Compromised Barrier Function Research Articles

A16 ALTERATIONS TO BOTH INTESTINAL BARRIER INTEGRITY AND THE MICROBIOTA CONSPIRE DURING AGING TO PROMOTE DISEASE PROGRESSION IN A MOUSE MODEL OF MULTIPLE SCLEROSIS

Abstract Background Multiple sclerosis (MS) is a chronic, inflammatory condition impacted by both genetic and environmental factors. Aged individuals have an increased risk of progressive multiple sclerosis (pMS), as well as altered microbiota community composition and elevated levels of circulating gut microbiota-derived metabolites due to compromised gut barrier function. Fecal microbiota transplantation (FMT) studies confirm that an MS-associated microbiome can potentiate disease onset and enhance neuroinflammation in experimental autoimmune encephalomyelitis (EAE), a murine model of MS. Aims 1) Assess whether an aged microbiota alone is sufficient to promote age-associated progressive-like disease in our mouse model of EAE 2) Determine whether altered intestinal barrier integrity results in central nervous system-specific immune changes and/or disease course during EAE Methods To assess the impact of an aged microbiota on EAE outcomes we performed age-disparate human-derived FMT from young vs aged healthy co-habiting, first degree relatives into recipient mice. Then, to further interrogate the role of the gut-brain axis in the association of age and pMS we isolated the compromised intestinal barrier seen in aged individuals as a variable via two methods – Muc2-/- mice, which exhibit a defective mucus barrier, and DSS treated mice. Results Following adoptive transfer of young EAE-primed T cells, ‘young’ FMT recipients exhibited disease resolution while an ‘aged’ FMT lead to severe, non-remitting EAE relative to both controls and ‘young’ FMT recipients. This difference in outcomes was not seen in all donor pairs, suggesting that worsened EAE is not a universal feature of the aged microbiota. We collected cecal material, blood, and central nervous system (CNS) tissue from an age-differential FMT mouse pair and performed metabolomic analysis. We found robust differences in the metabolomic profile of young vs aged FMT recipients before and after EAE induction. Further, in both models of altered barrier integrity, we observed altered myeloid cell trafficking to and immune activation in the CNS, suggesting that the intestinal barrier itself plays a critical role in immune activation during EAE. Conclusions These findings suggest specific age-related microbiota-derived metabolites, which have increased access to the host system due to a leaky intestinal barrier, may be associated with increased risk of progressive disease and may represent promising therapeutic targets for MS. Funding Agencies CIHRMS Canada

ADS024, a single-strain live biotherapeutic product of Bacillus velezensis alleviates dextran sulfate-mediated colitis in mice, protects human colonic epithelial cells against apoptosis, and maintains epithelial barrier function.

Epithelial cell apoptosis and compromised gut barrier function are features of inflammatory bowel disease. ADS024 is a single-strain live biotherapeutic product (LBP) of Bacillus velezensis under development for treating ulcerative colitis (UC). The cytoprotective effects of the sterile filtrate of ADS024's secreted products on UC patient-derived colonic tissues, human primary colonic epithelial cells (HPEC), and human colonic epithelial T84 cells were evaluated. ADS024 filtrate significantly inhibited apoptosis and inflammation with reduced Bcl-2 Associated X-protein (BAX) and tumor necrosis factor (TNF) mRNA expression in fresh colonic explants from UC patients. Exposure to UC patient-derived serum exosomes (UCSE) induced apoptosis with increased cleaved caspase 3 protein expression in HPECs. ADS024 filtrate diminished the UCSE-mediated apoptosis by inhibiting cleaved caspase 3. TNFα and interferon-gamma (IFNγ) damaged epithelial barrier integrity with reduced transepithelial electrical resistance (TEER). ADS024 filtrate partially attenuated the TEER reduction and restored tight junction protein 1 (TJP1) expression. Oral live ADS024 treatment reduced weight loss, disease activity, colonic mucosal injury, and colonic expression of interleukin 6 (IL-6) and TNFα in dextran sodium sulfate (DSS)-treated mice with colitis. Thus, ADS024 may protect the colonic epithelial barrier in UC via anti-inflammatory, anti-apoptotic, and tight-junction protection mechanisms.

High methionine intake alters gut microbiota and lipid profile and leads to liver steatosis in mice.

