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The vaginal microenvironment is key in mediating susceptibility to sexually transmitted infections. A polymicrobial environment with reduced Lactobacilllus spp. is characteristic of vaginal dysbiosis, associated with increased production of several short chain fatty acids (SCFAs), vaginal inflammation and an increased risk of HIV-1 acquisition. In contrast, a eubiotic vaginal microbiome (VMB), dominated by Lactobacillus spp. correlates with increased production of lactic acid (LA), an acidic milieu and protection against HIV-1. Vaginal metabolites, specifically LA and SCFAs including butyric, succinic and acetic acids are associated with modulation of HIV-1 risk. We assessed the impact of combined and individual SCFAs and LA on vaginal epithelial cells (VK2) grown in air–liquid interface cultures. Treatment of VK2 cells with eubiotic SCFA + LA mixture showed increased epithelial barrier integrity, reduced FITC dextran leakage and enhanced expression of cell–cell adhesion proteins. Treatment with dysbiotic SCFA + LA mixture diminished epithelial barrier integrity, increased NFκB activation and inflammatory mediators: TNF-α, IL-6, IL-8 and RANTES. LA was found to be the primary contributor of the beneficial effects. Eubiotic SCFA + LA mixture ameliorated HIV-1 mediated barrier disruption and HIV-1 leakage, whereas dysbiotic SCFA + LA treatment exacerbated HIV-1 effects. These findings indicate a key role for LA in future prophylactic strategies.

Inflammation is a known mechanism that facilitates HIV acquisition and the spread of infection. In this study, we evaluated whether curcumin, a potent and safe anti-inflammatory compound, could be used to abrogate inflammatory processes that facilitate HIV-1 acquisition in the female genital tract (FGT) and contribute to HIV amplification. Primary, human genital epithelial cells (GECs) were pretreated with curcumin and exposed to HIV-1 or HIV glycoprotein 120 (gp120), both of which have been shown to disrupt epithelial tight junction proteins, including ZO-1 and occludin. Pre-treatment with curcumin prevented disruption of the mucosal barrier by maintaining ZO-1 and occludin expression and maintained trans-epithelial electric resistance across the genital epithelium. Curcumin pre-treatment also abrogated the gp120-mediated upregulation of the proinflammatory cytokines tumor necrosis factor-α and interleukin (IL)-6, which mediate barrier disruption, as well as the chemokines IL-8, RANTES and interferon gamma-induced protein-10 (IP-10), which are capable of recruiting HIV target cells to the FGT. GECs treated with curcumin and exposed to the sexually transmitted co-infecting microbes HSV-1, HSV-2 and Neisseria gonorrhoeae were unable to elicit innate inflammatory responses that indirectly induced activation of the HIV promoter and curcumin blocked Toll-like receptor (TLR)-mediated induction of HIV replication in chronically infected T-cells. Finally, curcumin treatment resulted in significantly decreased HIV-1 and HSV-2 replication in chronically infected T-cells and primary GECs, respectively. All together, our results suggest that the use of anti-inflammatory compounds such as curcumin may offer a viable alternative for the prevention and/or control of HIV replication in the FGT.

Clinically, a rich vaginal microbiota (VMB) is considered optimal for reproductive outcomes, while a VMB populated by anaerobes is associated with dysbiosis and the clinical condition bacterial vaginosis (BV), which is linked to increased susceptibility to sexually transmitted infections and adverse reproductive outcomes. Mouse models that mimic eubiotic and dysbiotic VMB are currently lacking but could play a critical role in improving protective interventions. In this study, probiotic, eubiotic, and dysbiotic models were developed in C57BL/6 mice, using probiotic strains GR-1 and RC-14, eubiotic , or dysbiotic strains. Endogenous sex hormones were manipulated by either ovariectomizing (OVX) mice or administering 17β-estradiol or progesterone pellets in OVX mice. Hormone-altered mice were inoculated with probiotic species, , or , and colonization was tracked using quantitative plating assays. Glycogen and MUC-1 levels in hormone-treated mice were determined with ELISA and MUC-1 staining. Following a single administration, and persisted in the mouse vaginal tract for up to eight days, persisted for up to three days, and persisted for up to two days, as measured by quantitative plating assays and qPCR. Colonization of was facilitated by the presence of mucin. The lack of endogenous hormones in OVX mice dramatically decreased VMB bacterial load compared to normal mice. None of the exogenous bacteria including could colonize OVX mice for more than 24 hours. Treatment with 17β-estradiol but not progesterone restored the endogenous VMB and colonization with and . Interestingly, 17β-estradiol treated mice had significantly increased levels of glycogen compared to OVX and progesterone-treated mice. Based on the results, we have shown that estrogen played a significant role in the ability for human VMB species to colonize in our mouse models, potentially through a glycogen mediated mechanism. These results suggest there is a dynamic interaction between sex hormones and the VMB, which can affect bacterial diversity and the ability for a VMB to colonize.

