Distinct Effects of the Cervicovaginal Microbiota and Herpes Simplex Type 2 Infection on Female Genital Tract Immunology.

Background: Genital inflammation is a key determinant of human immunodeficiency virus (HIV) transmission, and may increase HIV-susceptible target cells and alter epithelial integrity. Several genital conditions that increase HIV risk are more prevalent in African, Caribbean, and other black (ACB) women, including bacterial vaginosis and herpes simplex virus type-2 (HSV-2) infection. Therefore, we assessed the impact of the genital microbiota on mucosal immunology in ACB women and microbiome-HSV-2 interactions. Methods: Cervicovaginal secretions and endocervical cells were collected by cytobrush and Instead Softcup, respectively. T cells and dendritic cells were assessed by flow cytometry, cytokines by multiplex enzyme-linked immunosorbent assay (ELISA), and the microbiota by 16S ribosomal ribonucleic acid gene sequencing. Results: The cervicovaginal microbiota of 51 participants were composed of community state types (CSTs) showing diversity (20/51; 39%) or predominated by Lactobacillus iners (22/51; 42%), L. crispatus (7/51; 14%), or L. gasseri (2/51; 4%). High-diversity CSTs and specific bacterial phyla (Gardnerella vaginalis and Prevotella bivia) were strongly associated with cervicovaginal inflammatory cytokines, but not with altered endocervical immune cells. However, cervical CD4+ T-cell number was associated with HSV-2 infection and a distinct cytokine profile. Conclusions: This suggests that the genital microbiota and HSV-2 infection may influence HIV susceptibility through independent biological mechanisms.

The impact of cervical cytobrush sampling on cervico-vaginal immune parameters and microbiota relevant to HIV susceptibility.

The immunology and microbiota of the female genital tract (FGT) are key determinants of HIV susceptibility. Cervical cytobrush sampling is a relatively non-invasive method permitting the longitudinal assessment of endocervical immune cells, but effects on FGT immunology are unknown. Blood, cervico-vaginal secretions and cervical cytobrushes were collected from sexually transmitted infection (STI)-free women at baseline and after either 6 hours or 48 hours. Endocervical immune cell subsets were assessed by flow cytometry, and pro-inflammatory cytokines by multiplex ELISA. The density of Lactobacillus species and key bacterial vaginosis-associated bacterial taxa were determined by qPCR. Paired changes were assessed before and after cytobrush sampling. After 6 hours there were significant increases in CD4 + T cell, antigen presenting cell (APC) and neutrophil numbers; APC elevations persisted at 48 hours, while neutrophil and CD4 + T cell numbers returned to baseline. In addition, pro-inflammatory cytokine levels were increased at 6 hours and returned to baseline by 48 hours. No significant changes were observed in the absolute abundance of Lactobacillus species or BV-associated bacteria at either time point. Overall, cytobrush sampling altered genital immune parameters at 6 hours, but only APC number increases persisted at 48 hours. This should be considered in longitudinal analyses of FGT immunology.

Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota.

Cervical human papillomavirus (HPV) infection may increase HIV risk. Since other genital infections enhance HIV susceptibility by inducing inflammation, we assessed the impact of HPV infection and clearance on genital immunology and the cervico-vaginal microbiome. Genital samples were collected from 65 women for HPV testing, immune studies and microbiota assessment; repeat HPV testing was performed after 6 months. All participants were HIV-uninfected and free of bacterial STIs. Cytobrush-derived T cell and dendritic cell subsets were assessed by multiparameter flow cytometry. Undiluted cervico-vaginal secretions were used to determine cytokine levels by multiplex ELISA, and to assess bacterial community composition and structure by 16S rRNA gene sequence analysis. Neither HPV infection nor clearance were associated with broad differences in cervical T cell subsets or cytokines, although HPV clearance was associated with increased Langerhans cells and HPV infection with elevated IP-10 and MIG. Individuals with HPV more frequently had a high diversity cervico-vaginal microbiome (community state type IV) and were less likely to have an L. gasseri predominant microbiome. In summary, HPV infection and/or subsequent clearance was not associated with inflammation or altered cervical T cell subsets, but associations with increased Langerhans cells and the composition of the vaginal microbiome warrant further exploration.

Identification of preferential CD4+ T-cell targets for HIV infection in the cervix.

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)ß(7), or α(4)ß(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)ß(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)ß(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)ß(7) still demonstrated enhanced entry into α(4)ß(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)ß(7) or α(4)ß(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)ß(7) or α(4)ß(1) with HIV envelope.

A role for mucosal IL-22 production and Th22 cells in HIV-associated mucosal immunopathogenesis.

Interleukin-22 (IL-22) is a cytokine with epithelial reparative and regenerative properties that is produced by Th22 cells and by other immune cell subsets. Therefore, we explored the hypothesis that disruption of the gut barrier during HIV infection involves dysregulation of these cells in the gastrointestinal mucosa. Sigmoid IL-22-producing T cell and Th22 cells were dramatically depleted during chronic HIV infection, epithelial integrity was compromised, and microbial translocation was increased. These alterations were reversed after long-term antiretroviral therapy. While all mucosal IL-22-producing T-cell subsets were also depleted very early during HIV infection, at these early stages IL-22 production by non-T-cell populations (including NKp44+ cells) was increased and gut epithelial integrity was maintained. Circulating Th22 cells expressed a higher level of the HIV co-receptor/binding molecules CCR5 and α4ß7 than CD4+ T-cell subsets in HIV-uninfected participants, but this was not the case after HIV infection. Finally, recombinant IL-22 was protective against HIV and tumor necrosis factor-α-induced gut epithelial damage in a validated in vitro gut epithelial system. We conclude that reduced IL-22 production and Th22 depletion in the gut mucosa are important factors in HIV mucosal immunopathogenesis.

Immune reconstitution in the sigmoid colon after long-term HIV therapy.

Early and profound CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) may drive Human Immunodeficiency Virus (HIV) immunopathogenesis, and GALT immune reconstitution on highly active antiretroviral therapy (HAART) may be suboptimal. Blood and sigmoid colon biopsies were collected from HAART-treated individuals with undetectable blood HIV RNA for > or =4 years and from uninfected controls. HIV proviral levels and T-cell phenotype/function were examined in both compartments. CD4+ T-cell reconstitution in the sigmoid, including CD4+ T cells expressing CCR5, exceeded that in blood and did not differ from uninfected controls. Sigmoid HIV proviral load was not correlated with CD4+ reconsitution, but was correlated with the degree of mucosal CD8+ T-cell immune activation. Colonic Gag-specific T-cell responses were common, but were not associated with proviral load or immune activation. In this select study population, long-term HAART was associated with complete CD4+ T-cell reconstitution in sigmoid colon. However, colonic immune activation may drive ongoing HIV replication.

Complement fixation test for the diagnosis of Lyme disease.

Sera from 43 patients were tested for complement-fixing antibodies to Borrelia burgdorferi; these patients included 8 with confirmed Lyme disease, 21 who were serologically positive but not likely to have Lyme disease, and 14 who were serologically negative. Seven individuals, all confirmed Lyme disease patients, had complement-fixing antibodies. Complement fixation may be a useful confirmatory test for Lyme disease.