We have identified a CD11b–/loLy6ChiCX3CR1+ population in BM with dual OCP and MDSC function. This population is the predominant source of osteoclasts in BM cultures and is distinct from other myeloid precursors based on its cell-surface phenotype. We further show that CD11b–/loLy6Chi OCPs give rise to bone adherent, cathepsin K, and TRAP-expressing multinucleated osteoclasts in vivo and are thus are a bona fide OCPs.
The OCPs described in this study are phenotypically distinct from previously described MDP, CDP, and monocyte populations, suggesting that OCPs are a unique precursor population analogous to CDPs. It is likely, however, that some plasticity exists between OCPs and other myeloid phenotypes, as suggested by the in vitro findings that immature DCs can give rise to osteoclasts and our findings that OCPs can differentiate into macrophages or DCs in the appropriate cytokine environment. Interestingly, MDSCs are reported to appear transiently in BM cultures for DCs under some conditions. Somewhat similar to OCPs in this study, the MDSCs in DC cultures are Ly-6C+
). It would be of interest to determine whether in DC cultures this cell type gives rise to both MDSCs and osteoclasts.
It has previously been suggested that increased OCP frequency contributes to bone loss and erosions in inflammatory arthritis. Consistent with this, we observe that OCP cells increase in the BM in mouse models of inflammatory erosive arthritis. However, adoptive transfer experiments demonstrated that cotransfer of OCPs with SKG CD4+ T cells to a Rag2–/– recipient markedly decreases arthritis severity. These findings suggest that the CD11b–/loLy6Chi OCP population is also immunomodulatory. Phenotypically CD11b–/loLy6Chi OCPs share many characteristics of M-MDSCs, including a mixed M1- and M2-like phenotype and the ability to suppress T cells using a NO-dependent mechanism.
The OCP population identified here, however, is not phenotypically uniform, as demonstrated by heterogeneity of expression for CD11b, CD117, and Ki67, and variability in the osteoclastogenic potential of single-cell clones. Thus, we cannot rule out the possibility that while both functions reside within the OCP population, they do not reside in a single cell. Using current cell-surface markers, we have not been able to further subdivide this population into distinct functional subsets, and the maintenance of T cell suppression after differentiation to osteoclasts suggests that one cell type has the capacity to develop multiple functional activities. Consistent with this, a recent study found that mature human osteoclasts can suppress T cell proliferation in vitro (46
The inflammatory microenvironment affects osteoclast differentiation via the actions of multiple cytokines. TNF-α and IL-6 produced by synovial macrophages and Th17-derived IL-17 all act to increase RANKL on osteoblasts and synovial fibroblasts, and TNF-α, IL-1, and IL-6 can directly affect osteoclast differentiation (22
). Several studies suggest that OCPs are also altered by inflammation. Increased circulating OCPs can be detected in psoriatic arthritis and RA patients (20
). In the hTNF-α–Tg murine arthritis model, both BM and peripheral blood OCPs were increased in arthritic mice, as we see in the present study. Thus, bone loss and erosions in inflammatory arthritis may result from a synergy between increased precursors and a more prodifferentiation microenvironment. Interestingly, osteoclast differentiation is inhibited by IFN-γ, yet IFN-γ is required for OCPs to suppress T cells. Thus, exposure to IFN-γ may function to skew CD11b–/lo
cells toward a primarily MDSC function. In contrast, exposure of the OCP population to M-CSF and RANKL stimulates osteoclastogenesis, but does not eliminate MDSC function.
IFN-γ likely has complex effects on basal bone turnover beyond pure osteoclast inhibition. Although bone mass is decreased in Ifngr–/–
mice, this appears to be primarily a defect in bone formation, and osteoclast number is not increased (49
). Neither IFN-γ nor IFN-γR deficiency substantially affected BM OCP frequency or number (J.F. Charles, unpublished observations). In inflammatory conditions, however, a clear proosteoclast effect of IFN-γR deficiency is seen, with increased osteoclast formation and erosion after LPS injection (50
). The effect of IFN-γR deficiency may be most evident after an inflammatory stimulus, where the OCP population is both increased and biased toward an osteoclast fate rather than MSDC function due to the inability to respond to IFN-γ.
Whether the CD11b–/lo
myeloid precursor population differentiates toward an MDSC or osteoclast functionality may also be dependent on other innate signals in the environment. Interestingly, deficiency in PIR-B was recently shown to decrease MDSC function in the periphery in a tumor model (39
) and has also been shown to increase osteoclast differentiation in vitro (31
). Although no effect of PIR-B deficiency on basal bone parameters was seen, it is possible that the role of innate immune receptors such as PIR-B in regulating osteoclast differentiation is context dependent.
