Invariant NKT cells, a small subset of immunoregulatory T cells restricted by the glycolipid-presenting non-polymorphic CD1d molecule, are able to modulate a variety of innate and adaptive immune responses. Osteoclasts (OC) are bone resorbing polykaryons of hematopoietic lineage, that have the capacity to regulate myeloid cell egress from bone marrow (BM) thus making them an integral part of the innate immune response. We and others previously showed that NKT cells regulate hematopoiesis in mice as well as humans. In this work, we investigate the role of NKT cells in OC development and function in homeostasis and after their specific activation by the model glycolipid alpha-galactosylceramide (aGC). Using quantitative back scattered electron scanning microscopy, we found that TCR Ja18 −/− mice which selectively lack development of NKT cells, exhibit a moderate osteopetrotic phenotype affecting trabecular as well as cortical bone. Histologically, these mice had the same number of TRAP+ OC as WT mice suggesting a maturation rather developmental defect in the TCR Ja18 −/−-derived OC. In vitro differentiation in the presence of RANKL and M-CSF showed that while TCR Ja18 −/− BM cells are capable of forming multinucleated OC, these, as assessed by confocal microscopy, fail to form F-actin rings and sealing zone and thus are unable to resorb bone. Further underscoring the effect of NKT cells in this process, CD45.1+ BM cells highly purified from CD45.2+ WT/CD45.1+ TCRJa18 −/− mixed BM chimeras displayed restoration of their OC F-actin rings. Next we investigated whether in vivo activated NKT cells regulate OC function. We found that a single injection of aGC dramatically increased the number of CD3-B220-CD11b-c-fmshighc- kithigh BM OC progenitors and accelerated the in vitro development of OC in WT but not TCR Ja18 −/− mice. Furthermore, this resulted in high serum levels of IFN-g and IL-4 but not IL-1 or IL-17. An aGC-mediated increase of OC progenitors was observed in IFN-g −/− but not IL-4 −/−mice suggesting that NKT cell-derived IL-4 is the main cytokine promoting osteoclastogenesis in this context. Taken together, our data demonstrate a novel role of NKT cells in homeostatic bone mass regulation and in the orchestration of innate immune responses through regulation of OC development and function.

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