HTLV-1 infected T cells cause bone loss via small extracellular vesicles

Abstract

Adult T cell leukaemia (ATL), caused by infection with human T- lymphotropic virus type 1 (HTLV-1), is often complicated by hypercalcemia and osteolytic lesions. Therefore, we studied the communication between patient-derived ATL cells (ATL-PDX) and HTLV-1 immortalized CD4+ T cell lines (HTLV/T) with osteoclasts and their effects on bone mass in mice. Intratibial inoculation of some HTLV/T leads to a profound local decrease in bone mass similar to marrow-replacing ATL-PDX, despite the fact that few HTLV/T cells persisted in the bone. To study the direct effect of HTLV/T and ATL-PDX on osteoclasts, supernatants were added to murine and human osteoclast precursors. ATL-PDX supernatants from hypercalcemic patients promoted the formation of mature osteoclasts, while those from HTLV/T were variably stimulatory, but had largely consistent effects between human and murine cultures. Interestingly, this osteoclastic activity did not correlate with expression of osteoclastogenic cytokine receptor activator of nuclear factor kappa-B ligand (RANKL), suggesting an alternative mechanism. HTLV/T and ATL-PDX produce small extracellular vesicles (sEV), known to facilitate HTLV-1 infection. We hypothesized that these sEV also mediate bone loss by targeting osteoclasts. We isolated sEV from both HTLV/T and ATL-PDX, and found they carried most of the activity found in supernatants. In contrast, sEV from uninfected activated T cells had little effect. Analysis of sEV (both active and inactive) by mass spectrometry and electron microscopy confirmed absence of RANKL and intact virus. Viral proteins Tax and Env were only present in sEV from the active, osteoclast-stimulatory group, along with increased representation of proteins involved in osteoclastogenesis and bone resorption. sEV from osteoclast-active HTLV/T injected over mouse calvaria in the presence of low-dose RANKL caused more osteolysis than osteoclast-inactive sEV or RANKL alone. Thus, HTLV-1 infection of T cells can cause release of sEV with strong osteolytic potential, providing a mechanism beyond RANKL production that modifies the bone microenvironment, even in the absence of overt leukaemia.

Keywords: adult T cell leukaemia; extracellular vesicles; osteoclast; osteolysis; proteomics.

FIGURE 1

Implantation of HTLV‐1 infected T cell lines into bone causes osteolysis even in the absence of overt leukaemia. (a) NCG mice were injected intra‐tibially with 250,000 ATL‐PDX RB in the right tibia and trabecular bone volume fraction (BV/TV) was examined by viva‐CT every 2 weeks (n = 5). Percent change from baseline (prior to inoculation) is indicated. (b) Genomic DNA PCR for amount of human cells in bone marrow of injected tibiae after 8 weeks of injection for HTLV/T lines, together with RB bone marrow and spleen after 6 weeks of injection, and mouse bone marrow as negative control. (c) NCG mice were injected intra‐tibially with 250,000 HTLV/T lines C7 (grey), CJ2 (black) and C8 (green). Control mice were injected with PBS (open circles) or were uninjected (closed circles). BV/TV of the injected leg was determined by viva‐CT before and 4‐ and 8‐week post injection. Data represented as %BV/TV change from baseline (n = 11–18 mice per group). (d) Representative micro‐CT images from mice in (c). Scale bar 100µm. (a), (c); Linear mixed‐effects model with Tukey's method for multiplicity adjustment, ^** p < 0.01, ^*** p < 0.001 or ^**** p < 0.0001 versus control; ^# p < 0.05 or ^## p < 0.01 versus C7. HTLV/T, HTLV‐1 immortalized CD4+ T cell lines.

