Intestinal Epithelial Cells Research Articles

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Tissue-, region-, and gene-specific induction of microsomal epoxide hydrolase expression and activity in the mouse intestine by arsenic in drinking water.

This study aimed to characterize the effects of arsenic exposure on the expression of microsomal epoxide hydrolase (mEH or Ephx1) and soluble epoxide hydrolase (sEH or Ephx2) in the liver and small intestine. C57BL/6 mice were exposed to sodium arsenite in drinking water at various doses for up to 28 days. Intestinal, but not hepatic, mEH mRNA and protein expression was induced by arsenic at 25 ppm, in both males and females, whereas hepatic mEH expression was induced by arsenic at 50 or 100 ppm. The induction of mEH was gene specific, as the arsenic exposure did not induce sEH expression in either tissue. Within the small intestine, mEH expression was induced only in the proximal, but not the distal segments. The induction of intestinal mEH was accompanied by increases in microsomal enzymatic activities toward a model mEH substrate, cis-stilbene oxide, and an epoxide-containing drug, oprozomib, in vitro, and by increases in the levels of PR-176, the main hydrolysis metabolite of oprozomib, in the proximal small intestine of oprozomib-treated mice. These findings suggest that intestinal mEH, playing a major role in converting xenobiotic epoxides to less reactive diols, but not sEH, preferring endogenous epoxides as substrates, is relevant to the adverse effects of arsenic exposure, and that further studies of the interactions between drinking water arsenic exposure and the disposition or possible adverse effects of epoxide-containing drugs and other xenobiotic compounds in the intestine are warranted. Significance Statement Consumption of arsenic-contaminated water has been associated with increased risks of various adverse health effects, such as diabetes, in humans. The small intestinal epithelial cells are the main site of absorption of ingested arsenic, but they are not well characterized for arsenic exposure-related changes. This study identified gene expression changes in the small intestine that may be mechanistically linked to the adverse effects of arsenic exposure and possible interactions between arsenic ingestion and the pharmacokinetics of epoxide-containing drugs in vivo.

Modified Lichong decoction intervenes in colorectal cancer by modulating the intestinal flora and the Wnt/β-catenin signaling pathway

BackgroundThe pathogenesis and treatment of colorectal cancer (CRC) continue to be areas of ongoing research, especially the benefits of traditional Chinese medicine (TCM) in slowing the progression of CRC. This study was conducted to investigate the effectiveness and mechanism of action of modified Lichong decoction (MLCD) in inhibiting CRC progression.MethodsWe established CRC animal models using azoxymethane/dextran sodium sulfate (AOM/DSS) and administered high, medium, or low doses of MLCD or mesalazine (MS) for 9 weeks to observe MLCD alleviation of CRC. The optimal MLCD dose group was then subjected to metagenomic and RNA sequencing (RNA-seq) to explore the differentially abundant flora and genes in the control, model and MLCD groups. Finally, the mechanism of action was verified using WB, qRT‒PCR, immunohistochemistry and TUNEL staining.ResultsMLCD inhibited the progression of CRC, and the optimal effect was observed at high doses. MLCD regulated the structure and function of the intestinal flora by decreasing the abundance of harmful bacteria and increasing that of beneficial bacteria. The differentially expressed genes were mainly associated with the Wnt/β-catenin pathway and the cell cycle. Molecular biology analysis indicated that MLCD suppressed the Wnt/β-catenin pathway and the epithelial–mesenchymal transition (EMT), inhibited abnormal cell proliferation and promoted intestinal epithelial cell apoptosis.ConclusionMLCD mitigated the abnormal growth of intestinal epithelial cells and promoted apoptosis, thereby inhibiting the progression of CRC. This inhibition was accomplished by modifying the intestinal microbiota and disrupting the Wnt/β-catenin pathway and the EMT. Therefore, MLCD could serve as a potential component of TCM prescriptions for CRC treatment.

Extracellular release of damaged mitochondria induced by prehematopoietic stem cell transplant conditioning exacerbates GVHD

AbstractDespite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.

Assessment of Ingested Micro- and Nanoplastic (MNP)-Mediated Genotoxicity in an In Vitro Model of the Small Intestinal Epithelium (SIE).

