CD14-derived osteoclasts are susceptible to HIV infection in vitro
We characterized human osteoclasts generated by culturing with recombinant M-CSF plus RANKL the CD14+ monocytes purified from PBMCs [9]. After 6 to 7 days, a large number of osteoclast-like multinucleated cells (MNCs) (Figure 1A). Further incubation with M-CSF plus RANKL induced formation of larger MNCs after 7 to 14 days. The MNCs expressed tartrate-resistant acid phosphatase (TRAP), a specific osteoclast marker [10] (Figure 1A). Furthermore, the MNCs were able to resorb bone in the pit formation assay (Figure 1B). These results indicate that the MNCs were osteoclasts. In contrast, CD14+ cells cultured with M-CSF alone expressed a monocyte marker, CD14 at the same level as osteoclasts, and a macrophage specific marker, CD71 at a higher level than osteoclasts (Figure 1C), but not forming any TRAP-positive MNCs (Figure 1A).
We examined expression of CD4, CXCR4, and CCR5, receptors for HIV-1 infection, in CD14-derived osteoclasts and macrophages. Flow cytometry analysis showed that CD14-derived osteoclasts expressed all of the receptors on their cell surface, each at the similar level to that in the macrophages (Figure 1C).
We then exposed CD14-derived osteoclasts or macrophages to a CCR5-tropic (R5) HIV-1 strain, JR-FL or a CXCR4-tropic (X4) HIV-1 strain, NL4-3, in order to examine whether or not human osteoclasts are infected by HIV-1. The cells were immunostained with anti-HIV-1 p24 and anti-TRAP antibodies, 40 hours after the infection. In both cases, some macrophages and TRAP+-MNCs were found positive with p24 staining (Figure 2A). CD14-derived osteoclasts were infected by JR-FL and NL4-3 as efficiently as the macrophages, though JR-FL infected both CD14-derived macrophages and osteoclasts more efficiently than NL4-3 (Figure 2B). The treatment of the cells with tenofovir (TFV), a reverse-transcriptase inhibitor, prevented JR-FL and NL4-3 infection of CD14-derived macrophages and osteoclasts in a dose-dependent manner (Figure 2A,C).
Furthermore, the levels of p24 in the supernatants of osteoclast cultures rised in a time-dependent manner, although the p24 levels in CD14-derived osteoclasts were lower then those in macrophages (Figure 3A). The rises of the p24 levels were suppressed by TFV treatment (Figure 3B), and the supernatants had infectivity (Figure 3C), indicating that JR-FL and NL4-3 replicates in CD14-derived macrophages and osteoclasts. Taken together, these data (Figures 2 and 3) indicate that HIV-1 can infect CD14-derived osteoclasts and replicate in them.
HIV-1 infection enhances osteoclast differentiation
To elucidate possible link between HIV-1 infection and osteolytic disease, we tested whether HIV-1 infection has any effects on osteoclast differentiation. Microscopic analyses of the TRAP-stained cells revealed that MNCs incubated with JR-FL were significantly increased in size and number of nuclei per cell, in comparison with normal CD14-derived osteoclasts (Figure 4A). In contrast, MNCs treated with TFV or incubated with aldrithiol-2 (AT-2)-inactivated JR-FL were similar to normal CD14-derived osteoclasts.
We further analyzed mRNA expression of specific osteoclast markers, such as acid phosphatase 5 (ACP5) /TRAP, cathepsin K (CTSK), and the calcitonin receptor (CALCR) [11], in the cells infected with different doses of JR-FL or AT-2-inactivated JR-FL (Figure 4B). In the macrophages, these marker expression levels were very low and there was no significant difference in their expressions between the uninfected and infected cells. In contrast, all of the markers were expressed at high levels in the uninfected CD14-derived osteoclasts and further up-regulated by the viral infection in a dose-dependent manner. However, infection with AT-2-inactivated JR-FL did not significantly affect the maker expressions in CD14-derived osteoclasts. TFV treatment suppressed the enhancement of the maker expressions by JR-FL infection in a dose-dependent manner without any effect on the maker expression levels in the uninfected CD14-derived osteoclasts (Figure 4C).
On the other hand, NL4-3 infection also increased in size and number of nuclei, and enhanced the osteoclast marker expessions, although its effects were less marked as compared with JR-FL infection (Figures 4A,B and 5A,B). Infection with AT-2-inactivated NL4-3 did not affect the cell size and the marker expressions (Figure 5A,B). In addition, TFV treatment inhibited the increase in size and number of nuclei per cell and the enhancement of the marker expression levels (Figure 5A,C).
We finally performed a pit formation assay examining the effect of HIV-1 infection on osteoclast bone resorption activity. Microscopic images showed that pit areas produced by JR-FL or NL4-3-infected osteoclasts were larger than those by the uninfected osteoclasts (Figure 6A). Pit formation was enhanced by JR-FL infection and, to a lesser extent, by NL4-3 (Figure 6A,B). Furthermore, TFV treatment suppressed an increase in pit formation by viral infection (Figure 6A,C). Accordingly, these results strongly suggest that HIV-1 infection enhances osteoclast differentiation and bone resoption activity.