Massively HIV-1-infected macrophages exhibit a severely hampered ability to differentiate into osteoclasts

Abstract

Introduction: Osteoclasts play a crucial role in bone resorption, and impairment of their differentiation can have significant implications for bone density, especially in individuals with HIV who may be at risk of altered bone health. The present study aimed to investigate the effects of HIV infection on osteoclast differentiation using primary human monocyte-derived macrophages as precursors. The study focused on assessing the impact of HIV infection on cellular adhesion, cathepsin K expression, resorptive activity, cytokine production, expression of co-receptors, and transcriptional regulation of key factors involved in osteoclastogenesis.

Methods: Primary human monocyte-derived macrophages were utilized as precursors for osteoclast differentiation. These precursors were infected with HIV, and the effects of different inoculum sizes and kinetics of viral replication were analyzed. Subsequently, osteoclastogenesis was evaluated by measuring cellular adhesion, cathepsin K expression, and resorptive activity. Furthermore, cytokine production was assessed by monitoring the production of IL-1β, RANK-L, and osteoclasts. The expression levels of co-receptors CCR5, CD9, and CD81 were measured before and after infection with HIV. The transcriptional levels of key factors for osteoclastogenesis (RANK, NFATc1, and DC-STAMP) were examined following HIV infection.

Results: Rapid, massive, and productive HIV infection severely impaired osteoclast differentiation, leading to compromised cellular adhesion, cathepsin K expression, and resorptive activity. HIV infection resulted in an earlier production of IL-1β concurrent with RANK-L, thereby suppressing osteoclast production. Infection with a high inoculum of HIV increased the expression of the co-receptor CCR5, as well as the tetraspanins CD9 and CD81, which correlated with deficient osteoclastogenesis. Massive HIV infection of osteoclast precursors affected the transcriptional levels of key factors involved in osteoclastogenesis, including RANK, NFATc1, and DC-STAMP.

Conclusions: The effects of HIV infection on osteoclast precursors were found to be dependent on the size of the inoculum and the kinetics of viral replication. These findings underscore the importance of understanding the underlying mechanisms to develop novel strategies for the prevention and treatment of bone disorders in individuals with HIV.

Keywords: HIV; TRAP; bone; macrophage; osteoclast.

Figure 1

HIV replication kinetics during osteoclastogenesis. (A)Representation of the experimental timeline schedule. (B) Kinetics of HIV replication using low (0.01 pg/cell) and high (1.0 pg/cell) viral inoculum, measuring p24 antigen in culture supernatants by ELISA during osteoclast formation. (C) The efficiency of HIV infection (measured by flow cytometry as a percentage of cells expressing intracellular p24 antigen) at 12 dpi. (D) Representative dot plots were obtained at 12 dpi by flow cytometry using KC57 monoclonal antibody against gag p24 in control and HIV-infected cells. (E) Measurement of cell death (as a percentage of cells stained with Annexin V/7-AAD) upon HIV infection with high inoculum at two different time points, 3 dpi, and 12 dpi. (F) Representative dot plots obtained by flow cytometry measuring Annexin V/7-AAD staining at time points represented in (E) Data are expressed as mean ± SD obtained from 4-6 independent experiments performed with cells from different donors. ns, not significant.

Figure 2

HIV R5-tropic strain modulates osteoclast formation. Quantification of the number of TRAP-positive osteoclasts after culturing for 12 days and the number of TRAP-positive osteoclasts obtained from monocytes after 15 days of incubation with M-CSF, and 9 days with RANK-L (A). Representative images of A (x200) (B). Measurement of HIV infection efficiency (as a percentage of cells expressing p24-capsid antigen measured by flow cytometry using PE-KC57) at 12-dpi for each inoculum in the absence or presence of the reverse transcriptase inhibitor nevirapine (1μM) (C). Morphology of TRAP-positive osteoclast formation in cell cultures at 12 dpi, using the two HIV inoculums (low and high), and two different R5-tropic HIV strains (AD8, and BaL), in the presence or absence of nevirapine (only for the AD8 strain) (x200) (D). Scale bar: 200 µm. Data are expressed as mean ± SD obtained from 4-6 independent experiments performed with cells from different donors. *p < 0.05, **p < 0.01.

Figure 3

Macrophages activation profile and cytokines released after HIV infection. Surface marker expression and cytokine secretion during HIV infection of monocyte-derived macrophages. Surface marker expression (CD80, CD206, and HLA-DR) was determined by flow cytometry in cultured monocyte-derived macrophages after 6 days with M-CSF and 3 days post-inoculation (dpi) using the two HIV inoculums, in the absence of LPS stimulus (A). Surface marker expression was determined in the presence of LPS stimulus (B). IL-6 secretion was measured by ELISA at different times during the osteoclast differentiation process after HIV infection with each viral inoculum, both prior to and following RANK-L addition. The internal bracket depicts the fold change (C). IL-1b secretion was measured by ELISA at different times during the osteoclast differentiation process after HIV infection with each viral inoculum, both prior to and following RANK-L addition, throughout the experimental timeline (D). Data are expressed as mean ± SD for 2 to 4 independent experiments. MFI, Mean Fluorescence Intensity.

