of TJ proteins, inhibited intestinal epithelial development, and further aggravated the intestinal barrier function damage, leading to disturbing the small intestinal microenvironment balance.

Differences in MUC2 Gene Expression Based on the Clinical Severity of Colitis and the Degree of Histopathological Damage to the Colonic Mucosa in Colitis-induced Rat

BACKGROUND: Inflammatory bowel disease (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC), is characterized by intestinal inflammation and epithelial damage. Impaired mucosal cell barrier function mainly associated with thinning of the mucin layer may be the initial events underlying injury and inflammation in UC. Impaired expression of specific mucins is closely associated with IBD. MUC2 is a gene that produces mucin, which is predominant in the colon in humans and rats. METHODS: This study is an experimental study with a posttest-only design. The sample comprised 16 colitis-induced rats. Induction of colitis was done by giving a solution of dextran sodium sulfate (DSS) 2.5% 1 mL/day orally for 7 days. MUC2 gene expression was measured by rtPCR. The clinical severity of colitis was classified based on the disease activity index (DAI) score. The degree of histopathological damage was classified based on the score of colonic histology observations. The statistical analysis was done by the Shapiro–Wilk normality test and continued with an independent samples t-test. RESULTS: There were differences in MUC2 gene expression in mild and moderate colitis (1.81 vs. 2.99) but the difference was not significant (p &gt; 0.05). MUC2 gene expression also differed in mild and severe histopathological damage degrees (2.32 vs. 2.1) but the difference was not significant (p &gt; 0.05). CONCLUSION: It was concluded in this study that MUC2 gene expression did not have significant differences based on the clinical severity of colitis and the degree of histopathological damage to the colonic mucosa in colitis-induced rats.

Milk-Derived Small Extracellular Vesicles Promote Recovery of Intestinal Damage by Accelerating Intestinal Stem Cell-Mediated Epithelial Regeneration.

Milk-derived small extracellular vesicles (M-sEVs) are critical bioactive components in milk. They are considered to be regulators in milk that may have promising applications. Understanding their biological effects would be important in nutrition. Intestinal organoids and mice are used to explore the effects of M-sEVs on intestinal regeneration. M-sEVs could be absorbed by intestinal epithelia and upregulate expression of the microRNAs (miRNAs) expressed in milk: miR-148a, miR-22, miR-30, and miR-29a. Interestingly, M-sEVs promote proliferation of intestinal epithelia and repairs the epithelial damage that is caused by tumor necrosis factor-α in intestinal organoids. M-sEVs ameliorate intestinal mucosa damage in mice caused by treatment with dextran sulfate sodium, as well as increasing expression of the intestinal stem cells (ISC) markers leucine-rich repeat containing G-protein-coupled receptor 5 (Lgr5), olfactomedin 4 (Olfm4), and Achaete-Scute Family BHLH Transcription Factor 2 (Ascl2) and stimulating intestinal epithelial proliferation to repair epithelial damage. Furthermore, miR-29 is more abundant in M-sEVs-treated mice, and miR-29 could upregulate expression of ISC marker genes and accelerates intestinal regeneration to recover damaged intestinal epithelia. We reveal thatM-sEVs and miR-29 can accelerate intestinal stem cell-mediated epithelial regeneration and repair epithelial damage.

Inhibition of LncRNA-NEAT1 alleviates intestinal epithelial cells (IECs) dysfunction in ulcerative colitis by maintaining the homeostasis of the glucose metabolism through the miR-410-3p-LDHA axis

