Cell tropism of simian immunodeficiency virus in culture is not predictive of in vivo tropism or pathogenesis

Abstract

SIVmac239/316 is a molecular clone derived from SIVmac239 that differs from the parental virus by nine amino acids in env. This virus, unlike the parental SIVmac239, is able to replicate well in alveolar macrophages in culture. We have not however, observed macrophage-associated inflammatory disease in any animal infected with SIVmac239/316. Therefore, we sought to examine the cell tropism of this virus in vivo in multiple tissues using in situ hybridization combined with immunohistochemistry and multilabel confocal microscopy for viral nucleic acid and multiple cell-type-specific markers for macrophages and T lymphocytes. Tissues examined included brain, heart, lung, lymph nodes, spleen, thymus, and small and large intestine. Matched tissues from macaques infected with the parental SIVmac239 and uninfected macaques were also examined. Many infected cells were detected in the tissues of animals infected with SIVmac239 and SIVmac239/316 although there appeared to be fewer positive cells in animals infected with SIVmac239/316. Surprisingly, in light of the cell culture observations, nearly every simian immunodeficiency virus-infected cell in animals inoculated with SIVmac239/316 was a T lymphocyte rather than a macrophage. This was true both during early infection (first 2 months) and in terminal disease. In contrast, as previously described, SIVmac239 was found in both T cells and macrophages in tissues as early as 21 days after infection. These studies indicate that during both acute and chronic SIVmac239/316 infection T lymphocytes rather than macrophages are the principal targets in vivo. These data combined with the absence of macrophage-associated lesions in SIVmac239/316-infected animals indicate that in vitro cell tropism is not predictive of in vivo tropism or disease pathogenesis.

Figure 1

Principal lesions in SIVmac239/316-infected macaques. A: Follicular hyperplasia and dysplasia in lymph node of an animal during acute infection. C and E: Enlarged hypocellular germinal centers containing eosinophilic material without discernible mantle zones in chronically infected animals in lymph node (C) and spleen (E). Several chronically infected animals also had extensive myocardial fibrosis (B), arterial thrombi (D), and arteriopathy (F) involving small- to medium-sized vessels in heart, lung, brain, and kidney. Examples from brain (D) and heart (F) are shown.

Figure 2

Localization of virus in macaques infected with SIVmac239/316. A: SIV in situ hybridization reveals a dendritic staining pattern at 21 days after infection in lymph node consistent with antigen/antibody trapping on follicular dendritic cells. B: Numerous in situ hybridization-positive cells at 50 days after infection in lymph node. The higher magnification inset illustrates the cell morphology. C: In situ hybridization for SIV (blue) followed by immunohistochemistry for the macrophage marker CD68 (red) in LN at 50 days after infection. No co-localization of the two labels (would appear purple) is evident.

Figure 3

Immunophenotype of SIV-infected cells in SIVmac239/316 infection. A: Double-label confocal microscopy (three channel) in LN at 50 days after infection. Images for individual channels (CD3 with Alexa 488, green; SIV in situ hybridization with Fast Red, red, and differential interference contrast, DIC) are shown on the left and a large merged image containing two channels plus DIC showing many double-positive cells on the right. At higher magnification (inset with scale bar) the infected cells can be seen to be morphologically consistent with lymphocytes. B: In lymph node in long-term infected animals, images for individual channels (SIV-ISH, red; Ham56-macrophages, green; and DIC) are shown on the left. No evidence of macrophage infection (co-localization of the two markers) is present in the larger merged image. C: Infection of macrophages in vivo is extremely rare in SIVmac239/316 infection. C shows the only instance in which macrophage infection could be demonstrated (CD68 with Alexa 488, green; SIV-ISH, red; and DIC). The double-positive cell is present in the subcapsular sinus of the axillary lymph node. D: Co-localization of SIV (red), macrophages (HAM56, green), and T cells (CD5, blue) in the lymph node of a chronically infected animal. In the larger merged image several SIV-infected T cells are present and appear pink/purple because of the mixing of red and blue labels.

Figure 4

Immunophenotype of SIV-infected cells in SIVmac239 infection. A: Double-label confocal microscopy in lymph node at 50 days after infection showing SIV infection of macrophages. Images for individual channels (CD68 with Alexa 488, green; SIV-ISH with Fast Red, red) are shown on the left with the larger merged image on the right. Several infected macrophages are present (arrow). B and C: Infection of macrophages is also observed in the spleen in a long-term infected animal (B) using the same markers and in the intestine (C) using HAM56 in place of CD68 for detection of macrophages. D: Co-localization of SIV (red), macrophages (HAM56, green), and T cells (CD3, blue) in the lymph node of an animal chronically infected with SIVmac239. Infection of both T cells (pink/purple) and macrophages (yellow) can be seen.

Figure 5

Multilabel confocal microscopy in lung of an animal infected with SIVmac239 that died with giant cell pneumonia. The individual channels (SIV in situ hybridization, red; Ham56-macrophages with Alexa 488, green; all cellular nuclei with Topro3, blue and DIC) are shown on the left with the larger merged image on the right. Several infected macrophages and multinucleated giant cells are present.

Figure 6

Total numbers of SIV-infected cells/mm² (left axis) versus numbers of SIV-infected macrophages/mm² (right axis) detected by combined RNA in situ hybridization/immunohistochemistry for SIV and macrophages using HAM56 in LN and spleen. Bars represent averages of five 1-mm² areas per slide from each tissue in two animals per time point. In SIVmac239/316-infected animals both the total numbers of infected cells and infected macrophages were lower than animals inoculated with SIVmac239. All animals infected with SIVmac239 for 21 days or more had evidence of SIV-infected macrophages. In terminal SIVmac239-infected animals, the number of macrophages was primarily represented by multinucleated giant cells. These cells were not observed in terminal SIVmac239/316-infected animals.