HT-29 Intestinal Epithelial Cells Research Articles

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. Epithelial activation and/or barrier effects upon exposure to 2-50 μg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 μg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

Inhibition of ESCRT-independent extracellular vesicles biogenesis suppresses enterovirus 71 replication and pathogenesis in mice

Enterovirus 71 (EV71) causes hand-foot-and-mouth disease (HFMD), neurological complications, and even fatalities in infants. Clinically, the increase of extracellular vesicles (EVs) in EV71 patients' serum was highly associated with the severity of HFMD. EV71 boosts EVs biogenesis in an endosomal sorting complex required for transport (ESCRT)-dependent manner to facilitate viral replication. Yet, the impact of EVs-derived from ESCRT-independent pathway on EV71 replication and pathogenesis is highly concerned. Here, we assessed the effects of EV71-induced EVs from ESCRT-independent pathway on viral replication and pathogenesis by GW4869, a neutral sphingomyelinase inhibitor. Detailly, in EV71-infected mice, blockade of the biogenesis of tissue-derived EVs in the presence of GW4869 restored body weight loss, attenuated clinical scores, and improved survival rates. Furthermore, GW4869 dampens EVs biogenesis to reduce viral load and pathogenesis in multiple tissues of EV71-infected mice. Consistently, GW4869 treatment in a human intestinal epithelial HT29 cells decreased the biogenesis of EVs, in which the progeny EV71 particle was cloaked, leading to the reduction of viral infection and replication. Collectively, GW4869 inhibits EV71-induced EVs in an ESCRT-independent pathway and ultimately suppresses EV71 replication and pathogenesis. Our study provides a novel strategy for the development of therapeutic agents in the treatment for EV71-associated HFMD.

RNA-Based Anti-Inflammatory Effects of Membrane Vesicles Derived from Lactiplantibacillus plantarum.

Bacteria generally release extracellular membrane vesicles (MVs), which are nanoparticles that play important roles in bacterial-bacterial and bacterial-host communication. As probiotics, lactic acid bacteria provide diverse health benefits to their hosts. In this study, we found that the Gram-positive lactic acid bacteria Lactiplantibacillus plantarum subsp. plantarum NBRC 15891 produce high amounts of MVs (LpMVs), and that LpMVs inhibit interleukin (IL)-8 production induced by lipopolysaccharide in intestinal epithelial HT29 cells. Heat- or UV-killed bacterial cells did not exhibit anti-inflammatory effects, and there was no uptake of these bacterial cells; contrarily, LpMVs were taken up into the cytoplasm of HT29 cells. Small RNAs extracted from LpMVs also suppressed IL-8 production in HT29 cells, suggesting that RNAs in the cytoplasm of bacterial cells are encapsulated in the MVs and released from the cells, which may be delivered to HT29 cells to exert their anti-inflammatory effects. In addition, administration of LpMVs to mice with dextran sodium sulfate-induced colitis alleviated colitis-induced weight loss and colon length shortening, indicating that LpMV intake is likely to be effective in preventing or ameliorating colitis.

Distribution of P2Y and P2X purinergic receptor expression within the intestine.

Nucleotides are potent extracellular signaling molecules during homeostasis, infection, and injury due to their ability to activate purinergic receptors. The nucleotide ATP activates P2X receptors (P2RXs), whereas the nucleotides ADP, ATP, UTP, and UDP-glucose selectively activate different P2Y receptors (P2RYs). Several studies have established crucial roles for P2 receptors during intestinal inflammatory and infectious diseases, yet the most extensive characterization of purinergic signaling has focused on immune cells and the central and enteric nervous systems. As epithelial cells serve as the first barrier against irritants and infection, we hypothesized that the gut epithelium may express multiple purinergic receptors that respond to extracellular nucleotide signals. Using the Human Protein Atlas and Gut Cell Survey, we queried single-cell RNA sequencing (RNAseq) data for the P2 purinergic receptors in the small and large intestines. In silico analysis reveals robust mRNA expression of P2RY1, P2RY2, P2RY11, and P2RX4 throughout the gastrointestinal tract. Human intestinal organoids exhibited a similar expression pattern with a prominent expression of P2RY1, P2RY2, and P2RX4, but this purinergic receptor repertoire was not conserved in T84, Caco2, and HT29 intestinal epithelial cell lines. Finally, P2YR1 and P2YR2 agonists elicited robust calcium responses in human intestinal organoids, but calcium responses were weaker or absent in the cell lines. These findings suggest that the gastrointestinal epithelia respond to extracellular purinergic signaling via P2RY1, P2RY2, P2RY11, and P2RX4 receptors and highlight the benefit of using intestinal organoids as a model of intestinal purinergic signaling.NEW & NOTEWORTHY Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, however, these have largely been demonstrated in immune cells and the enteric nervous system. Although epithelial cells serve as the first barrier against infection and inflammation, the role of purinergic signaling within the gastrointestinal tract remains largely unknown. This work expands our knowledge of purinergic receptor distribution and relative expression along the intestine.

