Vaginal Epithelial Cell Line Research Articles

Probiotic lactobacillus and estrogen effects on vaginal epithelial gene expression responses to Candida albicans

BackgroundVaginal epithelial cells have receptors, signal transduction mechanisms, and cytokine secretion capabilities to recruit host defenses against Candida albicans infections. This research evaluates how probiotic lactobacilli affect the defensive epithelial response.MethodsThis study used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14® and challenged with C. albicans. Data were statistically evaluated by repeated measures analysis of variance and paired t-tests where appropriate.ResultsC. albicans induced mRNA expression of genes related to inflammatory cytokine responses associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways. 17β-estradiol suppressed expression of interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha (TNFα) mRNA. Probiotic lactobacilli suppressed C. albicans-induced nuclear factor-kappa B inhibitor kinase kinase alpha (Iκκα), Toll-like receptor-2 (TLR2), TLR6, IL-8, and TNFα, also suggesting inhibition of NF-κB signaling. The lactobacilli induced expression of IL-1α, and IL-1β mRNA, which was not inhibited by curcumin, suggesting that they induce an alternate inflammatory signal transduction pathway to NF-κB, such as the mitogen activated protein kinase and activator protein-1 (MAPK/AP-1) signal transduction pathway. Curcumin inhibited IL-13 secretion, suggesting that expression of this cytokine is mainly regulated by NF-κB signaling in VK2 cells.ConclusionsThe results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes. Probiotic lactobacilli may induce IL-1α and IL-1β expression by an alternate signal transduction pathway, such as MAPK/AP-1. Activation of alternate signaling mechanisms by lactobacilli to modify epithelial cell cytokine production may be a mechanism for probiotic modulation of morbidity in vulvovaginal candidiasis.

HIV gp120 Binds to Mannose Receptor on Vaginal Epithelial Cells and Induces Production of Matrix Metalloproteinases

BackgroundDuring sexual transmission of HIV in women, the virus breaches the multi-layered CD4 negative stratified squamous epithelial barrier of the vagina, to infect the sub-epithelial CD4 positive immune cells. However the mechanisms by which HIV gains entry into the sub-epithelial zone is hitherto unknown. We have previously reported human mannose receptor (hMR) as a CD4 independent receptor playing a role in HIV transmission on human spermatozoa. The current study was undertaken to investigate the expression of hMR in vaginal epithelial cells, its HIV gp120 binding potential, affinity constants and the induction of matrix metalloproteinases (MMPs) downstream of HIV gp120 binding to hMR.Principal FindingsHuman vaginal epithelial cells and the immortalized vaginal epithelial cell line Vk2/E6E7 were used in this study. hMR mRNA and protein were expressed in vaginal epithelial cells and cell line, with a molecular weight of 155 kDa. HIV gp120 bound to vaginal proteins with high affinity, (Kd = 1.2±0.2 nM for vaginal cells, 1.4±0.2 nM for cell line) and the hMR antagonist mannan dose dependently inhibited this binding. Both HIV gp120 binding and hMR exhibited identical patterns of localization in the epithelial cells by immunofluorescence. HIV gp120 bound to immunopurified hMR and affinity constants were 2.9±0.4 nM and 3.2±0.6 nM for vaginal cells and Vk2/E6E7 cell line respectively. HIV gp120 induced an increase in MMP-9 mRNA expression and activity by zymography, which could be inhibited by an anti-hMR antibody.ConclusionhMR expressed by vaginal epithelial cells has high affinity for HIV gp120 and this binding induces production of MMPs. We propose that the induction of MMPs in response to HIV gp120 may lead to degradation of tight junction proteins and the extracellular matrix proteins in the vaginal epithelium and basement membrane, leading to weakening of the epithelial barrier; thereby facilitating transport of HIV across the vaginal epithelium.

Serine-Rich Repeat Proteins and Pili Promote Streptococcus agalactiae Colonization of the Vaginal Tract

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.