Figure 4

CCR5 expression in osteoclast precursors. Expression of CCR5 on the cell surface (as a percentage of positive cells) in osteoclast precursors after HIV infection (using both inoculums, 0.01 and 1.0 pg/cell) at 3 and 6 dpi measured by flow cytometry (A). The efficiency of HIV infection (measured by flow cytometry as the percentage of cells expressing intracellular p24 antigen) and change in CCR5 expression level (as MFI) between paired samples (control vs. HIV infected) in osteoclast precursors infected with each HIV inoculum at 3 dpi and 6 dpi (B). Quantification of the number of TRAP-positive osteoclasts after CCR5 antagonism using TAK-779 (C) or CCR5 blocking using three different concentrations of recombinant HIV (AD8)-gp120 (D). The efficiency of VSV-G-pseudotyped HIV infection (measured by flow cytometry as the percentage of cells expressing intracellular p24 antigen) and the number of TRAP-positive osteoclasts (x200) at 12 dpi using the two viral inoculums in the absence and presence of nevirapine (E). Scale bar: 200 μm. Data are expressed as mean ± SD obtained from 2-4 independent experiments performed with cells from different donors. *p < 0.001, **p < 0.001, and ***p < 0.0001. ns, not significant.

Figure 5

mRNA levels of RANK, NFATc1, and DC-STAMP during osteoclastogenesis after HIV infection. The mRNA levels of RANK (A), NFATc1 (B), and DC-STAMP (C) were quantified using real-time quantitative PCR (RQ-PCR) in paired osteoclast precursor samples obtained from the same donor that were infected using two HIV inoculum and measured at 3 days post-infection (dpi) and/or 6 dpi, as indicated. The values of mRNA levels are relative to GAPDH (a housekeeping gene). *p< 0.001. ns, not significant.

Figure 6

Expression of tetraspanins CD9 and CD81 during osteoclast differentiation and HIV infection. The expression level of CD9 and CD81 was measured using flow cytometry (mean fluorescence intensity per cell, or MFI) at 6 days post-infection (dpi) using both HIV inoculums. The results are representative of two independent experiments performed using cells from different donors (A). Osteoclasts and their precursors at 6 dpi of HIV infection with the low-inoculum (upper panel) and control (non-infected) (lower panel) were stained with DAPI (for cell nuclei), PE (for HIV-p24 capsid antigen), FITC (for CD81), and merged using immunofluorescence staining and deconvolution microscopy to examine changes in the cellular localization of tetraspanins during maturation and HIV infection (x400) (B). Osteoclasts and their precursors at 6 dpi of HIV infection with the low-inoculum showing CD81 expression (green) and HIV-infected giant multinuclear cells (red, with nuclei in blue). (C) Yellow arrows show perinuclear structures resembling viral-containing compartments (VCC). Scale bars: 20 μm. *<0.05 indicates a statistically significant difference.

Figure 7

Adhesion of osteoclasts and their precursors. The adhesive properties of osteoclasts vary with the size of the HIV infection inoculum after 10 days of differentiation. The graph represents the average of five fields per condition from a total of six donors. **p ≤ 0.01 and ***p ≤ 0.001 indicate statistically significant differences.

Figure 8

Cathepsin K expression in osteoclasts and precursors after HIV infection. The level of cellular expression was measured using flow cytometry at 12 days post-infection (dpi) with two different viruses: wild-type HIV (AD8) and pseudotyped HIV (AD8)-VSV, each at two different inoculum levels (low and high). Mean fluorescence intensity (MFI) values are expressed as mean ± SD obtained from independent experiments performed using cells from 2-4 different donors. *p < 0.05 indicates a statistically significant difference, while “ns” indicates that the difference is not significant.

Figure 9

Bone resorption by osteoclasts. Representative data from a donor were obtained using a light microscope to show TRAP-positive (red) osteoclasts differentiated from macrophages during 12 days of culture with RANK-L and M-CSF on bone slices obtained from control (non-infected precursors) and HIV-infected precursors exposed to different viral inoculum (A). The bone resorption pits formed by osteoclasts from precursors (control, and HIV-infected using high and low-inoculum) cultured for 9 days were visualized by staining the slices with toluidine blue. Zoomed-in views of red dashed squares provide more detailed observation (B). The resorption area was observed under a light microscope (x100) and analyzed using Image-J (C). Scale bar: 100 μm. The data represent the mean ± SD of two independent donors, and **p < 0.01 indicates a statistically significant difference.