Micro- and nanoplastics (MNPs) have become ubiquitous contaminants of water and foods, resulting in high levels of human ingestion exposure. MNPs have been found in human blood and multiple tissues, suggesting that they are readily absorbed by the gastrointestinal tract (GIT) and widely distributed. Growing toxicological evidence suggests that ingested MNPs may pose a serious health threat. The potential genotoxicity of MNPs, however, remains largely unknown. In this study, genotoxicity of primary and environmentally relevant secondary MNPs was assessed in a triculture small intestinal epithelium (SIE) model using the CometChip assay. Aqueous suspensions of 25 and 1000 nm carboxylated polystyrene spheres (PS25C and PS1KC), and incinerated polyethylene (PEI PM0.1) were subjected to simulated GIT digestion to create physiologically relevant exposures (digestas), which were applied to the SIE model at final MNP concentrations of 1, 5, and 20 μg/mL for 24 or 48 h. PS25C and PS1KC induced DNA damage in a time- and concentration-dependent manner. To our knowledge, this is one of the first assessment of MNP genotoxicity in an integrated in vitro ingestion platform including simulated GIT digestion and a triculture SIE model. These findings suggest that ingestion of high concentrations of carboxylated PS MNPs could have serious genotoxic consequences in the SIE.

Targeting programmed cell death in inflammatory bowel disease through natural products: New insights from molecular mechanisms to targeted therapies.

Inflammatory bowel disease (IBD) is an autoimmune disorder primarily characterized by intestinal inflammation and recurrent ulceration, leading to a compromised intestinal barrier and inflammatory infiltration. This disorder's pathogenesis is mainly attributed to extensive damage or death of intestinal epithelial cells, along with abnormal activation or impaired death regulation of immune cells and the release of various inflammatory factors, which contribute to the inflammatory environment in the intestines. Thus, maintaining intestinal homeostasis hinges on balancing the survival and functionality of various cell types. Programmed cell death (PCD) pathways, including apoptosis, pyroptosis, autophagy, ferroptosis, necroptosis, and neutrophil extracellular traps, are integral in the pathogenesis of IBD by mediating the death of intestinal epithelial and immune cells. Natural products derived from plants, fruits, and vegetables have shown potential in regulating PCD, offering preventive and therapeutic avenues for IBD. This article reviews the role of natural products in IBD treatment by focusing on targeting PCD pathways, opening new avenues for clinical IBD management.

PSI-19 Growth performance of weaned pigs fed Jerusalem Artichoke tubers

Abstract Recent studies have shown that prebiotics have been able to positively affect growth of beneficial bacteria, indices of gut health, and growth performance in pigs. This type of research is important to identify alternative methods for maintaining herd health other than antimicrobial usage. Jerusalem Artichoke tubers were chosen due to their relatively greater amount of inulin (~60%) compared with other prebiotic crops (i.e., chicory pulp - ~20% inulin). The objective of this work was to evaluate growth performance of weaned pigs fed Jerusalem Artichoke tubers. Sixty-four pigs approximately 21 d of age, weighing 6 kg each were used to test four dietary treatments with four pens per treatment (two barrows and two gilts per pen). Dietary treatments were randomly allocated to pens following a completely randomized design in a three-phase feeding trial. Diet treatments consisted of a corn-soybean meal basal diet (control) meeting the nutrient requirements for weanling pigs and three treatment diets with ground Jerusalem Artichoke tubers replacing corn at the rate of 0.05%, 0.5%, and 5.0% (JA-low, JA-mid, and JA-high, respectively) through all three phases. The pigs were allowed 3 d to acclimate to the facility following arrival (d -3 to -1). The trial ran from d 0 to d 35. Growth performance data were collected on d 0, 7, 14, 21, 28, and 35. Blood samples were obtained on d 0, 7, 14, and 28. Fecal samples were collected on d 0, 14, and 28. PROC GLIMMIX in SAS was utilized to analyze the growth performance data. Results indicate greater (P < 0.001) gain to feed ratio (G:F) at wk 2 for JA-high and JA-mid pigs compared with all other treatments. No differences were observed for overall body weight (BW; 25.14, 25.48, 21.8, 22.83 kg), average daily gain (ADG; 0.52, 0.52, 0.51, 0.47 kg/d), or average daily feed intake (ADFI; 0.73, 0.74, 0.71, 0.72 kg/d), respectively, for control, JA-low, JA-mid, and JA-high fed pigs. The growth data suggests that Jerusalem Artichoke tubers may be included in weaned pig diets without negative effects on growth performance; however, these results need to be interpreted in conjunction with the pending analyses to provide a comprehensive look at the role of prebiotic fiber in the gastrointestinal tract of the pig. Future analyses will include evaluation of serum biomarkers (e.g., intestinal fatty acid binding protein as a measure of intestinal epithelial cell damage) and microbiome profiling via 16S microbial sequencing to assess gut health. It is important to note that our study population experienced morbidity and mortality (Trueperella pyogenes was cultured from one pig submitted for necropsy) and the decision was made to prophylactically treat the herd with ceftiofur on d 7 following sample collection to prevent additional loss.