Transcriptional responses of human intestinal epithelial HT-29 cells to spore-displayed p40 derived from Lacticaseibacillus rhamnosus GG

BackgroundsThe aims of this study were to construct spore-displayed p40, a Lacticaseibacillus rhamnosus GG-derived soluble protein, using spore surface display technology and to evaluate transcriptional responses in human intestinal epithelial cells.Resultsp40 was displayed on the surface of Bacillus subtilis spores using spore coat protein CotG as an anchor protein. Effects of spore-displayed p40 (CotG-p40) on gene expression of intestinal epithelial cell line HT-29 were evaluated by transcriptome analysis using RNA-sequencing. As a result of differentially expressed gene (DEG) analysis, 81 genes were up-regulated and 82 genes were down-regulated in CotG-p40 stimulated cells than in unstimulated cells. Gene ontology enrichment analysis showed that CotG-p40 affected biological processes such as developmental process, metabolic process, cell surface receptor linked signaling pathway, and retinoic acid metabolic process. Gene-gene network analysis suggested that 10 DEGs (EREG, FOXF1, GLI2, PTGS2, SPP1, MMP19, TNFRSF1B, PTGER4, CLDN18, and ALDH1A3) activated by CotG-p40 were associated with probiotic action.ConclusionsThis study demonstrates the regulatory effects of CotG-p40 on proliferation and homeostasis of HT-29 cells. This study provided comprehensive insights into the transcriptional response of human intestinal epithelial cells stimulated by CotG-p40.

High-Molecular-Weight Dextran-Type Exopolysaccharide Produced by the Novel Apilactobacillus waqarii Improves Metabolic Syndrome: In Vitro and In Vivo Analyses.

Metabolic syndrome is a leading medical concern that affects one billion people worldwide. Metabolic syndrome is defined by a clustering of risk factors that predispose an individual to cardiovascular disease, diabetes and stroke. In recent years, the apparent role of the gut microbiota in metabolic syndrome has drawn attention to microbiome-engineered therapeutics. Specifically, lactic acid bacteria (LAB) harbors beneficial metabolic characteristics, including the production of exopolysaccharides and other microbial byproducts. We recently isolated a novel fructophilic lactic acid bacterium (FLAB), Apilactobacillus waqarii strain HBW1, from honeybee gut and found it produces a dextran-type exopolysaccharide (EPS). The objective of this study was to explore the therapeutic potential of the new dextran in relation to metabolic syndrome. Findings revealed the dextran’s ability to improve the viability of damaged HT-29 intestinal epithelial cells and exhibit antioxidant properties. In vivo analyses demonstrated reductions in body weight gain and serum cholesterol levels in mice supplemented with the dextran, compared to control (5% and 17.2%, respectively). Additionally, blood glucose levels decreased by 16.26% following dextran supplementation, while increasing by 15.2% in non-treated mice. Overall, this study displays biotherapeutic potential of a novel EPS to improve metabolic syndrome and its individual components, warranting further investigation.

Lacticaseibacillus rhamnosus R0011 secretome attenuates Salmonella enterica serovar Typhimurium secretome-induced intestinal epithelial cell monolayer damage and pro-inflammatory mediator production in intestinal epithelial cell and antigen-presenting cell co-cultures.

