Tissue-resident macrophages are productively infected ex vivo by primary X4 isolates of human immunodeficiency virus type 1

Abstract

Infection of macrophages has been implicated as a critical event in the transmission and persistence of human immunodeficiency virus type 1 (HIV-1). Here, we explore whether primary X4 HIV-1 isolates can productively infect tissue macrophages that have terminally differentiated in vivo. Using immunohistochemistry, HIV-1 RNA in situ hybridization, and confocal immunofluorescence microscopy, we demonstrate that macrophages residing in human tonsil blocks can be productively infected ex vivo by primary X4 HIV-1 isolates. This challenges the model in which macrophage tropism is a key determinant of the selective transmission of R5 HIV-1 strains. Infection of tissue macrophages by X4 HIV-1 may be highly relevant in vivo and contribute to key events in HIV-1 pathogenesis.

FIG. 1.

Primary X4 isolates UG021 and J130, but not TCLA-X4 strain NL4-3, productively infect tissue macrophages in human tonsil histocultures. Tonsil blocks were either mock infected or infected with 80 50% tissue culture infective doses of NL4-3, 7/86, J130, or UG021. At day 11 postchallenge, tissue blocks (6 to 12 blocks per donor) were harvested. Sections were costained for the p24 antigen (red staining) and the macrophage marker CD68 (brown staining). Cells showing red staining only, representing p24 staining, are labeled p24. Cells showing brown staining only, representing CD68 macrophage marker staining, are labeled M. Double-labeled cells are highlighted by black arrows. Magnifications: left micrograph column, ×20; middle and right micrograph columns, ×63. Hematoxylin-and-eosin staining was used.

FIG. 2.

Tissue-resident macrophages in tonsil histocultures infected with primary X4 isolates are HIV-1 RNA positive. Viral infection was detected by HIV-1 RNA in situ hybridization (35). In parallel, CD68-positive macrophages were identified with MAb HAM56 and an immunohistochemical staining reaction (brown staining). HIV-1 RNA and CD68 double-positive macrophages are highlighted by black arrows. Magnifications: left column, ×20; right column, ×63.

FIG. 3.

Confocal double-immunofluorescence microscopy allows determination of frequency of infected tissue macrophages. Productive HIV-1 infection was detected by staining with anti-p24 MAb Kal-1, and CD68-positive macrophages were detected with MAb PG-M1. Quantitative analysis, by a pathologist (I.B.), of macrophage infection was based on evaluation of individual fluorochrome images, as well as the digital merge. Slides were viewed with a laserscan microscope (Leica spectral confocal microscope TCS-SL). Magnification, ×20.

FIG. 4.

Markers for T cells and macrophages do not colocalize in infected tonsil histocultures. The CD3 antigen was detected with a rabbit polyclonal antiserum, and CD68-positive macrophages were detected with MAb PG-M1. Confocal digital images for Cy2 and Cy5 were acquired independently. Cy5 images are presented in red to allow better assessment of merged images. Images were evaluated by a pathologist (F.A.). Magnification, ×63.