While several clinical studies have shown that HIV-1 infection is associated with increased permeability of the intestinal tract, there is very little understanding of the mechanisms underlying HIV-induced impairment of mucosal barriers. Here we demonstrate that exposure to HIV-1 can directly breach the integrity of mucosal epithelial barrier, allowing translocation of virus and bacteria. Purified primary epithelial cells (EC) isolated from female genital tract and T84 intestinal cell line were grown to form polarized, confluent monolayers and exposed to HIV-1. HIV-1 X4 and R5 tropic laboratory strains and clinical isolates were seen to reduce transepithelial resistance (TER), a measure of monolayer integrity, by 30-60% following exposure for 24 hours, without affecting viability of cells. The decrease in TER correlated with disruption of tight junction proteins (claudin 1, 2, 4, occludin and ZO-1) and increased permeability. Treatment of ECs with HIV envelope protein gp120, but not HIV tat, also resulted in impairment of barrier function. Neutralization of gp120 significantly abrogated the effect of HIV. No changes to the barrier function were observed when ECs were exposed to Env defective mutant of HIV. Significant upregulation of inflammatory cytokines, including TNF-alpha, were seen in both intestinal and genital epithelial cells following exposure to HIV-1. Neutralization of TNF-alpha reversed the reduction in TERs. The disruption in barrier functions was associated with viral and bacterial translocation across the epithelial monolayers. Collectively, our data shows that mucosal epithelial cells respond directly to envelope glycoprotein of HIV-1 by upregulating inflammatory cytokines that lead to impairment of barrier functions. The increased permeability could be responsible for small but significant crossing of mucosal epithelium by virus and bacteria present in the lumen of mucosa. This mechanism could be particularly relevant to mucosal transmission of HIV-1 as well as immune activation seen in HIV-1 infected individuals.

Clinically, a dysbiotic vaginal microbiota (VMB) colonized with anaerobic species such as has been linked to increased susceptibility to viral sexually transmitted infections (STIs) such as Herpes Simplex Virus Type 2 (HSV-2). The mechanism is poorly understood due to the lack of small animal models. Mice were inoculated with 10 CFU of the eubiotic bacteria , the dysbiotic bacteria , or PBS as a negative control every 48 h for ten days. On day ten, mice were inoculated with 10 PFU WT HSV-2 333 and survival, pathology, and viral titers were assessed. To elucidate changes in the vaginal microenvironment following bacterial inoculations, vaginal tissue and washes were collected following ten days of inoculations. To assess barrier integrity, tissue was fixed and stained for the barrier protein Desmoglein-1 (DSG-1). To evaluate the immune microenvironment, tissue was processed for flow cytometry to examine tissue-resident T cells and cytokine production by T cells. Vaginal washes were used for multiplex cytokine/chemokine analysis. inoculated mice infected with HSV-2 had significantly decreased survival rates, increased pathology, and higher viral titers than PBS and inoculated mice. The vaginal epithelium of inoculated mice showed decreased DSG-1 staining compared to other groups, indicating compromised barrier function. Decreased total numbers of CD4+ and CD8+ T cells expressing activated mucosal immune markers CD44, CD69, and CD103 were observed in the vaginal tract of inoculated mice. They also showed increased proportions of T cells expressing inflammatory cytokines TNF-α and IFN-γ, while inoculated mice had increased proportions and absolute counts of T cells expressing the regulatory cytokine IL-10. In the multiplex assay, vaginal washes from mice had increased inflammatory cytokines and chemokines compared to and PBS groups. These results suggest inoculation may be increasing HSV-2 infection by disrupting the epithelial barrier, decreasing protective immune responses and increasing tissue inflammation in the vaginal tract.

Approximately 40% of global HIV-1 transmission occurs in the female genital tract (FGT) through heterosexual transmission. Epithelial cells lining the FGT provide the first barrier to HIV-1 entry. Previous studies have suggested that certain hormonal contraceptives or a dysbiosis of the vaginal microbiota can enhance HIV-1 acquisition in the FGT. We examined the effects of lactobacilli and female sex hormones on the barrier functions and innate immune responses of primary endometrial genital epithelial cells (GECs). Two probiotic strains, Lactobacillus reuteri RC-14 and L. rhamnosus GR-1, were tested, as were sex hormones estrogen (E2), progesterone (P4), and the hormonal contraceptive medroxyprogesterone acetate (MPA). Our results demonstrate that probiotic lactobacilli enhance barrier function without affecting cytokines. Treatment of GECs with MPA resulted in reduced barrier function. In contrast, E2 treatment enhanced barrier function and reduced production of proinflammatory cytokines. Comparison of hormones plus lactobacilli as a pre-treatment prior to HIV exposure revealed a dominant effect of lactobacilli in preventing loss of barrier function by GECs. In summary, the combination of E2 and lactobacilli had the best protective effect against HIV-1 seen by enhancement of barrier function and reduction in proinflammatory cytokines. These studies provide insights into how probiotic lactobacilli in the female genital microenvironment can alter HIV-1-mediated barrier disruption and how the combination of E2 and lactobacilli may decrease susceptibility to primary HIV infection.