Certain lactic acid bacteria (LAB) are associated with immune modulatory activities including down-regulation of pro-inflammatory gene transcription and expression. While host antigen-presenting cells (APCs) and intestinal epithelial cells (IEC) can interact directly with both pathogenic and commensal bacteria through innate immune pattern recognition receptors, recent evidence indicates indirect communication through secreted molecules is an important inter-domain communication mechanism. This communication route may be especially important in the context of IEC and APC interactions which shape host immune responses within the gut environment. We have previously shown that the Lacticaseibacillus rhamnosus R0011 secretome (LrS) dampens pro-inflammatory gene transcription and mediator production from Tumor Necrosis Factor-α and Salmonella enterica serovar Typhimurium secretome (STS)-challenged HT-29 IECs through the induction of negative regulators of innate immunity. However, many questions remain about interactions mediated through these bacterial-derived soluble components and the resulting host immune outcomes in the context of IEC and APC interactions. In the present study, we examined the ability of the LrS to down-regulate pro-inflammatory gene transcription and cytokine production from STS-challenged T84 human IEC and THP-1 human monocyte co-cultures. Cytokine and chemokine profiling revealed that apically delivered LrS induces apical secretion of macrophage inhibitory factor (MIF) and down-regulates STS-induced pro-inflammatory mediator secretion into the apical and basolateral chambers of the T84/THP-1 co-culture. Transcriptional profiling confirmed these results, as the LrS attenuated STS challenge-induced CXCL8 and NFκB1 expression in T84 IECs and THP-1 APCs. Interestingly, the LrS also reversed STS-induced damage to monolayer transepithelial resistance (TER) and permeability, results which were confirmed by ZO-1 gene expression and immunofluorescence visualization of ZO-1 expression in T84 IEC monolayers. The addition of a MIF-neutralizing antibody abrogated the ability of the LrS to reverse STS-induced damage to T84 IEC monolayer integrity, suggesting a novel role for MIF in maintaining IEC barrier function and integrity in response to soluble components derived from LAB. The results presented here provide mechanistic evidence for indirect communication mechanisms used by LAB to modulate immune responses to pathogen challenge, using in vitro approaches which allow for IEC and APC cell communication in a context which more closely mimics that which occurs in vivo.

Functional screen of inflammatory bowel disease genes reveals key epithelial functions

BackgroundGenetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited.MethodsWe identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions.ResultsAnalysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota.ConclusionsThis functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell’s transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

Design, synthesis and biological evaluation of 1H-indazole derivatives as novel ASK1 inhibitors.

Apoptosis signal-regulating kinase 1 (ASK1, MAP3K5), a member of the mitogen-activated protein kinase (MAPK) signaling pathway, is involved in cell survival, differentiation, stress response, and apoptosis. ASK1 kinase inhibition has emerged as a promising therapeutic strategy for inflammatory disease. A series of novel ASK1 inhibitors with 1H-indazole scaffold were designed, synthesized and evaluated for their ASK1 kinase activity and AP1-HEK293cell inhibitory effect. Systematic structure-activity relationship (SAR) efforts led to the discovery of promising compound 15, which showed excellent invitro ASK1 kinase activity and potent inhibitory effects on ASK1 in AP1-HEK293cells. In a tumor necrosis factor-α (TNF-α)-induced HT-29 intestinal epithelial cell model, compound 15 exhibited a significantly protective effect on cell viability comparable to that of GS-4997; moreover, compound 15 exhibited no obvious cytotoxicity against HT-29cells at concentrations up to 25μM. Mechanistic research demonstrated that compound 15 suppresses phosphorylation in the ASK1-p38/JNK signaling pathway in HT-29cells, and regulates the expression levels of apoptosis-related proteins. Altogether, these results show that compound 15 may serve as a potential candidate compound for the treatment of inflammatory bowel disease (IBD).