Influence of Lactic Acid on Endogenous and Viral RNA-Induced Immune Mediator Production by Vaginal Epithelial Cells

To evaluate the influence of lactic acid on immune mediator release from vaginal epithelial cells. The human vaginal epithelial cell line, VK2/E6E7, was cultured in the presence or absence of physiological concentrations of lactic acid, and in the presence or absence of the viral Toll-like receptor 3 agonist, poly (inosinic acid:cytidylic acid). Supernatants were assayed by enzyme-linked immunosorbent assay (ELISA) for interleukin (IL)-1β, IL-6, IL-8, IL-23, transforming growth factor (TGF)-β and secretory leukocyte protease inhibitor. Vaginal epithelial cells spontaneously released IL-1β (25.9 pg/mL), IL-8 (1.0 ng/mL), TGF-β (175 pg/mL), and secretory leukocyte protease inhibitor (33.8 ng/mL). Only TGF-β production was marginally enhanced (49%) by addition of lactic acid alone. Poly (inosinic acid:cytidylic acid) by itself stimulated the release of IL-6 (305 pg/mL) and enhanced IL-8 production (2.8 ng/mL). The combination of poly (inosinic acid:cytidylic acid) and lactic acid markedly increased IL-8 production (5.0 ng/mL) and induced the release of IL-1β (96.2 pg/mL). The poly (inosinic acid:cytidylic acid)-mediated lactic acid effect on IL-1β and IL-8 release was abrogated when the lactic acid was neutralized or if acetic acid was substituted for lactic acid. Lactic acid enhances the release of selective mediators from vaginal epithelial cells and stimulates antiviral immune responses.

A Rabbit Vaginal Cell-Derived Antimicrobial Peptide, RVFHbαP, Blocks Lipopolysaccharide-Mediated Inflammation in Human Vaginal CellsIn Vitro

Antimicrobial peptides (AMPs) constitute a phylogenetically ancient form of innate immunity that provides host defense at various mucosal surfaces, including the vagina. Recently, we have identified one such AMP, rabbit vaginal fluid hemoglobin alpha peptide (RVFHbαP), from the vaginal lavage of rabbits (Oryctolagus cuniculus). The recent demonstration of a protective role of this peptide in erythrocytes and vaginal cells led us to investigate (i) the lipopolysaccharide (LPS) interactive domain in RVFHbαP and (ii) whether RVFHbαP of rabbit origin modulates the cellular immune responses of another species (humans) in vitro. HeLa-S3, a human vaginal epithelial cell line (hVEC), was exposed to LPS alone (10 μg/ml for 6 h), or LPS-induced cells were treated with RVFHbαP (70.45 μM for 1 h) and cultured for 24 h, and the results obtained were compared with the medium control. We show here that RVFHbαP exerts an anti-inflammatory activity in hVECs, as suggested by the prevention of LPS-induced production of extracellular (supernatant) and intracellular (lysate) levels of cytokines (interleukin 6 [IL-6] and IL-1α) and chemokines (IL-8 and monocyte chemoattractant protein 1 [MCP-1]). The demonstration of Toll-like receptor 4 (TLR4) and NF-κB expression in hVECs and the observations of RVFHbαP suppression of human β-defensin-1 (hBD1) mRNA expression further support the hypothesis of a genomic activity of RVFHbαP. Confocal microscopy and flow cytometry results demonstrate that RVFHbαP inhibits LPS-induced phagocytosis of Escherichia coli by macrophages. The chemotaxis studies performed using the Boyden chamber Transwell method showed the increased migration of U937 cells when supernatants of LPS-induced hVECs were used, and this effect was inhibited by RVFHbαP. In conclusion, our study proposes a novel explanation for the protective role of RVFHbαP in inflammation-associated infections, which not only may provide the new cellular targets for the screening of RVFHbαP ligands acting in the vaginal tissue but also has the potential to develop RVFHbαP as a therapeutic agent for reproductive tract infections.

Formulation of tenofovir‐loaded functionalized solid lipid nanoparticles intended for HIV prevention

The objective of this study is to engineer polylysine-heparin functionalized solid lipid nanoparticles (fSLNs) for the use of a vaginal microbicide delivery template for HIV prevention. The fSLNs are prepared using a modified phase-inversion technique followed by a layer-by-layer deposition method. The Box-Behnken experimental design is used to analyze the influence of three factors (X(1) = bovine serum albumin concentration, X(2) = pH of the aqueous phase, and X(3) = lipid amount) on the particle mean diameter (PMD) measured by dynamic light scattering (DLS). Tenofovir is used as a model anti-HIV microbicide. The SLNs are also characterized for morphology, zeta potential (ζ ), percent drug encapsulation efficiency (EE%), and cytotoxicity on a human vaginal epithelial cell line by electron microscopy, DLS, ultraviolet, and fluorescence spectroscopy, respectively. The statistical model predicts particle size (Y) with 90% confidence and the Y values are significantly affected by X(1) and X(2) . The produced fSLNs appear noncytotoxic and exhibit a platelet-like shape with respective PMD, EE%, and ζ value of 153 nm, 8.3%, and - 51 mV. These fSLNs intended to be administered topically have the potential to enhance cellular uptake of hydrophobic microbicides and outdistance the virus during the HIV/AIDS infection process, possibly leading to more effective prevention of the disease transmission.

