Intestinal Epithelial Cell Line Research Articles

Klebsiella pneumoniae increases invasion inintestinal epithelial cells by disrupting thecytoskeleton.

Klebsiella pneumoniae is an opportunistic pathogen and it can cause human mucosal lesions through the intestine, leading to bacteremia and abscess formation in liver and spleen. Previous studies have shown that K. pneumoniae can enter or cross cells through the intestinal epithelium, but the mechanism is unknown. In this study, we treated the intestinal epithelial cell line Caco-2 with KP1195, a clinically isolated strain with high adhesion and invasion of intestinal epithelial cells. The results showed that thetreatment of K. pneumoniae could increase the expression of integrin gene and further disrupt the changes of cytoskeleton. Treating Caco-2 with cytoskeletal inhibitor cytorelaxin D can significantly increase the efficiency of K. pneumoniae invading Caco-2 cells. These data suggest that disruption of the cytoskeleton through integrins may be one of the mechanisms by which K. pneumoniae increases intracellular invasion. This study provides a theoretical basis for further understanding of the mechanism of K. pneumoniae entering intestinal epithelial cells.

New insights into the mechanisms of iron absorption: Iron dextran uptake in the intestines of weaned pigs through glucose transporter 5 (GLUT5) and divalent metal transporter 1 (DMT1) transporters

The purpose of this study was to gain insight into the mechanism of iron dextran (DexFe) absorption in the intestines. A total of 72 piglets (average BW = 7.12 ± 0.75 kg, male to female ratio = 1:1) weaned at 28 d of age were randomly divided into two treatment groups with six replicates for each group. The experimental diets included the basal diet supplemented with 100 mg/kg iron dextran (DexFe group) and the basal diet supplemented with 100 mg/kg FeSO4•H2O (CON group). The experiment lasted for 28 d. The piglets' intestinal iron transport was measured in vitro using an Ussing chamber. Porcine intestinal epithelial cell line (IPEC-J2) cells were used to develop a monolayer cell model that explored the molecular mechanism of DexFe absorption. Results showed that compared to the CON group, the ADG of pigs in the DexFe group was improved (P = 0.022), while the F/G was decreased (P = 0.015). The serum iron concentration, apparent iron digestibility, and iron deposition in the duodenum, jejunum, and ileum were increased (P < 0.05) by dietary DexFe supplementation. Piglets in the DexFe group had higher serum red blood count, hemoglobin, serum iron content, serum ferritin and transferrin levels and lower total iron binding capacity (P < 0.05). In the Ussing chamber test, the iron absorption rate of the DexFe group was greater (P < 0.001) than the CON group, and there was no significant difference between the DexFe group and the glucose group (P > 0.05). Furthermore, when compared to the CON group, DexFe administration improved (P < 0.05) SLC2A5 gene and glucose transporter 5 (GLUT5) protein expression but had no effect (P > 0.05) on SLC11A2 gene or divalent metal transporter 1 (DMT1) protein expression. Once the GLUT5 protein was suppressed, the iron transport rate and apparent permeability coefficient were decreased (P < 0.05) in IPEC-J2 monolayer cell models. The findings suggest the effectiveness of DexFe application in weaned piglets and revealed for the first time that DexFe absorption in the intestine is closely related to the glucose transporter GLUT5 protein channel.

Modulation of miR-155-5p signalling via 5-ASA for the prevention of high microsatellite instability: an in vitro study using human epithelial cell lines.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug's chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA's prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.

Spirobenzofuran Mitigates Ochratoxin A-Mediated Intestinal Adverse Effects in Pigs through Regulation of Beta Defensin 1.

Antimicrobial peptides (AMPs) function to extensively suppress various problematic factors and are considered a new alternative for improving livestock health and enhancing immunomodulation. In this study, we explored whether AMP regulation has positive influences on Ochratoxin A (OTA) exposure using a porcine intestinal epithelial cell line (IPEC-J2 cells). We constructed a beta-defensin 1 (DEFB1) expression vector and used it to transfection IPEC-J2 cells to construct AMP overexpression cell lines. The results showed that OTA induced cytotoxicity, decreased cell migration, and increased inflammatory markers mRNA in IPEC-J2 cells. In DEFB1 overexpressing cell lines, OTA-induced reduced cell migration and increased inflammatory markers mRNA were alleviated. Additionally, a natural product capable of inducing DEFB1 expression, which was selected through high-throughput screening, showed significant alleviation of cytotoxicity, cell migration, and inflammatory markers compared to OTA-treated IPEC-J2 cells. Our finding provides novel insights and clues for the porcine industry, which is affected by OTA exposure.

