Effect of Lactobacillus.salivarius HHuMin-U on gene expression of VK2/E6E7 juman vaginal cell lines.
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ABSTRACT: Vulvovaginal candidiasis (VVC) is a common mucosal infection caused by Candida albicans, characterized by inflammation and disruption of epithelial immunity. Probiotic therapy has emerged as a promising approach for managing VVC by modulating host immune responses and enhancing mucosal defenses. In this study, we investigated the therapeutic potential of Lactobacillus salivarius HHuMin-U (HMU) against VVC using in vitro and in vivo models. HMU treatment enhanced the expression of antimicrobial peptides (AMPs) such as DEFB1 and S100A8 and modulated cytokine production (e.g., IL-6, IL-8) in vaginal epithelial cells. Furthermore, RNA sequencing of HMU-treated VK2/E6E7 cells revealed significant transcriptional changes, including upregulation of epithelial barrier function and immune-enhancing pathways. Gene Set Enrichment Analysis (GSEA) identified the NF-κB pathway as a critical regulator of HMU-induced immunity. In vivo, HMU administration significantly reduced fungal burden, mitigated tissue inflammation, and improved epithelial integrity in a murine model of VVC. The sequencing data presented in this report provide a comprehensive transcriptomic landscape of HMU-treated vaginal epithelial cells and offer insights into its underlying mechanisms. These findings support the therapeutic potential of HMU as a probiotic agent for VVC management.
Project description:During sexual transmission of HIV-1 from male to female partners, the vagina is the initial site of contact with HIV infected semen. The mechanism of HIV traversing the CD4 negative multi-layered stratified squamous epithelial barrier of the vagina to infect sub-epithelial susceptible immune cells, is hitherto unknown. HIV gp120 binds to several host proteins on vaginal epithelial cells. To gain an insight into the physiologic changes that may occur in vaginal epithelial cells in response to interactions with HIV gp120, and obtain an understanding of the molecular mechanisms by which HIV breaches the vaginal epithelium, a global snap shot of gene expression profiles in the vaginal epithelial cell line Vk2/E6E7, treated with HIV gp120 was determined. The vaginal epithelial cell line Vk2/E6E7 was treated with HIV gp120 (83nM) for 24 hr, and Agilent one colour, microarrays were performed. Agilent one-color experiment,Organism: Human ,Agilent-Custom Whole Genome Human 8x60k designed by Genotypic Technology Pvt. Ltd. (AMADID: 027114), Labeling kit: Agilent Quick-Amp labeling Kit (p/n5190-0442)
Project description:During sexual transmission of HIV-1 from male to female partners, the vagina is the initial site of contact with HIV infected semen. The mechanism of HIV traversing the CD4 negative multi-layered stratified squamous epithelial barrier of the vagina to infect sub-epithelial susceptible immune cells, is hitherto unknown. HIV gp120 binds to several host proteins on vaginal epithelial cells. To gain an insight into the physiologic changes that may occur in vaginal epithelial cells in response to interactions with HIV gp120, and obtain an understanding of the molecular mechanisms by which HIV breaches the vaginal epithelium, a global snap shot of gene expression profiles in the vaginal epithelial cell line Vk2/E6E7, treated with HIV gp120 was determined. The vaginal epithelial cell line Vk2/E6E7 was treated with HIV gp120 (83nM) for 24 hr, and Agilent one colour, microarrays were performed.
Project description:Inflammation and immune activation of the cervicovaginal mucosa are considered factors that increase susceptibility to HIV infection. It is essential to screen candidate anti-HIV microbicides for potential mucosal immunomodulatory/inflammatory effects prior to further clinical development. The goal of this study was to develop an in vitro method for preclinical evaluation of the inflammatory potential of new candidate microbicides. We compared transcriptomes of human vaginal cells (Vk2/E6E7) treated with well-characterized pro-inflammatory (PIC) and non-inflammatory (NIC) compounds. Microarraray comparative analysis allowed us to generate a panel of 20 genes that were consistently deregulated by all PICs compared to NICs, thus distinguishing between these two groups.
Project description:The female menopause, characterised by reduced estrogen associates with an increased risk of recurrent UTIs caused by uropathogenic Escherichia coli (UPEC). Clinically such infections can be countered by topical vaginal estrogen treatment and the aim of this study was to investigate, in vitro, the effects of topical estrogen treatment on vaginal epithelial responses following challenge with E.coli flagellin used to mimic UPEC. Immortalised vaginal epithelial cells (VK2 E6/E7), modelling the vaginal epithelium were treated with either 4nM 17β-estradiol (E) for seven days, 50ng/ml E.coli flagellin (F) for 12h, or 4nM 17β-estradiol plus 50ng/ml flagellin (E + F(12h)). RNA was analysed by microarray gene profiling using the Illumina HumanHT-12 v 4 Expression Beadchip. Following E + F treatments expression of genes encoding host defence molecules including DEFβ4A, DEFB103A, LCN2 as well as those associated with keratinisation e.g. CNFN and SPRR family genes were significantly enhanced (P<0.05) compared to either E or F treatments alone. Mutation of EREs identified in the DEFβ4 gene promoter abolished the augmented gene expression suggesting estrogen functioned directly through a transcriptional regulatory mechanism involving ESR1/2. Ingenuity pathway analyses also suggested the pro-inflammatory cytokine IL-17A to regulate the vaginal host defences during infection. Pre-treating VK2 E6/E7 cells with estrogen (4nM) and challenging with 1L-17A & F (12h) significantly enhanced DEFβ4, DEF103A and S100A7 expression (P<0.05). Origins of vaginal IL-17 in vivo remain unclear, but vaginal biopsy material suggests gd T cells located within the vaginal epithelium. These data suggest that the vaginal antimicrobial response induced by flagellin activation of TLR5 cell signalling is augmented significantly by topical estrogen treatment.
