Tight Junction Gene Expression Research Articles

The impact of exogenous vasoactive intestinal polypeptide (VIP) on inflammatory responses and mRNA expression of tight junction genes in lambs fed a high-grain diet.

This study assessed the impact of administering vasoactive intestinal polypeptide (VIP) on inflammation and intestinal VIP and tight junction mRNA expression in lambs fed grain-based finishing diets. Sixteen wether lambs (69.6 ± 1.9kg) were individually housed, adapted to a corn-based diet containing no forage, and randomly assigned to 2 treatment groups. Lambs were intraperitoneally injected every other day for 28 d with either saline (0.9% NaCl) with no VIP (n = 8; control) or saline with VIP (n = 8; 1.3 nmol/kg BW). Blood samples were collected weekly for analysis of cytokine concentrations, and on day 0 and 28 for lipopolysaccharide (LPS), and LPS binding protein (LBP) concentrations. Upon completion of the treatment period, lambs were euthanized and gastrointestinal tissues, including rumen, jejunum, cecum, and colon samples, collected for analysis of the expression of tight junction mRNA (claudin-1, claudin-4, occludin, and ZO-1), endogenous VIP, and VIP receptor (VPAC-1). No treatment effects (P ≥ 0.38) were observed for VIP and VPAC-1 mRNA expression in colon. Supplementation with VIP did not influence (P ≥ 0.28) the expression of claudin-1, claudin-4, occludin, and ZO-1 tight junction mRNA in the rumen, jejunum, cecum, and colon. Lambs treated with VIP had greater (P ≤ 0.01) plasma concentrations of the anti-inflammatory cytokines, IL-10 and IL-36RA. There were treatment by day interactions observed (P ≤ 0.02) for concentrations of the proinflammatory cytokines, MIP-1α and MIP-1β. Lambs that did not receive VIP had greater serum concentrations of LPS (P = 0.05) than the lambs receiving VIP. These data suggest that VIP administration may not influence tight junction mRNA expression but may decrease LPS concentrations and thus inflammation in lambs fed a grain-based diet.

Circadian control of polycyclic aromatic hydrocarbon-induced dysregulation of endothelial tight junctions and mitochondrial bioenergetics

The study evaluates the impact of environmental toxicants, such as polycyclic aromatic hydrocarbons (PAHs), on circadian regulations and functions of brain endothelial cells, which form the main structural element of the blood-brain barrier (BBB). PAH are lipophilic and highly toxic environmental pollutants that accumulate in human and animal tissues. Environmental factors related to climate change, such as an increase in frequency and intensity of wildfires or enhanced strength of hurricanes or tropical cyclones, may lead to redistribution of these toxicants and enhanced human exposure. These natural disasters are also associated with disruption of circadian rhythms in affected populations, linking increased exposure to environmental toxicants to alterations of circadian rhythm pathways. Several vital physiological processes are coordinated by circadian rhythms, and disruption of the circadian clock can contribute to the development of several diseases. The blood-brain barrier (BBB) is crucial for protecting the brain from blood-borne harmful substances, and its integrity is influenced by circadian rhythms. Exposure of brain endothelial cells to a human and environmentally-relevant PAH mixture resulted in dose-dependent alterations of expression of critical circadian modulators, such as Clock, Bmal1, Cry1/2, and Per1/2. Moreover, silencing of the circadian Clock gene potentiated the impact of PAHs on the expression of the main tight junction genes and proteins (namely, claudin-5, occludin, JAM-2, and ZO-2), as well as mitochondrial bioenergetics. Findings from this study contribute to a better understanding of pathological influence of PAH-induced health effects, especially those related to circadian rhythm disruption.

Evaluating the Impact of the PoultryStar®Bro Probiotic on the Incidence of Bacterial Chondronecrosis with Osteomyelitis Using the Aerosol Transmission Challenge Model.

