Murine modeling of menstruation identifies immune correlates of protection during Chlamydia muridarum challenge.

The menstrual cycle influences the risk of acquiring sexually transmitted infections (STIs), including Chlamydia trachomatis (C. trachomatis), although the underlying immune contributions are poorly defined. A mouse model simulating the immune-mediated process of menstruation could provide valuable insights into tissue-specific determinants of protection against chlamydial infection within the cervicovaginal and uterine mucosae comprising the female reproductive tract (FRT). Here, we used the pseudopregnancy approach in naïve C57Bl/6 mice and performed vaginal challenge with Chlamydia muridarum (C. muridarum) at decidualization, endometrial tissue remodeling, or uterine repair. This strategy identified that the time frame comprising uterine repair correlated with robust infection and greater bacterial burden as compared with mice on hormonal contraception, while challenges during endometrial remodeling were least likely to result in a productive infection. By comparing the infection site at early time points following chlamydial challenge, we found that a greater abundance of innate effector populations and proinflammatory signaling, including IFNγ correlated with protection. FRT immune profiling in uninfected mice over pseudopregnancy or in pig-tailed macaques over the menstrual cycle identified NK cell infiltration into the cervicovaginal tissues and lumen over the course of endometrial remodeling. Notably, NK cell depletion over this time frame reversed protection, with mice now productively infected with C. muridarum following challenge. This study shows that the pseudopregnancy murine menstruation model recapitulates immune changes in the FRT as a result of endometrial remodeling and identifies NK cell localization at the FRT as essential for immune protection against primary C. muridarum infection.

The menstrual cycle regulates migratory CD4 T-cell surveillance in the female reproductive tract via CCR5 signaling.

Despite their importance for immunity against sexually transmitted infections, the composition of female reproductive tract (FRT) memory T-cell populations in response to changes within the local tissue environment under the regulation of the menstrual cycle remains poorly defined. Here, we show that in humans and pig-tailed macaques, the cycle determines distinct clusters of differentiation 4 T-cell surveillance behaviors by subsets corresponding to migratory memory (TMM) and resident memory T cells. TMM displays tissue-itinerant trafficking characteristics, restricted distribution within the FRT microenvironment, and distinct effector responses to infection. Gene pathway analysis by RNA sequencing identified TMM-specific enrichment of genes involved in hormonal regulation and inflammatory responses. FRT T-cell subset fluctuations were discovered that synchronized to cycle-driven CCR5 signaling. Notably, oral administration of a CCR5 antagonist drug blocked TMM trafficking. Taken together, this study provides novel insights into the dynamic nature of FRT memory CD4 T cells and identifies the menstrual cycle as a key regulator of immune surveillance at the site of STI pathogen exposure.

Proinflammatory oscillations over the menstrual cycle drives bystander CD4 T cell recruitment and SHIV susceptibility from vaginal challenge.

BACKGROUND: The menstrual cycle influences HIV infection-risk in women, although the timing and underlying mechanism are unclear. Here we investigated the contribution of the menstrual cycle to HIV susceptibility through evaluating immune behavior with infection-risk over time. METHODS: Blood and vaginal lavage samples were collected from 18 pig-tailed macaques to evaluate immune changes over reproductive cycles, and from 5 additional animals undergoing repeated vaginal exposures to simian HIV (SHIV). Peripheral blood mononuclear cell (PBMC) samples from healthy women (n = 10) were prospectively collected over the course of a menstrual cycle to profile T cell populations. Immune properties from PBMC and vaginal lavage samples were measured by flow cytometry. Plasma progesterone was measured by enzyme immunoassay. The oscillation frequency of progesterone concentration and CCR5 expression on CD4 T cells was calculated using the Lomb-Scargle periodogram. SHIV infection was monitored in plasma by RT-PCR. Immune measures were compared using generalized estimating equations (GEE). FINDINGS: Macaques cycle-phases were associated with fluctuations in systemic immune properties and a type-1 inflammatory T cell response with corresponding CCR5+ memory CD4 T cell (HIV target cell) infiltration into the vaginal lumen at the late luteal phase. Power spectral analysis identified CCR5 oscillation frequencies synchronized with reproductive cycles. In a repetitive low-dose vaginal challenge model, productive SHIV163P3 infection only occurred during intervals of mounting type-1 T cell responses (n = 5/5). Finally, we identify similar type-1 inflammatory T cell responses over the menstrual cycle are occurring in healthy women. INTERPRETATION: These data demonstrate that periodic shifts in the immune landscape under menstrual cycle regulation drives bystander CCR5+ CD4 T cell recruitment and HIV susceptibility in the female reproductive tract. FUNDING: This study was supported by the U.S. Centers for Disease Control and Prevention, Atlanta, GA 30329 and NIH grants to Emory University (K23AI114407 to A.N.S., the Emory University Center for AIDS research [P30AI050409], and Atlanta Clinical and Translational Sciences Institute [KLR2TR000455, UL1TR000454]). DISCLAIMER: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the U.S. Centers for Disease Control and Prevention or the Department of Health and Human Services.

