Human Intestinal Epithelial Cell Line Research Articles

Expression of HMGCS2 in intestinal epithelial cells is downregulated in inflammatory bowel disease associated with endoplasmic reticulum stress.

The Unfolded Protein Response, a mechanism triggered by the cell in response to Endoplasmic reticulum stress, is linked to inflammatory responses. Our aim was to identify novel Unfolded Protein Response-mechanisms that might be involved in triggering or perpetuating the inflammatory response carried out by the Intestinal Epithelial Cells in the context of Inflammatory Bowel Disease. We analyzed the transcriptional profile of human Intestinal Epithelial Cell lines treated with an Endoplasmic Reticulum stress inducer (thapsigargin) and/or proinflammatory stimuli. Several genes were further analyzed in colonic biopsies from Ulcerative Colitis patients and healthy controls. Lastly, we generated Caco-2 cells lacking HMGCS2 by CRISPR Cas-9 and analyzed the functional implications of its absence in Intestinal Epithelial Cells. Exposure to a TLR ligand after thapsigargin treatment resulted in a powerful synergistic modulation of gene expression, which led us to identify new genes and pathways that could be involved in inflammatory responses linked to the Unfolded Protein Response. Key differentially expressed genes in the array also exhibited transcriptional alterations in colonic biopsies from active Ulcerative Colitis patients, including NKG2D ligands and the enzyme HMGCS2. Moreover, functional studies showed altered metabolic responses and epithelial barrier integrity in HMGCS2 deficient cell lines. We have identified new genes and pathways that are regulated by the Unfolded Protein Response in the context of Inflammatory Bowel Disease including HMGCS2, a gene involved in the metabolism of Short Chain Fatty Acids that may have an important role in intestinal inflammation linked to Endoplasmic Reticulum stress and the resolution of the epithelial damage.

Metabolism of Acetaminophen by Enteric Epithelial Cells Mitigates Hepatocellular Toxicity In Vitro.

The gut-liver axis is defined by dietary and environmental communication between the gut, microbiome and the liver with its redox and immune systems, the overactivation of which can lead to hepatic injury. We used media preconditioning to mimic some aspects of the enterohepatic circulation by treating the human Caco-2 intestinal epithelial cell line with 5, 10 and 20 mM paracetamol (N-acetyl-para-aminophenol; APAP) for 24 h, after which cell culture supernatants were transferred to differentiated human hepatic HepaRG cells for a further 24 h. Cell viability was assessed by mitochondrial function and ATP production, while membrane integrity was monitored by cellular-based impedance. Metabolism by Caco-2 cells was determined by liquid chromatography with tandem mass spectrometry. Caco-2 cell viability was not affected by APAP, while cell membrane integrity and tight junctions were maintained and became tighter with increasing APAP concentrations, suggesting a reduction in the permeability of the intestinal epithelium. During 24 h incubation, Caco-2 cells metabolised 64-68% of APAP, leaving 32-36% of intact starting compound to be transferred to HepaRG cells. When cultured with Caco-2-preconditioned medium, HepaRG cells also showed no loss of cell viability or membrane integrity, completely in contrast to direct treatment with APAP, which resulted in a rapid loss of cell viability and membrane integrity and, ultimately, cell death. Thus, the pre-metabolism of APAP could mitigate previously observed hepatotoxicity to hepatic tight junctions caused by direct exposure to APAP. These observations could have important implications for the direct exposure of hepatic parenchyma to APAP, administered via the intravenous route.