Bacterial chondronecrosis with osteomyelitis (BCO) lameness is a major welfare issue for broiler production worldwide affecting approximately 1.5% of broilers over 42 days old. Excessive body weight gain causes mechanical stress on long bones, leading to micro-fractures. This condition induces a bacterial infection of fractures, resulting in bone necrosis and eventual BCO lameness. Increasing gut integrity and supporting Calcium metabolism contribute to the optimal bone structure and subsequently reduce BCO lameness. Probiotics thus provide an excellent strategy for alleviating BCO due to the improvement of intestinal integrity and barrier function. Accordingly, the present study investigated the lameness reduction through the feed supplementation of a selected probiotic. Broiler chickens were assigned to three treatments, including a control litter group (FL), a PoultryStar®Bro probiotic fed group (BRO), and a control wire-flooring group (CW) designed to induce BCO lameness. The probiotic significantly decreased lameness by 46% compared to the control group (p < 0.05). The most predominant bacteria identified from the BCO lesions were Staphylococcus cohnii and Staphylococcus lentus. Moreover, significant increments of tight junction gene expression in jejunum and ileum, plus numerical improvements of body weight gain (BW; +360 g) and feed conversion ratio (FCR; -12 pts) were observed in BRO-supplemented birds.

Molecular Mechanisms Underlying Loss of Vascular and Epithelial Integrity in Irritable Bowel Syndrome

The pathophysiology of irritable bowel syndrome (IBS) is multifactorial and includes epithelial barrier dysfunction, a key element at the interface between the gut lumen and the deeper intestinal layers. Beneath the epithelial barrier there is the vascular one representing the last barrier to avoid luminal antigen dissemination The aims of this study were to correlate morpho-functional aspects of epithelial and vascular barriers with symptom perception in IBS. Seventy-eight healthy subjects (controls) and 223 patients with IBS were enrolled in the study and phenotyped according to validated questionnaires. Sugar test was used to evaluate invivo permeability. Immunohistochemistry, western blot, and electron microscopy were used to characterize the vascular barrier. Vascular permeability was evaluated by assessing the mucosal expression of plasmalemma vesicle-associated protein-1 and vascular endothelial cadherin. Caco-2 or human umbilical vein endothelial cell monolayers were incubated with soluble mediators released by mucosal biopsies to highlight the mechanisms involved in permeability alteration. Correlation analyses have been performed among experimental and clinical data. The intestinal epithelial barrier was compromised in patients with IBS throughout the gastrointestinal tract. IBS-soluble mediators increased Caco-2 permeability via a downregulation of tight junction gene expression. Blood vessel density and vascular permeability were increased in the IBS colonic mucosa. IBS mucosal mediators increased permeability in human umbilical vein endothelial cell monolayers through the activation of protease-activated receptor-2 and histone deacetylase 11, resulting in vascular endothelial cadherin downregulation. Permeability changes correlated with intestinal and behavioral symptoms and health-related quality of life of patients with IBS. Epithelial and vascular barriers are compromised in patients with IBS and contribute to clinical manifestations.

Essential oils ameliorate the intestinal damages induced by nonylphenol exposure by modulating tryptophan metabolism and activating aryl hydrocarbon receptor via gut microbiota regulation

Nonylphenol (NP) is a ubiquitous endocrine disruptor that persists in the environment and can significantly contribute to serious health hazards, particularly intestinal barrier injury. Plant essential oils (EOs) have recently gained widespread interest due to their potential for improving intestinal health. However, the precise mechanism and protective effects of EOs ameliorating the intestinal damages induced by NP exposure remain unclear. To clarify the potential mechanism and protective impact of EOs against intestinal injury induced by NP, a total of 144 one-day-old male ducks were randomly allocated to four groups: CON (basal diet), EO (basal diet + 200 mg/kg EOs), NP (basal diet + 40 mg/kg NP), and NPEO (basal diet + 200 mg/kg EOs + 40 mg/kg NP). The data revealed that NP exposure significantly damaged intestinal barrier, as evidenced by a reduction in the levels of tight junction gene expression and an increase in intestinal permeability. Additionally, it disturbed gut microbiota, as well as interfered with tryptophan (Trp) metabolism. The NP-induced disorder of Trp metabolism restrained the activation of aryl hydrocarbon receptor (AhR) and resulted in decreased the expression levels of CYP1A1, IL-22, and STAT3 genes, which were alleviated after treatment with EOs. Taken together, NP exposure resulted in impairment of the intestinal barrier function, disruption of gut microbiota, and disturbances in Trp metabolism. Dietary EOs supplementation alleviated the intestinal barrier injury induced by NP through the Trp/AhR/IL-22 signaling pathway.

The impact of microplastics polystyrene on the microscopic structure of mouse intestine, tight junction genes and gut microbiota.