Methionine is an important sulfur-containing amino acid. Health effects of both methionine restriction (MR) and methionine supplementation (MS) have been studied. This study aimed to investigate the impact of a high-methionine diet (HMD) (1.64% methionine) on both the gut and liver functions in mice through multi-omic analyses. Hepatic steatosis and compromised gut barrier function were observed in mice fed the HMD. RNA-sequencing (RNA-seq) analysis of liver gene expression patterns revealed the upregulation of lipid synthesis and degradation pathways, cholesterol metabolism and inflammation-related nucleotide-binding oligomerization domain (NOD)-like receptor signaling pathway. Metagenomic sequencing of cecal content demonstrated a shift in gut microbial composition with an increased abundance of opportunistic pathogens and gut microbial functions with up-regulated lipopolysaccharide (LPS) biosynthesis in mice fed HMD. Metabolomic study of cecal content showed an altered gut lipid profile and the level of bioactive lipids, including docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), palmitoylethanolamide (PEA), linoleoyl ethanolamide (LEA) and arachidonoyl ethanolamide (AEA), that carry anti-inflammatory effects significantly reduced in the gut of mice fed the HMD. Correlation analysis demonstrated that gut microbiota was highly associated with liver and gut functions and gut bioactive lipid content. In conclusion, this study suggested that the HMD exerted negative impacts on both the gut and liver, and an adequate amount of methionine intake should be carefully determined to ensure normal physiological function without causing adverse effects.

Increased Mortality Risk at Septic Condition in Inflammatory Skin Disorders and the Effect of High-Fat Diet Consumption.

In recent years, attention has increasingly focused on various infectious diseases. Although some fatalities are directly attributed to the causative virus, many result from complications and reactive inflammation. Patients with comorbidities are at a higher risk of mortality. Refractory skin conditions such as atopic dermatitis, psoriasis, and epidermolysis bullosa, known for an elevated risk of sepsis, partly owe this to compromised surface barrier function. However, the detailed mechanisms underlying this phenomenon remain elusive. Conversely, although the detrimental effects of a high-fat diet on health, including the onset of metabolic syndrome, are widely recognized, the association between diet and susceptibility to sepsis has not been extensively explored. In this study, we examined the potential causes and pathogenesis of increased sepsis susceptibility in inflammatory skin diseases using a mouse dermatitis model: keratin 14-driven caspase-1 is overexpressed (KCASP1Tg) in mice on a high-fat diet. Our findings reveal that heightened mortality in the dermatitis mouse model is caused by the inflamed immune system due to the chronic inflammatory state of the local skin, and administration of LPS causes a rapid increase in inflammatory cytokine levels in the spleen. Intake of a high-fat diet exacerbates these cytokine levels. Interestingly, we also observed a reduced expression of Toll-like receptor 4 (TLR4) in monocytes from KCASP1Tg mice, potentially predisposing these animals to heightened infection risks and associated complications. Histological analysis showed a clear decrease in T and B cells in the spleen of KCASP1Tg mice fed a high-fat diet. Thickening of the alveolar wall, inflammatory cell infiltration, and alveolar hemorrhage were more prominent in the lungs of KCASP1Tg and KCASP1Tg with fat mice. We postulate that the chronic, non-infectious inflammation induces a negative feedback loop within the inflammatory cascade, and the suppressed expression of TLR4 renders the mice more susceptible to infections. Therefore, it is imperative for individuals with chronic skin inflammation to closely monitor disease progression upon infection and seek timely and appropriate treatment. Additionally, chronic inflammation of adipose tissue, induced by high-fat food intake, combined with dermatitis inflammation, may exacerbate infections, necessitating a review of dietary habits.

Cellulitis caused by Roseomonas mucosa in a child: a case report

BackgroundRoseomonas mucosa (R. mucosa) is a pink-pigmented, Gram-negative short rod bacterium. It is isolated from moist environments and skin, resistant to multiple drugs, including broad-spectrum cephalosporins, and a rare cause of infection with limited reports. R. mucosa mostly causes catheter-related bloodstream infections, with even fewer reports of skin and soft tissue infections.Case presentationA 10-year-old boy received topical steroid treatment for sebum-deficient eczema. A few days before the visit, he was bitten by an insect on the front of his right lower leg and scratched it due to itching. The day before the visit, redness, swelling, and mild pain in the same area were observed. Based on his symptoms, he was diagnosed with cellulitis. He was treated with sulfamethoxazole/trimethoprim, and his symptoms improved. Pus culture revealed R. mucosa.ConclusionsWe report a rare case of cellulitis caused by R. mucosa. Infections caused by rare organisms that cause opportunistic infections, such as R. mucosa, should be considered in patients with compromised skin barrier function and regular topical steroid use. Gram stain detection of organisms other than Gram-positive cocci should be considered.

