Human vaginal microbiota colonization is regulated by female sex hormones in a mouse model.

Introduction: Clinically, a Lactobacillus 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. Methods: In this study, probiotic, eubiotic, and dysbiotic models were developed in C57BL/6 mice, using probiotic strains Lactobacillus rhamnosus GR-1 and Lactobacillus reuteri RC-14, eubiotic Lactobacillus crispatus, or dysbiotic Gardnerella vaginalis strains. Endogenous sex hormones were manipulated by either ovariectomizing (OVX) mice or administering 17ß-estradiol or progesterone pellets in OVX mice. Hormone-altered mice were inoculated with probiotic Lactobacillus species, L. crispatus, or G. vaginalis, 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. Results: Following a single administration, L. rhamnosus and L. reuteri persisted in the mouse vaginal tract for up to eight days, L. crispatus persisted for up to three days, and G. vaginalis persisted for up to two days, as measured by quantitative plating assays and qPCR. Colonization of G. vaginalis 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 Lactobacilli could colonize OVX mice for more than 24 hours. Treatment with 17ß-estradiol but not progesterone restored the endogenous VMB and colonization with Lactobacilli and G. vaginalis. Interestingly, 17ß-estradiol treated mice had significantly increased levels of glycogen compared to OVX and progesterone-treated mice. Discussion: 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.

Lactic acid enhances vaginal epithelial barrier integrity and ameliorates inflammatory effects of dysbiotic short chain fatty acids and HIV-1.

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.

ASK1 inhibitors are potential pan-antiviral drugs, which dampen replication of diverse viruses including SARS-CoV2.

Apoptosis signal-regulating kinase 1 (ASK1)/MAP3K5 is a stress response kinase that is activated by various stimuli. It is known as an upstream activator of p38- Mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) that are reactive oxygen species (ROS)-induced kinases. Accumulating evidence show that ROS accumulate in virus-infected cells. Here, we investigated the relationship between viruses and ASK1/p38MAPK or ASK1/JNK pathways. Our findings suggest that virus infection activates ASK1 related pathways. In parallel, ASK1 inhibition led to a remarkable reduction in the replication of a broad range of viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccinia virus (VV), vesicular stomatitis virus (VSV), Herpes Simplex Virus (HSV), and Human Immunodeficiency virus (HIV) in different human cell lines. Our work demonstrates the potential therapeutic use of Selonsertib, an ASK1 inhibitor, as a pan-antiviral drug in humans. Surprisingly, we observed differential effects of Selonsertib in in vitro and in vivo hamster models, suggesting caution in using rodent models to predict clinical and therapeutic outcomes in humans.

Metabolic signature for a dysbiotic microbiome in the female genital tract: A systematic review and meta-analysis.

BACKGROUND: The vaginal microbiome (VMB) is a critical determinant of reproductive health, where a microbial shift towards a dysbiotic environment has implications for susceptibility to, and clinical presentation of sexually transmitted infections (STIs). Metabolomic profiling of the vaginal microenvironment has led to the identification of metabolic responses to clinical conditions of dysbiosis. However, no studies have examined metabolic markers that are common across conditions and can serve as a signature for vaginal dysbiosis. METHOD OF STUDY: We have conducted a comprehensive systematic review and meta-analysis to identify consistently deregulated metabolites along with their impact on host and microbial metabolism during dysbiosis. We employed two complementary approaches including a vote counting analysis for all eligible studies identified in the systematic review, in addition to a meta-analysis for a subset of studies with sufficient available data. Significantly deregulated metabolites were then selected for pathway enrichment analysis. RESULTS: Our results revealed a total of 502 altered metabolites reported across 10 dysbiotic conditions from 16 studies. Following a rigorous, collective analysis, six metabolites which were consistently downregulated and could be generalized to all dysbiotic conditions were identified. In addition, five downregulated and one upregulated metabolite was identified from a bacterial vaginosis (BV) focused sub-analysis. These metabolites have the potential to serve as a metabolic signature for vaginal dysbiosis. Their role in eight altered metabolic pathways indicates a disruption of amino acid, carbohydrate, and energy metabolism during dysbiosis. CONCLUSION: Based on this analysis, we propose a schematic model outlining the common metabolic perturbations associated with vaginal dysbiosis, which can be potential targets for therapeutics and prophylaxis.

LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection.

Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. IMPORTANCE Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.

Comparing Current and Next-Generation Humanized Mouse Models for Advancing HIV and HIV/Mtb Co-Infection Studies.

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.

TRIM26 Facilitates HSV-2 Infection by Downregulating Antiviral Responses through the IRF3 Pathway.

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-ß 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-ß 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.

Transcriptional response of vaginal epithelial cells to medroxyprogesterone acetate treatment results in decreased barrier integrity.