Development of a Novel Prognostic Panel for Colorectal Cancer Based on Cancer Functional Status, and Validation of STC2 as a Promising Biomarker.

Improving the clinical outcome of colorectal cancer (CRC) patients remains a major challenge. This study aimed to develop a new predictive classifier for CRC and to examine its relationship with the immune environment and therapeutic response. A comprehensive bioinformatics analysis was applied to develop a risk panel comprised of cancer function status-related genes (CFSRGs). This panel was evaluated for prognostic utility by Area Under the Curve (AUC) and Kaplan-Meier (KM) analyses. Differences between high- and low-risk groups were subsequently investigated using multi-omics data. Immunohistochemistry (IHC), quantitative real-time polymerase chain reaction (qRT-PCR), and cell phenotype assays were also employed to ascertain the clinical value of STC2 expression. Significant differences were observed in the survival rate between high- and low-risk groups defined by our 7-CFSRG panel, both in internal and external CRC patient cohorts. The AUC for prediction of survival at 1-, 3- and 5-years was satisfactory in all cohorts. Detailed analysis revealed that tumor mutation burden, drug sensitivity, and pathological stage were closely associated with the risk score. Elevated expression of STC2 in CRC tissues relative to normal paraneoplastic tissues was associated with less favorable patient outcomes. qRT-PCR experiments confirmed that STC2 expression was significantly upregulated in several CRC cell lines (HCT116, SW480, and LOVO) compared to a normal intestinal epithelial cell line (NCM460). The proliferation, migration, and invasion of CRC cells were all significantly inhibited by knockdown of STC2. Our 7-CFSRG panel is a promising classifier for assessing the prognosis of CRC patients. Moreover, the targeting of STC2 may provide a novel therapeutic approach for improving patient outcomes.

Effects of Luteolin in an In Vitro Model of Porcine Intestinal Infections.

Intestinal infections caused by Escherichia coli and Salmonella enterica pose a huge economic burden on the swine industry that is exacerbated by the development of antimicrobial resistance in these pathogens, thus raising the need for alternative prevention and treatment methods. Our aim was to test the beneficial effects of the flavonoid luteolin in an in vitro model of porcine intestinal infections. We infected the porcine intestinal epithelial cell line IPEC-J2 with E. coli and S. enterica subsp. enterica serovar Typhimurium (106 CFU/mL) with or without previous, concurrent, or subsequent treatment with luteolin (25 or 50 µg/mL), and measured the changes in the reactive oxygen species and interleukin-6 and -8 levels of cells. We also tested the ability of luteolin to inhibit the adhesion of bacteria to the cell layer, and to counteract the barrier integrity damage caused by the pathogens. Luteolin was able to alleviate oxidative stress, inflammation, and barrier integrity damage, but it could not inhibit the adhesion of bacteria to IPEC-J2 cells. Luteolin is a promising candidate to be used in intestinal infections of pigs, however, further studies are needed to confirm its efficacy. The use of luteolin in the future could ultimately lead to a reduced need for antibiotics in pig production.

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

Characteristics and Functions of Infection-enhancing Antibodies to the N-terminal Domain of SARS-CoV-2.

Fcγ-receptor (FcγR)-independent enhancement of SARS-CoV-2 infection mediated by N-terminal domain (NTD)-binding monoclonal antibodies (mAbs) has been observed in vitro, but the functional significance of these antibodies in vivo is less clear. We characterized 1,213 SARS-CoV-2 spike (S)-binding mAbs derived from COVID-19 convalescent patients for binding specificity to the SARS-CoV-2 S protein, VH germ-line usage, and affinity maturation. Infection enhancement in a vesicular stomatitis virus (VSV)-SARS-CoV-2 S pseudovirus (PV) assay was characterized in respiratory and intestinal epithelial cell lines, and against SARS-CoV-2 variants of concern (VOC). Proteomic deconvolution of the serum antibody repertoire was used to determine functional attributes of secreted NTD-binding mAbs. We identified 72/1213 (5.9%) mAbs that enhanced SARS-CoV-2 infection in a PV assay. The majority (68%) of these mAbs recognized the NTD, were identified in patients with mild and severe disease, and persisted for at least 5 months post-infection. Infection enhancement by NTD-binding mAbs was not observed in intestinal and respiratory epithelial cell lines and was diminished or lost against SARS-CoV-2 VOC. Proteomic deconvolution of the serum antibody repertoire from 2 of the convalescent patients identified, for the first time, NTD-binding, infection-enhancing mAbs among the circulating immunoglobulins directly isolated from serum. Functional analysis of these mAbs demonstrated robust activation of FcγRIIIa associated with antibody binding to recombinant S proteins. Functionally active NTD-specific mAbs arise frequently during natural infection and can last as major serum clonotypes during convalescence. These antibodies display functional attributes that include FcγR activation, and may be selected against by mutations in NTD associated with SARS-CoV-2 VOC.