Microplastics, which are tiny plastic particles less than 5 mm in diameter, are widely present in the environment, have become a serious threat to aquatic life and human health, potentially causing ecosystem disorders and health problems. The present study aimed to investigate the effects of microplastics, specifically microplastics-polystyrene (MPs-PS), on the structural integrity, gene expression related to tight junctions, and gut microbiota in mice. A total of 24 Kunming mice aged 30 days were randomly assigned into four groups: control male (CM), control female (CF), PS-exposed male (PSM), and PS-exposed female (PSF)(n = 6). There were significant differences in villus height, width, intestinal surface area, and villus height to crypt depth ratio (V/C) between the PS group and the control group(C) (p <0.05). Gene expression analysis demonstrated the downregulation of Claudin-1, Claudin-2, Claudin-15, and Occludin, in both duodenum and jejunum of the PS group (p < 0.05). Analysis of microbial species using 16S rRNA sequencing indicated decreased diversity in the PSF group, as well as reduced diversity in the PSM group at various taxonomic levels. Beta diversity analysis showed a significant difference in gut microbiota distribution between the PS-exposed and C groups (R2 = 0.113, p<0.01), with this difference being more pronounced among females exposed to MPs-PS. KEGG analysis revealed enrichment of differential microbiota mainly involved in seven signaling pathways, such as nucleotide metabolism(p<0.05). The relative abundance ratio of transcriptional pathways was significantly increased for the PSF group (p<0.01), while excretory system pathways were for PSM group(p<0.05). Overall findings suggest that MPs-PS exhibit a notable sex-dependent impact on mouse gut microbiota, with a stronger effect observed among females; reduced expression of tight junction genes may be associated with dysbiosis, particularly elevated levels of Prevotellaceae.

Dietary Polycan, a β-glucan originating from Aureobasidium pullulansSM-2001, attenuates high-fat-diet-induced intestinal barrier damage in obese mice by modulating gut microbiota dysbiosis.

Various metabolic diseases caused by a high-fat diet (HFD) are closely related to gut microbiota dysbiosis and epithelial barrier dysfunction. Polycan, a type of β-glucan, is effective in treating anti-obesity and metabolic diseases caused by HFD. However, the effect of Polycan on dysbiosis and epithelial barrier damage is still unknown. In this study, the effects of Polycan on dysbiosis and intestinal barrier damage were investigated using HFD-induced obese model mice. C57BL/6 mice were fed a HFD for 12 weeks and treated with two different doses of Polycan (250 and 500 mg/kg) orally administered during weeks 9 to 12. Polycan supplementation increased the expression of tight junction genes (zonula occludens-1, occludin, and claudin-3) and short-chain fatty acid (SCFA) content while reducing toxic substances (phenol, p-cresol, and skatole). Most significantly, Polycan enriched SCFA-producing bacteria (i.e., Phocaeicola, Bacteroides, Faecalibaculum, Oscillibacter, Lachnospiraceae, and Muribaculaceae), and decreased the Firmicutes/Bacteroidetes ratio and toxic substances-producing bacteria (i.e., Olsenella, Clostridium XVIII, and Schaedlerella). Furthermore, microbial functional capacity prediction of the gut microbiota revealed that Polycan enriched many SCFA-related KEGG enzymes while toxic substance-related KEGG enzymes were depleted. These findings indicated that Polycan has the potential to alleviate HFD-induced intestinal barrier damage by modulating the function and composition of the gut microbiota.

149 Development of a leaky gut model: Impact of 24-h feed restriction on ileal expression of tight junction proteins in weaned pigs