PSII-6 The interactive effects of deoxynivalenol and lipopolysaccharides on gut integrity and inflammation in IPEC-J2 cells

Abstract The objective of this research is to investigate the interactive effects of deoxynivalenol (DON) and lipopolysaccharides (LPS) on gut integrity and inflammation using porcine intestinal epithelial cell line (IPEC-J2). Three experiments were conducted in this study: Exp 1: cells were treated with control, DON (0.5 ppm) or LPS (10 ug/mL) individually and combination of DON and LPS for 24 h, then measure the Lactate dehydrogenase (LDH) release and interleukin 8 (IL8) production in medium and measure the transepithelial electric resistance (TEER) and FITC-dextran passage rate at the end of each experiment. 2) Treat the cells with control or DON for 24 h, then split the two groups for control or LPS treatment for additional 24 h; same measurements were taken as in experiment 1; 3) Treat the cells with control or LPS for 24 h, then split the two groups for control or DON treatment for additional 24 h; same measurements were taken as in previous experiments. In each experiment, six replications were done. Data were analyzed for the effect of DON, LPS and their interaction. Treatments did not affect the LDH in all experiments except for the high LDH release in the treatment of LPS followed by DON in experiment 3. In experiment 1, DON, LPS or combination of DON and LPS significantly reduced the TEER value by 15.1%, 21.7% and 23.9% respectively with only DON has effect on FITC-dextran passage rate (P < 0.001). LPS significantly increased IL8 secretion in the medium (P < 0.001). In experiment 2, DON or LPS decreased TEER value whereas the cells treated with DON before were more susceptible to LPS (23.6% more reduction compared without DON treatment before). Similarly, the FITC-dextran passage rate was also increased by LPS in the cells exposed to DON before (P < 0.05). In addition, LPS significantly increased IL8 secretion no matter the cells was exposed to DON before. In experiment 3, DON or LPS decreased TEER value (P < 0.05) and cells treated with LPS before were more susceptible to DON (11.8% more reduction compared with no LPS treatment before). However, only DON increased FITC-dextran passage rate. The secretion of IL8 increased by DON or LPS with greatest secretion when cells treated with DON which exposed to LPS before (P < 0.05). In conclusion, the results suggested that cells were more susceptible to LPS when the cells were exposed to DON before or vice versa, and the synergetic effect is limited when LPS and DON were added to cells simultaneously.

Cellular evidence and spatial distribution of endosomal biosynthesis and autophagy in intestinal immune barrier cells of crucian carp (Carassiuscarassius)

Crucian carp (Carassius carassius) is an important aquatic economic animal, and the immune barrier function of its intestine has been a focus of research into oral vaccines and drugs. However, the histological structures of the intestinal barrier and its adjacent areas have not been clearly established, and little subcellular evidence is available to elucidate the spatial distribution of intracellular biological processes. In this study, the spatial distribution of autophagy and endosome formation in the intestinal epithelial cells (IECs) of crucian carp were analyzed. These two biological activities are closely related to intestinal homeostasis, immunity, and cell communication. Periodic acid-Schiff (PAS) and Masson's trichrome staining were employed to elucidate the distinctive histological framework of the Crucian carp's myoid cell network, which resides within the subepithelial layer and is characterized by gap junctions. Transmission electron microscopy (TEM), immunohistochemistry (IHC), and immunofluorescence (IF) were used to detect the structural and functional aspects of the IEC in different intestinal segments. TEM and immunohistochemical analyses captured the biogenesis and maturation of early and late endosomes as well as multivesicular bodies (MVBs), as well as the initiation and progression of autophagy, including macroautophagy and mitophagy. The endosome and MVBs-specific marker CD63 and autophagy-related protein LC3 were highly expressed in IECs and were correlated with autophagy and endosome biosynthesis in the apical and basal regions of individual cells, and differed between different intestinal segments. In summary, this study elucidated the ubiquity and morphological characteristics of autophagy and endosome formation across different intestinal segments of crucian carp. A unique myoid cell network beneath the intestinal epithelium in crucian carp was also identified, expanding the histological understanding of this animal's intestinal tract.