FIGURE 2

sEV from osteolytic HTLV/T cell lines stimulate in vitro osteoclastogenesis. (a) mBMM were treated with either vehicle or 10% supernatant together with low‐dose RANKL for 4 days. TRAP positive multinuclear cells were counted as osteoclasts. n = 9, from at least three independent experiments. (b) mBMM were treated with ∼ 6 × 10⁶ sEV isolated from culture supernatants or vehicles for 4 days. TRAP positive multinuclear cells were counted as osteoclasts. Data from three independent experiments, n = 9. (c) HTLV/T cells were pre‐treated with GW4869 (10 µM) or DMSO for 5 days and 10% supernatant was added during 4 days of differentiation. Data from three biological repeats, n = 9. (d) sEV were isolated from IL‐2 expanded human PBMC, stimulated (or not) with PMA (50 ng/mL) and ionomycin (1 nM), and added (∼6 × 10⁶ sEV) to osteoclastogenic cultures for 4 days. Results from two independent experiments, n = 6. Scale bars 500 µm. All statistics one‐way ANOVA, _* p < 0.05_, ^** p < 0.01, ^*** p < 0.001. HTLV/T, HTLV‐1 immortalized CD4+ T cell lines; PBMC, peripheral blood mononuclear cells; RANKL, receptor activator of nuclear factor kappa‐B ligand; sEV, small extracellular vehicle.

FIGURE 3

HTLV/T cells modulate maturation of osteoclasts. (a), (b) HTLV/T supernatants were added to osteoclastogenic mBMM cultures during the first 2 days (a) or last 2 days (b). Cells were fixed and TRAP stained after 4 days, and osteoclasts were counted. (c) Pre‐osteoclasts were generated by treating mBMM with 50 ng/mL of RANKL for 2 days. These were then cultured with low‐dose RANKL with or without sEV (from HTLV/T C8) for an additional 24 h. (d) Pre‐osteoclasts were lifted and replated on bone slices, treated with supernatant for 5 days, and resorption pits were stained. Resorption area was measured using ImageJ. n = 6–9, from 2 to 3 independent experiments. Scale bars: 500 µm—(a), (b), and (c) (upper panel); 200 µm—(c) (lower panel), (d) all statistics one‐way ANOVA, ^* p < 0.05, ^** p < 0.01_, ^*** p < 0.001, ^**** p < 0.0001. HTLV/T, HTLV‐1 immortalized CD4+ T cell lines; RANKL, receptor activator of nuclear factor kappa‐B ligand; sEV, small extracellular vesicles.

FIGURE 4

Proteomics of sEV suggests multiple possible candidates modulating osteoclastogenesis. (a) Representative transmission electron micrograph, of sEV isolated from C8. Scale bar: 100 nm. (b)–(h) LC‐MS/MS analysis was performed on proteins isolated from sEV with positive (n = 4, brown), and negligible (n = 3, blue) effect on osteoclast differentiation. (b) Analysis of viral proteins shows presence of gag‐pro in all sEV, but Env (gp62) and Tax only in sEV that stimulate osteoclasts. HBZ was not detected in any. (c)–(h) Analysis of cellular proteins, (c) heat map, (d) PCA plot, (e) Pearson's correlation, (f) gene ontogeny analysis. (g) STRING network analysis displaying enriched proteins in sEV with high osteoclastogenic effect, (h) STRING network analysis displaying enriched proteins in sEV without osteoclastogenic effect. sEV, small extracellular vesicles.

FIGURE 5

HTLV/T sEV causes bone loss in calvarial osteolysis model. Mice were injected with RANKL together with PBS or sEV (9 × 10⁷) from HTLV/T C7 or C8 subcutaneously over the calvaria. (a) Calvaria were scanned using micro‐CT and bone volume fraction (BV/TV) was determined in two regions, as indicated. Scale bar: 1 mm. (b) Calvaria were decalcified, and paraffin embedded sections were subjected to TRAP staining. NOc/BPm was counted. n = 6–10/group. Scale bar: 100 µm. One‐way ANOVA, p‐values indicated. HTLV/T, HTLV‐1 immortalized CD4+ T cell lines; NOc/BPm, osteoclast number per bone perimeter; RANKL, receptor activator of nuclear factor kappa‐B ligand; sEV, small extracellular vesicles.