Abstract Weanling barrows, 26 to 28 d old [n = 90; Duroc terminal line Fast genetics, initial body weight (BW)= 5.63 ± 0.43 kg] were used to evaluate the effect of 24-h complete feed restriction on growth and intestinal integrity. Upon arrival at the NutriQuest modeling center (Fergus Falls, MN), pigs were weighed, blocked by BW, and randomly assigned to 1 of 30 pens. Five treatments were randomly assigned to 6 pens: 1) Positive Control (PC), and 4 treatments that had 24-hr feed restriction (FR); 2) Negative control (NC), 3) Direct Fed Microbial (DFM) A, 4) DFM B, and 5) DFM C. Feed intake and BW were determined weekly and before and after FR. All pigs were fed a common diet. Fifteen pigs were removed prior to FR due to enteric disease. On d 18, feed was removed from pigs on treatments 2 to 5 for 24 h. After 24 h, 8 pigs per treatment were harvested for tissue collection. Feed access was restored to the remaining pigs who were harvested 3 d later. Ileal samples were collected and immediately frozen. Plasma and fecal samples were collected upon arrival, at FR, and 24 h and 4-d post-FR. Ileal expression of claudin-3, occludin, and tight-junction-1 genes was determined using QuantStudio 3 Real Time PCR system. Fold-change in gene expression compared with NC was calculated for each treatment. Plasma samples were analyzed for LPS binding protein (LBP) concentration, and fecal samples were analyzed for myeloperoxidase (MPO) concentration. Pig (n = 75) was the experimental unit for BW, LBP, MPO, and gene expression data. Pen (n = 30) was the experimental unit for feed intake. Data were analyzed using PROC GLIMMIX of SAS. Preplanned orthogonal contrasts were performed to determine the effect of PC vs. 4 FR treatments. To determine the relationship of tight junction gene expression and LBP and MPO, PROC REG of SAS was used. Data are described as PC vs. FR treatments to focus on model development. Final BW for pigs on PC was greater compared with the FR treatments (P &amp;lt; 0.05; Table 1). From 1 to 4 d post-FR, average daily gain (ADG) for FR treatments was greater than PC (P &amp;lt; 0.01). There was no effect of treatment on feed intake or feed efficiency. Plasma LBP and fecal MPO concentrations were greater 24 h post-FR for pigs on PC compared with FR treatments (P &amp;lt; 0.01). Fold change in occludin and claudin-3 expression was greater for PC compared with FR treatments (P ≤ 0.05). For pigs euthanized 24 h post-FR, there was a positive relationship between LBP concentrations and occludin expression (P &amp;lt; 0.02). Twenty-four hour FR in weaned pigs alters ileal tight junction gene expression and plasma LBP concentrations; therefore, a timed FR event may serve as a model for leaky gut.

150 Development of a leaky gut model: Meta-analysis of plasma and fecal markers of leaky gut

Abstract Intestinal integrity can be compromised in livestock and poultry species during stress events, including off-feed events. To develop a leaky gut model, weanling barrows 26 to 28 d old [n = 307; Duroc terminal line Fast genetics, initial body weight (BW) = 5.90 ± 0.47 kg), were used in a total of 6 randomized complete block experiments at the NutriQuest modeling center (Fergus Falls, MN). All trials included positive control (PC) and negative control (NC) treatments. The other treatments evaluated (n = 8) were various strains and levels of direct-fed microbial (DFM) products. With the exception of PC, all treatments had complete 24-h feed restriction (FR) approximately 18 d after arrival. Pigs assigned to PC had ad libitum access to feed. Pigs were housed 3 pigs per pen in 1 of 30 pens, and all pigs were fed a common high-quality basal diet. For 3 trials, pigs received Excede (Zoetis; Parsipanny, NJ) IM upon arrival and were provided gentamycin via water for 3 d post-arrival. For 3 trials, no prophylactic antibiotics were administered. Eighteen days post-arrival, fecal and plasma samples were collected from all pigs and feed was removed from FR pens. After 24 h, plasma and fecal samples were collected from all pigs, and one-half of the pigs per trial were harvested for tissue collection. Four days after FR, samples were collected similarly, and the remaining pigs were harvested. Plasma samples were analyzed for LPS binding protein concentrations (LBP), and fecal samples were analyzed for myeloperoxidase (MPO) concentrations. Data from the PC (n = 90) and NC (n = 92) treatments only were analyzed as Individual Participant Data meta-analysis using PROC MIXED of SAS. Data from all pigs (n = 307) were analyzed using PROC REG of SAS with forward step-wise selection to determine relationships between response variables. Plasma LBP concentrations did not differ between PC and NC 24 h after FR (P = 0.14). However, change in LBP concentrations and the ratio of LBP immediately before and after FR, differed between PC and NC (P &amp;lt; 0.001). For NC, LBP concentrations decreased, and for PC, LBP concentrations increased (Table 1). There was no effect of treatment on fecal MPO concentrations. Using the broader dataset (n = 307), variables that were predictive of plasma LBP concentrations immediately following FR were trial (P &amp;lt; 0.001), treatment (P &amp;lt; 0.001), and fecal MPO concentrations (P &amp;lt; 0.05). Variables that were predictive of change in LBP concentrations before and after FR were trial (P &amp;lt; 0.001), treatment (P &amp;lt; 0.001), and initial BW (P &amp;lt; 0.01). These data, taken with data presented in a companion abstract on ileal tight junction gene expression, suggest that plasma LBP concentrations immediately following a FR event are a useful marker of leaky gut. Fecal MPO concentrations do not consistently respond to FR.

