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P. Giannoni et al.
Involvement of TNFα, IL-6 and IL-11 in enhanced osteoclast maturation by CLL-conditioned media
To assess whether TNFα, IL-6 and IL-11 could be involvedinenhancedformationofosteoclastsbyCLL-cm, we added neutralizing monoclonal antibodies for TNFα and IL-11 or anti-IL-6R and anti-GP130 monoclonal anti- bodies to cultures of monocytes stimulated with MCSF+CLL-cm, with or without previous RANKL activa- tion. Anti-IL-6R and anti-IL-11 monoclonal antibodies reduced monocyte osteoclastogenesis in the absence of RANKL pre-stimulation only, while the anti-GP-130 monoclonal antibody also acted in the presence of RANKL (Figure 6A). Infliximab (anti-TNFα), on the other hand, inhibited differentiation of large, multinucleated osteo- clasts, previously activated with MCSF+RANKL (Figure 6B, C). Together, these observations suggest that IL-6 and IL-11 drive monocytes toward an initial stage of osteoclast maturation, while TNFα, in synergy with RANKL, plays a key role in the differentiation of fully competent, multi- nucleated osteoclasts. Conditioned media from TNFα- silenced MEC-1 cells weakly decreased the formation of large osteoclasts and increased the number of small trinu- cleated TRAP+ cells (Online Supplementary Figure S7). Higher levels of TNFα were also detected in media recov- ered from MCSF+RANKL-activated monocytes cultured in the presence of CLL-cm than in any of the other exper- imental conditions (Figure 6D). Data derived from experi- ments in a CLL mouse model, which results in derange- ment of bone structure following leukemic cell engraft- ment,2 appear in line with these observations. Moreover TNFα levels, found in mouse sera but of human source, appeared increased from the third to the sixth weeks after CLL cell injection (Online Supplementary Figure S8). Collectively these observations suggest that TNFα may have a key role in impairment of physiological bone remodeling in CLL patients. Of note, the concentration of TNFα in sera directly correlated with the degree of bone erosion in CLL patients.2
Osteoclasts support CLL viability in vitro
To investigate whether osteoclasts support leukemic cell
viability we generated fully differentiated osteoclasts and then co-cultured them with CLL cells, while measuring apoptosis during time-course experiments. Osteoclasts significantly prevented CLL cells from spontaneous apop- tosis (Figure 7A). Interestingly also small, TRAP+, tri- nucleated cells, derived by treating monocytes with MCSF and CLL-cm, without RANKL, displayed analo- gous effects (Figure 7B). Bright field images (Figure 7C) show viable leukemic B cells surrounding large classical osteoclasts or smaller osteoclast precursors in in vitro co- culture. While assessing whether osteoclasts could stimu- late CLL-cell proliferation, we determined that, after co- culture with osteoclasts, Ki-67 was upregulated on the whole leukemic population but scored markedly positive in a limited fraction of cells (Ki-67 bright: Figure 7D, E). Accordingly, only a limited number of cells was induced by the osteoclasts to progress through the cell cycle along the experiment (Figure 7F).
Bone biopsies from CLL patients show the presence of small and large TRAP+ osteoclasts
The presence of osteoclasts was further investigated in bone marrow biopsies from two CLL patients. Classical large cells, immuno-challenged for TRAP positivity,
appeared in close contact with bone trabeculae, but much smaller TRAP+ cells were also detectable (Figure 8A); the former, canonical and differentiated osteoclasts, were located on bone surfaces, while the latter, activated TRAP+ monocytes possibly representing immature osteo- clasts, were mostly observable within the bone marrow stroma of the biopsies (Figure 8Ai). Moreover, in co- immunostaining experiments, CD79a+ leukemic B cells often appeared in strict vicinity of large TRAP+ cells (Figure 8Aii), further indicating that direct crosstalk between the two cell types may take place in vivo. Additionally, as shown in Figure 8B, the number of osteo- clasts found in bone biopsies from three CLL patients appeared higher than in normal controls.
Ibrutinib inhibits increased osteoclastogenesis induced by CLL-conditioned media
B-cell receptor tyrosine kinase inhibitors, such as the BTK inhibitor ibrutinib, represent the gold standard ther- apy for CLL patients. BTK, a member of the TEC kinase family, integrates RANK/RANKL and ITAM pathways along osteoclast formation and activity.27 We determined that ibrutinib significantly inhibited the enhanced gener- ation of large, mature osteoclasts when added contem- porarily with CLL-cm to previously activated monocyte cultures (Online Supplementary Figure S9). Ibrutinib treat- ment may thus counteract survival stimuli provided by the bone microenvironment.
Discussion
In recent years it has become increasingly clear that the bi-directional crosstalk28 between CLL cells and cellular components of the microenvironment plays a relevant role in disease progression. We have demonstrated here that CLL cells affect the differentiation of the two major bone components: osteoblasts and osteoclasts. Indeed CLL cells, through the release of soluble factors, on the one hand inhibit osteoblast differentiation and on the other hand they increase osteoclastogenesis and bone resorption. TNFα, IL-6 and IL-11 appear to be involved in the inhibition of osteoblast differentiation: cytokine knockdown, the use of neutralizing monoclonal antibod- ies or blocking binding to their receptors significantly counteracted the inhibition of extracellular matrix deposi- tion when osteo-induced BMSC were cultured in the presence of CLL-cm. Restoration of bone matrix deposi- tion, particularly evident after the addition of the anti- TNFα monoclonal antibody infliximab, suggests a major role of TNFα in the CLL cell-driven impairment of osteoblastogenesis. Indeed TNFα affects osteoblast func- tion and differentiation through the inhibition of the pro- duction of extracellular matrix components (i.e., type I collagen), or downmodulating the expression of osteocal- cin and alkaline phosphatase.29 TNFα also inhibits the expression of the osteoblast differentiation factor RUNX2.30 Accordingly, neutralization of TNFα in CLL-cm rescued levels of RUNX2 and osteocalcin mRNA in osteo- induced BMSC, paralleling the increase of matrix miner- alization. Moreover, a contribution of pSTAT3 and AKT, as downstream molecules involved in bone remodeling signals31 by leukemic cells, may be envisaged through decreased levels of these molecules in BMSC treated with CLL-cm only, as compared with TNFα-neutralized CLL-
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haematologica | 2021; 106(10)