The Impact of Niclosamide Exposure on the Activity of Antioxidant Enzymes and the Expression of Glucose and Lipid Metabolism Genes in Black Carp (Mylopharyngodon piceus).

Niclosamide (NIC, 2',5-dichloro-4'-nitrosalicylanilide) is a salicylanilide molluscicide, and the extensive utilization and environmental pollution associated with NIC engender a potential hazard to both human health and the wellbeing of aquatic organisms. However, the mechanism of the chronic toxicity of NIC at environmentally relevant concentrations in terms of oxidative stress, metabolic disorder, and barrier functions in black carp (Mylopharyngodon piceus) is unknown. Therefore, healthy juvenile black carp (M. piceus) (average weight: 38.2 ± 2.5 g) were exposed to NIC at an environmentally realistic concentration (0, 10, and 50 μg/L) for 28 days. The findings of this study indicate that exposure to NIC resulted in reductions in weight gain, decreased activity of antioxidant enzymes, and increased expression of the Nrf2 gene. Furthermore, the liver demonstrated a greater accumulation of NIC than that in the gut and gills, as determined with a chemical analysis. Additionally, NIC exposure led to a significant reduction in ATP content and the activity of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the gut. Meanwhile, exposure to NIC resulted in a decrease in the liver glucose (Glu) level, gut cholesterol (CHO), and glycogen (Gln) and triglyceride (TG) content in all examined tissues. Conversely, it led to an increase in tissue lactic acid (LA) and acetyl-CoA levels, as well as LDH activity. Furthermore, NIC exposure at environmentally relevant concentrations demonstrated an upregulation in the expression of genes associated with glycolysis, such as PK and GK, while concurrently downregulating the gluconeogenesis gene G6Pase. Additionally, NIC exhibited an upregulation in the expression of genes related to β-oxidation, such as CPT1 and ACOX, while downregulating genes involved in triglyceride synthesis, including SREBP1, GPAT, FAS, and ACC1. Moreover, NIC facilitated fatty acid transportation through the overexpression of FATP and Fat/cd36. These results suggest that chronic exposure to NIC is associated with oxidative stress, compromised barrier function, and metabolic disorder. Moreover, these results underscore the significance of assessing the potential consequences of NIC for black carp and aquatic environments for aquaculture.

Effects of a multispecies direct-fed microbial on gastrointestinal permeability during feed restriction in a growing heifer model

The objectives were to evaluate the effects of a 4-strain direct-fed microbial (DFM) on gastrointestinal tract (GIT) permeability and inflammation during feed restriction (FR) in heifers. Holstein heifers (n = 32; mean ± standard deviation; 295 ± 25 kg body weight; 287 ± 17 d of age) were used in an experiment conducted in 2 replicates (16/replicate). Heifers were randomly assigned to 1 of 2 top-dressed dietary treatments: (1) control (CON; 10 g/d dried lactose; n = 16) or (2) DFM containing a commercial blend of Lactobacillus animalis, Propionibacterium freudenreichii, Bacillus licheniformis, and Bacillus subtilis at 11.8 × 109 cfu/d (PRO; 10 g/d 4-strain DFM; n = 16). The trial consisted of 2 experimental periods (P): P1 (14 d) served as baseline for P2 (5 d), when all heifers were restricted to 40% of their P1 dry matter intake (DMI). On P1 d 12 and P2 d 2 and 5, GIT permeability was evaluated using oral chromium (Cr)-EDTA. By design, FR decreased DMI (60%) and body weight (∼18 kg) in all heifers. Regardless of treatment, during FR, all heifers had decreased circulating glucose, β-hydroxybutyrate, insulin, and l-lactate (4, 14, 45, and 19%, respectively), but increased nonesterified fatty acids, serum amyloid A, and haptoglobin (3.0-, 1.7-, and 5.0-fold, respectively). Circulating white blood cells, neutrophils, lymphocytes, and basophils decreased (4, 7, 5, and 6%, respectively), whereas eosinophils increased (41%) during P2 irrespective of dietary treatment. Circulating IFN-γ inducible protein-10 increased (23%) during FR compared with P1 regardless of treatment. Plasma Cr area under the curve increased in all heifers on d 2 and 5 (10 and 14%, respectively) of P2 relative to P1, but this was unaltered by dietary treatment. In summary, FR compromised GIT barrier function and stimulated an inflammatory response, but this did not appear to be ameliorated by PRO.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