Engineering tenofovir loaded chitosan nanoparticles to maximize microbicide mucoadhesion

The objective of this study was to engineer a model anti-HIV microbicide (tenofovir) loaded chitosan based nanoparticles (NPs). Box–Behnken design allowed to assess the influence of formulation variables on the size of NPs and drug encapsulation efficiency (EE%) that were analyzed by dynamic light scattering and UV spectroscopy, respectively. The effect of the NPs on vaginal epithelial cells and Lactobacillus crispatus viability and their mucoadhesion to porcine vaginal tissue were assessed by cytotoxicity assays and fluorimetry, respectively. In the optimal aqueous conditions, the EE% and NPs size were 5.83% and 207.97 nm, respectively. With 50% (v/v) ethanol/water as alternative solvent, these two responses increased to 20% and 602 nm, respectively. Unlike small size (182 nm) exhibiting burst release, drug release from medium (281 nm) and large (602 nm)-sized NPs fitted the Higuchi ( r 2 = 0.991) and first-order release ( r 2 = 0.999) models, respectively. These NPs were not cytotoxic to both the vaginal epithelial cell line and L. crispatus for 48 h. When the diameter of the NPs decreased from 900 to 188 nm, the mucoadhesion increased from 6% to 12%. However, the combinatorial effect of EE% and percent mucoadhesion for larger size NPs was the highest. Overall, large-size, microbicide loaded chitosan NPs appeared to be promising nanomedicines for the prevention of HIV transmission.

Effect of Lactobacillus rhamnosus GR‐1 and Lactobacillus reuteri RC‐14 on the ability of Candida albicans to infect cells and induce inflammation

Vulvovaginal candidiasis, a high prevailing infection worldwide, is mainly caused by Candida albicans. Probiotic Lactobacillus reuteri RC-14 and Lactobacillus rhamnosus GR-1 have been previously shown to be useful as adjuvants in the treatment of women with VVC. In order to demonstrate and better understand the anti-Candida activity of the probiotic microorganisms in an in vitro model simulating vaginal candidiasis, a human vaginal epithelial cell line (VK2/E6E7) was infected with C.albicans 3153a and then challenged with probiotic L. rhamnosus GR-1 and/or L. reuteri RC-14 or their respective CFS (alone or in combination). At each time point (0, 6, 12 and 24 hr), numbers of yeast, lactobacilli and viable VK2/E6E7 cells were determined and, at 0, 6 and 12 hr, the supernatants were measured for cytokine levels. We found that C. albicans induced a significant increase in IL-1alpha and IL-8 production by VK2/E6E7 cells. After lactobacilli challenge, epithelial cells did not alter IL-6, IL-1alpha, RANTES and VEGF levels. However, CFS from the probiotic microorganisms up-regulated IL-8 and IP-10 levels secreted by VK2/E6E7 cells infected with C. albicans. At 24 hr of co-incubation, L. reuteri RC-14 alone and in combination with L. rhamnosus GR-1 decreased the yeast population recoverable from the cells. In conclusion, L. reuteri RC-14 alone and together with L. rhamnosus GR-1 have the potential to inhibit the yeast growth and their CFS may up-regulate IL-8 and IP-10 secretion by VK2/E6E7 cells, which could possibly have played an important role in helping to clear VVC in vivo.