Medroxyprogesterone acetate (MPA) is a frequently used hormonal contraceptive that has been shown to significantly increase HIV-1 susceptibility by approximately 40 %. However, the underlying mechanism by which this occurs remains unknown. Here, we examined the biological response to MPA by vaginal epithelial cells, the first cells to encounter HIV-1 during sexual transmission, in order to understand the potential mechanism(s) of MPA-mediated increase of HIV-1 infection. Using microarray analysis and in vitro assays, we characterized the response of vaginal epithelial cells, grown in biologically relevant air-liquid interface (ALI) cultures, to physiological levels of female sex hormones, estradiol (E2), progesterone (P4), or MPA. Transcriptional profiling of E2, P4 or MPA-treated vaginal epithelial cells indicated unique transcriptional profiles associated with each hormone. MPA treatment increased transcripts of genes related to cholesterol/sterol synthesis and decreased transcripts related to cell division and cell-cell adhesion, results not seen with E2 or P4 treatments. MPA treatment also resulted in unique gene expression indicative of decreased barrier integrity. Functional assays confirmed that MPA, but not E2 or P4 treatments, resulted in increased epithelial barrier permeability and inhibited cell cycle progression. The effects of MPA on vaginal epithelial cells seen in this study may help explain the increase of HIV-1 infection in women who use MPA as a hormonal contraceptive.

Protective Effect of Probiotic Bacteria and Estrogen in Preventing HIV-1-Mediated Impairment of Epithelial Barrier Integrity in Female Genital 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.

Suppressive and Gut-Reparative Functions of Human Type 1 T Regulatory Cells.

BACKGROUND & AIMS: T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. METHODS: We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function. RESULTS: Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. CONCLUSIONS: Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.

Interferon-ß induced in female genital epithelium by HIV-1 glycoprotein 120 via Toll-like-receptor 2 pathway acts to protect the mucosal barrier.

More than 40% of HIV infections occur via female reproductive tract (FRT) through heterosexual transmission. Epithelial cells that line the female genital mucosa are the first line of defense against HIV-1 and other sexually transmitted pathogens. These sentient cells recognize and respond to external stimuli by induction of a range of carefully balanced innate immune responses. Previously, we have shown that in response to HIV-1 gp120, the genital epithelial cells (GECs) from upper reproductive tract induce an inflammatory response that may facilitate HIV-1 translocation and infection. In this study, we report that the endometrial and endocervical GECs simultaneously induce biologically active interferon-ß (IFNß) antiviral responses following exposure to HIV-1 that act to protect the epithelial tight junction barrier. The innate antiviral response was directly induced by HIV-1 envelope glycoprotein gp120 and addition of gp120 neutralizing antibody inhibited IFNß production. Interferon-ß was induced by gp120 in upper GECs through Toll-like receptor 2 signaling and required presence of heparan sulfate on epithelial cell surface. The induction of IFNß was dependent upon activation of transcription factor IRF3 (interferon regulatory factor 3). The IFNß was biologically active, had a protective effect on epithelial tight junction barrier and was able to inhibit HIV-1 infection in TZM-bl indicator cells and HIV-1 replication in T cells. This is the first report that recognition of HIV-1 by upper GECs leads to induction of innate antiviral pathways. This could explain the overall low infectivity of HIV-1 in the FRT and could be exploited for HIV-1 prophylaxis.

Transcriptional profiling of primary endometrial epithelial cells following acute HIV-1 exposure reveals gene signatures related to innate immunity.

PROBLEM: Genital epithelial cells (GECs) line the mucosal surface of the female genital tract (FGT) and are the first cells that interface with both commensal microbiota and sexually transmitted pathogens. Despite the protective barrier formed by GECs, the FGT is a major site of HIV-1 infection. This highlights the importance of studying the interaction of HIV-1 and GECs. METHOD OF STUDY: Using microarray analysis, we characterized the transcriptional profile of primary endometrial GECs grown in the presence or absence of physiological levels of E2 (10-9  mol/L) or P4 (10-7  mol/L) following acute exposure to HIV-1 for 6 hours. RESULTS: Acute exposure of primary endometrial GECs to HIV-1 resulted in the expression of genes related to inflammation, plasminogen activation, adhesion and diapedesis and interferon response. Interestingly, exposure to HIV-1 in the presence of E2 and P4 resulted in differential transcriptional profiles, suggesting that the response of primary endometrial GECs to HIV-1 exposure is modulated by female sex hormones. CONCLUSION: The gene expression signature of endometrial GECs indicates that the response of these cells may be key to determining host susceptibility to HIV-1 and that sex hormones modulate these interactions. This study allows us to explore possible mechanisms that explain the hormone-mediated fluctuation of HIV-1 susceptibility in women.

A mutation in the TMEM65 gene results in mitochondrial myopathy with severe neurological manifestations.