ABSTRACT Dysfunction of intestinal epithelial cells (IECs) leads to intestinal epithelial barrier damage and critically involves in the pathogenesis and development of ulcerative colitis (UC). Accumulating studies revealed essential functions of non-coding RNAs in UC. LncRNA NEAT1 (long non-coding RNA nuclear paraspeckle assembly transcript 1) is frequently dysregulated in diverse human diseases. Currently, the precise roles of NEAT1 in the dysfunction of IECs during UC remain unclear. We report NEAT1 was significantly upregulated in IECs from UC patients. In addition, microRNA-410-3p was remarkedly suppressed in IECs from UC patients. Silencing NEAT1 effectively ameliorates the LPS-induced IECs dysfunction. Bioinformatical analysis, RNA pull-down and luciferase assays illustrated that NEAT1 sponged miR-410-3p to downregulate its expression in IECs. Interestingly, the glucose metabolism was obviously elevated in IECs from UC compared with normal colon tissues. Furthermore, NEAT1 promoted and miR-410-3p suppressed glucose metabolism of IECs. We identified lactate dehydrogenase A (LDHA), a glucose metabolism key enzyme, was a direct target of miR-410-3p in IECs. Rescue experiments verified that restoration of miR-410-3p in NEAT1-overexpressing IECs successfully overcame the NEAT1-promoted cell death under LPS treatment by targeting LDHA. In summary, these results unveiled new roles and molecular mechanisms for the NEAT1-mediated IECs dysfunction during the ulcerative colitis.

Adipose-derived mesenchymal stem cells may reduce intestinal epithelial damage in ulcerative colitis by communicating with macrophages and blocking inflammatory pathways: an analysis in silico.

Ulcerative colitis is a chronic, non-specific inflammatory disease that affects mainly the colonic mucosa and submucosa. The pathogenesis of ulcerative colitis is unclear, which limits the development of effective treatments. In this study, single-cell sequencing data from 18 ulcerative colitis samples and 12 healthy controls were downloaded from the Single Cell Portal database, cell types were defined through cluster analysis, and genes in each cluster that were differentially expressed in ulcerative colitis were identified. These genes were enriched in functional pathways related to apoptosis, immunity and inflammation. Analysis using iTALK software suggested extensive communication among immune cells. Single-cell sequencing data from adipose-derived mesenchymal stem cells from three healthy female donors were obtained from the Sequence Read Archive database. The SingleR package was used to identify different cell types, for each of which a stemness score was calculated. Pseudotime analysis was performed to infer the trajectory of cells. SCENIC software was used to identify the gene regulatory network in adipose-derived mesenchymal stem cells, and iTALK software was performed to explore the relationship among macrophages, adipose-derived mesenchymal stem cells and enterocytes. Molecular docking confirmed the possibility of cell-cell interactions via binding between surface receptors and their ligands. The bulk data were downloaded and analyzed to validate the expression of genes. Our bioinformatics analyses suggest that ulcerative colitis involves communication between macrophages and enterocytes via ligand-receptor pairs. Our results further suggest that adipose-derived mesenchymal stem cells may alleviate ulcerative colitis by communicating with macrophages to block inflammation.

Microbial signals, MyD88, and lymphotoxin drive TNF-independent intestinal epithelial tissue damage.

Anti-TNF antibodies are effective for treating patients with inflammatory bowel disease (IBD), but many patients fail to respond to anti-TNF therapy, highlighting the importance of TNF-independent disease. We previously demonstrated that acute deletion of 2 IBD susceptibility genes, A20 (Tnfaip3) and Abin-1 (Tnip1), in intestinal epithelial cells (IECs) sensitized mice to both TNF-dependent and TNF-independent death. Here we show that TNF-independent IEC death after A20 and Abin-1 deletion was rescued by germ-free derivation or deletion of MyD88, while deletion of Trif provided only partial protection. Combined deletion of Ripk3 and Casp8, which inhibits both apoptotic and necroptotic death, completely protected against death after acute deletion of A20 and Abin-1 in IECs. A20- and Abin-1–deficient IECs were sensitized to TNF-independent, TNFR1-mediated death in response to lymphotoxin α (LTα) homotrimers. Blockade of LTα in vivo reduced weight loss and improved survival when combined with partial deletion of MyD88. Biopsies of inflamed colon mucosa from patients with IBD exhibited increased LTA and IL1B expression, including a subset of patients with active colitis on anti-TNF therapy. These data show that microbial signals, MyD88, and LTα all contribute to TNF-independent intestinal injury.