Microbial compounds induce the expression of pro-inflammatory cytokines, chemokines and human β-defensin-2 in vaginal epithelial cells

Vaginal epithelium has a powerful innate immune system that protects the female reproductive organs from bacterial and fungal infections. In the present study, we aimed to explore whether the Toll-like receptor (TLR) signaling pathway and the induction of pro-inflammatory cytokines and antimicrobial peptides could contribute to the protection against pathogenic microorganisms in vaginal epithelia, using an immortalized vaginal epithelial cell line PK E6/E7 as a model. We found that TLR2 and TLR4 receptors are expressed in vivo in the vaginal epithelia and in vitro in PK E6/E7 vaginal epithelial cell line. The Gram-negative cell wall compound lipopolysaccharide (LPS), the Gram-positive compound peptidoglycan (PGN), heat-killed Candida albicans and zymosan significantly ( P < 0.05) induced the expression of pro-inflammatory cytokines and chemokines such as TNF-α and IL-8/ CXCL8 in vaginal epithelial cells. Furthermore, the expression and production of human β-defensin-2 (hBD2), an antimicrobial peptide with chemotactic functions, was also up-regulated in PK E6/E7 cells after treatment with LPS, PGN or C. albicans. Treatment of vaginal epithelial cells with microbial compounds induced the activation and nuclear translocation of NF-κB transcription factor, a key element of innate and adaptive immune responses. In our work, we provide evidence that microbial compounds induce the production of pro-inflammatory cytokines, chemokines and antimicrobial peptides in vaginal epithelial cells. In vivo, vaginal epithelial cell-derived inflammatory mediators and antimicrobial peptides may play important roles in vaginal immune responses and in the elimination of pathogens from the female reproductive tract.

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction

Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1beta through the nuclear factor-kappaB pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

Cytokine and chemokine production by human oral and vaginal epithelial cells in response to Candida albicans.

Oropharyngeal and vaginal candidiases are the most common forms of mucosal fungal infections and are primarily caused by Candida albicans, a dimorphic fungal commensal organism of the gastrointestinal and lower female reproductive tracts. Clinical and experimental observations suggest that local immunity is important in host defense against candidiasis. Accordingly, cytokines and chemokines are present at the oral and vaginal mucosa during C. albicans infections. Since mucosal epithelial cells produce a variety of cytokines and chemokines in response to microorganisms and since C. albicans is closely associated with mucosal epithelial cells as a commensal, we sought to identify cytokines and/or chemokines produced by primary oral and vaginal epithelial cells and cell lines in response to C. albicans. The results showed that proinflammatory cytokines were produced by oral and/or vaginal epithelial cells at various levels constitutively with considerable interleukin-1alpha (IL-1alpha) and tumor necrosis factor alpha, but not IL-6, produced in response to C. albicans. In contrast, Th1-type (IL-12 and gamma interferon) and Th2-type-immunoregulatory (IL-10 and transforming growth factor beta) cytokines and the chemokines monocyte chemoattractant protein 1 and IL-8 were produced in low to undetectable concentrations with little additional production in response to C. albicans. Taken together, these results indicate that cytokines and chemokines are variably produced by oral and vaginal epithelial cells constitutively, as well as in response to C. albicans, and are predominated by proinflammatory cytokines.

Secretory I gA differentially promotes adherence of type 1-piliated Escherichia coli to immortalized vaginal epithelial cell lines

Objectives. To elucidate the factors that influence bacterial adherence to vaginal epithelium. We developed an in vitro model to examine the interaction of type 1-piliated Escherichia coli, strain HB101/p1-17, with immortalized vaginal epithelial cells (VEC) from postmenopausal donors with (patient 1) and without (patient 2) clinical histories of urinary tract infections (UTI). Methods. The VEC were incubated in microtiter plates in the presence of E. coli HB101/p1-17, and factors such as time, mannose concentration, and secretory immunoglobulin A (sIgA) concentration were assessed. After incubation, the numbers of bacteria bound per VEC were counted on a scintillation counter. Results. The E. coli adhered to the VEC and the adherence was inhibited in the presence of 100-mM mannose solution. Clinical donor UTI histories were reflected in the binding characteristics of the VEC cell lines, with cells from patient 1 having a 30% higher baseline binding capacity than cells from patient 2 and an enhanced binding response in the presence of increasing sIgA concentrations. The sIgA concentration did not affect the patient 2 cell–bacterial binding. Conclusions. These results indicate that our in vitro model is suitable for studying the factors influencing bacterial adherence to vaginal mucosa, and that after immortalization, vaginal mucosa maintains clinically relevant characteristics that can be studied. Our data suggest that E. coli adherence to vaginal mucosa from postmenopausal women susceptible to UTI is affected by intrinsic baseline bacterial binding capacity of the mucosa cells as well as by increased sIgA concentration in the women’s vaginal fluid. Increasing vaginal fluid sIgA concentration in this population may have a deleterious rather than beneficial effect.