LINC00460 contributes to colorectal cancer cell invasion

ABSTRACT Research background: Colorectal cancer (CRC) is one of the most prevalent malignant tumors in the world. Research on long noncoding RNAs (LncRNAs) may illuminate tumorigenesis and progression of CRC. Methods: We screened long non-coding RNA LINC00460 as a new candidate, which promoted the development of CRC in two independent datasets (GSE39582 and GSE21510) from the Gene Expression Omnibus (GEO). In 98 CRC tissues, expression levels of LINC00460 were significantly increased in cancerous tissues compared to paired adjacent normal tissues (P < 0.001). In addition, in the most common CRC cell lines. LINC00460 expression was up-regulated compared to normal human intestinal epithelial cell line NCM460. siRNA was transfected into CRC cell lines. LINC00460 knockdown reduced cell invasion ability and did not affect cell proliferation. The association between LINC00460 expression and clinical pathological features and prognosis were also analyzed. Results: This increased expression was found to significantly correlate with lymph node metastasis (P = 0.002), distant metastasis (P = 0.045) and TNM stage (P < 0.001); but not related to age, gender, location of tumor, and histological grade. The overall survival (OS) in CRC patients with overexpression of LINC00460 was inferior to that with low expression (P = 0.0167). Multivariate Cox regression analyses indicated that LINC00460 expression, as well as TNM stage was an independent prognostic risk factor for patients with CRC. Conclusion: These results showed that a higher expression level of LINC00460 might play an oncogenic role in colorectal cancer invasion and metastasis. It also proved that LINC00460 might be used as a potential diagnostic and prognostic biomarker in CRC patients.

Visible light induced photocatalytic degradation of direct red 80 dye molecules using CuxFeSx nanocomposites

The discharge of azo-dye molecules in wastewater has raised significant environmental and health concerns. Therefore, the development of visible light-active photocatalysts to degrade the dye molecules in wastewater can be a promising solution. In this study, CuxFeSx derivatives were synthesized by solvo-hydrothermal method with different solvents (deionized water (Wt), ethanol-Wt (Et-Wt) and Et) to investigate their photocatalytic activity towards Direct Red 80 (DR-80) and safety in human cell lines. The physicochemical properties of the synthesized products, including crystallinity, surface morphology and chemical properties were characterized. The results showed that CuxFeSx-Wt and CuxFeSx-Et-Wt produced flower-like morphology interconnected with nanosheets, while CuxFeSx-Et is produced a mixture of edged particles and nanosheets. Among them, CuxFeSx-Et exhibited the highest photocatalytic efficiency of DR-80 at 98.76% in 195 min, compared to CuxFeSx-Et-Wt (96.17% in 240 min) and CuxFeSx-Wt (93.32% in 285 min). CuxFeSx-Et also showed a high recyclability of 94.93%. The degradation of DR-80 dye followed the Langmuir adsorption model. Furthermore, the low cytotoxicity of CuxFeSx nanocomposites to human intestinal epithelial cell line (Caco-2) and human macrophage-like cell line (differentiated THP-1) suggests that these materials are benign. This study provides new insights into the benign-by-design of CuxFeSx as photocatalysts for the degradation of dye molecules in environmental remediation.

MicroRNA-483-3p Inhibitor Ameliorates Sepsis-Induced Intestinal Injury by Attenuating Cell Apoptosis and Cytotoxicity Via Regulating HIPK2.

Sepsis is a life-threatening syndrome that can result in multi-organ dysfunction. MicroRNA (miR)-483-3p was previously demonstrated to be upregulated in sepsis patients; however, its specific functions in sepsis-triggered intestinal injury remain unclarified. Human intestinal epithelial NCM460 cell line was stimulated with lipopolysaccharide (LPS) to mimic sepsis-induced intestinal injury in vitro. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) staining was utilized for examining cell apoptosis. Western blotting and real time quantitative polymerase chain reaction (RT-qPCR) were used for detecting molecular protein and RNA levels. LPS-induced cytotoxicity was determined by measuring concentrations of lactate dehydrogenase (LDH), diamine oxidase (DAO) and fatty acid binding protein 2 (FABP2). Luciferase reporter assay was utilized for verifying the interaction between miR-483-3p and homeodomain interacting protein kinase 2 (HIPK2). Inhibiting miR-483-3p alleviates LPS-triggered NCM460 cell apoptosis and cytotoxicity. miR-483-3p targeted HIPK2 in LPS-stimulated NCM460 cells. Knockdown of HIPK2 reversed the above effects mediated by miR-483-3p inhibitor. Inhibiting miR-483-3p ameliorates LPS-triggered apoptosis and cytotoxicity by targeting HIPK2.