Distinct cellular immune properties in cerebrospinal fluid are associated with cognition in HIV-infected individuals initiating antiretroviral therapy.

We examined the relationship between CSF immune cells and neurocognition and neuronal damage in HIV+ individuals before and after initiating antiretroviral therapy. Multivariate analysis at baseline indicated that greater CD4+ T cell abundance was associated with better cognition (p = .017), while higher CSF HIV RNA was associated with increased neuronal damage (p = .014). Following 24 weeks of antiretroviral therapy, CD8+ T cells, HLA-DR expressing CD4+ and CD8+ T cells, B cells, NK cells, and non-classical monocyte percentage decreased in CSF. Female gender was negatively associated with cognitive performance over time, as was higher percentage of HLA-DR expressing CD8+ T cells at baseline.

Impact of etonogestrel implant use on T-cell and cytokine profiles in the female genital tract and blood.

BACKGROUND: While prior epidemiologic studies have suggested that injectable progestin-based contraceptive depot medroxyprogesterone acetate (DMPA) use may increase a woman's risk of acquiring HIV, recent data have suggested that DMPA users may be at a similar risk for HIV acquisition as users of the copper intrauterine device and levonorgestrel implant. Use of the etonogestrel Implant (Eng-Implant) is increasing but there are currently no studies evaluating its effect on HIV acquisition risk. OBJECTIVE: Evaluate the potential effect of the Eng-Implant use on HIV acquisition risk by analyzing HIV target cells and cytokine profiles in the lower genital tract and blood of adult premenopausal HIV-negative women using the Eng-Implant. METHODS: We prospectively obtained paired cervicovaginal lavage (CVL) and blood samples at 4 study visits over 16 weeks from women between ages 18-45, with normal menses (22-35 day intervals), HIV uninfected with no recent hormonal contraceptive or copper intrauterine device (IUD) use, no clinical signs of a sexually transmitted infection at enrollment and who were medically eligible to initiate Eng-Implant. Participants attended pre-Eng-Implant study visits (week -2, week 0) with the Eng-Implant inserted at the end of the week 0 study visit and returned for study visits at weeks 12 and 14. Genital tract leukocytes (enriched from CVL) and peripheral blood mononuclear cells (PBMC) from the study visits were evaluated for markers of activation (CD38, HLA-DR), retention (CD103) and trafficking (CCR7) on HIV target cells (CCR5+CD4+ T cells) using multicolor flow cytometry. Cytokines and chemokines in the CVL supernatant and blood plasma were measured in a Luminex assay. We estimated and compared study endpoints among the samples collected before and after contraception initiation with repeated-measures analyses using linear mixed models. RESULTS: Fifteen of 18 women who received an Eng-Implant completed all 4 study visits. The percentage of CD4+ T cells in CVL was not increased after implant placement but the percentage of CD4+ T cells expressing the HIV co-receptor CCR5 did increase after implant placement (p = 0.02). In addition, the percentage of central memory CD4+ T-cells (CCR7+) in CVL increased after implant placement (p = 0.004). The percentage of CVL CD4+, CCR5+ HIV target cells expressing activation markers after implant placement was either reduced (HLA-DR+, p = 0.01) or unchanged (CD38+, p = 0.45). Most CVL cytokine and chemokine concentrations were not significantly different after implant placement except for a higher level of the soluble lymphocyte activation marker (sCD40L; p = 0.04) and lower levels of IL12p70 (p = 0.02) and G-CSF (p<0.001). In systemic blood, none of the changes noted in CVL after implant placement occurred except for decreases in the percentage CD4 T-cells expressing HLA-DR+ T cells (p = 0.006) and G-CSF (p = 0.02). CONCLUSIONS: Eng-Implant use was associated with a moderate increase in the availability of HIV target cells in the genital tract, however the percentage of these cells that were activated did not increase and there were minimal shifts in the overall immune environment. Given the mixed nature of these findings, it is unclear if these implant-induced changes alter HIV risk.

Interleukin-36γ Is Elevated in Cervicovaginal Epithelial Cells in Women With Bacterial Vaginosis and In Vitro After Infection With Microbes Associated With Bacterial Vaginosis.