Azathioprine promotes intestinal epithelial cell differentiation into Paneth cells and alleviates ileal Crohn’s disease severity

Paneth cells (PCs), a subset of intestinal epithelial cells (IECs) found at the base of small intestinal crypts, play an essential role in maintaining intestinal homeostasis. Altered PCs function is associated with diverse intestinal pathologies, including ileal Crohn’s disease (CD). CD patients with ileal involvement have been previously demonstrated to display impairment in PCs and decreased levels of anti-microbial peptides. Although the immunosuppressive drug Azathioprine (AZA) is widely used in CD therapy, the impact of AZA on IEC differentiation remains largely elusive. In the present study, we hypothesized that the orally administered drug AZA also exerts its effect through modulation of the intestinal epithelium and specifically via modulation of PC function. AZA-treated CD patients exhibited an ileal upregulation of AMPs on both mRNA and protein levels compared to non-AZA treated patients. Upon in vitro AZA stimulation, intestinal epithelial cell line MODE-K exhibited heightened expression levels of PC marker in concert with diminished cell proliferation but boosted mitochondrial OXPHOS activity. Moreover, differentiation of IECs, including PCs differentiation, was boosted in AZA-treated murine small intestinal organoids and was associated with decreased D-glucose consumption and decreased growth rates. Of note, AZA treatment strongly decreased Lgr5 mRNA expression as well as Ki67 positive cells. Further, AZA restored dysregulated PCs associated with mitochondrial dysfunction. AZA-dependent inhibition of IEC proliferation is accompanied by boosted mitochondria function and IEC differentiation into PC.

Rapamycin and Starvation Mitigate Indomethacin-Induced Intestinal Damage through Preservation of Lysosomal Vacuolar ATPase Integrity.

Nonsteroidal anti-inflammatory drugs (NSAIDs) possess anti-inflammatory, antipyretic, and analgesic properties and are among the most commonly used drugs. Although the cause of NSAID-induced gastric ulcers is well understood, the mechanism behind small intestinal ulcers remains elusive. In this study, we examined the mechanism through which indomethacin (IM), a prominent NSAID, induces small intestinal ulcers, both in vitro and in vivo. In IEC6 cells, a small intestinal epithelial cell line, IM treatment elevated levels of LC3-II and p62. These expression levels remained unaltered after treatment with chloroquine or bafilomycin, which are vacuolar ATPase (V-ATPase) inhibitors. IM treatment reduced the activity of cathepsin B, a lysosomal protein hydrolytic enzyme, and increased the lysosomal pH. There was a notable increase in subcellular colocalization of LC3 with Lamp2, a lysosome marker, post IM treatment. The increased lysosomal pH and decreased cathepsin B activity were reversed by pretreatment with rapamycin (Rapa) or glucose starvation, both of which stabilize V-ATPase assembly. To validate the in vitro findings in vivo, we established an IM-induced small intestine ulcer mouse model. In this model, we observed multiple ulcerations and heightened inflammation following IM administration. However, pretreatment with Rapa or fasting, which stabilize V-ATPase assembly, mitigated the IM-induced small intestinal ulcers in mice. Coimmunoprecipitation studies demonstrated that IM binds to V-ATPase in vitro and in vivo. These findings suggest that IM induces small intestinal injury through lysosomal dysfunction, likely due to the disassembly of lysosomal V-ATPase caused by direct binding. Moreover, Rapa or starvation can prevent this injury by stabilizing the assembly. SIGNIFICANCE STATEMENT: This study elucidates the largely unknown mechanisms behind small intestinal ulceration induced by indomethacin and reveals the involvement of lysosomal dysfunction via vacuolar ATPase disassembly. The significance lies in identifying potential preventative interventions, such as rapamycin treatment or glucose starvation, offering pivotal insights that extend beyond nonsteroidal anti-inflammatory drugs-induced ulcers to broader gastrointestinal pathologies and treatments, thereby providing a foundation for novel therapeutic strategies aimed at a wide array of gastrointestinal disorders.

