HIV Modulates Osteoblast Differentiation via Upregulation of RANKL and Vitronectin

Abstract

Bone loss is a prevalent characteristic among people with HIV (PWH). We focused on mesenchymal stem cells (MSCs) and osteoblasts, examining their susceptibility to different HIV strains (R5- and X4-tropic) and the subsequent effects on bone tissue homeostasis. Our findings suggest that MSCs and osteoblasts are susceptible to R5- and X4-tropic HIV but do not support productive HIV replication. HIV exposure during the osteoblast differentiation process revealed that the virus could not alter mineral and organic matrix deposition. However, the reduction in runt-related transcription factor 2 (RUNX2) transcription, the increase in the transcription of nuclear receptor activator ligand kappa B (RANKL), and the augmentation of vitronectin deposition strongly suggested that X4- and R5-HIV could affect bone homeostasis. This study highlights the HIV ability to alter MSCs' differentiation into osteoblasts, critical for maintaining bone and adipose tissue homeostasis and function.

Keywords: HIV; bone; osteoblasts; vitronectin.

Figure 1

Expression of CD4, CCR5, and CXCR4 on the cell surface. Representative dot plots obtained by flow cytometry indicating the surface marker expression of CD4, CCR5, and CXCR4 in mesenchymal stem cells (MSCs) (A), and at 7 and 14 days of the osteoblast differentiation process (B). The bars indicate the percentage of positive cells in A (C) and in B (D). Data are expressed as mean ± SD obtained from 3 independent experiments.

Figure 2

Exposure of MSCs and osteoblasts to X4- and R5-tropic HIV. MSCs and 14 days’ differentiated osteoblasts were infected with an inoculum of 1 pg of p24/cell with CXCR4-tropic HIV (HIV (X4)) and CCR5-tropic HIV (HIV (R5)). Representative microscopy images showing the kinetics of HIV replication at 3, 5, and 7 days post-infection (dpi) by the immunostaining of HIV-p24 capsid antigen (A). Bars express the percentage of HIV-infected MSCs (B) and osteoblasts (C). Ten microscopic fields per condition were quantified for each experiment. Scale bar: 50 µm. Data are expressed as mean ± SD obtained from 4 independent experiments.

Figure 3

HIV was unable to modulate osteoblast differentiation. Effect of CXCR4-tropic HIV (HIV (X4)) and CCR5-tropic HIV (HIV (R5)) exposure on osteoblast differentiation. Representative microscopy images reveal alkaline phosphatase (ALP) activity by deposition of BCIP-NTB substrate, calcium deposition by alizarin red S staining, and collagen deposition by Sirius red staining at 7, 14, and 21 days post-differentiation (A). Spectrophotometric quantification of ALP activity, calcium, and collagen deposition (B). NI (noninfected). d (days post-differentiation). Ten microscopic fields per condition were quantified for each experiment. Scale bar: 200 µm. Data are expressed as mean ± SD obtained from 4 independent experiments.

Figure 4

HIV modulates RUNX2, vitronectin, and RANKL expression during osteoblast differentiation. RUNX2 transcription was determined by RT-qPCR at day 1, 7, 14, and 21 post-differentiation (A). Vitronectin expression revealed by immunofluorescence with a specific antibody at 14 and 21 days post-differentiation (B). Quantification of median fluorescence intensity (MFI) using ImageJ from images in A at 14 (C) and 21 days post-differentiation (D). Adhesion of CellTrace^TM yellow-labeled U937 monocytes to differentiated osteoblasts previously infected or not with HIV-R5 or HIV-X4, uninfected osteoblast, and osteoblast without U937 (negative control, NC). The presence of adherent cells was determined by fluorescence microscopy (RED). DIC, differential interference contrast (E). Quantification of VPD-positive cells from images in E (F). *** p < 0.0001; ** p < 0.001; * p < 0.01 vs. non-infected (NI). RANKL transcription determined by RT-qPCR (G). RANKL expression in culture supernatants (H). NI (noninfected). d (days post-differentiation). Ten microscopic fields per condition were quantified for each experiment. Scale bar: 50 µm. Data are expressed as mean ± SD obtained from 3 independent experiments. * p < 0.01, ** p < 0.001, *** p < 0.0001 vs. NI.