A Novel IRAK4 Inhibitor DW18134 Ameliorates Peritonitis and Inflammatory Bowel Disease.

IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.

Milk and Lacticaseibacillus paracasei BL23 effects on intestinal responses in a murine model of colitis.

Probiotic-containing fermented dairy foods have the potential to benefit human health, but the importance of the dairy matrix for efficacy remains unclear. We investigated the capacity of Lacticaseibacillus paracasei BL23 in phosphate-buffered saline (BL23-PBS), BL23-fermented milk (BL23-milk), and milk to modify intestinal and behavioral responses in a dextran sodium sulfate (DSS, 3% wt/vol) mouse model of colitis. Significant sex-dependent differences were found such that female mice exhibited more severe colitis, greater weight loss, and higher mortality rates. Sex differences were also found for ion transport ex vivo, colonic cytokine and tight junction gene expression, and fecal microbiota composition. Measurements of milk and BL23 effects showed BL23-PBS consumption improved weight recovery in females, whereas milk resulted in better body weight recovery in males. Occludin and Claudin-2 gene transcript levels indicated barrier function was impaired in males, but BL23-milk was still found to improve colonic ion transport in those mice. Proinflammatory and anti-inflammatory gene expression levels were increased in both male and female mice fed BL23, and to a more variable extent, milk, compared with controls. The female mouse fecal microbiota contained high proportions of Akkermansia (average of 18.1%) at baseline, and females exhibited more changes in gut microbiota composition following BL23 and milk intake. Male fecal microbiota harbored significantly more Parasutterella and less Blautia and Roseburia after DSS treatment, independent of BL23 or milk consumption. These findings show the complex interplay between dietary components and sex-dependent responses in mitigating inflammation in the digestive tract.NEW & NOTEWORTHY Sex-dependent responses to probiotic Lacticaseibacillus paracasei and milk and the potential of the dairy matrix to enhance probiotic protection against colitis in this context have not been previously explored. Female mice were more sensitive than males to colonic injury, and neither treatment effectively alleviated inflammation in both sexes. These sex-dependent responses may result from differences in the higher baseline proportions of Akkermansia in the gut microbiome of female mice.

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

The effects of Pediococcus acidilactici MA18/5M on growth performance, gut integrity, and immune response using in vitro and in vivo Pacific salmonid models.

Microbial management is central to aquaculture's efficiency. Pediococcus acidilactici MA18/5M has shown promising results promoting growth, modulation of the immune response, and disease resistance in many fishes. However, the mechanisms through which this strain confers health benefits in fish are poorly understood, particularly in Pacific salmonid models. Briefly, the aims of this study were to i) assess the protective effects of P. acidilactici MA18/5M by examining gut barrier function and the expression of tight junction (TJ) and immune genes in vitro and in vivo, and ii) to determine the protective effects of this strain against a common saltwater pathogen, Vibrio anguillarum J382. An in vitro model of the salmonid gut was employed utilizing the cell line RTgutGC. Barrier formation and integrity assessed by TEER measurements in RTgutGC, showed a significant decrease in resistance in cells exposed only to V. anguillarum J382 for 24 h, but pre-treatment with P. acidilactici MA18/5M for 48 h mitigated these effects. While P. acidilactici MA18/5M did not significantly upregulate tight junction and immune molecules, pre-treatment with this strain protected against pathogen-induced insults to the gut barrier. In particular, the expression of ocldn was significantly induced by V. anguillarum J382, suggesting that this molecule might play a role in the host response against this pathogen. To corroborate these observations in live fish, the effects of P. acidilactici MA18/5M was evaluated in Chinook salmon reared in real aquaculture conditions. Supplementation with P. acidilactici MA18/5M had no effect on Chinook salmon growth parameters after 10 weeks. Interestingly, histopathological results did not show alterations associated with P. acidilactici MA18/5M supplementation, indicating that this strain is safe to be used in the industry. Finally, the expression pattern of transcripts encoding TJ and immune genes in all the treatments suggest that variation in expression is more likely to be due to developmental processes rather than P. acidilactici MA18/5M supplementation. Overall, our results showed that P. acidilactici MA18/5M is a safe strain for use in fish production, however, to assess the effects on growth and immune response previously observed in other salmonid species, an assessment in adult fish is needed.