Differential Effects of Iron Chelates vs. Iron Salts on Induction of Pro-Oncogenic Amphiregulin and Pro-Inflammatory COX-2 in Human Intestinal Adenocarcinoma Cell Lines

We previously showed that two iron compounds that are orally ingested by humans, namely ferric EDTA and ferric citrate, can induce an oncogenic growth factor (amphiregulin) in human intestinal epithelial adenocarcinoma cell lines. Here, we further screened these iron compounds, plus four other iron chelates and six iron salts (i.e., 12 oral iron compounds in total), for their effects on biomarkers of cancer and inflammation. Ferric pyrophosphate and ferric EDTA were the main inducers of amphiregulin and its receptor monomer, IGFr1. Moreover, at the maximum iron concentrations investigated (500 µM), the highest levels of amphiregulin were induced by the six iron chelates, while four of these also increased IGfr1. In addition, we observed that ferric pyrophosphate promoted signaling via the JAK/STAT pathway by up-regulating the cytokine receptor subunit IFN-γr1 and IL-6. For pro-inflammatory cyclooxygenase-2 (COX-2), ferric pyrophosphate but not ferric EDTA elevated intracellular levels. This, however, did not drive the other biomarkers based on COX-2 inhibition studies and was probably downstream of IL-6. We conclude that of all oral iron compounds, iron chelates may particularly elevate intracellular amphiregulin. Ferric pyrophosphate additionally induced COX-2, probably because of the high IL-6 induction that was observed with this compound.

Adrenomedullin alleviates mucosal injury in experimental colitis and increases claudin-4 expression in the colonic epithelium.

Adrenomedullin (AM) is a peptide with pleiotropic physiological functions that attenuates intestinal mucosal inflammation. However, the mechanism underpinning mucosal protection by AM is not fully understood, and its effect on intestinal epithelial cells remains unclear. Here, we investigated the effects of AM on junctional molecules in primary-cultured murine intestinal epithelial cells and discovered that AM upregulates claudin-4 expression. In a mouse model of dextran sulfate sodium-induced colitis, AM administration also enhanced claudin-4 expression and accelerated mucosal regeneration. Furthermore, AM reversed TNFα-mediated downregulation of claudin-4 and loss of cell-cell adhesion of the HCT116 human intestinal epithelial cell line in vitro. These results indicate that AM may enhance intestinal epithelial integrity by upregulating claudin-4 expression.

Synergistic Inhibitory Effect of Lactobacillus Cell Lysates and Butyrate on Poly I:C-Induced IL-8 Production in Human Intestinal Epithelial Cells.

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Sodium Butyrate Induces CRC Cell Ferroptosis via the CD44/SLC7A11 Pathway and Exhibits a Synergistic Therapeutic Effect with Erastin.

Human normal intestinal epithelial cell line FHT and colorectal tumor cell line HCT-116 were treated with NaB alone or in combination with different programmed cell death inhibitors. Cell activity was then assessed with MTT assays and PI staining; ferroptosis with Fe2+, glutathione (GSH), and lipid peroxidation assays; signaling pathway screening with PCR arrays; and CD44, SCL7A11, and GPX4 expression with Western blotting. A CD44-overexpressing HCT-116 cell line was constructed to determine the effect of the overexpression of CD44 on NaB-induced ferroptosis. The synergistic effect of co-treatment with NaB and Erastin was assessed by isobolographic analysis. NaB induced apoptosis and ferroptosis in HCT-116 cells but only induced low-level apoptosis in FHC cells. Moreover, NaB significantly increased intracellular Fe2+ and promoted GSH depletion and lipid peroxidation in HCT-116 cells. Ferroptosis-related qPCR array analysis identified CD44/SLC7A11 as a potential effector molecular of NaB-induced ferroptosis. NaB significantly inhibited the expression of CD44 and SLC7A11 in mouse CRC tissues. A CD44 overexpressed HCT-116 cell line was used to verify that CD44/SLC7A11 was a key signaling pathway that NaB-induced GSH depletion, lipid peroxidation accumulation, and ferroptosis in HCT-116 cells. Examination of whether NaB can increase the effect of ferroptosis agents showed that NaB, in combination with Erastin, a ferroptosis inducer, further promoted HCT-116 cell death and increased changes of ferroptosis markers. Our results suggest that NaB induces ferroptosis in CRC cells through the CD44/SLC7A11 signaling pathway and has synergistic effects with Erastin. These results may provide new insights into CRC prevention and the combined use of NaB and ferroptosis-inducing agents.

