To achieve efficient bone resorption by osteoclasts, the specialized innate immune cells, it is important not only to promote osteoclast differentiation and activation but also to maintain their survival. C-type lectin (CLEC) receptors recognize pathogen ligands and altered self-tissues, comprising activating and inhibitory types whose balance eliminates pathogens while preventing excessive immune responses. However, roles of CLEC receptors in osteoclast differentiation, function, and survival remain unclear. We established knockout (KO) mice of CLEC receptor genes highly expressed by osteoclast and analyzed osteoclast features and bone morphology. We conducted comprehensive in silico screening of osteoclast lineage-specific CLEC receptors utilizing a mouse gene expression dataset and generated single and double KO (DKO) mice of Clec4a2 and Clec4d using a multi-targeted CRISPR-Cas9 system. Clec4a2 KO and DKO enhanced osteoclast differentiation in vitro, and Clec4a2 KO also stimulated enlargement of osteoclasts. Clec4d KO slightly reduced trabecular bone thickness in the femur, while Clec4a2 KO and DKO did not affect bone morphology under physiological conditions. Contrary to conventional understanding that enhanced osteoclast differentiation leads to increased bone resorption, our time-lapse analysis revealed that Clec4a2 KO paradoxically increased osteoclast formation while reducing resorption efficiency due to cell death of osteoclasts and its daughter cells after fission. Clec4a2 KO provided protection against inflammatory bone loss induced by lipopolysaccharide, demonstrating the first evidence that Clec4a2 could serve as therapeutic targets for inflammatory osteolytic diseases. This study introduces a novel paradigm that osteoclast survival regulation by Clec4a2 is fundamental for efficient bone resorption.