Intestinal permeability before and after albendazole treatment in low and high socioeconomic status schoolchildren in Makassar, Indonesia

Intestinal helminths are highly prevalent in low-SES children and could contribute to poor health outcomes either directly or via alteration of the gut microbiome and gut barrier function. We analysed parasitic infections and gut microbiota composition in 325 children attending high- and low-SES schools in Makassar, Indonesia before and after albendazole treatment. Lactulose/Mannitol Ratio (LMR, a marker of gut permeability); I-FABP (a surrogate marker of intestinal damage) as well as inflammatory markers (LBP) were measured. Helminth infections were highly prevalent (65.6%) in low-SES children. LMR and I-FABP levels were higher in low-SES children (geomean (95%CI): 4.03 (3.67–4.42) vs. 3.22 (2.91–3.57); p. adj < 0.001; and 1.57 (1.42–1.74) vs. 1.25 (1.13–1.38); p. adj = 0.02, respectively) while LBP levels were lower compared to the high-SES (19.39 (17.09–22.01) vs. 22.74 (20.07–26.12); p.adj = 0.01). Albendazole reduced helminth infections in low-SES and also decreased LMR with 11% reduction but only in helminth-uninfected children (estimated treatment effect: 0.89; p.adj = 0.01). Following treatment, I-FABP decreased in high- (0.91, p.adj < 0.001) but increased (1.12, p.adj = 0.004) in low-SES children. Albendazole did not alter the levels of LBP. Microbiota analysis showed no contribution from specific bacterial-taxa to the changes observed. Intestinal permeability and epithelial damage are higher while peripheral blood inflammatory marker is lower in children of low-SES in Indonesia. Furthermore, treatment decreased LMR in helminth-uninfected only.

Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis.

Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions. Here, we aimed to investigate the contribution of intestinal epithelial AMPK to the initiation, progression and resolution of acute colitis. We also tested the hypothesis that protection mediated by metformin administration on intestinal epithelium damage required AMPK activation. A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell (IEC)-specific AMPK KO mice. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and pro-inflammatory cytokines and tight junction protein expression. The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS exposure and was associated with a slower re-epithelization of the intestinal mucosa coupled with severe ulceration and inflammation, and altered barrier function. Following intestinal injury, IEC AMPK KO mice displayed a lower goblet cell counts with concomitant decreased Muc2 gene expression, unveiling an impaired restitution of goblet cells and contribution to wound healing process. Metformin administration during the recovery phase attenuated the severity of DSS-induced colitis through improvement in intestinal repair capacity in both WT and IEC AMPK KO mice. Taken together, these findings demonstrate a critical role for IEC-expressed AMPK in regulating mucosal repair and epithelial regenerative capacity following acute colonic injury. Our studies further underscore the therapeutic potential of metformin to support repair of the injured intestinal epithelium, but this effect is conferred independently of intestinal epithelial AMPK.

Extracellular histones cause intestinal epithelium injury and disrupt its barrier function in vitro and in vivo

Extracellular histones are cytotoxic to various cells and have been extensively proven a vital mediator of multiple organ injuries. However, the effect of extracellular histones on the intestine remains largely unknown. This study aimed to clarify the effect of extracellular histones on the intestine. IEC-6, a cell line of rat small intestinal epithelial crypt, and C57BL/6 or ICR mice were treated with histones. The IEC-6 cells treated with histones from 20 μg/mL to 200 μg/mL for 0−24 h displayed a decline of cell viability and an increase of cell death in a concentration- and time-dependent manner. Moreover, histones (100 μg/mL) induced IEC-6 apoptosis through activating caspase 3 and necroptosis through up-regulation of receptor-interacting serine/threonine protein kinase 1 and 3 (RIPK1 and RIPK3), phosphorylated mixed-lineage kinase domain-like protein (p-MLKL) along with the decrease of caspase-8. Histones treatment disturbed zonular occludens 1 (ZO-1) expression and increased permeability of IEC-6 cell monolayer. In vivo, histones 50 mg/kg injection caused mice intestinal edema, loss apex of villus, epithelial lifting down the sides of the villi, and increased neutrophil infiltration. Elevation of serum intestinal fatty acid binding protein (I-FABP), d-lactate, or Diamine oxidase (DAO) and loss of tight junction protein, ZO-1, at 3 h and 6 h after histones injection strongly indicated severe intestinal epithelium injury, which led to increased permeability of the intestine. In conclusion, extracellular histones cause intestinal epithelial damage via direct cytotoxicity. Consequently, intestinal epithelial tight junction and barrier integrity are disrupted, which may play pivotal roles in diverse diseases.