Functional Properties of Dunaliella salina and Its Positive Effect on Probiotics.

The unicellular green microalga Dunaliella is a potential source of a wide range of nutritionally important compounds applicable to the food industry. The aim of this study was to assess the effect of Dunaliella salina dried biomass on the growth and adherence of 10 strains of Lactobacillus, Lacticaseibacillus, and Bifidobacterium. The immunomodulatory, antioxidant, and cytotoxic effects of D. salina on human peripheral mononuclear cells and simulated intestinal epithelial cell lines Caco-2 and HT-29 were evaluated. Furthermore, the hypocholesterolemic effects of the microalgae on lipid metabolism in rats fed a high-fat diet were analyzed. The addition of D. salina biomass had a positive effect on the growth of nine out of 10 probiotics and promoted the adherence of three bifidobacteria strains to human cell lines. The antioxidant and immunomodulatory properties of D. salina were concentration-dependent. The inflammatory cytokines (TNF-α and IL-6) were significantly increased following Dunaliella stimulation at the lowest concentration (0.5% w/v). Eight week supplementation of D. salina to the diet of hypercholesteromic rats significantly decreased the serum concentrations of LDL-C, VLDL, IDL-B, and IDL-C. D. salina is not cytotoxic in intestinal cell models; it promotes adherence of selected bifidobacteria, it affords immunomodulatory and antioxidant effects, and its addition to diets may help decrease atherosclerosis risk factors.

Cytotoxicity of a Novel Compound Produced in Foods via the Reaction of Amino Acids with Acrolein along with Formaldehyde.

Acrolein (ACR) and formaldehyde (FA) are toxic aldehydes co-produced in foods. This work found that amino acids, the nucleophiles ubiquitously existing in foods, can react simultaneously with them. Six amino acids, including γ-aminobutyric acid (GABA), glycine, alanine, serine, threonine, and glutamine, can scavenge ACR and FA at 37, 85, and 160 °C. GABA had the highest scavenging capacity for ACR and FA, by 79 and 13% at 37 °C for 2 h, and 99 and 48% at 160 °C for 30 min, respectively. Moreover, a new type of compound with a basic structure of 5-formyl-3-methylene-3,6-dihydropyridin was identified in all reactions and formed by 1 molecule of FA and amino acid and 2 molecules of ACR. The content of this compound was higher than that of free ACR in typical thermally processed foods. Moreover, the compounds produced from different amino acids showed different cytotoxicity values. In gastric epithelial and human intestinal epithelial cell lines, the cytotoxicity values of serine-sourced and threonine-sourced products were lower than that of ACR but higher than that of FA, whereas others had less toxicity compared with the two aldehydes. Considering that the content of serine-sourced products was the highest in almost all tested foods, their safety needs to be evaluated.

Transient receptor potential melastatin 7 aggravates necrotizing enterocolitis by promoting an inflammatory response in children.

