Macrophages in vaginal but not intestinal mucosa are monocyte-like and permissive to human immunodeficiency virus type 1 infection

Abstract

Mucosal surfaces play a major role in human immunodeficiency virus type 1 (HIV-1) transmission and pathogenesis, and yet the role of lamina propria macrophages in mucosal HIV-1 infection has received little investigative attention. We report here that vaginal and intestinal macrophages display distinct phenotype and HIV-1 permissiveness profiles. Vaginal macrophages expressed the innate response receptors CD14, CD89, CD16, CD32, and CD64 and the HIV-1 receptor/coreceptors CD4, CCR5, and CXCR4, similar to monocytes. Consistent with this phenotype, green fluorescent protein-tagged R5 HIV-1 entered macrophages in explanted vaginal mucosa as early as 30 min after inoculation of virus onto the epithelium, and purified vaginal macrophages supported substantial levels of HIV-1 replication by a panel of highly macrophage-tropic R5 viruses. In sharp contrast, intestinal macrophages expressed no detectable, or very low levels of, innate response receptors and HIV-1 receptor/coreceptors and did not support HIV-1 replication, although virus occasionally entered macrophages in intestinal tissue explants. Thus, vaginal, but not intestinal, macrophages are monocyte-like and permissive to R5 HIV-1 after the virus has translocated across the epithelium. These findings suggest that genital and gut macrophages have different roles in mucosal HIV-1 pathogenesis and that vaginal macrophages play a previously underappreciated but potentially important role in mucosal HIV-1 infection in the female genital tract.

FIG. 1.

Vaginal, but not intestinal, macrophages retain a monocyte-like phenotype. Gradient sedimentation purified blood and mucosal MNLs were stained with fluorescence-conjugated antibodies to the myeloid markers HLA-DR and CD13 and the innate receptors CD14, CD89, CD16, CD32, CD64 and analyzed by flow cytometry by gating on the macrophage population. Profiles are representative of cells from four separate vaginal and intestinal specimens.

FIG. 2.

Vaginal macrophages express higher levels of CD4, CCR5, and CXCR4 protein and mRNA than intestinal macrophages. (A) Gradient sedimentation purified mucosal MNLs were stained with the indicated fluorescence-conjugated antibodies, and the macrophages were analyzed by flow cytometry by gating on the macrophage population. Insets show staining with the isotype control antibodies. Profiles are representative of macrophages in four vaginal and four intestinal tissue specimens. (B) Purified vaginal and intestinal macrophages were analyzed by real-time PCR for CD4, CCR5, and CXCR4 mRNA. The mean (± the standard deviation) levels of CD4, CCR5, and CXCR4 mRNA were 4.9-, 2.3-, and 2.6-fold higher, respectively, in vaginal macrophages than in intestinal macrophages (n = 3).

FIG. 3.

Localization of potential mononuclear target cells and detection of HIV-1 in macrophages in vaginal and intestinal mucosa. (A) In noninflamed vaginal mucosa, HAM56⁺ macrophages were detected in the basal region of the epithelium and bordering the dermal papillae and scattered throughout the lamina propria; few CD3⁺ lymphocytes were identified in the epithelium, concentrating mainly at the dermal papillae, but were present predominantly in the lamina propria. In noninflamed intestinal mucosa, dense populations of HAM56⁺ macrophages and CD3⁺ lymphocytes were present throughout the lamina propria. Insets show sections stained with irrelevant, isotype-matched control antibodies. (Sections representative of vaginal and intestinal tissues were from four separate donors [magnification, ×20].) (B) GFP-tagged YU2 particles were inoculated onto the apical surface of vaginal and intestinal mucosal explants and, after 30 min, the explants were harvested, sectioned, stained, and analyzed by confocal microscopy for HAM56⁺ macrophages containing GFP-YU2. GFP-tagged viruses were stained with anti-GFP Alexa Fluor 488, macrophages were stained with anti-HAM56-Cy3, and cell nuclei were stained with DAPI.

FIG. 4.

Vaginal, not intestinal, macrophages in isolated mucosal MNLs support R5 HIV-1 replication. Cultures of gradient sedimentation-purified vaginal and intestinal MNLs were inoculated with YU-2env pseudotyped GFP-expressing HIV-1 and incubated at 37°C. The ability of macrophages to support virus replication was analyzed 2 days postinfection by GFP expression using flow cytometry. The results are representative of two separate experiments.

FIG. 5.

Purified vaginal, but not intestinal, macrophages support HIV-1 replication. Cultures of purified vaginal and intestinal macrophages were inoculated with YU2 and three highly macrophage-tropic viruses (NA420 B33, NA20 B59, and NA353 B27) at an MOI of 1. The levels of p24 production in media were assayed by ELISA at the indicated time points postinoculation. The inset shows cultures of purified vaginal and intestinal lymphocytes that had been isolated from the same tissue specimens as the macrophages were inoculated in parallel with the same viruses and analyzed for p24 production. Values are mean ± the standard deviation p24 levels for two to four wells/time point/virus in a representative experiment (n = 3).