Herpes simplex virus type 2 (HSV-2) is the primary cause of genital herpes which results in significant morbidity and mortality, especially in women, worldwide. HSV-2 is transmitted primarily through infection of epithelial cells at skin and mucosal surfaces. Our earlier work to examine interactions between HSV-2 and vaginal epithelial cells demonstrated that infection of the human vaginal epithelial cell line (VK2) with HSV-2 resulted in increased expression of TRIM26, a negative regulator of the Type I interferon pathway. Given that upregulation of TRIM26 could negatively affect anti-viral pathways, we decided to further study the role of TRIM26 in HSV-2 infection and replication. To do this, we designed and generated two cell lines derived from VK2s with TRIM26 overexpressed (OE) and knocked out (KO). Both, along with wildtype (WT) VK2, were infected with HSV-2 and viral titres were measured in supernatants 24 h later. Our results showed significantly enhanced virus production by TRIM26 OE cells, but very little replication in TRIM26 KO cells. We next examined interferon-β production and expression of two distinct interferon stimulated genes (ISGs), MX1 and ISG15, in all three cell lines, prior to and following HSV-2 infection. The absence of TRIM26 (KO) significantly upregulated interferon-β production at baseline and even further after HSV-2 infection. TRIM26 KO cells also showed significant increase in the expression of MX1 and ISG15 before and after HSV-2 infection. Immunofluorescent staining indicated that overexpression of TRIM26 substantially decreased the nuclear localization of IRF3, the primary mediator of ISG activation, before and after HSV-2 infection. Taken together, our data indicate that HSV-2 utilizes host factor TRIM26 to evade anti-viral response and thereby increase its replication in vaginal epithelial cells.

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of Th1 and Th17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher Th17 and Th1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c+ DCs in the vagina were the predominant APC population responsible for priming these Th17 responses, and a potent source of IL-6 and IL-1β, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored Th17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient Th1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4+ T cell anti-viral immunity by priming vaginal DCs to induce Th17 responses through an IL-1-dependent pathway.

In people living with HIV, Mycobacterium tuberculosis (Mtb) is the major cause of death. Due to the increased morbidity/mortality in co-infection, further research is urgently required. A limiting factor to research in HIV and HIV/Mtb co-infection is the lack of accessible in vivo models. Next-generation humanized mice expressing HLA transgenes report improved human immune reconstitution and functionality, which may better recapitulate human disease. This study compares well-established huNRG mice and next-generation HLA I/II-transgenic (huDRAG-A2) mice for immune reconstitution, disease course, and pathology in HIV and TB. HuDRAG-A2 mice have improved engraftment of key immune cell types involved in HIV and TB disease. Upon intravaginal HIV-1 infection, both models developed significant HIV target cell depletion in the blood and tissues. Upon intranasal Mtb infection, both models sustained high bacterial load within the lungs and tissue dissemination. Some huDRAG-A2 granulomas appeared more classically organized, characterized by focal central necrosis, multinucleated giant cells, and foamy macrophages surrounded by a halo of CD4+ T cells. HIV/Mtb co-infection in huNRG mice trended towards worsened TB pathology and showed potential for modeling co-infection. Both huNRG and huDRAG-A2 mice are viable options for investigating HIV and TB, but the huDRAG-A2 model may offer advantages.

The lower female reproductive tract (FRT) is comprised of the cervix and vagina, surfaces that are continuously exposed to a variety of commensal and pathogenic organisms. Sexually transmitted viruses, such as herpes simplex virus type 2 (HSV-2), have to traverse the mucosal epithelial lining of the FRT to establish infection. The majority of current culture systems that model the host-pathogen interactions in the mucosal epithelium have limitations in simulating physiological conditions as they employ a liquid-liquid interface (LLI), in which both apical and basolateral surfaces are submerged in growth medium. We designed the current study to simulate in vivo conditions by growing an immortalized vaginal epithelial cell line (Vk2/E6E7) in culture with an air-liquid interface (ALI) and examined the effects of female sex hormones on their growth, differentiation, and susceptibility to HSV-2 under these conditions, in comparison to LLI cultures. ALI conditions induced Vk2/E6E7 cells to grow into multi-layered cultures compared to the monolayers present in LLI conditions. Vk2 cells in ALI showed higher production of cytokeratin in the presence of estradiol (E2), compared to cells grown in progesterone (P4). Cells grown under ALI conditions were exposed to HSV-2-green fluorescent protein (GFP) and the highest infection and replication was observed in the presence of P4. Altogether, this study suggests that ALI cultures more closely simulate the in vivo conditions of the FRT compared to the conventional LLI cultures. Furthermore, under these conditions P4 was found to confer higher susceptibility to HSV-2 infection in vaginal cells. The vaginal ALI culture system offers a better alternative to study host-pathogen interactions.