As a rare disease in children, necrotizing enterocolitis (NEC) leads to high morbidity and mortality. However, its pathophysiology is largely unclear. Transient receptor potential melastatin 7 (TRPM7) is a membrane protein, which plays key roles in the inflammatory response. This study sought to examine the promoting effect of TRPM7 on NEC in children and explore the therapeutic effect of a TRPM7 inhibitor NS8593. First, we detected TRPM7 and NLR family pyrin domain containing 3 (NLRP3) expression and the state of inflammation in children with NEC through quantitative real-time polymerase chain reaction (RT-PCR), Western blot, and enzyme-linked immunosorbent assays. Next, Human intestinal epithelial cell lines were induced to NEC by lipopolysaccharides (LPSs). The level of cytokines and reactive oxygen species (ROS) were tested by RT-PCR and flow cytometry. The TRPM7 mediated calcium flux were determined by fluorescence. In addition, we used the TRPM7 inhibitor NS8593 to treat the in vivo rat model. The mRNA and protein expression were determined by real-time PCR and Elisa analysis, respectively. TRPM7 and NLRP3 expression was more increased in the samples from children with NEC compared to the control samples. Additionally, the elevated secretion of interleukin-1β, interleukin-6, and tumor necrosis factor alpha was also detected in the serum of children with NEC. These results showed that TRPM7 had a promoting effect on NEC development, possibly via the activation of NLRP3. To test our hypothesis, the TRPM7 inhibitor NS8593 was used to treat the LPS-stimulated IEC-6 cells. We found that the TRPM7 inhibitor NS8593 inhibited LPS-induced cytokine production and exhibited an anti-inflammatory effect by alleviating TRPM7-mediated NLRP3 inflammasome activation. Through in-vivo experiments, we found that TRPM7 was involved in the occurrence of NEC, and its inhibitor NS8593 played a certain therapeutic role in the rat model. Our study revealed TRPM7 inhibitors attenuated LPS-induced ROS and reduced the release of pro-inflammatory cytokines. It also exhibited protective effects on the NEC model.

Acute cytomegalovirus infection modulates the intestinal microbiota and targets intestinal epithelial cells.

Primary and recurrent cytomegalovirus (CMV) infections frequently cause CMV colitis in immunocompromised as well as inflammatory bowel disease (IBD) patients. Additionally, colitis occasionally occurs upon primary CMV infection in patients who are apparently immunocompetent. In both cases, the underlying pathophysiologic mechanisms are largely elusive - in part due to the lack of adequate access to specimens. We employed the mouse cytomegalovirus (MCMV) model to assess the association between CMV and colitis. During acute primary MCMV infection of immunocompetent mice, the gut microbial composition was affected as manifested by an altered ratio of the Firmicutes to Bacteroidetes phyla. Interestingly, these microbial changes coincided with high-titer MCMV replication in the colon, crypt hyperplasia, increased colonic pro-inflammatory cytokine levels, and a transient increase in the expression of the antimicrobial protein Regenerating islet-derived protein 3 gamma (Reg3γ). Further analyses revealed that murine and human intestinal epithelial cell lines, as well as primary intestinal crypt cells and organoids represent direct targets of CMV infection causing increased cell death. Accordingly, in vivo MCMV infection disrupted the intestinal epithelial barrier and increased apoptosis of intestinal epithelial cells. In summary, our data show that CMV transiently induces colitis in immunocompetent hosts by altering the intestinal homeostasis.

Ginsenoside Rc attenuates DSS-induced ulcerative colitis, intestinal inflammatory, and barrier function by activating the farnesoid X receptor.