RNA surveillance by ADAR1 is required for vascular endothelial resilience

Abstract Introduction Long dsRNAs are recognised as danger-associated molecular patterns by the cytosolic innate immune sensors inducing thus innate immune responses. RNA editing is a post-transcriptional modification induced by the binding of Adenosine Deaminases Acting on RNA (ADARs) to double-stranded RNA structures (dsRNAs). We have recently reported that ADAR1 is the main RNA editor in endothelial cells (ECs) but the role of ADAR1 in vascular homeostatic processes remains yet elusive. Methods Genetic deletion strategies were employed by crossing mice carrying a conditional (floxed) Adar1 , Ifh1 or Tlr3 allele with either a Tie2-Cre or with a tamoxifen-inducible VE-Cadherin-CreERT2 mouse line. Primary human and murine vascular EC culture assays, dsRNA metabolism studies, gene-silencing and expression techniques, immunohistochemistry and confocal microscopy were used to assess the EC-specific ADAR1 effects. Results EC-specific ADAR1 deletion resulted in prenatal embryonic lethality evidencing an absolute requirement of EC-ADAR1 in life. Inducible EC-ADAR1 ablation in adult mice elicited premature sudden death due to a widespread lung vascular leakage and pleural effusion, reflecting a compromised EC barrier function. Mechanistically, silencing of endothelial ADAR1 disrupted long-to-short dsRNA metabolism witnessed by the striking accumulation of cytoplasmic long dsRNAs. Subsequently, the cytoplasmic accumulation of endogenous long dsRNAs induced aberrant levels of interferon-β and activation of innate immune system. Finally, activation of the cytosolic dsRNA sensors inflicted the dissociation of β-catenin from VE-cadherin in EC junctions, while, co-silencing of the dsRNA sensor, MDA5, was sufficient to restore the integrity of the ADAR1-deficient endothelium. While double EC-specific ADAR1 and TLR3 did not fully rescue the lethality of the EC-ADAR1 animals, in vivo experiments using double EC-specific ADAR1 and MDA5 KO mice fully rescued the phenotype. Validation experiments using murine vascular ECs isolated from the mouse strains of our studies confirmed our mechanistic and phenotypic findings. Conclusion Endogenous suppression of double-stranded RNA-induced MDA5 autoinflammatory signaling by the RNA editor ADAR1 is essential for the maintenance of vascular integrity in adult mice.

Roles unveiled for membrane-associated mucins at the ocular surface using a Muc4 knockout mouse model

Membrane-associated mucins (MAMs) are proposed to play critical roles at the ocular surface; however, in vivo evidence has been lacking. Here we investigate these roles by phenotyping of a Muc4 KO mouse. Histochemical analysis for expression of the beta-galactosidase transgene replacing Muc4 revealed a spiraling ribbon pattern across the corneal epithelium, consistent with centripetal cell migration from the limbus. Depletion of Muc4 compromised transcellular barrier function, as evidenced by an increase in rose bengal staining. In addition, the corneal surface was less smooth, consistent with disruption of tear film stability. While surface cells presented with well-developed microprojections, an increase in the number of cells with fewer microprojections was observed. Moreover, an increase in skin-type keratin K10 and a decrease in transcription factor Pax6 was observed, suggesting an incipient transdifferentiation. Despite this, no evidence of inflammatory dry eye disease was apparent. In addition, Muc4 had no effect on signaling by toll-like receptor Tlr4, unlike reports for MUC1 and MUC16. Results of this study provide the first in vivo evidence for the role of MAMs in transcellular barrier function, tear film stability, apical epithelial cell architecture, and epithelial mucosal differentiation at the ocular surface.

Secretomics reveals gelatinase substrates at the blood-brain barrier that are implicated in astroglial barrier function.