Probiotics enhance alveolar bone microarchitecture, intestinal morphology and estradiol levels in osteoporotic animals.

Osteoporosis is associated with bone microarchitecture alterations, and the depletion of estrogen during menopause is a major contributing factor to its development. The literature highlights the noteworthy role of gut microbiota in bone metabolism, particularly in the progression of osteoporosis. Periodontal disease leads to alveolar bone loss, which may be influenced by estrogen deficiency, and this mechanism is intricately associated with an imbalance in systemic microbiota. The aim of this study was to evaluate the effects of Bifidobacterium animalis subsp. lactis HN019 (B. lactis HN019) and Lacticaseibacillus casei 01 (L. casei 01) administrations on an osteoporosis animal model. Thirty-three female rats were randomly divided into three groups: control (C-OVX), C-OVX-HN019 and C-OVX-LC01. All animals were ovariectomized. In groups C-OVX-HN019 and C-OVX-LC01, the probiotics were administered for 4 months. All animals were euthanized after 16 weeks from ovariectomy. Microtomographic, histopathological and immunohistochemical examinations were conducted on periodontal tissues, whereas histomorphometry, histopathological and immunohistochemical analyses were carried out on the intestine. The levels of estradiol were assessed in blood using an immunoenzymatic assay. The data were subjected to statistical analyses (p < .05). The C-OVX-LC01 group exhibited a significant reduction in alveolar bone porosity and an increase in connective tissue density compared to C-OVX (p < .05). The C-OVX-HN019 and C-OVX-LC01 groups presented reduced expression of TRAP and RANKL compared to the C-OVX (p < .05). The C-OVX group presented villi defects, mild neutrophil infiltration, decrease in both villous height and intestinal crypts and reduced expression of intestinal junctional epithelium markers e-cadherin and claudin 01 compared to C-OVX-HN019 and C-OVX-LC01 (p < .05). The C-OVX group had lower estradiol levels than C-OVX-HN019 and C-OVX-LC01 (p < .05). The probiotic therapy promoted a reduction in alveolar bone destruction and intestinal permeability as well as an increase in estradiol levels in ovariectomized rats. Specifically, the probiotic strain Lacticaseibacillus casei 01 exhibited greater effectiveness compared to Bifidobacterium animalis subsp. lactis HN019, indicating strain-dependent outcomes.

4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study.

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4β,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4β,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Differential Effect of JAK1/2 and TYK2 Inhibitors on Type III Interferon Induced Antiviral Defence in Human Intestinal Epithelial Cells

Plain Language SummaryCytokines are small molecules that play a crucial role in causing inflammatory diseases. Janus kinase (JAK) and tyrosine kinase (TYK) are proteins that help these messengers communicate with cells. JAK inhibitors (JAKis) are drugs that block these proteins and are becoming more and more commonly used to treat inflammatory diseases. A common side effect of JAKis is an increased risk for viral infections. In this study, it is shown that a TYK2 inhibitor drug may not weaken our defences against viruses in our intestines as much as a JAK1/2 inhibitor. As this inhibitor blocks the disease causing cytokines, this inhibitor drug may be better than the other and be associated with lower risk of infections with viruses that enter the body through the gut.

