Chemokine Levels in the Penile Coronal Sulcus Correlate with HIV-1 Acquisition and Are Reduced by Male Circumcision in Rakai, Uganda

Abstract

Individual susceptibility to HIV is heterogeneous, but the biological mechanisms explaining differences are incompletely understood. We hypothesized that penile inflammation may increase HIV susceptibility in men by recruiting permissive CD4 T cells, and that male circumcision may decrease HIV susceptibility in part by reducing genital inflammation. We used multi-array technology to measure levels of seven cytokines in coronal sulcus (penile) swabs collected longitudinally from initially uncircumcised men enrolled in a randomized trial of circumcision in Rakai, Uganda. Coronal sulcus cytokine levels were compared between men who acquired HIV and controls who remained seronegative. Cytokines were also compared within men before and after circumcision, and correlated with CD4 T cells subsets in foreskin tissue. HIV acquisition was associated with detectable coronal sulcus Interleukin-8 (IL-8 aOR 2.26, 95%CI 1.04-6.40) and Monokine Induced by γ-interferon (MIG aOR 2.72, 95%CI 1.15-8.06) at the visit prior to seroconversion, and the odds of seroconversion increased with detection of multiple cytokines. Coronal sulcus chemokine levels were not correlated with those in the vagina of a man's female sex partner. The detection of IL-8 in swabs was significantly reduced 6 months after circumcision (PRR 0.59, 95%CI 0.44-0.87), and continued to decline for at least two years (PRR 0.29, 95%CI 0.16-0.54). Finally, prepuce IL-8 correlated with increased HIV target cell density in foreskin tissues, including highly susceptible CD4 T cells subsets, as well as with tissue neutrophil density. Together, these data suggest that penile inflammation increases HIV susceptibility and is reduced by circumcision.

Fig 1. Risk of seroconversion increased when multiple coronal sulcus cytokines detected.

IL-8, GM-CSF, MCP-1, MIP3α, IL-1a and RANTES were measured in coronal sulcus swabs taken at the visit immediately prior to seroconversion in men who acquired HIV (n = 60, cases). Cytokines were also measured in time-matched swabs from men who remained persistently seronegative (n = 120, controls). The proportion of men with either no, one, or two or more cytokines detected who were either seroconverters or controls is presented. Adjusted odds ratio (aOR) of being a seroconverter given two or more cytokines was detected (compared to no cytokines detected) is presented.

Fig 2. Impact of male circumcision on coronal sulcus IL-8 detection.

IL-8 was measured in coronal sulcus swabs taken at enrolment (time = 0), and at month 6, 12 and 24 follow-up visits. Enrolment swabs were collected prior to surgery for men randomized to receive immediate circumcision (n = 80); control men remained uncircumcised (n = 80). The proportion of men with detectable IL-8 and 95% CIs (Fisher’s exact) is presented, with prevalence risk ratio (PRR) compared to baseline, and adjusted PRR (aPRR), below. * p-value for PRR comparing prevalence of IL-8 detection between controls and circumcised men is <0.001.

Fig 3. Association of cytokine levels with foreskin T cell density.

IL-8 and MIG were measured in coronal sulcus swabs (n = 89) taken immediately prior to circumcision, and densities of T cell subsets were measured in foreskin tissue by flow cytometry and IHC. A-F: Correlation of IL-8 concentration with the following densities of T cell populations are displayed: (A) CD8+ T cells; (B) CD4+ T cells; (C) CCR5+ CD4 T cells; (D) Th17 cells; (E) Th1 cells; and (F) TNFα+ CD4 T cells. G: Median and range of total foreskin CD4 and CD8 T cell density stratified by detection of MIG and compared using Mann-Whitney U test. All data are log₁₀ transformed.

Fig 4. Association of cytokine levels with foreskin neutrophil and dendritic cell density.

Immunofluorescence was used to measure neutrophil and dendritic cell densities in foreskin tissues from men with either high (n = 5) or low (n = 5) coronal sulcus IL-8 and MIG levels. A-C: Representative images of: (A) CD15+ neutrophils (green); (B) CD11c+ dermal dendritic cells (red); and (C) CD207+ Langerhans cells (green). Nuclei are counterstained with DAPI (blue) in all images. D: Median and range of foreskin neutrophil density in total foreskin tissue, Langerhans density in epidermal tissue, and dermal dendritic cell density in dermal tissue, from men with high (hatched bars) or low (open bars) cytokine levels. Groups compared using Mann-Whitney U test.