The gelatinases, matrix metalloproteinase 2 (MMP-2) and MMP-9, are key for leukocyte penetration of the brain parenchymal border in neuroinflammation and the functional integrity of this barrier; however, it is unclear which MMP substrates are involved. Using a tailored, sensitive, label-free mass spectrometry-based secretome approach, not previously applied to nonimmune cells, we identified 119 MMP-9 and 21 MMP-2 potential substrates at the cell surface of primary astrocytes, including known substrates (β-dystroglycan) and a broad spectrum of previously unknown MMP-dependent events involved in cell-cell and cell-matrix interactions. Using neuroinflammation as a model of assessing compromised astroglial barrier function, a selection of the potential MMP substrates were confirmed in vivo and verified in human samples, including vascular cell adhesion molecule-1 and neuronal cell adhesion molecule. We provide a unique resource of potential MMP-2/MMP-9 substrates specific for the astroglia barrier. Our data support a role for the gelatinases in the formation and maintenance of this barrier but also in astrocyte-neuron interactions.

Restoration of the gut barrier integrity and restructuring of the gut microbiome in aging by angiotensin-(1-7).

Compromised barrier function of colon epithelium with aging is largely due to gut microbial dysbiosis. Recent studies implicate an important role for angiotensin converting enzymes, ACE and ACE2, angiotensins, and the receptors, AT1 receptor (AT1R) and Mas receptor (MasR), in the regulation of colon functions. The present study tested the hypothesis that leaky gut in aging is associated with an imbalance in ACE2/ACE and that the treatment with angiotenisn-(1-7) (Ang-(1-7)) will restore gut barrier integrity and microbiome. Studies were carried out in Young (3-4 months) and old (20-24 months) male mice. Ang-(1-7) was administered by using osmotic pumps. Outcome measures included expressions of ACE, ACE2, AT1R, and MasR, intestinal permeability by using FITC-dextran, and immunohistochemistry of claudin 1 and occludin, and intestinal stem cells (ISCs). ACE2 protein and activity were decreased in Old group while that of ACE were unchanged. Increased intestinal permeability and plasma levels of zonulin-1 in the Old group were normalized by Ang-(1-7). Epithelial disintegrity, reduced number of goblet cells and ISCs in the old group were restored by Ang-(1-7). Expression of claudin 1 and occludin in the aging colon was increased by Ang-(1-7). Infiltration of CD11b+ or F4/80+ inflammatory cells in the old colons were decreased by Ang-(1-7). Gut microbial dysbiosis in aging was evident by decreased richness and altered beta diversity that were reversed by Ang-(1-7) with increased abundance of Lactobacillus or Lachnospiraceae. The present study shows that Ang-(1-7) restores gut barrier integrity and reduces inflammation in the aging colon by restoring the layer of ISCs and by restructuring the gut microbiome.

Matrix stiffness regulates the tight junction phenotypes and local barrier properties in tricellular regions in an iPSC-derived BBB model

The blood-brain barrier (BBB) can respond to various mechanical cues such as shear stress and substrate stiffness. In the human brain, the compromised barrier function of the BBB is closely associated with a series of neurological disorders that are often also accompanied by the alteration of brain stiffness. In many types of peripheral vasculature, higher matrix stiffness decreases barrier function of endothelial cells through mechanotransduction pathways that alter cell-cell junction integrity. However, human brain endothelial cells are specialized endothelial cells that largely resist changes in cell morphology and key BBB markers. Therefore, it has remained an open question how matrix stiffness affects barrier integrity in the human BBB. To gain insight into the effects of matrix stiffness on BBB permeability, we differentiated brain microvascular endothelial-like cells from human induced pluripotent stem cells (iBMEC-like cells) and cultured the cells on extracellular matrix-coated hydrogels of varying stiffness. We first detected and quantified the junction presentation of key tight junction (TJ) proteins. Our results show matrix-dependent junction phenotypes in iBMEC-like cells, where cells on softer gels (1 kPa) have significantly lower continuous and total TJ coverages. We also determined that these softer gels also lead to decreased barrier function in a local permeability assay. Furthermore, we found that matrix stiffness regulates the local permeability of iBMEC-like cells through the balance of continuous ZO-1 TJs and no junction regions ZO-1 in tricellular regions. Together, these findings provide valuable insights into the effects of matrix stiffness on TJ phenotypes and local permeability of iBMEC-like cells. Statement of SignificanceBrain mechanical properties, including stiffness, are particularly sensitive indicators for pathophysiological changes in neural tissue. The compromised function of the blood-brain barrier is closely associated with a series of neurological disorders often accompanied by altered brain stiffness. In this study, we use polymeric biomaterials and provide new evidence that biomaterial stiffness regulates the local permeability in iPSC-derived brain endothelial cells in tricellular regions through the tight junction protein ZO-1. Our findings provide valuable insights into the changes in junction architecture and barrier permeability in response to different substrate stiffnesses. Since BBB dysfunction has been linked to many diseases, understanding the influence of substrate stiffness on junction presentations and barrier permeability could lead to the development of new treatments for diseases associated with BBB dysfunction or drug delivery across BBB systems.