Although initially described as a tumor-induced population, an increase in MDSCs was recently found in mouse models of autoimmune disease and in humans with ulcerative colitis (43
splenic MDSCs are induced in EAE (44
). Similarly, CD11b+
MDSCs with both in vitro and in vivo suppressive activity are induced in spleen and intestine in a mouse model of colitis (43
). Expansion of MDSC populations in chronic inflammatory conditions may be one of many mechanisms for fine-tuning immune system activation, although the physiologic importance of this mechanism for autoimmune disease is only beginning to be defined. In the SKG autoimmune arthritis model, induction of an OCP population with MDSC activity in arthritic mice was not sufficient for disease control. MDSC function of OCPs may be incompletely effective for a number of reasons, including inadequate number, lack of colocalization with pathogenic T cells, resistance of SKG T cells to suppression, insufficient T cell production of IFN-γ, or some combination of these factors.
MDSCs have been demonstrated to induce expansion of Tregs in a tumor model, a function that is diminished if MDSCs are skewed toward an M1 phenotype by loss of PIR-B (39
). It is possible that high levels of TNF-α, IL-1, and IL-6 produced by synovial fibroblasts bias the CD11b–/lo
OCP population toward a more M1 phenotype in the periphery, altering their peripheral MDSC functionality without changing the suppressive properties of BM OCPs. The cytokine environment could also bias OCPs toward osteoclast differentiation such that systemic bone loss and erosive disease is a by-product of the immune system’s failed attempt at homeostasis.
Artificially increasing CD11b–/lo
OCPs to approximately 5-fold their normal number decreased joint inflammation in the SKG adoptive transfer model. Skin inflammation was not ameliorated, however, and was even exacerbated. Skin inflammation after adoptive transfer of naive T cells into scid/scid
mice has been previously described (37
). As cell-cell contact is required for full T cell suppression by OCPs, the differential effect on inflammation at distinct sites may relate to localization of adoptively transferred OCPs and/or the local cytokine environment that may influence the suppressive capacity of OCPs.
In contrast with the adoptive transfer model, the increase in CD11b–/loLy6Chi OCPs after zymosan injection does not suppress the development of SKG inflammatory arthritis in response to zymosan. It may be that the OCP number is inadequate in the physiologic setting or that they do not localize in adequate proximity to T cells to substantially alter disease activity. In the adoptive transfer model of inflammatory arthritis using SKG T cells, OCPs and T cells were transferred to the RAG2-deficient recipient together, ensuring colocalization that may have been critical for the observed immunomodulatory effect. Although SKG OCPs are equivalent to BALB/c OCPs in T cell–suppressive activity in vitro, we cannot rule out the possibility that, particularly in vivo, SKG T cells may be less susceptible to suppression, may not consistently produce the IFN-γ required for MDSC activity, or that additional cell types prevent OCPs from suppressing the development of inflammatory arthritis.
The MDSC function of the OCP population is similar to monocytic MDSCs described in other autoimmune models. However, OCPs likely represent only a subset of MDSCs. In the OCP population, suppression of T cell proliferation is dependent on IFN-γ and NO. MDSCs are a heterogeneous population, and other MDSCs suppress T cells via arginase-dependent pathways or inhibitory cytokine secretion. In addition, the OCP population was not the only suppressive myeloid population seen in the BM of mice with inflammatory arthritis. We also observed that a CD11b+Ly6Chi population could suppress T cell proliferation in vitro, though these cells did not show osteoclastogenic potential (J.F. Charles, unpublished observations). Thus while the CD11b–/loLy6C+ OCP population has MDSC function, not all MDSCs are OCPs.
The outcome of the interaction between OCPs and T cells will also differ depending on the subset of T cells and the environment in which they are interrogated. Th17 cells promote osteoclast differentiation via induction of RANKL on stromal cells, as well as surface RANKL on Th17 cells themselves, whereas Th1 cell production of IFN-γ results in inhibited osteoclast differentiation, although they also express RANKL (42
). Our results suggest that Th1 cells producing IFN-γ would activate T cell–suppressive activity by OCP cells. The effect of Th17 cells and IL-17 on MDSC function is not yet known, but may be an additional factor influencing the fate of the OCP population.
Our results demonstrate a dual OCP and MDSC function for the BM CD11b–/loLy6Chi population in vitro and in vivo. It is possible that induction of CD11b–/loLy6Chi OCPs under inflammatory conditions represents a homeostatic mechanism aimed at dampening T cell activation, which is ineffective in the inflammatory microenvironment, but successfully increases a pool of precursors that can be shunted toward osteoclast differentiation and the formation of erosions. Thus,a more complete understanding of the regulation of the OCP population likely has significance for the regulation of bone loss and autoimmunity.