Differential effects of oligosaccharides on the effectiveness of ampicillin against Escherichia coli in vitro

BackgroundThe mounting antibiotic resistance emphasizes an urgent need for alternatives. Recent investigations indicate that non-digestible oligosaccharides (NDOs), besides their prebiotic properties, can directly interact with pathogenic bacteria. In this study, the protective effect of alginate-oligosaccharides (AOS), chitosan-oligosaccharides (COS), galacto-oligosaccharides (GOS) and fructo-oligosaccharides, against enteropathogenicEscherichia. coli was investigated. MethodsThe effect of these NDOs onE. coli growth, adhesion and E. coli-induced inflammatory response (IL-8 release) of HT-29 intestinal epithelial cells were determined in vitro in the presence or absence of ampicillin, using minimum inhibitory concentration (MIC) assay, anti-adhesion assay and ELISA, respectively. ResultsAt low concentrations 0.5 % and 1%, AOS decreased theE. coli growth, while high GOS concentrations (6%, 8%, 10 %) were effective. Interestingly, the combination of the low concentrations of AOS with ampicillin (2 μg/mL) exerted a 2-fold decrease in the MIC level of ampicillin against E. coli. AOS also concentration dependently reduced the adherence of E. coli to HT-29 cells. The combination of AOS with ampicillin further increased these anti-adhesive properties. Pre-incubation of HT-29 cells with AOS, COS or GOS significantly hampered the E. coli-induced IL-8 release. ConclusionCurrent study highlights the direct effects of NDOs onE. coli growth, adhesion and inflammatory responses of HT-29 cells in vitro.

Protective Role of Natural and Semi-Synthetic Tocopherols on TNFα-Induced ROS Production and ICAM-1 and Cl-2 Expression in HT29 Intestinal Epithelial Cells.

Vitamin E, a fat-soluble compound, possesses both antioxidant and non-antioxidant properties. In this study we evaluated, in intestinal HT29 cells, the role of natural tocopherols, α-Toc and δ-Toc, and two semi-synthetic derivatives, namely bis-δ-Toc sulfide (δ-Toc)2S and bis-δ-Toc disulfide (δ-Toc)2S2, on TNFα-induced oxidative stress, and intercellular adhesion molecule-1 (ICAM-1) and claudin-2 (Cl-2) expression. The role of tocopherols was compared to that of N-acetylcysteine (NAC), an antioxidant precursor of glutathione synthesis. The results show that all tocopherol containing derivatives used, prevented TNFα-induced oxidative stress and the increase of ICAM-1 and Cl-2 expression, and that (δ-Toc)2S and (δ-Toc)2S2 are more effective than δ-Toc and α-Toc. The beneficial effects demonstrated were due to tocopherol antioxidant properties, but suppression of TNFα-induced Cl-2 expression seems not only to be related with antioxidant ability. Indeed, while ICAM-1 expression is strongly related to the intracellular redox state, Cl-2 expression is TNFα-up-regulated by both redox and non-redox dependent mechanisms. Since ICAM-1 and Cl-2 increase intestinal bowel diseases, and cause excessive recruitment of immune cells and alteration of the intestinal barrier, natural and, above all, semi-synthetic tocopherols may have a potential role as a therapeutic support against intestinal chronic inflammation, in which TNFα represents an important proinflammatory mediator.

Campylobacter concisus upregulates PD-L1 mRNA expression in IFN-γ sensitized intestinal epithelial cells and induces cell death in esophageal epithelial cells

ABSTRACT Introduction: Campylobacter concisus is an oral bacterium that is associated with inflammatory bowel disease (IBD) and Barrett’s esophagus (BE). Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein that is used by tumor cells for immune evasion and has increased expression in patients with IBD and BE. We examined whether C. concisus upregulates PD-L1 expression in intestinal and esophageal epithelial cells. Methods: Human intestinal epithelial HT-29 cells and esophageal epithelial FLO-1 cells with and without interferon (IFN)-γ sensitization were incubated with C. concisus strains. The level of PD-L1 mRNA was quantified using quantitative real-time PCR. Cytokines were measured using Enzyme-Linked Immunosorbent Assay (ELISA). Apoptosis of HT-29 and FLO-1 cells were measured using caspase 3/7 assay. Results: We found that intestinal epithelial cells with IFN-γ sensitization incubated with C. concisus significantly upregulated PD-L1 expression and significantly increased the production of interleukin (IL)-8. Whereas, PD-L1 expression was significantly inhibited in IFN-γ sensitized FLO-1 cells incubated with C. concisus strains. Furthermore, FLO-1 cells with and without IFN-γ sensitization incubated with C. concisus strains both had significantly higher levels of cell death. Conclusion: C. concisushas the potential to cause damage to both intestinal and esophageal epithelial cells, however, with different pathogenic effects.