CXCR2, as a key regulatory gene of HDP-PG-1, maintains intestinal mucosal homeostasis

The intestine defends against pathogenic microbial invasion via the secretion of host defense peptides (HDPs). Nutritional immunomodulation can stimulate the expression of endogenous HDPs and enhance the body's immune defense, representing a novel non-antibiotic strategy for disease prevention. The project aims to explore the regulatory mechanism of protegrin-1 (PG-1) expression using sodium phenylbutyrate (PBA) by omics sequencing technology and further investigate the role of key regulatory genes on intestinal health. The results showed that PBA promoted PG-1 expression in intestinal epithelial cells based on cell density through epidermal growth factor receptor (EGFR) and G protein-coupled receptor (GPR43). Transcriptome sequencing and microRNA sequencing revealed that C-X-C motif chemokine receptor 2 (CXCR2) exhibited interactions with PG-1. Pre-treatment cells with a CXCR2 inhibitor (SB225002) effectively suppressed the induction of PG-1 by PBA. Furthermore, SB225002 significantly suppressed the gene expression of HDPs in the jejunum of mice without influencing on the morphology, number of goblet cells, and proliferation of the intestine. CXCR2 inhibition significantly reduced the expression of HDPs during E. coli infection, and resulted in the edema of jejunal epithelial cells. The 16S rDNA analysis of cecal contents showed that the E. coli and SB225002 treatments changed gut microbiota diversity and composition at different taxonomic levels. Correlation analysis suggested a potential regulatory relationship between gut microbiota and HDPs. To that end, a gene involved in the HDP expression, CXCR2, has been identified in the study, which contributes to improving intestinal immune function. PBA may be used as a functional additive to regulate intestinal mucosal function, thereby enhancing the health of the intestinal and host.

Probiotic Characteristics of Lactiplantibacillus plantarum CECT 9435 and Its Survival and Competitive Properties Under Simulated Conditions of the Child Gut Microbiota.

Probiotics are valuable microorganisms effective in reducing malnutrition-related infections in children. In this work, a collection of lactobacilli strains representative of traditional Andean fermented beverages was in vitro screened for their capability to survive the gastrointestinal transit, to adhere to the intestinal epithelium and to compete under simulated conditions of the child gut microbiota. The results allowed the selection of the riboflavin overproducing strain Lactiplantibacillus plantarum CECT 9435 based on its good rate of survival under in vitro gastrointestinal conditions when included in a food matrix representing the fortified food supplement Incaparina. The strain also showed good adhesion to HT29 cells producing mucus and outstanding performance in E. coli competition for the adhesion to this epithelial cell line. L. plantarum CECT 9435 gut performance was also evaluated in the child intestinal microbiota simulated in a dynamic gut model (BFBL simulator). The viability of the probiotic candidate in the gut conditions was high during the 7-day intervention period, reaching over 1 × 107 counts in each of the reactors simulating the three colonic regions. The transient viability of L. plantarum CECT 9435 within the child gut microbiota and its adhesion capacity to intestinal cells could facilitate the strain potential benefits as probiotic added to fortified supplementary foods destined to malnourished children.

Arginase 2 attenuates ulcerative colitis by antioxidant effects of spermidine.

Spermidine suppress oxidative stress and is involved in various disease pathogenesis including ulcerative colitis (UC). Arginase 2 (ARG2) plays a central role in the synthesis of spermidine. This study aimed to clarify the effect of endogenously produced spermidine on colitis. The physiological role of ARG2 and spermidine was investigated using Arg2-deficient mice with reduced spermidine. Immunohistochemical staining of the rectum was used to analyze ARG2 expression and spermidine levels in healthy controls and UC patients. In mice with dextran sulfate sodium-induced colitis, ARG2 and spermidine levels were increased in the rectal epithelium. Spermidine protects colonic epithelial cells from oxidative stress and Arg2 knockdown cells reduced antioxidant activity. Organoids cultured from the small intestine and colon of Arg2-deficient mice both were more susceptible to oxidative stress. Colitis was exacerbated in Arg2-deficient mice compared to wild-type mice. Supplementation with spermidine result in comparable severity of colitis in both wild-type and Arg2-deficient mice. In the active phase of UC, rectal ARG2 expression and spermidine accumulation were increased compared to remission. ARG2 and spermidine levels were similar in healthy controls and UC remission patients. ARG2 produces spermidine endogenously in the intestinal epithelium and has a palliative effect on ulcerative colitis. ARG2 and spermidine are potential novel therapeutic targets for UC.