Dietary L-Methionine modulates the gut microbiota and improves the expression of tight junctions in an in vitro model of the chicken gastrointestinal tract

BackgroundThe poultry industry encounters a number of factors that affect growth performance and productivity; nutrition is essential for sustaining physiological status and protecting against stressors such as heat, density, and disease. The addition of vitamins, minerals, and amino acids to the diet can help restore productivity and support the body’s defense mechanisms against stress. Methionine (Met) is indispensable for poultry’s energy metabolism, physiology, performance, and feed utilization capacity. Through this study, we aimed to examine the physiological effects of methionine supplementation on poultry as well as alterations of intestinal microbiome.MethodsWe utilized the DL- and L- form of methionine on Caenorhabditis elegans and the FIMM (Fermentor for intestine microbiota model) in-vitro digesting system. A genomic-analysis of the transcriptome confirmed that methionine supplementation can modulate growth-related physiological metabolic pathways and immune responses in the host poultry. The C. elegans model was used to assess the general health benefits of a methionine supplement for the host.ResultsRegardless of the type or concentration of methionine, supplementation with methionine significantly increased the lifespan of C. elegans. Feed grade L-Methionine 95%, exhibited the highest lifespan performance in C. elegans. Methionine supplementation increased the expression of tight junction genes in the primary intestinal cells of both broiler and laying hens, which is directly related to immunity. Feed grade L-Methionine 95% performed similarly or even better than DL-Methionine or L-Methionine treatments with upper doses in terms of enhancing intestinal integrity. In vitro microbial cultures of healthy broilers and laying hens fed methionine revealed changes in intestinal microflora, including increased Clostridium, Bacteroides, and Oscillospira compositions. When laying hens were given feed grade L-Methionine 95% and 100%, pathogenic Campylobacter at the genus level was decreased, while commensal bacteria were increased.ConclusionsSupplementation of feed grade L-Methionine, particularly L-Methionine 95%, was more beneficial to the host poultry than supplementing other source of methionine for maintaining intestinal integrity and healthy microbiome.

Effects of dietary binder sources on growth, digestive enzymes activity, and gut barrier function and microbial composition of larval largemouth bass (Micropterus salmoides)

A 4-week feeding trail was conducted to explore the impacts of dietary binder sources on growth performance, digestive enzymes activities, intestinal microbiota, and expression of genes involved in tight junctions and nutrient sensing pathway of larval largemouth bass with initial body weight of 8.43 ± 0.01 mg (13 day after hatching). Five isolipidic micro-diets were formulated with different binder sources including sodium alginate (SA), carrageenan (CG), hydroxypropyl methyl cellulose (HPMC), gelatin (GT), and starch (SC). The results revealed that the highest growth performance was observed in the CG group, and meanwhile, the promotion effects of SA and GT was statistically higher than that of HPMC and SC groups. SA, CG and GT had positive effects on digestive enzymes activity compared to other groups. Gene expression results showed that SA, CG and GT groups up-regulated the expression of tight junction genes, zo-1, claudin-4 and claudin-7, and activated the genes involved in the TOR signaling pathway, including tor, akt1, s6kβ1 and s6. Among all the groups, the CG had the most significant activation effect on TOR signaling pathway. Bacterial 16S rRNA V3–4 region analysis showed that the addition of different dietary binders influenced the diversity of intestinal microbial communities. The inclusion of SA and CG groups reduced the relative abundance of Proteobacteria and Actinobacteria at the phylum level, while at the genus level, the relative abundance of Rhodococcus and Achromobacter increased and the abundance of Plesiomonas was inhibited. In summary, dietary binder sources affected the growth, digestive function, gut barrier function and microbial composition, and nutrient sensing, and SA and CG could be suggested as the optimal dietary binders for formulation of micro-diets for largemouth bass larvae.