A clostripain-like protease plays a major role in generating the secretome of enterotoxigenic Bacteroides fragilis.

Bacteroides fragilis toxin (BFT) is a protein secreted by enterotoxigenic (ETBF) strains of B. fragilis. BFT is synthesized as a proprotein (proBFT) that is predicted to be a lipoprotein and that is cleaved into two discrete fragments by a clostripain-like protease called fragipain (Fpn). In this study, we obtained evidence that Fpn cleaves proBFT following its transport across the outer membrane. Remarkably, we also found that the disruption of the fpn gene led to a strong reduction in the level of>100 other proteins, many of which are predicted to be lipoproteins, in the culture medium of an ETBF strain. Experiments performed with purified Fpn provided direct evidence that the protease releases at least some of these proteins from the cell surface. The observation that wild-type cells outcompeted an fpn- strain in co-cultivation assays also supported the notion that Fpn plays an important role in cell physiology and is not simply dedicated to toxin biogenesis. Finally, we found that purified Fpn altered the adhesive properties of HT29 intestinal epithelial cells. Our results suggest that Fpn is a broad-spectrum protease that not only catalyzes the protein secretion on a wide scale but that also potentially cleaves host cell proteins during colonization.

Escherichia coli K12 Upregulates Programmed Cell Death Ligand 1 (PD-L1) Expression in Gamma Interferon-Sensitized Intestinal Epithelial Cells via the NF-κB Pathway.

Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein which is used by tumor cells for immune evasion. PD-L1 is upregulated in inflamed intestinal tissues. The intestinal tract is colonized by millions of bacteria, most of which are commensal bacterial species. We hypothesized that under inflammatory conditions, some commensal bacterial species contribute to increased PD-L1 expression in intestinal epithelium and examined this hypothesis. Human intestinal epithelial HT-29 cells with and without interferon (IFN)-γ sensitization were incubated with six strains of four enteric bacterial species. The mRNA and protein levels of PD-L1 in HT-29 cells were examined using quantitative real-time PCR and flow cytometry, respectively. The levels of interleukin (IL)-1β, IL-18, IL-6, IL-8, and tumor necrosis factor (TNF)-α secreted by HT-29 cells were measured using enzyme-linked immunosorbent assay. Apoptosis of HT-29 cells was measured using a caspase 3/7 assay. We found that Escherichia coli K12 significantly upregulated both PD-L1 mRNA and protein in IFN-γ-sensitized HT-29 cells. E. coli K12 induced the production of IL-8 in HT-29 cells, however, IL-8 did not affect HT-29 PD-L1 expression. Inhibition of the nuclear factor-kappa B pathway significantly reduced E. coli K12-induced PD-L1 expression in HT-29 cells. The other two E. coli strains and two enteric bacterial species did not significantly affect PD-L1 expression in HT-29 cells. Enterococcus faecalis significantly inhibited PD-L1 expression due to induction of cell death. Data from this study suggest that some gut bacterial species have the potential to affect immune function under inflammatory conditions via upregulating epithelial PD-L1 expression.

Probiotic Lactobacillus and Bifidobacterium Strains Counteract Adherent-Invasive Escherichia coli (AIEC) Virulence and Hamper IL-23/Th17 Axis in Ulcerative Colitis, but Not in Crohn’s Disease

Hypersecretion of proinflammatory cytokines and dysregulated activation of the IL-23/Th17 axis in response to intestinal microbiota dysbiosis are key factors in the pathogenesis of inflammatory bowel diseases (IBD). In this work, we studied how Lactobacillus and Bifidobacterium strains affect AIEC-LF82 virulence mechanisms and the consequent inflammatory response linked to the CCR6–CCL20 and IL-23/Th17 axes in Crohn’s disease (CD) and ulcerative colitis (UC) patients. All Lactobacillus and Bifidobacterium strains significantly reduced the LF82 adhesion and persistence within HT29 intestinal epithelial cells, inhibiting IL-8 secretion while not affecting the CCR6–CCL20 axis. Moreover, they significantly reduced LF82 survival within macrophages and dendritic cells, reducing the secretion of polarizing cytokines related to the IL-23/Th17 axis, both in healthy donors (HD) and UC patients. In CD patients, however, only B. breve Bbr8 strain was able to slightly reduce the LF82 persistence within dendritic cells, thus hampering the IL-23/Th17 axis. In addition, probiotic strains were able to modulate the AIEC-induced inflammation in HD, reducing TNF-α and increasing IL-10 secretion by macrophages, but failed to do so in IBD patients. Interestingly, the probiotic strains studied in this work were all able to interfere with the IL-23/Th17 axis in UC patients, but not in CD patients. The different interaction mechanisms of probiotic strains with innate immune cells from UC and CD patients compared to HD suggest that testing on CD-derived immune cells may be pivotal for the identification of novel probiotic strains that could be effective also for CD patients.