Global Transcriptome Analysis Reveals Corresponding Genes and Key Pathways Involved in Oxidative Stress in Mouse Small Intestinal Cells

Abstract Understanding the molecular mechanisms involved in the effects of oxidative stress in humans and animals is important to minimize the damage it causes, leading to various intestinal diseases. Our aim is to study the genes and pathways involved in oxidative stress in the gut using mouse small intestinal epithelial cells (MODE-K) as a model. The MODE-K cell line was divided into two different groups: one group was treated with hydrogen peroxide (H2O2) and the other group was not. To analyze the effects of H2O2 exposure, cell viability, apoptosis rate and reactive oxygen species (ROS) levels were determined. Next, transcriptome sequencing was performed, differentially expressed genes (DEGs) were identified and function annotation was performed, followed by a series of bioinformatics analyses. Real-time PCR was used to confirm the transcriptome data. Our results showed that H2O2-induced oxidative stress significantly increased ROS synthesis and promoted cell apoptosis in mouse small intestinal epithelial cells. During oxidative stress, 1207 DEGs (859 up-regulated, 348 down-regulated) were identified. According to GO analysis, DEGs are annotated into 51 different GO classifications including 22 biological processes, 15 cellular components and 14 molecular functions. In addition, using KEGG, PPI and correlation analysis, the two most significant subnetworks were identified. Ten correlated nodal DEGs of the first subnetwork correspond to MAPK, NF-kappa B and PI3K-AKT signaling pathways, and six correlated DEGs of the second subnetwork are associated with mitochondria. KDM6B was found to link these two subnetworks. The results suggest that oxidative stress affects epithelial growth, metabolism and apoptosis in a mouse model of intestinal cells through signaling pathways such as MAPK and PI3K/AKT/NF-kappa B, and mitochondria-related genes that are interconnected through the PTGS2-KDM6B-MT-ATP6 pathway.

Sorbitol Destroyed Intestinal Microfold Cells (M Cells) Development through Inhibition of PDE4-Mediated RANKL Expression.

Microfold cells (M cells) are specific intestinal epithelial cells for monitoring and transcytosis of antigens, microorganisms, and pathogens in the intestine. However, the mechanism for M-cell development remained elusive. Real-time polymerase chain reaction, immunofluorescence, and western blotting were performed to analyze the effect of sorbitol-regulated M-cell differentiation in vivo and in vitro, and luciferase and chromatin Immunoprecipitation were used to reveal the mechanism through which sorbitol-modulated M-cell differentiation. Herein, in comparison to the mannitol group (control group), we found that intestinal M-cell development was inhibited in response to sorbitol treatment as evidenced by impaired enteroids accompanying with decreased early differentiation marker Annexin 5, Marcksl1, Spib, sox8, and mature M-cell marker glycoprotein 2 expression, which was attributed to downregulation of receptor activator of nuclear factor kappa-В ligand (RANKL) expression in vivo and in vitro. Mechanically, in the M-cell model, sorbitol stimulation caused a significant upregulation of phosphodiesterase 4 (PDE4) phosphorylation, leading to decreased protein kinase A (PKA)/cAMP-response element binding protein (CREB) activation, which further resulted in CREB retention in cytosolic and attenuated CREB binds to RANKL promoter to inhibit RANKL expression. Interestingly, endogenous PKA interacted with CREB, and this interaction was destroyed by sorbitol stimulation. Most importantly, inhibition of PDE4 by dipyridamole could rescue the inhibitory effect of sorbitol on intestinal enteroids and M-cell differentiation and mature in vivo and in vitro. These findings suggested that sorbitol suppressed intestinal enteroids and M-cell differentiation and matured through PDE4-mediated RANKL expression; targeting to inhibit PDE4 was sufficient to induce M-cell development.