IL-22 regulates MASTL expression in intestinal epithelial cells.

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as an oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic colorectal cancer and colitis-associated cancer (CAC). CAC is one of the most severe complications of chronic inflammatory bowel disease (IBD), but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL levels in patients with IBD and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from patients with late-stage ulcerative colitis compared with controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, which facilitates IL-22-mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Furthermore, CAIX associates with and stabilizes MASTL in response to IL-22 stimulation.NEW & NOTEWORTHY MASTL is upregulated in colorectal cancer; however, its role in colitis and colitis-associated cancer is poorly understood. This study is the first to draw a link between MASTL and IL-22, a proinflammatory/intestinal epithelial recovery-promoting cytokine that is also implicated in colon tumorigenesis. We propose that IL-22 increases MASTL protein stability by promoting its association with CAIX potentially via AKT signaling to promote cell survival and proliferation.

Alleviating Pentatrichomonas hominis-induced damage in IPEC-J2 cells: the beneficial influence of porcine-derived lactobacilli.

Pentatrichomonas hominis is a common intestinal parasitic protozoan that causes abdominal pain and diarrhea, and poses a zoonotic risk. Probiotics, known for enhancing immunity and pathogen resistance, hold promise in combating parasitic infections. This study aimed to evaluate two porcine-derived probiotics, Lactobacillus reuteri LR1 and Lactobacillus plantarum LP1, against P. hominis infections in pigs. Taxonomic identity was confirmed through 16S rRNA gene sequencing, with L. reuteri LR1 belonging to L. reuteri species and L. plantarum LP1 belonging to L. plantarum species. Both probiotics exhibited robust in vitro growth performance. Co-culturing intestinal porcine epithelial cell line (IPEC-J2) with these probiotics significantly improved cell viability compared with the control group. Pre-incubation probiotics significantly enhanced the mRNA expression of anti-oxidative response genes in IPEC-J2 cells compared with the PHGD group, with L. reuteri LR1 and L. plantarum LP1 significantly up-regulating CuZn-SOD、CAT and Mn-SOD genes expression (p < 0.05). The anti-oxidative stress effect of L. reuteri LR1 was significantly better than that of L. plantarum LP1 (p < 0.05). Furthermore, pre-incubation with the probiotics alleviated the P. hominis-induced inflammatory response. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated IL-6、IL-8 and TNF-α gene expression(p < 0.05) compared with the PHGD group. The probiotics also mitigated P. hominis-induced apoptosis. L. reuteri LR1 and L. plantarum LP1 significantly down-regulated Caspase3 and Bax gene expression (p < 0.05), significantly up-regulated Bcl-2 gene expression (p < 0.05) compared with the PHGD group. Among them, L. plantarum LP1 showed better anti-apoptotic effect. These findings highlight the probiotics for mitigating P. hominis infections in pigs. Their ability to enhance anti-oxidative responses, alleviate inflammation, and inhibit apoptosis holds promise for therapeutic applications. Simultaneously, probiotics can actively contribute to inhibiting trichomonal infections, offering a novel approach for preventing and treating diseases such as P. hominis. Further in vivo studies are required to validate these results and explore their potential in animal and human health.

Alternariol Monomethyl-Ether Induces Toxicity via Cell Death and Oxidative Stress in Swine Intestinal Epithelial Cells.

Alternariol monomethyl-ether (AME), together with altenuene and alternariol, belongs to the Alternaria mycotoxins group, which can contaminate different substrates, including cereals. The aim of the present study was to obtain a deeper understanding concerning the effects of AME on pig intestinal health using epithelial intestinal cell lines as the data concerning the possible effects of Alternaria toxins on swine are scarce and insufficient for assessing the risk represented by Alternaria toxins for animal health. Our results have shown a dose-related effect on IPEC-1 cell viability, with an IC50 value of 10.5 μM. Exposure to the toxin induced an increase in total apoptotic cells, suggesting that AME induces programmed cell death through apoptosis based on caspase-3/7 activation in IPEC-1 cells. DNA and protein oxidative damage triggered by AME were associated with an alteration of the antioxidant response, as shown by a decrease in the enzymatic activity of catalase and superoxide dismutase. These effects on the oxidative response can be related to an inhibition of the Akt/Nrf2/HO-1 signaling pathway; however, further studies are needed in order to validate these in vitro data using in vivo trials in swine.