Recent research has suggested that transmembrane protein 65 (TMEM65) is localized within the inner mitochondrial membrane. Little else is known about its function. In this study we investigated the location and function of TMEM65. Further, we report the functional consequences of a novel homozygous splice variant (c.472+1G>A) in the TMEM65 gene in a patient with mitochondrial encephalomyopathy. Here we investigated the location of TMEM65 by immunofluorescence staining of the protein and by immunoblotting of the isolated mitochondrial fractions in healthy fibroblasts and those from the patient. To study the function of TMEM65 we knocked down mRNA using TMEM65-specific siRNA, and measured mitochondrial function by enzymology, protein abundance and oxygen consumption rate in fibroblasts. Subcellular fractionation confirmed that the TMEM65 protein was present in the inner mitochondrial membrane. Knocking down TMEM65 expression in dermal fibroblasts severely affected mitochondrial content and respiration rate. Further evidence for the essential role of TMEM65 in mitochondrial function came from the demonstration of severe cellular and clinical consequences resulting from the novel TMEM65 gene mutation. In conclusion, these findings suggest that TMEM65, an inner mitochondrial membrane protein, plays a significant role in mitochondrial respiratory chain function. We also provide the first evidence that a mutation in the TMEM65 gene results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype.

Effects of Female Sex Hormones on Susceptibility to HSV-2 in Vaginal Cells Grown in Air-Liquid Interface.

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.

Correction: Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

[This corrects the article DOI: 10.1371/journal.ppat.1005589.].

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway.

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-1ß, important factors for Th17 differentiation. Th17 responses were abrogated in APC-T cell co-cultures containing IL-1ß KO, but not IL-6 KO vaginal DCs, showing that IL-1ß is a critical factor for Th17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1ß in vaginal DCs, and addition of IL-1ß restored Th17 induction by IL-1ß 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.

The anti-inflammatory activity of curcumin protects the genital mucosal epithelial barrier from disruption and blocks replication of HIV-1 and HSV-2.

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.

Polymerase gamma mutator mice rely on increased glycolytic flux for energy production.

Several studies have illustrated that the polymerase gamma mutator (PolG) mice have reduced mitochondrial content secondary to systemic mitochondrial dysfunction, and subsequently a lower capacity to perform aerobic respiration and endurance exercise. We sought to delineate the extent of glycolysis as a means of energy production in the PolG mice in the absence of optimal mitochondrial function. PolG mice display an enhanced reliance on glycolysis as compared to their wild-type counterparts. This is evident by the resting hypoglycemia, higher PFK content, and elevated plasma lactate levels in the PolG mice. In vitro experiments provide further proof that PolG derived dermal fibroblasts have a higher rate of, and capacity for, glycolysis. PolG mice also have enhanced capacity to perform hepatic gluconeogenesis that is likely enhancing the Cori cycle capacity.

Latent TGF-ß1 is compartmentalized between blood and seminal plasma of HIV-positive men and its activation in semen is negatively correlated with viral load and immune activation.

PROBLEM: Semen is the primary medium for sexual transmission of HIV-1 and contains high concentrations of TGF-ß1, but its role in regulating HIV-mediated immune activation is unclear. METHOD OF STUDY: TGF-ß1 and sCD14 were compared in blood plasma (BP) and seminal plasma (SP) from HIV-uninfected and infected, antiretroviral therapy (ART)-naive and ART-treated men and in THP-1 cells following exposure to HIV-1. The relationship between TGF-ß1 and sCD14 was determined by Spearman correlation. RESULTS: Active and latent forms of TGF-ß1 were compartmentalized between BP and SP. Highest active TGF-ß1 levels were present in SP of ART-naïve chronic-infected men and decreased following ART treatment. Latent TGF-ß1 was upregulated in BP following HIV infection, and highest levels were observed in BP of acute-infected men. Similar expression trends were observed between latent TGF-ß1 and sCD14 in BP. A significant negative correlation was observed between active TGF-ß1, sCD14, and semen viral load in ART-naive men. CONCLUSION: TGF-ß1 is compartmentalized between blood and semen, possibly co-expressed with sCD14 by activated monocytes/macrophages in BP as a result of HIV infection. Conversion of latent TGF-ß1 into its active form could contribute to regulation of viral load and immune activation in the male genital tract, but depends on the stage of infection.

Medroxyprogesterone Acetate Regulates HIV-1 Uptake and Transcytosis but Not Replication in Primary Genital Epithelial Cells, Resulting in Enhanced T-Cell Infection.

Although clinical and experimental evidence indicates that female sex hormones and hormonal contraceptives regulate susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, the underlying mechanism remains unknown. Genital epithelial cells (GECs) are the first cells to encounter HIV during sexual transmission and their interaction with HIV may determine the outcome of exposure. This is the first report that HIV uptake by GECs increased significantly in the presence of the hormonal contraceptive medroxyprogesterone acetate (MPA) and progesterone and that uptake occurred primarily via endocytosis. No productive infection was detected, but endocytosed virus was released into apical and basolateral compartments. Significantly higher viral transcytosis was observed in the presence of MPA. In GEC and T-cell cocultures, maximum viral replication in T cells was observed in the presence of MPA, which also broadly upregulated chemokine production by GECs. These results suggest that MPA may play a significant role in regulating susceptibility to HIV.