Dysregulated B cell expression of RANKL and OPG correlates with loss of bone mineral density in HIV infection

Abstract

HIV infection is associated with high rates of osteopenia and osteoporosis, but the mechanisms involved are unclear. We recently reported that bone loss in the HIV transgenic rat model was associated with upregulation of B cell expression of the key osteoclastogenic cytokine receptor-activator of NF-κB ligand (RANKL), compounded by a simultaneous decline in expression of its physiological moderator, osteoprotegerin (OPG). To clinically translate these findings we performed cross-sectional immuno-skeletal profiling of HIV-uninfected and antiretroviral therapy-naïve HIV-infected individuals. Bone resorption and osteopenia were significantly higher in HIV-infected individuals. B cell expression of RANKL was significantly increased, while B cell expression of OPG was significantly diminished, conditions favoring osteoclastic bone resorption. The B cell RANKL/OPG ratio correlated significantly with total hip and femoral neck bone mineral density (BMD), T- and/or Z-scores in HIV infected subjects, but revealed no association at the lumbar spine. B cell subset analyses revealed significant HIV-related increases in RANKL-expressing naïve, resting memory and exhausted tissue-like memory B cells. By contrast, the net B cell OPG decrease in HIV-infected individuals resulted from a significant decline in resting memory B cells, a population containing a high frequency of OPG-expressing cells, concurrent with a significant increase in exhausted tissue-like memory B cells, a population with a lower frequency of OPG-expressing cells. These data validate our pre-clinical findings of an immuno-centric mechanism for accelerated HIV-induced bone loss, aligned with B cell dysfunction.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. Increased frequency of RANKL-expressing B cells and decreased frequency of OPG-expressing B cells in HIV infection.

Intracellular expression of: A) OPG and B) RANKL, by circulating peripheral blood B cells were quantified by flow cytometry in 56 HIV-negative and 57 HIV-infected individuals. Comparisons between HIV sero status were performed using Wilcoxon rank-sum test.

Figure 2. B cell subset RANKL and OPG expression in HIV-negative and HIV-positive individuals.

A) Total B cells (CD3−CD20+ cells) were identified in the lymphocyte gate. B) B cells were further divided into 4 distinct subsets: naïve (CD21hiCD27−), resting memory (CD21hiCD27+), activated memory (CD21−CD27+), and exhausted tissue-like memory (CD21−CD27−). C) B cell subset distribution in HIV-negative (N = 28) and seropositive (N = 24) individuals. Intracellular expression of D) OPG and E) RANKL in B cell subsets of HIV-negative and seropositive individuals combined. Graphs reflect individual individuals with bars at the mean (parametric data) or median [non-parametric data, Activated and tissue-like memory subset (C) and naïve and resting memory (E)]. Simple comparisons were done using Student's t test (for parametric data) or Wilcoxon rank sum test (for non-parametric data) for each of the subsets (C) and one-way ANOVA was used to compare multiple groups (D). Actual P values are reported for simple comparisons. For ANOVA *P<0.05 or ***P<0.001 or P =  not significant (ns).

Figure 3. Differential B cell subset RANKL and OPG expression in HIV-negative and HIV-positive individuals.

Representative histograms (alternate top panels) of B cell subset staining for RANKL (A) and OPG (B) for HIV negative individuals (solid line) and HIV positive individuals (dashed line). Scatter plots (alternate bottom panels) represent cumulative data for B cell subset RANKL (A) or OPG (B) expression from individual HIV-negative (HIV−, N = 17) or seropositive (HIV+, N = 15) individuals with bars at the mean (parametric data) or median [non-parametric data, RANKL: tissue-like memory (HIV-); OPG: All HIV- subsets]. Comparisons were done using two-way ANOVA, followed by pairwise comparisons of individual B cell subsets using student's t-test (parametric data) or Wilcoxon rank sum test (non-parametric data).

Figure 4. Immunocentric model of HIV induced bone loss.

We propose a model for HIV- induced bone loss whereby HIV infection, either through direct effects on B cells, or though disruption of T cell costimulation of the humoral system, leads to a decline in the frequency of B cells secreting OPG, coupled with an increase in the frequency of B cells secreting RANKL. This disruption of the immuno-skeletal interface results in an increase in the RANKL/OPG ratio that is permissive for osteoclast formation and enhanced bone resorption, which ultimately contributes to bone loss and the elevated bone fracture risk characteristic of HIV-infected individuals.