Resveratrol alleviates enterotoxigenic Escherichia coli K88-induced damage by regulating SIRT-1 signaling in intestinal porcine epithelial cells.

This study found that resveratrol pretreatment attenuated porcine intestinal epithelial cell damage caused by enterotoxigenic Escherichia coli (ETEC) K88 in vitro and the protective effects of resveratrol were associated with SIRT-1 signaling. ETEC K88 is a main intestinal pathogen for post-weaning diarrhea (PWD) in piglets. With the strict ban on antibiotics in animal feed, people are seeking effective antibiotic substitutes to protect the intestinal system against harmful pathogenic bacteria. This study was conducted to evaluate the effects of resveratrol, a natural plant polyphenol, on ETEC K88-induced cellular damage in porcine enterocytes and underlying mechanisms. Intestinal porcine epithelial cell line 1 (IPEC-1) cells, pretreated with or without resveratrol (30 μM, 4 h), were challenged with ETEC K88 (MOI = 1 : 10) for 3 h. The results showed that ETEC K88 infection induced severe damage and dysfunction in IPEC-1 cells, as evidenced by a reduced cell viability, decreased tight junctions, mitochondrial dysfunction, and autophagy. It is noteworthy that IPEC-1 cells pre-treated with resveratrol improved their capacity for resistance to most of these abnormal phenotypes caused by ETEC K88 infection. Furthermore, we found that the activation of SIRT-1 signaling was associated with the benefits of resveratrol, as demonstrated by EX-527, an inhibitor of SIRT-1, which reversed most of the protective effects of resveratrol. In conclusion, these results indicated that resveratrol could protect intestinal epithelial cells against ETEC K88 infection by activating SIRT-1 signaling. These findings provide new insights into the role of resveratrol in maintaining intestinal physiological functions.

Intestinal epithelial damage due to herbal compounds – an in vitro study

Intestinal epithelial injury from herbal products has rarely been reported, despite the gut being the first point of contact for oral preparations. These products often consist of multiple herbs, thereby potentially exposing consumers to higher levels of reactive phytochemicals than predicted due to pharmacokinetic interactions. The phytochemical coumarin, found in many herbal products, may be taken in combination with herbal medicines containing astragalosides and atractylenolides, purported cytochrome P450 (CYP) modulators. As herbal use increases, the need to predict interactions in multiple at-risk organ systems is becoming critical. Hence, to determine whether certain herbal preparations containing coumarin may cause damage to the intestinal epithelium, Caco2 cells were exposed to common phytochemicals. Coumarin, astragaloside IV (AST-IV) or atractylenolide I (ATR-I) solutions were exposed to Caco2 cultures in increasing concentrations, individually or combined. Coumarin produced a significant concentration-dependant fall in cell viability that was potentiated when CYP enzymes were induced with rifampicin and incubated with CYP3A4 inhibitor econazole, suggesting a role for other CYP enzymes generating toxic metabolites. ATR-I alone produced no toxicity in uninduced cells but showed significant toxicity in rifampicin-induced cells. ATR-I had no effect on coumarin-induced toxicity. AST-IV was nontoxic alone but produced significant toxicity when combined with nontoxic concentrations of coumarin. The combination of coumarin, ATR-I and AST-IV was significantly toxic, but no synergistic interaction was seen. This investigation was conducted to determine the likelihood for intestinal-based interactions, with the results demonstrating coumarin is potentially toxic to intestinal epithelium, and combinations with other phytochemicals can potentiate this toxicity.

