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Different from their primary roles in inflammation and immunity, these cells can differentiate into bone-resorbing osteoclasts. However, it remains unclear how macrophages determine which function to engage in bone, especially when exposed to the same stimulus, such as TNF, which can drive both pathways. Understanding these mechanisms is particularly important in inflammatory bone diseases, such as rheumatoid arthritis, where inflammation and bone resorption are hallmarks. Here, we identified an RBP-J\u2013Hs2st1\u20132-O-sulfated heparan sulfate (HS)\u2013IFN\u03b2 dependent molecular network that acts as a gatekeeper balancing macrophage fate inclinations in response to TNF. This network integrates cellular outside-in, inside-out, and relayed outside-in pathways. RBP-J deficiency in macrophages shifts TNF action from inflammatory regulation to osteoclastogenesis. Hs2st1, the exclusive biosynthetic enzyme for 2-O sulfation of HS, is a key target suppressed by RBP-J. Loss of Hs2st1 significantly reduces inflammatory osteoclastogenesis and arthritic bone erosion without affecting physiological bone mass. Elevated Hs2st1 expression and 2-O sulfation shift macrophages toward an osteoclastogenic fate, and vice versa. We further identified residues in IFN\u03b2 that are responsible for binding HS. Mutation of these sites diminished HS binding, augmented type I IFN response, and more strongly suppressed osteoclastogenesis. Our findings reveal a previously unrecognized molecular program fine-tuning macrophage fate and function, and highlight potential therapeutic strategies for inflammatory diseases with bone defects.<\/jats:p>","DOI":"10.64898\/2026.09.02.749003","type":"posted-content","created":{"date-parts":[[2026,9,7]],"date-time":"2026-09-07T17:10:19Z","timestamp":1788801019000},"source":"Crossref","is-referenced-by-count":0,"title":["An RBP-J-heparan sulfate-dependent gatekeeper directs macrophage fate between inflammatory regulation and osteoclastogenesis"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-7723-7653","authenticated-orcid":false,"given":"Ting","family":"Zheng","sequence":"first","affiliation":[{"name":"Arthritis and Tissue Degeneration Program and David Z. 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