Objectives: Farnesoid X receptor (FXR) activation is involved in ameliorating inflammatory bowel disease (IBD), such as ulcerative colitis (UC), and inflammatory regulation may be involved in its mechanism. Ginsenoside Rc (Rc) is a major component of Panax ginseng, and it plays an excellent role in the anti-inflammatory processes. Our aim is to explore the alleviative effect of Rc on dextran sulfate sodium (DSS)-induced inflammation and deficiencies in barrier function based on FXR signaling. Materials and Methods: In vitro, we treated human intestinal epithelial cell lines (LS174T) with LPS to explore the anti-inflammatory effect of Rc supplementation. In vivo, a DSS-induced IBD mice model was established, and the changes in inflammatory and barrier function in colons after Rc treatment were measured using the disease activity index (DAI), hematoxylin and eosin (H&E) staining, immunofluorescence, ELISA, and qPCR. Molecular docking analysis, luciferase reporter gene assay, and qPCR were then used to analyze the binding targets of Rc. DSS-induced FXR-knockout (FXR-/-) mice were used for further validation. Results: Rc significantly recovered the abnormal levels of inflammation indexes (TNF-α, IL-6, IL-1β, and NF-KB) induced by LPS in LS174T. DSS-induced C57BL/6 mice exhibited a significantly decreased body weight and elevated DAI, as well as a decrease in colon weight and length. Increased inflammatory markers (TNF-α, IL-6, IL-1β, ICAM1, NF-KB, F4/80, and CD11b displayed an increased expression) and damaged barrier function (Claudin-1, occludin, and ZO-1 displayed a decreased expression) were observed in DSS-induced C57BL/6 mice. Nevertheless, supplementation with Rc mitigated the increased inflammatory and damaged barrier function associated with DSS. Further evaluation revealed an activation of FXR signaling in Rc-treated LS174T, with FXR, BSEP, and SHP found to be upregulated. Furthermore, molecular docking indicated that there is a clear interaction between Rc and FXR, while Rc activated transcriptional expression of FXR in luciferase reporter gene assay. However, these reversal abilities of Rc were not observed in DSS-induced FXR-/- mice. Conclusion: Our findings suggest that Rc may ameliorate inflammation and barrier function in the intestine, which in turn leads to the attenuation of DSS-induced UC, in which Rc may potentially activate FXR signaling to protect the intestines from DSS-induced injury.

Roseburia intestinalis stimulates TLR5-dependent intestinal immunity against Crohn's disease.

SummaryBackgroundCrohn's disease (CD) is a chronic inflammatory disorder characterized by intestinal immune dysfunction. Multiple factors, including gut dysbiosis, are involved in the pathogenesis of CD. However, the effect of commensal bacteria on controlling the inflammatory response in individuals with CD remains unclear.MethodsWe detected Toll-like receptor 2 (TLR2), TLR4, and TLR5 expression in Roseburia intestinalis (R. intestinalis)-treated mice with 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Then, we quantified the signs of colonic inflammation, the proportion of regulatory T cells (Tregs) and the expression of thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β in TLR-5-deficient (Tlr5−/−) mice, bone marrow chimera mice (generated using wild-type (WT) and Tlr5−/− mice), and anti-TSLP/anti-TGFβ-treated C57BL/6 mice with colitis induced by TNBS. In vitro, we used the lipopolysaccharide (LPS)-stimulated human intestinal epithelial cell line Caco-2 as an inflammatory colon cell model treated with or without the TLR5-siRNA intervention in the presence of R. intestinalis and incubated human monocyte-derived dendritic cells (DCs) with the supernatant of Caco-2 cells. Then, we cocultured human CD4+ T cells with the aforementioned DCs to determine the differentiation of Tregs. Additionally, samples from patients with CD were collected to analyse the correlation between TLR5/TSLP/TGFβ expression and the percentage of R. intestinalis.FindingsHere, we show that R. intestinalis inhibits the development of CD by increasing the differentiation of anti-inflammatory Tregs. Mechanistically, R. intestinalis stimulates TSLP production in intestinal epithelial cells (IECs) through TLR5 but not TLR2 or TLR4. TSLP produced by IECs specifically induces the secretion of interleukin-10 (IL-10) and TGFβ from DCs, which is necessary for subsequent Treg differentiation. Consequently, the depletion of TLR5 (using Tlr5−/− mice) or inhibition of TSLP (using anti-TSLP neutralizing antibodies) attenuates the protective effect of R. intestinalis on experimental colitis in mice. Importantly, the expression of TSLP in patients with CD is positively correlated with the level of R. intestinalis.InterpretationThese findings reveal the previously unknown mechanism of R. intestinalis-mediated intestinal immune regulation, which may provide the basis for new therapeutic strategies for CD.FundingThis work was funded by the National Natural Science Foundation of China (81670504 and 81970494), the Key Project of Research and Development Plan of Hunan Province (2019SK2041) and the Changsha Municipal Natural Science Foundation (kq2014258).