Targeted deletion of keratin 8 in intestinal epithelial cells disrupts tissue integrity and predisposes to tumorigenesis in the colon

Keratin 8 (K8) is the main intestinal epithelial intermediate filament protein with proposed roles for colonic epithelial cell integrity. Here, we used mice lacking K8 in intestinal epithelial cells (floxed K8 and Villin-Cre1000 and Villin-CreERt2) to investigate the cell-specific roles of intestinal epithelial K8 for colonocyte function and pathologies. Intestinal epithelial K8 deletion decreased K8 partner proteins, K18–K20, 75–95%, and the remaining keratin filaments were located at the colonocyte apical regions with type II K7, which decreased 30%. 2-Deoxy-2-[18F]-fluoroglucose positron emission tomography in vivo imaging identified a metabolic phenotype in the lower gut of the conditional K8 knockouts. These mice developed intestinal barrier leakiness, mild diarrhea, and epithelial damage, especially in the proximal colon. Mice exhibited shifted differentiation from enterocytes to goblet cells, displayed longer crypts and an increased number of Ki67 + transit-amplifying cells in the colon. Significant proproliferative and regenerative signaling occurred in the IL-22, STAT3, and pRb pathways, with minor effects on inflammatory parameters, which, however, increased in aging mice. Importantly, colonocyte K8 deletion induced a dramatically increased sensitivity to azoxymethane-induced tumorigenesis. In conclusion, intestinal epithelial K8 plays a significant role in colonocyte epithelial integrity maintenance, proliferation regulation and tumor suppression.Graphical abstract

AMPK-PINK1/Parkin Mediated Mitophagy Is Necessary for Alleviating Oxidative Stress-Induced Intestinal Epithelial Barrier Damage and Mitochondrial Energy Metabolism Dysfunction in IPEC-J2.

The imbalance of redox biology and oxidative stress leads to intestinal barrier injury and mitophagy. However, much uncertainty still exists about the role of mitophagy in oxidative stress and intestinal function. Here, we showed the effects of hydrogen peroxide (H2O2)-induced oxidative stress on intestinal epithelial cell oxidation balance, intestinal barrier function and mitochondrial energy metabolism and its underlying mechanism. In this study, we found that H2O2-induced oxidative stress activated adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitophagy in intestinal porcine epithelial cells (IPEC-J2). While compound C (AMPK inhibitor) and mdivi-1 (mitophagy inhibitor) significantly reduced the activity of superoxide dismutase (SOD) and increased mitochondrial reactive oxygen species (ROS) levels in H2O2 treated cells. Moreover, compound C and mdivi-1 significantly reduced the trans-epithelium electrical resistant (TER) and increased the fluorescein isothiocyanate-dextran (FD4) flux in H2O2 treated IPEC-J2. Furthermore, compound C and mdivi-1 significantly reduced the activity of mitochondrial complex II. Seahorse XF96 data showed that compound C + mdivi-1+ H2O2 treatment significantly reduced maximum respiratory oxygen consumption and spare respiratory capacity. Additionally, compound C or mdivi-1 treatment reduced the formation of mitochondrial autophagosomes. These results unveiled that AMPK and PINK1/Parkin mediated mitophagy is necessary for alleviating oxidative stress induced intestinal epithelial barrier damage and mitochondrial energy metabolism dysfunction in IPEC-J2.

The Panda-Derived Lactobacillus plantarum G201683 Alleviates the Inflammatory Response in DSS-Induced Panda Microbiota-Associated Mice.