The effect of gut microbiota-derived carnosine on mucosal integrity and immunity in broiler chickens challenged with Eimeria maxima

In the first study, an in vitro culture system was developed to investigate the effects of carnosine on macrophage proinflammatory cytokine response using an established chicken macrophage cell line (CMC), gut integrity using a chicken intestinal epithelial cell line (IEC), muscle differentiation in quail muscle cells (QMCs) and primary chicken embryonic muscle cells (PMCs), and direct anti-parasitic effect against Eimeria maxima sporozoites. Cells to be tested were seeded in 24-well plates and treated with carnosine at 4 different concentrations (0.1, 1.0, and 10.0 µg). After 18 h of incubation, cells were harvested to measure gene expression of proinflammatory cytokines in CMC, tight junction (TJ) proteins in IECs, and muscle cell growth markers in QMCs and PMCs. In vivo trials were conducted to investigate the effect of dietary carnosine on disease parameters in broiler chickens challenged with E. maxima. One hundred and twenty male broiler chickens (0-day-old) were allocated into four treatment groups: (1) basal diet without infection (NC), (2) basal diet with E. maxima infection (PC), (3) carnosine at 10.0 mg/kg feed with PC (HCS), and (4) carnosine at 1.0 mg/kg feed with PC (LCS). All groups except NC were orally infected with E. maxima on day 14. Jejunal samples were collected for lesion scoring and jejunum gut tissues were used for transcriptomic analysis of cytokines and TJ proteins. In vitro, carnosine treatment significantly decreased IL-1β gene expression in CMC following LPS stimulation. In vivo feeding studies showed that dietary carnosine increased BW and ADG of chickens in E. maxima-infected groups and reduced the jejunal lesion score and fecal oocyst shedding in HCS group. Jejunal IL-1β, IL-8, and IFN-γ expression were suppressed in the HCS group compared to PC. The expression levels of claudin-1 and occludin in IECs were also increased in HCS following carnosine treatment. In conclusion, these findings highlight the beneficial effects of dietary carnosine supplementation on intestinal immune responses and gut barrier function in broiler chickens exposed to E. maxima infection.

Immunomodulatory properties of Bacillus subtilis extracellular vesicles on rainbow trout intestinal cells and splenic leukocytes.

Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules. Among EVs, outer membrane vesicles (OMVs), specifically produced by Gram-negative bacteria, have been extensively characterized and their potential as vaccines, adjuvants or immunotherapeutic agents, broadly explored in mammals. Nonetheless, Gram-positive bacteria can also produce bilayered spherical structures from 20 to 400 nm involved in pathogenesis, antibiotic resistance, nutrient uptake and nucleic acid transfer. However, information regarding their immunomodulatory potential is very scarce, both in mammals and fish. In the current study, we have produced EVs from the Gram-positive probiotic Bacillus subtilis and evaluated their immunomodulatory capacities using a rainbow trout intestinal epithelial cell line (RTgutGC) and splenic leukocytes. B. subtilis EVs significantly up-regulated the transcription of several pro-inflammatory and antimicrobial genes in both RTgutGC cells and splenocytes, while also up-regulating many genes associated with B cell differentiation in the later. In concordance, B. subtilis EVs increased the number of IgM-secreting cells in splenocyte cultures, while at the same time increased the MHC II surface levels and antigen-processing capacities of splenic IgM+ B cells. Interestingly, some of these experiments were repeated comparing the effects of B. subtilis EVs to EVs obtained from another Bacillus species, Bacillus megaterium, identifying important differences. The data presented provides evidence of the immunomodulatory capacities of Gram-positive EVs, pointing to the potential of B. subtilis EVs as adjuvants or immunostimulants for aquaculture.

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 &amp;lt; 0.001). LPS significantly increased IL8 secretion in the medium (P &amp;lt; 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 &amp;lt; 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 &amp;lt; 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 &amp;lt; 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.