Secretome-Mediated Interactions with Intestinal Epithelial Cells: A Role for Secretome Components from Lactobacillus rhamnosus R0011 in the Attenuation of Salmonella enterica Serovar Typhimurium Secretome and TNF-α-Induced Proinflammatory Responses.

Recent evidence suggests that lactic acid bacteria communicate with host cells via secretome components to influence immune responses but less is known about gut-pathogen secretomes, impact of lactic acid bacteria secretomes on host-pathogen interactions, and the mechanisms underlying these interactions. Genome-wide microarrays and cytokine profiling were used to interrogate the impact of the Lactobacillus rhamnosus R0011 secretome (LrS) on TNF-α and Salmonella enterica subsp. enterica serovar Typhimurium secretome (STS)-induced outcomes in human intestinal epithelial cells. The LrS attenuated both TNF-α- and STS-induced gene expression involved in NF-κB and MAPK activation, as well as expression of genes involved in other immune-related signaling pathways. Specifically, the LrS induced the expression of dual specificity phosphatase 1 (DUSP1), activating transcription factor 3 (ATF3), and tribbles pseudokinase 3 (TRIB3), negative regulators of innate immune signaling, in HT-29 intestinal epithelial cells challenged with TNF-α or STS. TNF-α- and STS-induced acetylation of H3 and H4 histones was attenuated by the LrS, as was the production of TNF-α- and STS-induced proinflammatory cytokines and chemokines. Interestingly, the LrS induced production of macrophage migration inhibitory factor (MIF), a cytokine involved in host-microbe interactions at the gut interface. We propose that the LrS attenuates proinflammatory mediator expression through increased transcription of negative regulators of innate immune activity and changes in global H3 and H4 histone acetylation. To our knowledge, these findings provide novel insights into the complex multifaceted mechanisms of action behind secretome-mediated interdomain communication at the gut-mucosal interface.

Melatonin controls microbiota in colitis by goblet cell differentiation and antimicrobial peptide production through Toll-like receptor 4 signalling

Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Melatonin improved dextran sulfate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3β, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Herein, we showed that melatonin significantly increases goblet cells, Reg3β, and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

Presence of PTPN2 SNP rs1893217 Enhances the Anti-inflammatory Effect of Spermidine.

The single nucleotide polymorphism (SNP) rs1893217 within the gene locus encoding PTPN2 represents a risk factor for inflammatory bowel disease (IBD). Our previous work demonstrated reduced PTPN2 activity and subsequently increased inflammatory signaling upon presence of SNP rs1893217. The naturally occurring polyamine spermidine reduces pro-inflammatory signaling via induction of PTPN2 activity; however, the effect of SNP rs1893217 on the anti-inflammatory potential of spermidine is still unknown. Here, we investigated how presence of SNP rs1893217 affects treatment efficacy of spermidine and whether it might serve as a potential biomarker for spermidine treatment. Human T84 (wild-type [WT] for PTPN2 SNP rs1893217) and HT29 (heterozygous for PTPN2 SNP rs1893217) intestinal epithelial cells (IECs) were treated with several polyamines from the putrescine-spermidine pathway. T84 and HT29 IECs, THP-1 monocytes (WT and transfected with a lentiviral vector expressing PTPN2 SNP rs1893217) and genotyped, patient-derived peripheral blood mononuclear cells were challenged with IFN-γ and/or spermidine. Among the analyzed polyamines, spermidine was the most efficient activator of PTPN2 phosphatase activity, regardless of the PTPN2 genotype. Spermidine suppressed IFN-γ-induced STAT1 and STAT3 phosphorylation, along with decreased mRNA expression of ICAM-1, NOD2, and IFNG in IECs and monocytes. Of note, these effects were clearly more pronounced when the disease-associated PTPN2 C-variant in SNP rs1893217 was present. Our data demonstrate that spermidine is the most potent polyamine in the putrescine-spermine axis for inducing PTPN2 enzymatic activity. The anti-inflammatory effect of spermidine is potentiated in the presence of SNP rs1893217, and this SNP might thus be a useful biomarker for possible spermidine-treatment in IBD patients.