Gut Microbial Metabolite Butyrate and Its Therapeutic Role in Inflammatory Bowel Disease: A Literature Review.

Background and objective: Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a chronic inflammatory disorder characterized by aberrant immune responses and compromised barrier function in the gastrointestinal tract. IBD is associated with altered gut microbiota and their metabolites in the colon. Butyrate, a gut microbial metabolite, plays a crucial role in regulating immune function, epithelial barrier function, and intestinal homeostasis. In this review, we aim to present an overview of butyrate synthesis and metabolism and the mechanism of action of butyrate in maintaining intestinal homeostasis and to discuss the therapeutic implications of butyrate in IBD. Methods: We searched the literature up to March 2023 through PubMed, Web of Science, and other sources using search terms such as butyrate, inflammation, IBD, Crohn's disease, and ulcerative colitis. Clinical studies in patients and preclinical studies in rodent models of IBD were included in the summary of the therapeutic implications of butyrate. Results: Research in the last two decades has shown the beneficial effects of butyrate on gut immune function and epithelial barrier function. Most of the preclinical and clinical studies have shown the positive effect of butyrate oral supplements in reducing inflammation and maintaining remission in colitis animal models and IBD patients. However, butyrate enema showed mixed effects. Butyrogenic diets, including germinated barley foodstuff and oat bran, are found to increase fecal butyrate concentrations and reduce the disease activity index in both animal models and IBD patients. Conclusions: The current literature suggests that butyrate is a potential add-on therapy to reduce inflammation and maintain IBD remission. Further clinical studies are needed to determine if butyrate administration alone is an effective therapeutic treatment for IBD.

The impact of aging on pulmonary microvascular endothelial cell barrier function

Elderly individuals have substantially elevated morbidity and mortality during conditions of lung injury. However, the precise underlying mechanisms leading to this elevated mortality are ill-defined. Lung injury is associated with damage to pulmonary microvascular endothelial cells (PMVEC) leading to a compromised vascular barrier, fluid and protein leakage into the tissue, and subsequent respiratory dysfunction. Cell-cell junctions, including adherens junctions and tight junctions, are crucial for the maintenance of the endothelial barrier and have been shown to be dysregulated in various disease models. Pilot data from our lab demonstrated increased pulmonary vascular leak in aged mice during lung injury compared to young mice. We hypothesized that aging contributes to PMVEC barrier dysfunction due to impaired cell-cell junction integrity.To address our hypothesis, PMVEC isolated from young and aged mice were cultured in vitro until a confluent monolayer was formed. Endothelial barrier integrity was assessed by Evans blue-labelled albumin flux across the monolayers, as well as immunofluorescence staining of the adherens junction protein, vascular endothelial (VE)-cadherin, and tight junction protein, claudin-5. Localization of leak in relation to junctional proteins was performed using the fluorescently labelled macromolecule, NeutrAvidin. Proteomics analysis was conducted to identify differentially enriched pathways in young and aged PMVEC.Endothelial monolayers from aged mice exhibited a significant increase in albumin leak associated with augmented VE-cadherin and claudin-5 disruption vs. endothelial monolayers form young mice. Furthermore, NeutrAvidin staining localized to paracellular regions with VE-cadherin discontinuity was increased in PMVEC monolayers from aged mice vs. young mice, suggesting that the junctional disruption in aged PMVEC was directly associated with leak. Proteomics analysis revealed pathways known to be implicated in endothelial barrier dysfunction, including regulation of oxidoreduction and protein processing and trafficking, that were altered in PMVEC from aged mice.In summary, PMVEC from aged mice exhibit compromised barrier function, which is directly associated with impaired cell-cell junction integrity. We will next validate and target protein pathways identified in our proteomics analysis that were dysregulated in aged PMVEC to potentially rescue age-induced barrier dysfunction. These findings may highlight molecular pathways involved in predisposing aged individuals to worsened outcomes during lung injury, which can help in the development of therapeutics. Funded by Lawson Health Research Institute, CIHR, and Western University This is the full abstract presented at the American Physiology Summit 2023 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 epidemiology of antimicrobial resistance and transmission of cutaneous bacterial pathogens in domestic animals.