Abstract 2044: Disruption Of DENND5B Attenuates Triglyceride-rich Lipoprotein Secretion From Human Enterocytes And Hepatocytes

Background and Hypothesis: Elevated plasma triacylglycerides (TAGs) are an established risk factor for atherosclerotic cardiovascular disease, and are regulated by dietary absorption via the small intestine and endogenous production by the liver. Our laboratory formerly reported that Dennd5b -deficient mice are protected from diet-induced hepatic steatosis, hyperlipidemia, and atherosclerosis. Electron microscopy imaging of the intestinal epithelium of Dennd5b -/- mice reveals significant lipid accumulation in intracellular chylomicron secretory vesicles, suggesting a post-Golgi chylomicron secretion defect. Given that Dennd5b is expressed by the two major triglyceride-rich lipoprotein (TRL)-secreting organs (liver and small intestine), we hypothesized that disruption of DENND5B attenuates TRL secretion in both of these cell types in humans. Methods and Results: To determine if DENND5B disruption impairs TRL secretion in human cells, we generated DENND5B -/- intestinal epithelial (Caco-2) and hepatocyte (HepG2) cell lines using CRISPR gene editing. Western blots verified DENND5B protein reduction in DENND5B -/- cells. The impact of DENND5B deficiency on TRL secretion was quantified by measuring the cellular and secreted TAG content of DENND5B +/+ and DENND5B -/- cells under standard growth conditions and after oleic acid loading. Caco-2 cells were differentiated on membrane supports for 21 days prior to experiments. DENND5B -/- Caco-2 cells secreted significantly less TAGs compared to wild-type Caco-2 cells (-67.8%, p&lt;0.0001). DENND5B -/- HepG2 cells also had significantly reduced TAG secretion (-91.0%, p&lt;0.0001). Disruption of DENND5B in both cell lines reduced TAG secretion without affecting cellular TAG content. Immunofluorescence microscopy showed intracellular DENND5B protein localization consistent with secretory pathway involvement with detection on a perinuclear structure (likely Golgi), cytosolic vesicles, and plasma membrane. Conclusions: These studies suggest that DENND5B plays a role in TRL secretion from human enterocytes and hepatocytes and provide new model systems for investigating the molecular function of DENND5B in TRL secretion from human cells.

Single-cell transcriptomics predicts colony stimulating factor-1 regulation of age-dependent intestinal epithelial restitution

Intestinal diseases associated with ischemic injury, which damages the principal barrier against noxious luminal contents, result in unacceptably poor outcomes in newborns. Ischemia-induced loss of the intestinal epithelial barrier predisposes patients to life-threatening sepsis unless that barrier is rapidly restored. There is an age-dependency of intestinal recovery in that neonates are the most susceptible to succumb to disease of the intestinal barrier versus older patients. While this age-dependence in repair has not been demonstrated in traditional rodent models, we have developed a highly translational pig model of intestinal ischemic injury and repair that does reflect this difference. We have shown that, while juvenile (weaned) pigs recover rapidly after ischemic intestinal injury, barrier repair is markedly underdeveloped in neonatal (nursing) pigs due to complete failure of epithelial restitution. Importantly, we found that restitution in neonates can be rescued by the direct application of homogenized mucosa from ischemia-injured small intestine from juvenile pigs. The mechanisms that allow for this restitution and rescue remain to be defined. We hypothesized that by identifying a subpopulation of restituting enterocytes by their expression of cell migration transcriptional pathways, we can then predict novel upstream regulators of age-dependent restitution response programs. Superficial mucosal epithelial cells collected from recovering ischemic jejunum of juvenile pigs were processed for single cell RNA sequencing, unbiased clustering and upstream regulator analysis. A porcine intestinal epithelial cell line (IPEC-J2) and banked tissues from prior rescue experiments were qualitatively and functionally assessed for activity of predicted upstream regulators. Single cell transcriptomics in recovering juvenile epithelium revealed a subcluster of absorptive enterocytes that express several cell migration pathways key to restitution. Differentially expressed genes in this subcluster predicted their upstream regulation by many potential molecules, including colony stimulating factor-1 (CSF-1) which is known to induce cell migration in non-intestinal epithelial tissues. To begin validating this prediction, we demonstrated that CSF-1 was enriched in the ischemic juvenile mucosa which rescues neonatal restitution and documented expression of the CSF-1 receptor (CSF1R) in both neonatal and juvenile epithelium, indicating that these cells are equipped to respond to CSF-1. CSF-1 and CSF1R co-localized in ischemic juvenile, but not neonatal, wound-adjacent epithelial cells and in the restituted epithelium of juveniles and rescued (but not control) neonates. Further, the CSF1R inhibitor BLZ945 reduced restitution in scratch wounded IPEC-J2 cells. Single cell transcriptomics have the power to inform potential novel therapeutic targets, such as CSF-1, to improve mucosal recovery in neonates with intestinal failure in this unique and powerful pig model. NIH K01 OD028207, NIH R01 HD095876, NIH U01 TR002953, NIH T32 OD011130, NIH P30 DK034987, USDA NIFA 1007263 and 07985. 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.

Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.