Elucidating the Role of SYK in the Intestinal Epithelium

Background: Inflammatory Bowel Disease (IBD) involves chronic and recurring inflammation of the gastrointestinal tract. We identified several patients presenting with immune dysregulation and systemic inflammation, including inflammation of the gut. Analysis of Whole Exome Sequencing data from pediatric IBD patients has identified five rare protein coding Spleen Tyrosine Kinase (SYK) variants. Functional studies have demonstrated increased kinase activity implicating them in disease pathophysiology. While the role of SYK is well characterized in immune cells, it is not understood what role it plays in the intestinal epithelium. Aims: The primary aim of this project is to understand the role SYK plays in the intestinal epithelium, what molecular pathways it signals through, and how its dysregulation leads to intestinal disease. Methods: To understand SYKs role in the intestinal epithelium, a BioID screen was used to identify novel SYK interacting proteins. BioID was done with wild type SYK as well as the patient derived variants in HEK293 cells. BioID hits were validated using co-immunoprecipitation (CoIP) and proximity ligation assay (PLA). Further functional analysis was done in Caco2 cells, an intestinal epithelial cell model, with western blot, ELISA, and luciferase assays to assess the effect of SYK activity on downstream inflammatory pathways. Finally, we investigated the effect of small molecule kinase inhibitors to explore possible novel treatment options. Results: The BioID screen revealed several high confidence interactors, with dysregulated interactions in the patient variants. USP25, a ubiquitin specific peptidase that is widely expressed, including in the intestinal epithelium, was identified as one of the strongest hits based on peptide counts. The interaction was validated with CoIP and PLA, and further analysis showed SYK activation decreased USP25 protein levels by up to eight-fold. The secretion of the proinflammatory chemokine, IL-8, in response to lipopolysaccharide (LPS) was found to be almost twice as high in Caco2 cells with patient SYK variants relative to WT. Cells expressing the patient variants of SYK showed increased NFkB and AP1 levels, indicating increased inflammatory gene expression when stimulated with 100ng/mL LPS. NFkB was also active for longer in cells expressing the patient variant of SYK post stimulation, with WT cells returning to baseline in under half an hour, while the patient variant stayed in an inflamed state for 36 to 48 hours after LPS was removed. Conclusions: Increased SYK activity is associated with the degradation of USP25. USP25 is known to cleave ubiquitin off TNF receptor associated factors (TRAFs). TRAFs are activated downstream of innate immune receptors such as TLR4, and need to be ubiquitinated for activation. USP25, which is responsible for the deubiquitination of these TRAFs, acts as a negative regulator of these pathways. The hyperactive SYK variants degrade USP25 and impair the cells’ ability to switch off inflammatory signaling pathways, indicated by the cells elongated period in an inflamed state. This research is funded by grants from Canadian Institute for Health Research (CIHR) and the Leona M. and Harry B. Helmsley Charitable Trust. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The IBD Risk gene, PTPN2, Is Required for Commensal-associated Th17 Responses to Protect Against Pathobiont Colonization