As the primary agents of skin and soft tissue infections in animals, Staphylococcus spp and Pseudomonas aeruginosa are among the most formidable bacterial pathogens encountered by veterinarians. Staphylococci are commensal inhabitants of the surfaces of healthy skin and mucous membranes, which may gain access to deeper cutaneous tissues by circumventing the stratum corneum's barrier function. Compromised barrier function occurs in highly prevalent conditions such as atopic dermatitis, endocrinopathies, and skin trauma. P aeruginosa is an environmental saprophyte that constitutively expresses virulence and antimicrobial resistance genes that promote its success as an animal pathogen. For both organisms, infections of the urinary tract, respiratory tract, joints, central nervous system, and body cavities may occur through ascension along epithelial tracts, penetrating injuries, or hematogenous spread. When treating infections caused by these pathogens, veterinarians now face greater therapeutic challenges and more guarded outcomes for our animal patients because of high rates of predisposing factors for infection and the broad dissemination of antimicrobial resistance genes within these bacterial species. This review considers the history of the rise and expansion of multidrug resistance in staphylococci and P aeruginosa and the current state of knowledge regarding the epidemiologic factors that underly the dissemination of these pathogens across companion animal populations. Given the potential for cross-species and zoonotic transmission of pathogenic strains of these bacteria, and the clear role played by environmental reservoirs and fomites, a one-health perspective is emphasized.

A172 THE LACK OF CHROMOGRANIN A PROTECTS THE COLONIC EPITHELIAL BARRIER FUNCTIONS FROM COLITIS IN MALE MICE AND EXACERBATES COLITIS IN FEMALE MICE

Abstract Background Ulcerative colitis (UC) is associated with compromised mucosal barrier function and colonic epithelial repair in a sex-dependent manner. Chromogranin A (CHGA), a pro-hormone, correlates positively with UC disease severity. In male mice, deletion of CHGA has been shown to decrease the inflammatory process; however, the effect of CHGA on mucosal barrier function and colonic epithelial repair between males and females is unknown. Purpose We investigated whether the lack of CHGA modulates gut barrier function, mucosa integrity, and colonic epithelial repair between males and females in a mice model of colitis. Method Male and female wild-type (WT) and CHGA (CHGA-/-) deficient mice (13-17 weeks old) were given 5% dextran sulfate sodium (DSS) to induce colitis or water for 5-days (n=5-8 mice per group). The disease activity index (DAI) was assessed. Colons were collected, and tumor necrosis factor (TNF)-α and IL-25 concentrations were measured by ELISA. Expression of structural and functional markers specific to epithelial cells, namely, colonocytes (Na-K-Cl cotransporter [Nkcc]1), goblet cells function (resistin-like molecule [Relm]β), and mucin [MUC]2) and stem cells (reserve Hopx, fast-cycling Lgr5, and fetal-like Ly6a cells) were evaluated by qRT-PCR. Result(s) Colitic male CHGA-/- did not show significant changes in DAI compared to WT mice. Conversely, female CHGA-/- mice demonstrated a trend toward higher susceptibility to colitis compared to female WT mice with increased weight loss and bleeding. This was associated with elevated levels of colonic TNF-α and IL-25 (p<0.05) in CHGA-/- females compared to CHGA-/- males. TNF-α levels were not different between female groups at baseline and during colitis. While colitic CHGA-/- female had elevated Relmβ expression (p<0.01) compared to WT mice. No significative change was noted in Relmβ expression between female WT mice at baseline and during colitis. Similarly, Nkcc1 and Muc2 expression was not different between female groups. By contrast, male CHGA-/- were less susceptible to colitis than male WT mice with elevated Nkcc1and a lower Relmβ and Muc2 expression (p<0.01). In colitis, expression of stem cell markers, Hopx and Lgr5, was markedly reduced in all groups, while male WT, CHGA-/-, and female WT had elevated Ly6a expression. However, the magnitude of Hopx and Ly6a expression was associated with sex. Thus, colitic male CHGA-/- mice had a higher Hopx expression than male WT and female CHGA-/ - mice, with a lower reduction of 1.9 compared to 4.9 and 6.6, respectively (p<0.05, 0.01, and 0.0001). While colitic male CHGA-/- mice had elevated Ly6a expression (p<0.05) in contrast to female CHGA-/- mice (p=0.5). The magnitude of the decrease in Lgr5 expression was not different between all groups. Conclusion(s) In the absence of CHGA, male mice preserved their colonic mucosa integrity and repair potential, while female mice suffered significant loss of mucosa integrity and repair potential during colitis. Disclosure of Interest None Declared