Intestinal diseases are one of the main causes of captive giant panda death. Their special dietary habits and gastrointestinal tract structure often lead to intestinal epithelium damage and secondary intestinal infection. The captive giant panda is predisposed to suffer from microbiota dysbiosis due to long-term artificial feeding and antibiotic misuse. However, there are few reported probiotics to treat giant panda enteritis and the associated dysbiosis. This study aims to elucidate the mechanism by which Lactobacillus plantarum G201683 (L. plantarum G83), a promising panda-derived probiotic, exerts a protective effect on intestinal inflammation in the dextran sulfate sodium- (DSS) induced panda microbiota-associated (DPMA) mouse model. The DPMA mouse was generated by antibiotic treatment and 5% DSS drinking water administration to assess the effect of L. plantarum G83 on intestinal inflammation and microbiota in vivo. Our results demonstrated the successful generation of a DPMA mouse model with Enterobacteriaceae enrichment, consistent with the giant panda intestinal microbiota. L. plantarum G83 decreased clinical and histological severity of intestinal inflammation, enhanced intestinal tight junction protein expression (ZO-1, Occludin) and alleviated inflammatory cytokine production (TNF-) in the colon of DPMA mice. The administration of L. plantarum G83 altered the microbiota composition by decreasing pathogen associated taxa such as E. coli and increasing abundance of beneficial bacteria including Bifidobacterium spp. These changes in microbiota composition were associated with an increased concentration of short chain fatty acids (SCFA), reduced NF-κB signaling, and an altered balance of T helper cell subsets. Our findings support L. plantarum G83 as a promising probiotic to treat intestinal inflammation in the giant panda.

Intestinal IL-33 promotes platelet activity for neutrophil recruitment during acute inflammation

Peripheral serotonin (5-HT) is mainly generated from the gastrointestinal tract and taken up and stored by platelets in the circulation. Although the gut is recognized as a major immune organ, how intestinal local immune responses control whole-body physiology via 5-HT remains unclear. Here, we show that intestinal inflammation enhances systemic platelet activation and blood coagulation. Intestinal epithelium damage induces elevated levels of the alarm cytokine interleukin-33 (IL-33), leading to platelet activation via promotion of gut-derived 5-HT release. More importantly, we found that loss of intestinal epithelial-derived IL-33 lowers peripheral 5-HT levels, resulting in compromised platelet activation and hemostasis. Functionally, intestinal IL-33 contributes to the recruitment of neutrophils to sites of acute inflammation by enhancing platelet activities. Genetic deletion of intestinal IL-33 or neutralization of peripheral IL-33 protects animals from lipopolysaccharide endotoxic shock through attenuated neutrophil extravasation. Therefore, our data establish a distinct role of intestinal IL-33 in activating platelets by promoting 5-HT release for systemic physiology and inflammation.

Bacteroides fragilis restricts colitis-associated cancer via negative regulation of the NLRP3 axis.

Patients with persistent ulcerative colitis (UC) are at a higher risk of developing colitis-associated cancer (CAC). Previous studies have reported that intestinal microbiota disturbance plays an important role in the process of CAC development in patients with UC, indicating that targeted intervention of intestinal microbiota and its metabolites may be a potential therapeutic strategy. Gut microbiota in the process of colorectal cancer development in UC patients was analyzed using the gutMEGA database and verified in fecal samples. The abundance of Bacteroides fragilis reduced significantly in the process of colitis associated cancer development. Broad-spectrum antibiotics (BSAB) intervene with the intestinal microbiota of mice and accelerate the process of colon cancer development. However, gavage transplantation with B. fragilis can effectively reverse the effects of BSAB. In the intestinal tract, B. fragilis promotes the secretion of short-chain fatty acids (SCFAs). Subsequently, SCFAs, especially butyrate, negatively regulate the inflammatory signaling pathway mediated by NLRP3 to inhibit the activation of macrophages and the secretion of proinflammatory mediators such as IL-18 and IL-1β, reducing the level of intestinal inflammation and restricting CAC development. In conclusion, colonization with B. fragilis has been shown to be effective in ameliorating intestinal epithelial damage caused by chronic inflammation and preventing the development of colonic tumors. Thus, it can be a therapeutic intervention strategy with good clinical application prospects.