Condomless receptive anal intercourse is associated with markers of mucosal inflammation in a cohort of men who have sex with men in Atlanta, Georgia

Abstract

Introduction: We previously showed that the rectal mucosal immune environment among men who have sex with men (MSM) engaging in condomless receptive anal intercourse (CRAI) is immunologically distinct from that of men who do not engage in anal intercourse (AI). Here, we further examined these differences with quantitative immunohistochemistry to better understand the geographic distribution of immune markers of interest.

Methods: We enrolled a cohort of MSM engaging in CRAI (n = 41) and men who do not engage in AI (n = 21) between October 2013 and April 2015. Participants were healthy, HIV-negative men aged 18-45 from the metro Atlanta area. We performed rectal mucosal sampling via rigid sigmoidoscopy during two study visits separated by a median of nine weeks and timed with sexual activity for MSM engaging in CRAI. We used standardized, automated immunohistochemistry and quantitative image analysis to investigate the rectal mucosal distribution of neutrophils (MPO), IL-17-producing cells (IL-17) and T_regs (FOXP3) in the lamina propria, and cellular proliferation (Ki67) and adherens junction protein (E-cadherin) in the epithelium. We examined associations between biomarker expression and the rectal mucosal microbiota composition by 16s rRNA sequencing.

Results: Relative to the colonic crypt base, IL-17, FOXP3, and MPO expression increased towards the rectal lumen, while Ki67 decreased and E-cadherin was more uniformly distributed. Throughout the rectal mucosa distribution examined, MSM engaging in CRAI had higher mean lamina propria MPO expression (p = 0.04) and epithelial Ki67 (p = 0.04) compared to controls. There were no significant differences in IL-17, FOXP3 or E-cadherin expression. We found no significant associations of the five biomarkers with the global rectal microbiota composition or the individual taxa examined.

Conclusions: Understanding the mucosal distribution of inflammatory mediators can enhance our knowledge of the earliest events in HIV transmission. Neutrophil enrichment and crypt epithelial cell proliferation likely represent sub-clinical inflammation in response to CRAI in the rectal mucosa of MSM, which could increase the risk for HIV acquisition. However, the contributory role of the microbiota in mucosal inflammation among MSM remains unclear. HIV prevention may be enhanced by interventions that reduce inflammation or capitalize on the presence of specific inflammatory mechanisms during HIV exposure.

Keywords: HIV transmission; men who have sex with men; microbiome; mucosal immunology; receptive anal intercourse; rectal mucosa.

Figure 1

Key structural components of the rectal mucosa. Crypts are glands found in the epithelial lining of the small and large intestine. The basal portion of the crypt contains multipotent stem cells that form new epithelium. The lamina propria underlies the epithelium. The lumen refers to the gut lumen, which is the centre of the intestine. Adapted from “Rectum epithelium (enterocytes only),” by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender‐templates.

Figure 2

Representative immunohistochemical staining for rectal crypt epithelium markers E‐cadherin and Ki67 and stromal markers MPO, FOXP3 and IL‐17 within human colorectal mucosa. Tissue is from MSM engaging in CRAI. Positive control tissues are breast cancer tissue for E‐cadherin, tonsillar tissue for Ki67, MPO and FOXP3 and upper GI tissue for IL‐17 as described in the Methods section. Negative control staining was done with non‐immune IgG as described in the Methods section. All histologic sections were counterstained with haematoxylin. Images also depict aspects of image analyses to measure the optical densities of the biomarkers in colonic hemicrypts (epithelial markers) and in the areas adjacent to the hemicrypts (lamina propria markers) outlined in green and separated into 50 equidistant segments from the base of the crypt area to the lumen. A minimum of three regions were scored per biomarker, per participant visit.

Figure 3

Distribution of lamina propria markers FOXP3, MPO and IL‐17, and epithelial markers Ki67 and E‐cadherin in the crypts of normal‐appearing rectal mucosa in MSM. Values for graphs were generated using automated immunohistochemistry and quantitative image analysis. Full‐length hemicrypts (crypt epithelium markers) and regions adjacent to full‐length hemicrypts (lamina propria markers) were analysed separately via tracing the region, followed by automated sectioning and quantification of the optical density of the immunohistochemically labelled biomarker within each section. Each hemicrypt was divided into 50 sub‐sections, denoted along the x‐axis, of average colonocyte width within which biomarker expression was quantified. Optical density values (y‐axis) were averaged across study visits for visual representation. MSM engaging in CRAI are represented by red circles and controls are represented by black circles.

Figure 4

Representative images of dual immunohistochemical detection of the biomarkers in rectal mucosal tissue. Tissue is from MSM engaging in CRAI. The biomarkers were CD4 (green) and FOXP3 (red; panels A–D) and CD4 (green) and IL‐17 (red; panels E–H). Black arrows (D) show the colocalization of CD4 and FOXP3 expression. Panels D and H are enlargements of the outlined boxes in panels C and G, respectively. Scale bars, 100 μm (A–C and E–G) or 50 μm (D and H).

Figure 5

Principal coordination analysis (PCoA) plots for Bray–Curtis distance, a measure of beta diversity of the microbiome, showing no separation of the low (blue circles) and high (red triangles) biomarker expression groups, which were dichotomized at median expression values. p‐Values represent PERMANOVA test of significance. PC1, or principal coordinate 1, refers to the direction that captures the most variation in microbiome beta diversity. PC2, or principal coordinate 2, refers to the direction that captures the second‐most variation in microbiome beta diversity.