Glucomannan from Aloe vera gel maintains intestinal barrier integrity via mitigating anoikis mediated by Nrf2-mitochondria axis

Impairment of intestinal epithelium barrier is a hallmark of gut pathology. Cell death can compromise barrier function and impair epithelial restitution directly or indirectly in inflammatory bowel disease (IBD). Our previous work demonstrated that glucomannan from Aloe vera gel (AGP) protected mice from DSS-induced colitis, with unclear mechanism of AGP-intestinal barrier interactions. Here, AGP maintained the integrity of intestinal barrier in colitis mice. RNA-Sequencing results indicated that pathways related to anoikis (apoptosis induced by loss of cell-matrix interaction), mitochondrial function and oxidative stress were significantly altered in the process of AGP-intestinal barrier interaction. Further experiments confirmed that AGP activated Nrf2, decreased ROS levels, mitigated mitochondrial dysfunction and anoikis of colonic epithelial cells in mice. Intriguingly, AGP reversed oxidative stress and mitochondrial dysfunction induced by knockdown or inhibitor (ML385) of Nrf2 in IEC-6 cells, which indicated the essential role of Nrf2-mitochondrial axis in the intestinal protective function of AGP. In addition, AGP alleviated anoikis caused by impaired mitochondrial function. Hence, this current work indicated that AGP might maintain intestinal barrier integrity by mitigating anoikis mediated by Nrf2-mitochondria axis. These findings provide new evidence into the effect of polysaccharides maintaining intestinal barrier integrity.

Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2

Aberrant mechanotransduction and compromised epithelial barrier function are associated with numerous human pathologies including inflammatory skin disorders. However, the cytoskeletal mechanisms regulating inflammatory responses in the epidermis are not well understood. Here we addressed this question by inducing a psoriatic phenotype in human keratinocytes and reconstructed human epidermis using a cytokine stimulation model. We show that the inflammation upregulates the Rho-myosin II pathway and destabilizes adherens junctions (AJs) promoting YAP nuclear entry. The integrity of cell-cell adhesion but not the myosin II contractility per se is the determinative factor for the YAP regulation in epidermal keratinocytes. The inflammation-induced disruption of AJs, increased paracellular permeability, and YAP nuclear translocation are regulated by ROCK2, independently from myosin II activation. Using a specific inhibitor KD025, we show that ROCK2 executes its effects via cytoskeletal and transcription-dependent mechanisms to shape the inflammatory response in the epidermis.

HnRNPH1 establishes Sertoli-germ cell crosstalk through cooperation with PTBP1 and AR, and is essential for male fertility in mice.

Spermatogenesis depends on the crosstalk of Sertoli cells (SCs) and germ cells. However, the gene regulatory network establishing the communications between SCs and germ cells remains unclear. Here, we report that heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) in SCs is essential for the establishment of crosstalk between SCs and germ cells. Conditional knockout of hnRNPH1 in mouse SCs leads to compromised blood-testis barrier function, delayed meiotic progression, increased germ cell apoptosis, sloughing of germ cells and, eventually, infertility of mice. Mechanistically, we discovered that hnRNPH1 could interact with the splicing regulator PTBP1 in SCs to regulate the pre-mRNA alternative splicing of the target genes functionally related to cell adhesion. Interestingly, we also found hnRNPH1 could cooperate with the androgen receptor, one of the SC-specific transcription factors, to modulate the transcription level of a group of genes associated with the cell-cell junction and EGFR pathway by directly binding to the gene promoters. Collectively, our findings reveal a crucial role for hnRNPH1 in SCs during spermatogenesis and uncover a potential molecular regulatory network involving hnRNPH1 in establishing Sertoli-germ cell crosstalk.