Infants' First Solid Foods: Impact on Gut Microbiota Development in Two Intercontinental Cohorts.

The introduction of solid foods is an important dietary event during infancy that causes profound shifts in the gut microbial composition towards a more adult-like state. Infant gut bacterial dynamics, especially in relation to nutritional intake remain understudied. Over 2 weeks surrounding the time of solid food introduction, the day-to-day dynamics in the gut microbiomes of 24 healthy, full-term infants from the Baby, Food & Mi and LucKi-Gut cohort studies were investigated in relation to their dietary intake. Microbial richness (observed species) and diversity (Shannon index) increased over time and were positively associated with dietary diversity. Microbial community structure (Bray-Curtis dissimilarity) was determined predominantly by individual and age (days). The extent of change in community structure in the introductory period was negatively associated with daily dietary diversity. High daily dietary diversity stabilized the gut microbiome. Bifidobacterial taxa were positively associated, while taxa of the genus Veillonella, that may be the same species, were negatively associated with dietary diversity in both cohorts. This study furthers our understanding of the impact of solid food introduction on gut microbiome development in early life. Dietary diversity seems to have the greatest impact on the gut microbiome as solids are introduced.

Investigating colonization patterns of the infant gut microbiome during the introduction of solid food and weaning from breastmilk: A cohort study protocol.

The first exposures to microbes occur during infancy and it is suggested that this initial colonization influences the adult microbiota composition. Despite the important role that the gut microbiome may have in health outcomes later in life, the factors that influence its development during infancy and early childhood have not been characterized fully. Guidelines about the introduction of solid foods and cessation of breastfeeding, which is thought to have a significant role in the transition to a more adult-like microbiota, are not based on microbiome research. There is even less understanding of approaches used to transition to solid food in the preterm population. The purpose of this study is to identify the impact of early life dietary events on gut microbiome community structures and function among infants born at term and pre-term. We plan to prospectively monitor the gut microbiome of infants during two critical timepoints in microbial development: the introduction of solid foods and cessation from breastmilk. A total of 35 participants from three primary observational birth cohorts (two full-term cohorts and one pre-term cohort) will be enrolled in this sub-study. Participants will be asked to collect stool samples and fill out a study diary before, during and after the introduction of solids and again during weaning from breastmilk. We will use frequent fecal sampling analyzed using 16S rRNA gene profiling, metagenomics, metabolomics, and targeted bacterial culturing to identify and characterize the microbial communities, as well as provide insight into the phenotypic characteristics and functional capabilities of the microbes present during these transitional periods of infancy. This study will provide a comprehensive approach to detailing the effects of dietary transition from breastmilk to a more adult-like solid food diet on the microbiome and in doing so will contribute to evidence-based infant nutrition guidance.

Persistence of Suspected Probiotic Organisms in Preterm Infant Gut Microbiota Weeks After Probiotic Supplementation in the NICU.

Probiotics are becoming a prevalent supplement to prevent necrotizing enterocolitis in infants born preterm. However, little is known about the ability of these live bacterial supplements to colonize the gut or how they affect endogenous bacterial strains and the overall gut community. We capitalized on a natural experiment resulting from a policy change that introduced the use of probiotics to preterm infants in a single Neonatal Intensive Care Unit. We used amplicon sequence variants (ASVs) derived from the v3 region of the 16S rRNA gene to compare the prevalence and abundance of Bifidobacterium and Lactobacillus in the gut of preterm infants who were and were not exposed to a probiotic supplement in-hospital. Infants were followed to 5 months corrected age. In the probiotic-exposed infants, ASVs belonging to species of Bifidobacterium appeared at high relative abundance during probiotic supplementation and persisted for up to 5 months. In regression models that controlled for the confounding effects of age and antibiotic exposure, probiotic-exposed infants had a higher abundance of the suspected probiotic bifidobacteria than unexposed infants. Conversely, the relative abundance of Lactobacillus was similar between preterm groups over time. Lactobacillus abundance was inversely related to antibiotic exposure. Furthermore, the overall gut microbial community of the probiotic-exposed preterm infants at term corrected age clustered more closely to samples collected from 10-day old full-term infants than to samples from unexposed preterm infants at term age. In conclusion, routine in-hospital administration of probiotics to preterm infants resulted in the potential for colonization of the gut with probiotic organisms post-discharge and effects on the gut microbiome as a whole. Further research is needed to fully discriminate probiotic bacterial strains from endogenous strains and to explore their functional role in the gut microbiome and in infant health.

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.

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.

IL-17 Production by γδ+ T Cells Is Critical for Inducing Th17 Responses in the Female Genital Tract and Regulated by Estradiol and Microbiota.

IL-17 can be produced by adaptive immune cells such as Th17 cells and by immune cells that produce IL-17 without prior priming. This latter category, which we will refer to as "innate," includes innate cells such as NK cells and innate lymphoid cells and innate-like T cell populations such as NKT cells and γδ+ T cells. Studies in mucosal tissues have shown that the induction of Th17 immunity is amplified by innate IL-17 produced within those tissues. However, the role of innate IL-17 and its effect on Th17 induction in the female genital tract (FGT) is largely unknown. In this study, we characterize the primary source of IL-17-secreting vaginal cells and show that innate IL-17 plays a critical role in priming adaptive Th17 responses in the FGT. Under homeostatic conditions, γδ+ T cells were the predominant source of innate IL-17 in the murine FGT, and this population was modulated by both the sex hormone estradiol and the presence of commensal microbiota. Compared with wild-type C57BL/6 mice, vaginal APCs isolated from IL-17A-deficient (IL-17A-/- ) mice were severely impaired at priming Th17 responses in APC-T cell cocultures. Furthermore, the defect in Th17 induction in the absence of innate IL-17 was associated with impairment of IL-1ß production by vaginal CD11c+ dendritic cells. Overall, our study describes a novel role for IL-17 in the FGT and further demonstrates the importance of factors in the vaginal microenvironment that can influence adaptive immune responses.