Project description:The intestinal ecosystem is balanced by dynamic interactions between resident and incoming microbes, the gastrointestinal barrier, and the mucosal immune system. However, in the context of inflammatory bowel diseases (IBD) where the integrity of the gastrointestinal barrier is compromised, resident microbes contribute to the development and perpetuation of inflammation and disease. In this context, probiotic bacteria exert beneficial effects enhancing epithelial barrier integrity. However, the mechanisms underlying these beneficial effects are only poorly understood. Here, we comparatively investigated the effects of four probiotic lactobacilli, namely L. acidophilus, L. fermentum, L. gasseri, and L. rhamnosus in a T84 cell epithelial barrier model. Results of DNA-microarray experiments indicating that lactobacilli modulate the regulation of genes encoding in particular adherence junction proteins such as E-cadherin and b-catenin were confirmed by qRT-PCR. Furthermore, we show that epithelial barrier function is modulated by Gram-positive probiotic lactobacilli via their effect on adherence junction protein expression and complex formation. In addition, incubation with lactobacilli differentially influences the phosphorylation of adherence junction proteins and of PKC isoforms such as PKCd which thereby positively modulates epithelial barrier function. Further insight into the underlying molecular mechanisms triggered by these probiotics might also foster the development of novel strategies for the treatment of gastrointestinal diseases (e.g. IBD).
Project description:Problem: Recurrent vulvovaginal candidiasis (RVVC) affects 5-10% of all women, negatively impacting their reproductive health and quality of life. Herein, we investigated the molecular effects of RVVC on the vaginal mucosa of otherwise healthy women. Methods: Gene expression analysis was performed on vaginal tissue biopsies from women with RVVC, including those with a current episode of vulvovaginal candidiasis (RVVC, n=19) and women between infections (CNR, n=8); women asymptomatically colonized with Candida albicans (AS, n=7); and healthy controls (n=18). Gene expression profiles were compared between groups and correlated with clinical data retrieved from questionnaires and gynecologic examinations. Results: Of 20,171 genes identified in vaginal biopsies, 6,506 were differentially expressed in the RVVC group, compared to healthy controls. Gene expression pathway analysis revealed an association between RVVC and pathways of inflammatory responses, especially genes involved in neutrophil recruitment and activation. Expression of genes involved in inflammation and neutrophil recruitment increased with increasing clinical severity of vulvovaginal candidiasis, whereas expression of some genes involved in epithelial integrity decreased with increasing clinical severity of infection. Gene expression profiles of both the CNR and AS groups were comparable to those of healthy controls. Conclusions: The clinical severity of RVVC during active infection correlates with increased expression of genes involved in molecular inflammation and neutrophil activation in the vaginal mucosa. The lack of differences between healthy controls and women with RVVC who were between acute infections indicates that the molecular effects observed in the RVVC group are only present during active infection.
Project description:A lactobacilli dominated microbiota in most pre and post-menopausal women is an indicator of vaginal health. A Nugent scoring system serves as a proxy for determining the ratio of lactobacilli to other vaginal inhabitants where a high score usually represents a diseased state, whilst an intermediate score represents a warning zone. The objective of this double blinded, placebo-controlled crossover study was to evaluate in 14 post-menopausal women with an intermediate score, the effect of vaginal administration of probiotic L. rhamnosus GR-1 and L. reuteri RC-14 on the microbiota and host response. The probiotic treatment did not result in changes to clinical parameters such as dryness, irritation and comfort, compared to when placebo was applied. Analysis using 16S rRNA sequencing and metabolomics profiling revealed that the proportional abundance of Lactobacillus was increased following probiotic administration as compared to placebo, which was weakly associated with an increase in lactate levels. Analysis of host responses by microarray showed the probiotics had an immune-modulatory response and multiplex cytokine analysis showed up-regulation of IL-5. This is the first study to use an interactomic approach for the study of vaginal probiotic administration in post-menopausal women. It shows that in some cases multifaceted approaches are required to detect the subtle trigger molecular changes induced by the host to instillation of probiotic strains. A total of 35 total RNA samples extracted from vaginal brushes were analyzed on Affymetrix Gene 2.0 ST arrays from 14 Participants collected over multiple visits including administration of either a probiotic supplement or placebo control.
Project description:Bacterial vaginosis (BV) is characterized by depletion of Lactobacillus and overgrowth of anaerobic and facultative bacteria, leading to increased mucosal inflammation, epithelial disruption, and poor reproductive health outcomes. However, the molecular mediators contributing to vaginal epithelial dysfunction are poorly understood. Here we utilized proteomic, transcriptomic and metabolomic analyses to characterize biological features underlying BV in 405 African women and explored functional mechanisms using bacterial co-culturesin vitro. We identified five major vaginal microbiome groups, (L.crispatus(21%), L.iners(18%), any non-specific Lactobacillus species(9%), Gardnerella species .vaginalis(30%), or polymicrobial(22%)). Using multi-‘omics we show that BV associated epithelial disruption and mucosal inflammation are linked to the mammalian target of rapamycin (mTOR) pathway and associate with Gardnerella.vaginalis, M.mulieris, and specific metabolites including imidazole propionate. Bacterial co-culturesExperiments in vitro confirmed that type strain G.vaginalis and, M.mulieris supernatants and, as well as, and imidazole propionate, directly affect epithelial barrier function and , accompanied by activation of mTOR pathways. These results establish the microbiome-mTOR axis as a central feature of epithelial dysfunction in BV.