A29 INVESTIGATING THE IMPACT OF PARKINSON’S DISEASE-ASSOCIATED GENES ON INTESTINAL HOMEOSTASIS

Abstract Background Intestinal epithelial cells (IECs) provide an essential physical barrier between harsh luminal contents and underlying host tissue. The maintenance of intestinal homeostasis must be intricately regulated through the proliferation and differentiation of intestinal stem cells (ISCs). Dysregulation of this system results in the loss of barrier function, causing pathologies in both intestinal and extra-intestinal diseases. While Parkinson’s Disease (PD) is primarily a neurodegenerative disorder, there is increasing evidence linking PD progression and gastrointestinal (GI) dysfunction. Constipation and increased bowel permeability are often observed years prior to development of motor dysfunction in PD, and people with inflammatory bowel disease are more likely to develop PD. Our group developed a model to investigate the role of the gut in PD, demonstrating that mice with genetic ablation of the PD-associated gene Pink1 exhibited motor phenotypes only when previously infected with Gram-negative Citrobacter rodentium intestinal bacteria. As Pink1 and other PD-associated genes are expressed in IECs, we hypothesize that PD-associated gene mutations directly affect the epithelium and impact early PD pathophysiology. Aims 1. Determine how Pink1 mutation affects the transcriptional profiles in IECs at steady state and in vivo C. rodentium infection. 2. Use colonoid systems to study the effect of Pink1 mutation on epithelial activity. Methods ScRNAseq was performed on IECs isolated from uninfected and infected Pink1 WT and KO mice, sacrificed at day 7 and 14 (early and peak infection) to elucidate transcriptional differences between epithelial lineages of each genotype. Additionally, colonoids were derived from primary mouse tissue and treated with lipopolysaccharide (LPS) to determine how Pink1 KO affects the inflammatory response of the epithelium. Results Our data revealed that Pink1 KO profoundly affected several epithelial lineages. ISCs from infected Pink1 KO mice had dysregulated cell cycle genes, transit amplifying cells showed dysregulated expression of tight junction genes, and enterocytes displayed dysregulation in oxidative damage and apoptotic genes. Preliminary data from colonoids showed that Pink1 KO mice, when stimulated with LPS, had altered pro-inflammatory cytokine gene expression. We are also evaluating colon motility differences between genotypes through transit time assays, counting fecal pellets per hour, and fecal water content. Conclusions In Pink1 KO IECs, there is indeed an altered cellular response upon infection, but more information is needed to decern the mechanistic role of IECs in PD. By investigating the role of PD genes in the GI tract, these studies carry important implications for understanding the initiation and progression of PD. Funding Agencies CIHR

Intestinal Permeability, Gut Inflammation, and Gut Immune System Response Are Linked to Aging-Related Changes in Gut Microbiota Composition: A Study in Female Mice.

Aging entails changes at the cellular level that increase the risk of various pathologies. An association between gut microbiota and age-related diseases has also been attributed. This study aims to analyze changes in fecal microbiota composition and their association with genes related to immune response, gut inflammation, and intestinal barrier impairment. Fecal samples of female mice at different ages (2 months, 6 months, 12 months, and 18 months) and gene expression in colon tissue were analyzed. Results showed that the older mice group had a more diverse microbiota than the younger group. Additionally, the abundance of Cyanobacteria, Proteobacteria, Flavobacteriaceae, Bacteroides, Parabacteroides, Prevotellaceae_UCG-001, Akkermansia, and Parabacteroides goldsteinii increased with age. In contrast, there was a notable decline in Clostridiaceae, Lactobacillaceae, Monoglobaceae, Ligilactobacillus, Limosilactobacillus, Mucispirillum, and Bacteroides faecichinchillae. These bacteria imbalances were positively correlated with increased inflammation markers in the colon, including Tnf-α, Ccl2, and Ccl12, and negatively with the expression of tight junction genes (Jam2, Tjp1, and Tjp2), as well as immune response genes (Cd4, Cd72, Tlr7, Tlr12, and Lbp). In conclusion, high levels of diversity did not result in improved health in older mice; however, the imbalance in bacteria abundance that occurs with aging might contribute to immune senescence, inflammation, and leaky gut disease.

Effect of Millet Polyphenol-Rich Extracts on Human Fecal Bacteria and Dextran Sulfate Sodium-Induced Human Intestinal Epithelial Cell Derangements

ABSTRACT There is a rejuvenated interest in functional food research on polyphenols due to their gut microbial modulation and capacity to alleviate clinical conditions. In this study, polyphenol-rich extracts (PREs) from Finger millet (FM) and Kodo millet (KM) were assessed for modulating effects on selected human gut bacteria during fecal batch fermentation along with the protective effects on Dextran Sulfate Sodium (DSS)-induced colonic aberrations in vitro. Fecal batch fermentation of FM-PRE and KM-PRE enhanced the abundance of selected beneficial bacteria, such as Bifidobacterium spp. Lactobacillus spp. and the production of SCFAs. Catechin and naringin were the main phenolic fractions of fermented FM-PRE and KM-PRE. Moreover, the millet polyphenols protected the intestinal cells against DSS-induced intestinal barrier dysfunction by reducing inflammatory cytokines, TEER by ~40%, FITC-dextran fluorescence and upregulating the expression of tight junction genes, signifying improved gut functions. The study also imply that FM and KM polyphenols may inhibit intestinal inflammation.