The magnitude of cell invasion and cell-to-cell spread of Listeria monocytogenes is correlated with serotype-specific traits

Listeriosis is a foodborne disease caused by the Gram-positive bacterium Listeria monocytogenes, a pathogen that modulates its intracellular survival via vacuolar escape and cytosolic replication. In the present study, we examined the ability of 58 L. monocytogenes isolates recovered in Brazil (beef, clinical and environmental samples, from 1978 to 2013) to invade, replicate and spread in a human intestinal epithelial cell line (Caco-2). Premature stop codons were common in the inlA gene of serotype 1/2c strains from beef and environment samples, associated with decreased Caco-2 cell invasion when compared to other serotypes. The isolates varied widely in their intracellular doubling times, and there was no clear relationship between serotypes and samples origin. Serotype 1/2a isolates were generally impaired in their ability to spread between Caco-2 cells, with an average 30 % smaller focus area than the 10403S reference strain. However, most isolates of serotype 1/2b exhibited enhanced cell-to-cell spread, with an average 35 % increase in focus area. Our findings are consistent with serotype being a better predictors of cell invasion potential and cell spread compared with sample origin of isolates, although the most invasive isolates were primarily isolated from beef. Additionally, we have identified isolates that could provide novel insight into the pathogenicity of L. monocytogenes that may not be revealed by studying common laboratory reference strains.

Endometrial Regenerative Cell-Derived Exosomes Attenuate Experimental Colitis through Downregulation of Intestine Ferroptosis.

Background Endometrial regenerative cells (ERCs) have been identified to ameliorate colitis in mice; however, whether exosomes derived from ERCs (ERC-exos) own similar effects on colitis remains unclear. Ferroptosis, an iron-dependent cell programmed death form, has been reported to promote inflammation in UC. Thus, in this study, whether ERC-exos can treat colitis and regulate intestine ferroptosis will be explored. Methods In this study, iron, malondialdehyde (MDA) production, glutathione (GSH) synthesis, and acyl-CoA synthetase long-chain family member (ACSL) 4 and glutathione peroxidase 4 (GPX4) expressions were measured in colon samples from healthy people and UC patients to explore the effects of ferroptosis. In vitro, ERC-exos were cocultured with ferroptosis inducer erastin-treated NCM460 human intestinal epithelial cell line, and ferroptotic parameters were measured. In vivo, colitis was induced by 3% dextran sulfate sodium (DSS) in BALB/c mice, and animals were randomly assigned to normal, untreated, and ERC-exos-treated groups. The Disease Activity Index (DAI) score, histological features, tissue iron, MDA, GSH, ACSL4, and GPX4 were measured to verify the role of ERC-exos in attenuating UC. Results Compared with healthy people, UC samples exhibited higher levels of iron, MDA, and ACSL4, while less levels of GSH and GPX4. In vitro, the CCK-8 assay showed that ERC-exos rescued erastin-induced cell death, and ERC-exos treatment significantly increased the levels of GSH and expression of GPX4, while markedly decreasing the levels of iron, MDA, and expression of ACSL4. In vivo, ERC-exos treatment effectively reduced DAI score, ameliorated colon pathological damage, and improved disease symptoms. Moreover, ERC-exos treatment further enhanced the levels of GSH and the expression of GPX4 but reduced the levels of iron, MDA, and expression of ACSL4 in the colon of colitis mice. Conclusions Ferroptosis was involved in the pathogenesis of UC, and ERC-exos attenuated DSS-induced colitis through downregulating intestine ferroptosis. This study may provide a novel insight into treating UC in the future.

CPT1 Mediated Ionizing Radiation-Induced Intestinal Injury Proliferation via Shifting FAO Metabolism Pathway and Activating the ERK1/2 and JNK Pathway.