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.

Impact of Standard Bacterial Vaginosis Treatment on the Genital Microbiota, Immune Milieu, and Ex Vivo Human Immunodeficiency Virus Susceptibility.

BACKGROUND: Genital immunology is a key determinant of human immunodeficiency virus (HIV) susceptibility. Both factors are modulated by bacterial vaginosis (BV) and, to some extent, by Lactobacillus iners, the genital Lactobacillus spp. that predominates in African, Caribbean, and other Black (ACB) women. We conducted a clinical trial to assess the impact of oral metronidazole treatment on the genital immune parameters of HIV acquisition risks in Kenyan women with BV. METHODS: The primary endpoint was ex vivo cervical CD4+ T-cell HIV susceptibility after 1 month; secondary endpoints included genital cytokine/chemokine levels, cervical immune cell populations, and the composition of the cervico-vaginal microbiota by 16S ribosomal RNA gene amplicon sequencing. RESULTS: BV resolved (Nugent score ≤ 3) at 1 month in 20/45 participants, and cervical CD4+ T-cell HIV entry was moderately reduced in all participants, regardless of treatment outcome. Resolution of BV and reduced abundances of BV-associated gram-negative taxa correlated with reduced genital interleukin (IL)-1α/ß. However, BV resolution and the concomitant colonization by Lactobacillus iners substantially increased several genital chemokines associated with HIV acquisition, including interferon-γ inducible protein (IP)-10, macrophage inflammatory protein (MIP)-3α, and monokine induced by gamma interferon (MIG). In an independent cohort of ACB women, most of whom were BV-free, vaginal chemokines were again closely linked with L. iners abundance, though not other Lactobacillus spp. CONCLUSIONS: BV treatment reduced genital CD4+ T-cell HIV susceptibility and IL-1 levels, but dramatically increased the genital chemokines that may enhance HIV susceptibility; the latter effect was related to the restoration of an Lactobacillus iners-dominated microbiota. Further studies are needed before treatment of asymptomatic BV can be recommended for HIV prevention in ACB communities.

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.

Medroxyprogesterone acetate-treated human, primary endometrial epithelial cells reveal unique gene expression signature linked to innate immunity and HIV-1 susceptibility.

PROBLEM: Medroxyprogesterone acetate (MPA), a progestin-based hormonal contraceptive designed to mimic progesterone, has been linked to increased human immunodeficiency virus (HIV-1) susceptibility. Genital epithelial cells (GECs) form the mucosal lining of the female genital tract (FGT) and provide the first line of protection against HIV-1. The impact of endogenous sex hormones or MPA on the gene expression profile of GECs has not been comprehensively documented. METHOD OF STUDY: Using microarray analysis, we characterized the transcriptional profile of primary endometrial epithelial cells grown in physiological levels of E2, P4, and MPA. RESULTS: Each hormone treatment altered the gene expression profile of GECs in a unique manner. Interestingly, although MPA is a progestogen, the gene expression profile induced by it was distinct from P4. MPA increased gene expression of genes related to inflammation and cholesterol synthesis linked to innate immunity and HIV-1 susceptibility. CONCLUSION: The analysis of gene expression profiles provides insights into the effects of sex hormones and MPA on GECs and allows us to posit possible mechanisms of the MPA-mediated increase in HIV-1 acquisition.

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.

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.

HIV-1 gp120 induces TLR2- and TLR4-mediated innate immune activation in human female genital epithelium.

Although women constitute half of all HIV-1-infected people worldwide (UNAIDS World AIDS Day Report, 2011), the earliest events in the female reproductive tract (FRT) during heterosexual HIV-1 transmission are poorly understood. Recently, we demonstrated that HIV-1 could directly impair the mucosal epithelial barrier in the FRT. This suggested that the HIV-1 envelope glycoprotein gp120 was being recognized by a membrane receptor on genital epithelial cells, leading to innate immune activation. In this study, we report that pattern-recognition receptors TLR2 and -4 bind to HIV-1 gp120 and trigger proinflammatory cytokine production via activation of NF-κB. The gp120-TLR interaction also required the presence of heparan sulfate (HS). Bead-binding assays showed that gp120 can bind to HS, TLR2, and TLR4, and studies in transfected HEK293 cells demonstrated that HS and TLR2 and -4 were necessary to mediate downstream signaling. Exposure to seminal plasma from HIV-1-infected and uninfected men with gp120 added to it induced a significant proinflammatory cytokine response from genital epithelial cells and disruption of tight junctions, indicating a role for gp120 in mucosal barrier disruption during HIV-1 heterosexual transmission. These studies provide, for the first time to our knowledge, a possible mechanism by which HIV-1 gp120 could directly initiate innate immune activation in the FRT during heterosexual transmission.