Background/Aim: Inflammatory bowel disease (IBD) involves multiple factors including genetic susceptibilities, intestinal permeability, microbiome dysbiosis, and expansion of intestinal pathobionts such as adherent-invasive E. coli (AIEC). Mice lacking the IBD risk gene, Ptpn2, exhibit expansion of murine adherent-invasive Escherichia coli ( mAIEC strain, UCR-PP2). Internal transcribed spacer analysis of luminal and mucosal-associated bacteria also indicated a decrease in the protective commensal bacteria, segmented filamentous bacteria (SFB). SFB adherence to intestinal epithelial cells (IEC) induces Th17 cell secretion of IL-17 and IL-22 to promote mucosal defense. The goal of this study was to determine if constitutive whole-body, or IEC-specific Ptpn2 loss in mice modulates SFB abundance, adherence, and downstream production of protective Th17 cytokine in mice. Methods: All mice were housed in specific pathogen free (SPF) vivarium. Distal ileum (2-3 cm) was collected from whole body Ptpn2-wild-type (WT), heterozygous (Het) and Ptpn2 constitutive knockout (KO) mice [Balb/c]; or tamoxifen (TMX)-induced IEC specific Ptpn2 knockout mice ( Ptpn2ΔIEC) and TMX-treated control littermates ( Ptpn2fl/fl) [C57Bl/6]. Tissues were fixed and observed using the Hitachi TM4000 Scanning Electron Microscope (SEM) to visualize SFB. qPCR was used to quantify relative abundance of SFB in the Ileum tissue obtained from Ptpn2ΔIEC and Ptpn2fl/fl mice. Confirmed SFB-free C57Bl/6 mice (10 weeks old; JAX labs; Stock# 000664) were infected for four consecutive days by 109 mAIEC bacteria in PBS. Cytokine protein levels were quantified by Luminex Multiplex Array of whole tissue lysates from cecum, proximal and distal colon regions from 18-21day old WT, HET or KO mice. Immune cells were isolated from the cecum, colon, lamina propria, the mesenteric lymph nodes and the spleen of WT, HET and KO mice and analyzed by flow cytometry for IL-22+, and IL-17+ T cells. Results: SFB adherence to IECs in Ptpn2 constitutive KO mice was visibly decreased by SEM compared to WT and Ptpn2 Het littermates. SEM showed a dramatic decrease in SFB abundance in Ptpn2ΔIEC mice compared to control littermates ( Ptpn2fl/fl). This was confirmed by PCR analysis showing a significant decrease in SFB in Ptpn2ΔIEC mice (p&lt;0.05; n=8). The absence of SFB permitted mAIEC to colonize and cause mild disease in SFB-free mice (JAX; p&lt;0.01; n=8). SFB loss in whole-body Ptpn2-KO mice correlated with a decrease in IL-17+ CD4+ cells in the colon compared to Ptpn2-Het (p&lt;0.001; n=7) and Ptpn2-WT littermates (p&lt;0.05; n=5). Ptpn2-KO mice exhibited higher IL-22 levels in the distal colon (p&lt;0.01; n=3), and increased IL-22+CD4+ T-cells in the cecum, mesenteric lymph nodes and spleen compared to Ptpn2-WT and Het littermates (p&lt;0.001). Conclusion: Loss of the IBD risk gene Ptpn2 in mice, either constitutively or in IECs, reduces the abundance of SFB a commensal inducer of Th17 responses. Consistent with reduced SFB, IL-17+CD4+ T cells were reduced in whole-body Ptpn2-KO mice indicating a loss of SFB-induced Th17 maturation. Despite reduced IL-17, we observed increased IL-22 expression in Ptpn2-KO mice suggesting that loss of PTPN2 provokes a compensatory SFB/IL-17-independent increase in IL-22 production. These findings identify how loss of PTPN2 activity disrupts homeostatic commensal-mucosal immune crosstalk to facilitate IBD-relevant pathobiont colonization. This study was supported by grants NIH-2R01-DK091281, 1R01AI153314-01, R21AI152017, 1R01DK130373, and R01AI165490 (D.F.M.) from the National Institutes of Health (NIH). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

NKCC1-dependent K+ influx inhibits NLRP3 inflammasome activation and prevents intestinal epithelial cell injury

Background: Loss of function mutations in SLC12A2, the gene encoding the Na+-K+-2Cl− cotransporter (NKCC1) are associated with autoinflammatory and severe chronic inflammation in multiple organs. NLRP(s) are a family of cytosolic sensors that initiate the innate immune response. Once activated, NLRP(s) form inflammasomes, which are multi-protein complex that sense danger signals, damaged and infected cells. Assembly of the inflammasome mediates caspases-1 activation and the production of the proinflammatory cytokines IL-1β and IL-18 and initiates an inflammatory cell death process called pyroptosis. Aberrant activation of NLRP3 and other inflammasomes leads to a variety of autoimmune and chronic inflammatory diseases. Here we ask whether loss of NKCC1 function affects inflammasome activation in intestinal epithelial cells. Methods: Wild-type intestinal epithelial cells (IEC) and CRISPR/Cas9 NKCC1-deleted clones were tested for inflammasome activation. WT and NKCC1-deficient clones were treated with nigericin, ATP or ouabain to activate the inflammasome. Conditioned media, whole cell lysate and fixed cells were collected at different time points and analyzed by immunoblotting, immunofluorescence, and ELISA (to measure cytokine release). Inflammasome-induced pyroptosis was assessed by measuring LDH release in the supernatant and gasdermine D cleavage by immunoblotting. Results: Loss of NKCC1 function induces inflammasome activation by increasing IL-1β secretion in intestinal epithelial cells. Prolong inflammatory cytokine secretion led to increased pyroptosis-mediated cell death. Loss of NKCC1 mediates NLRP3 inflammasome activation by compromising K+ influx. NLRP3 agonists increase the interaction and localization of WNK1 and SPAK kinases with the NLRP3 inflammasome complex. Conclusions: NKCC1 regulates inflammatory cytokines secretion. Loss of NKCC1 inhibits K+ and Cl− inward fluxes, which in turn activate NLRP3 inflammasome. Loss of NKCC1 function is a previously unknown mechanism that causes aberrant inflammasome activation. GM118944 DK093501. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.