The intestinal compensatory proliferative potential is a key influencing factor for susceptibility to radiation-induced intestinal injury. Studies indicated that the carnitine palmitoyltransferase 1 (CPT1) mediated fatty acid β-oxidation (FAO) plays a crucial role in promoting the survival and proliferation of tumor cells. Here, we aimed to explore the effect of 60Co gamma rays on CPT1 mediated FAO in the radiation-induced intestinal injury models, and investigate the role of CPT1 mediated FAO in the survival and proliferation of intestinal cells after irradiation. We detected the changed of FAO in the plasma and small intestine of Sprague Dawley (SD) rats at 24 h after 60Co gamma irradiation (0, 5 and 10 Gy), using target metabolomics, qRT-PCR, immunohistochemistry (IHC), western blot (WB) and related enzymatic activity kits. We then analyzed the FAO changes in radiation-induced intestinal injury models regardless of ex vivo (mice enteroids), or in vitro (normal human intestinal epithelial cell lines, HIEC-6). HIEC-6 cells were transduced with lentivirus vector GV392 and treated with puromycin for obtaining CPT1 stable knockout cell lines, named CPT1 KO. CPT1 enzymatic activities of HIEC-6 cells and mice enteroids were also inhibited by pharmaceutical inhibitor ST1326 and Etomoxir (ETO), to study the function of CPT1 in the survival and proliferation of HIEC-6 cells after 60Co gamma irradiation. We found that CPT1 mediated FAO was altered in the small intestine of the SD rats after irradiation, especially, the expression level and enzymatic activity of CPT1 were significantly increased. Similarly, the expression levels of CPT1 were also remarkably enhanced in mice enteroids and HIEC-6 cells after irradiation. CPT1 inhibition decreased the proliferation of the HIEC-6 cells and mice enteroids after irradiation partially by reducing the extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways activation, CPT1 inhibition also reduced the proliferation of mice enteroids after irradiation partially by down-regulating the Wnt/β-catenin signaling activity. In conclusion, our study indicated that CPT1 plays a crucial role in promoting intestinal epithelial cell proliferation after irradiation.

CPT1 Mediated Ionizing Radiation-Induced Intestinal Injury Proliferation via Shifting FAO Metabolism Pathway and Activating the ERK1/2 and JNK Pathway.

The intestinal compensatory proliferative potential is a key influencing factor for susceptibility to radiation-induced intestinal injury. Studies indicated that the carnitine palmitoyltransferase 1 (CPT1) mediated fatty acid β-oxidation (FAO) plays a crucial role in promoting the survival and proliferation of tumor cells. Here, we aimed to explore the effect of 60Co gamma rays on CPT1 mediated FAO in the radiation-induced intestinal injury models, and investigate the role of CPT1 mediated FAO in the survival and proliferation of intestinal cells after irradiation. We detected the changed of FAO in the plasma and small intestine of Sprague Dawley (SD) rats at 24 h after 60Co gamma irradiation (0, 5 and 10 Gy), using target metabolomics, qRT-PCR, immunohistochemistry (IHC), western blot (WB) and related enzymatic activity kits. We then analyzed the FAO changes in radiation-induced intestinal injury models regardless of ex vivo (mice enteroids), or in vitro (normal human intestinal epithelial cell lines, HIEC-6). HIEC-6 cells were transduced with lentivirus vector GV392 and treated with puromycin for obtaining CPT1 stable knockout cell lines, named CPT1 KO. CPT1 enzymatic activities of HIEC-6 cells and mice enteroids were also inhibited by pharmaceutical inhibitor ST1326 and Etomoxir (ETO), to study the function of CPT1 in the survival and proliferation of HIEC-6 cells after 60Co gamma irradiation. We found that CPT1 mediated FAO was altered in the small intestine of the SD rats after irradiation, especially, the expression level and enzymatic activity of CPT1 were significantly increased. Similarly, the expression levels of CPT1 were also remarkably enhanced in mice enteroids and HIEC-6 cells after irradiation. CPT1 inhibition decreased the proliferation of the HIEC-6 cells and mice enteroids after irradiation partially by reducing the extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK) pathways activation, CPT1 inhibition also reduced the proliferation of mice enteroids after irradiation partially by down-regulating the Wnt/β-catenin signaling activity. In conclusion, our study indicated that CPT1 plays a crucial role in promoting intestinal epithelial cell proliferation after irradiation.