Effects of pectin's degree of methyl esterification on TLR2-mediated IL-8 secretion and tight junction gene expression in intestinal epithelial cells: influence of soluble TLR2.

This study investigates the anti-inflammatory effects of pectins with different degrees of methyl esterification (DM) on intestinal epithelial cells (IECs) expressing low and high levels of TLR2. It also studies the influence of soluble TLR2 (sTLR2) which may be enhanced in patients with inflammatory bowel syndrome on the inflammation-attenuating effects of pectins. Also, it examines the impact of pectins on tight junction gene expression in IECs. Lemon pectins with DM18 and DM88 were characterized, and their effects on TLR2-1-induced IL8 gene expression and secretion were investigated in low-TLR2 expressing Caco-2 and high-TLR2 expressing DLD-1 cells. The results demonstrate that both DM18 and DM88 pectins can counteract TLR2-1-induced IL-8 expression and secretion, with more pronounced effects observed in DLD-1 cells expressing high levels of TLR2. Furthermore, the presence of sTLR2 does not interfere with the attenuating effects of low DM18 pectin and may even support its anti-inflammatory effects in Caco-2 cells. The impact of pectins and sTLR2 on tight junction gene expression also demonstrates cell-type-dependent effects. Overall, these findings suggest that low DM pectins possess potent anti-inflammatory properties and may influence tight junction gene expression in IECs, thereby contributing to the maintenance of gut homeostasis.

Aspirin eugenol ester affects ileal barrier function, inflammatory response and microbiota in broilers under lipopolysaccharide-induced immune stress conditions.

The aim of this study was to investigate the effects of aspirin eugenol ester (AEE) on ileal immune function in broilers under lipopolysaccharide (LPS)-induced immune stress. Two hundred and forty one-day-old male Arbor Acres chicks were randomly divided into four groups (saline, LPS, saline + AEE and LPS + AEE) with six replicates of ten broilers each. The saline group and LPS group were fed the normal diet, while the other two groups received normal diet plus 0.1 g/kg AEE. Broilers in the LPS and LPS + AEE groups were injected intraperitoneally with 0.5 mg/kg B.W LPS in saline for seven consecutive days beginning at 14 days of age, while broilers in the saline and saline + AEE groups were injected with saline only. The results showed that AEE improved the ileal morphology and increased the ratio of villus height to crypt depth of immune-stressed broilers. LPS-induced immune stress significantly reduced the expression of the genes for the tight junction proteins occludin, zonula occludens-1 (ZO-1), claudin-1 and claudin-2, in the ileum, while AEE significantly up-regulated the expression of these genes. Compared with the saline group, the LPS-treated chickens showed significantly increased mRNA expression of the inflammatory factors tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), cyclooxygenase-2 (COX-2), and microsomal Prostaglandin E Synthesase-1 (mPGES-1) in the ileum, while they were significantly decreased by AEE supplementation. In addition, analysis of the ileal bacterial composition showed that compared with saline and LPS + AEE groups, the proportion of Firmicutes and Lactobacillus in the LPS group was lower, while the proportion of Proteobacteria and Escherichia-Shigella was higher. Similarly, Line Discriminant Analysis Effect Size (LEfSe) analysis showed that compared with the LPS group, Brevibacillus was dominant in the saline group, while the LPS + AEE group was rich in Rhizobium, Lachnoclostridium, Ruminococcaceae, Faecalibacterium, Negativibacillus, Oscillospiraceae, and Flavonifractor. These results indicate that dietary supplementation with 0.1 g/kg AEE could protect the intestinal health by improving the intestinal villus morphology, enhancing the expression of tight junction genes and alleviating inflammation to resist the immune stress caused by LPS stimulation in broilers, and the mechanism may involve COX-2-related signal transduction and improved intestinal microbiota composition.