P041 Melatonin controls microbiota in colitis through Toll-like receptor 4 signalling

Abstract Background Microbial dysbiosis has long been postulated to be associated with the pathogenesis of inflammatory bowel disease (IBD). Although evidence supporting the anti-colitic effects of melatonin have been accumulating, it is not clear how melatonin affects the microbiota. Herein, we investigated the effects of melatonin on the microbiome in colitis and identified involvement of Toll-like receptor (TLR) 4 signalling in the effects. Methods Colitis was induced in 8-week-old wild-type (WT) and TLR4 KO mice by administration of 2.5% dextran sulphate sodium (DSS) in drinking water and intraperitoneal (i.p.) treatment with melatonin (Mel) or vehicle (Veh). Body weight changes were monitored daily. Results DSS-treatment for 10 days significantly increased body weight loss in the vehicle-treated group of both wild-type and TLR4 KO mice. Melatonin improved dextran sulphate sodium (DSS)-induced colitis and reverted microbial dysbiosis in wild-type (WT) mice but not in TLR4 knockout (KO) mice. Induction of goblet cells was observed with melatonin administration, which was accompanied by suppression of Il1b and Il17a and induction of melatonin receptor and Reg3β, an antimicrobial peptide (AMP) against Gram-negative bacteria. In vitro, melatonin treatment of HT-29 intestinal epithelial cells promotes mucin and wound healing and inhibits growth of Escherichia coli. Conclusion we showed that melatonin significantly increases goblet cells, Reg3β and the ratio of Firmicutes to Bacteriodetes by suppressing Gram-negative bacteria through TLR4 signalling. Our study suggests that sensing of bacteria through TLR4 and regulation of bacteria through altered goblet cells and AMPs is involved in the anti-colitic effects of melatonin. Melatonin may have use in therapeutics for IBD.

The JAK-Inhibitor Tofacitinib Rescues Human Intestinal Epithelial Cells and Colonoids from Cytokine-Induced Barrier Dysfunction.

Alterations to epithelial tight junctions can compromise the ability of the epithelium to act as a barrier between luminal contents and the underlying tissues, thereby increasing intestinal permeability, an early critical event in inflammatory bowel disease (IBD). Tofacitinib (Xeljanz), an orally administered pan-Janus kinase (JAK) inhibitor, was recently approved for the treatment of moderate to severe ulcerative colitis. Nevertheless, the effects of tofacitinib on intestinal epithelial cell functions are largely unknown. The aim of this study was to determine if JAK inhibition by tofacitinib can rescue cytokine-induced barrier dysfunction in intestinal epithelial cells (IECs). T84 IECs were used to evaluate the effects of tofacitinib on JAK-signal transducer and activator of transcription (STAT) activation, barrier permeability, and expression and localization of tight junction proteins. The impact of tofacitinib on claudin-2 promoter activity was assessed in HT-29 IECs. Tofacitinib rescue of barrier function was also tested in human colonic stem cell-derived organoids. Pretreatment with tofacitinib prevented IFN-γ-induced decreases in transepithelial electrical resistance (TER) and increases in 4 kDa FITC-dextran permeability (FD4), partly due to claudin-2 transcriptional regulation and restriction of ZO-1 rearrangement at tight junctions. Although tofacitinib administered after IFN-γ challenge only partially normalized TER and claudin-2 levels, FD4 permeability and ZO-1 localization were fully recovered. The IFN-γ-induced FD4 permeability in primary human colonoids was fully rescued by tofacitinib. These data suggest differential therapeutic efficacy of tofacitinib in the rescue of pore vs leak-tight junction barrier defects and indicate a potential contribution of improved epithelial barrier function to the beneficial effects of tofacitinib in IBD patients.