In recent studies, the interleukin (IL)-36 cytokines were shown to be elevated in women with non-Lactobacillus-dominated vaginal microbiomes. In this study, we evaluated IL36G expression in clinical samples from women with and without bacterial vaginosis (BV) and a human 3-dimensional cervical epithelial cell model. IL36G expression was significantly elevated in cervicovaginal epithelial cells isolated from BV-positive women and corresponded with increased neutrophil counts relative to BV-negative women. In addition, specific BV-associated bacterial species as well as a polymicrobial cocktail significantly induced IL36G expression in vitro. These findings suggest that IL-36γ may exhibit an important function in the host response to BV and other sexually transmitted infections.

IL-36γ Is a Key Regulator of Neutrophil Infiltration in the Vaginal Microenvironment and Limits Neuroinvasion in Genital HSV-2 Infection.

HSV-2 is a neurotropic virus that causes a persistent, lifelong infection that increases risk for other sexually transmitted infections. The vaginal epithelium is the first line of defense against HSV-2 and coordinates the immune response through the secretion of immune mediators, including the proinflammatory cytokine IL-36γ. Previously, we showed that IL-36γ treatment promoted transient polymorphonuclear cell infiltration to the vaginal cavity and protected against lethal HSV-2 challenge. In this report, we reveal that IL-36γ specifically induces transient neutrophil infiltration but does not impact monocyte and macrophage recruitment. Using IL-36γ-/- mice in a lethal HSV-2 challenge model, we show that neutrophil counts are significantly reduced at 1 and 2 d postinfection and that KC-mediated mature neutrophil recruitment is impaired in IL-36γ-/- mice. Additionally, IL-36γ-/- mice develop genital disease more rapidly, have significantly reduced survival time, and exhibit an increased incidence of hind limb paralysis that is linked to productive HSV-2 infection in the brain stem. IL-36γ-/- mice also exhibit a significant delay in clearance of the virus from the vaginal epithelium and a more rapid spread of HSV-2 to the spinal cord, bladder, and colon. We further show that the decreased survival time and increased virus spread observed in IL-36γ-/- mice are not neutrophil-dependent, suggesting that IL-36γ may function to limit HSV-2 spread in the nervous system. Ultimately, we demonstrate that IL-36γ is a key regulator of neutrophil recruitment in the vaginal microenvironment and may function to limit HSV-2 neuroinvasion.

Chronic immune barrier dysregulation among women with a history of violence victimization.

We explored the association between violence victimization and increased risk for acquiring sexually transmitted infections (STIs) in women by measuring cellular immune barrier properties from the female reproductive tract. STI-negative participants reporting repeated prior victimization occurrences through the lifetime trauma and victimization history (LTVH) instrument were more likely to exhibit alterations in barrier homeostasis and the composition of critical immune mediators irrespective of demographic parameters or presence of bacterial vaginosis. By combining cellular data with mixed-effect linear modeling, we uncovered differences in local T cells, MHCII+ antigen-presenting cells, and epithelial cells indicative of altered trafficking behavior, increased immunosuppressive function, and decreased barrier integrity at sites of STI exposure that correlate most strongly with LTVH score. These data evidence a biological link between a history of violence victimization and risk of STI acquisition through immune dysregulation in the female reproductive tract.

Progesterone Levels Associate with a Novel Population of CCR5+CD38+ CD4 T Cells Resident in the Genital Mucosa with Lymphoid Trafficking Potential.

The female genital tract (FGT) provides a means of entry to pathogens, including HIV, yet immune cell populations at this barrier between host and environment are not well defined. We initiated a study of healthy women to characterize resident T cell populations in the lower FGT from lavage and patient-matched peripheral blood to investigate potential mechanisms of HIV sexual transmission. Surprisingly, we observed FGT CD4 T cell populations were primarily CCR7(hi), consistent with a central memory or recirculating memory T cell phenotype. In addition, roughly half of these CCR7(hi) CD4 T cells expressed CD69, consistent with resident memory T cells, whereas the remaining CCR7(hi) CD4 T cells lacked CD69 expression, consistent with recirculating memory CD4 T cells that traffic between peripheral tissues and lymphoid sites. HIV susceptibility markers CCR5 and CD38 were increased on FGT CCR7(hi) CD4 T cells compared with blood, yet migration to the lymphoid homing chemokines CCL19 and CCL21 was maintained. Infection with GFP-HIV showed that FGT CCR7(hi) memory CD4 T cells are susceptible HIV targets, and productive infection of CCR7(hi) memory T cells did not alter chemotaxis to CCL19 and CCL21. Variations of resident CCR7(hi) FGT CD4 T cell populations were detected during the luteal phase of the menstrual cycle, and longitudinal analysis showed the frequency of this population positively correlated to progesterone levels. These data provide evidence women may acquire HIV through local infection of migratory CCR7(hi) CD4 T cells, and progesterone levels predict opportunities for HIV to access these novel target cells.