Fig 2: The specificity of anti-TREM2 antibodies. The specificity of seven commercially available anti-TREM2 antibodies was determined by Western blot analysis of recombinant human TREM2 protein expressed in HEK293 cells following transfection of the expression vector. The upper panels represent (a) HPA010917 (at the final concentration of 200 ng/mL), (b) AF1828 (100 ng/mL), (c) 2B5 (1 μg/mL), (d) bs-2723R (2 μg/mL), (e) ab69405 (400 ng/mL), (f) ab85851 (500 ng/mL), and (g) PA5-18763 (500 ng/mL). The lower panels indicate the corresponding blots of the upper panels with an antibody against HSP60, an internal control of protein loading. The lanes represent the protein extract (80 μg each) of (1) nontransfected and (2) transfected HEK293 cells. The position of TREM2 is indicated by an arrowhead.

Fig 3: TREM2 immunohistochemistry of Alzheimer's disease (AD) brains. The panels represent serial sections of (a) AD, the hippocampus, Iba1; (b) AD, the same region as (a), HPA010917; (c) AD, the same region as (a), DAP12; (d) AD, the frontal white matter, Iba1; (e) AD, the same region as (d), HPA010917; and (f) AD, the same region as (d), DAP12. In (b), an arrow indicates an intravascular TREM2-expressing monocyte/macrophage.

Fig 4: IL‐33 induces macrophage differentiation through STAT6 signaling. A) GO and KEGG analysis of upregulated genes in the IL‐33 group compared to the PBS group. B) Double IF staining of TREM2 and p‐STAT6 in hind paws of the CAIA model and in spinal ligament tissues from patients with AS, n = 6 per group. Scale bar: 3mm. Scale bar: 200 µm. C) Quantitative analysis of TREM2 and p‐STAT6 colocalization. n = 6 per group. D) Immunoblot analysis of STAT6, p‐STAT6 in BMDMs after IL‐33 treatment for 1 h with or without ST2 antibody. E) ICC staining of BMDMs by p‐STAT6 after IL‐33 treatment for 1 h with or without ST2 antibody (shown is one representative result from n = 5 BRs). Scale bar: 20 µm. F) Flow cytometry analysis of TREM2 expression in IL‐33–induced macrophages from BMDMs with the application of AS1517499 or DMSO for 48 h G) Quantitative analysis of TREM2+ macrophages in (F). H) Flow cytometry analysis of TREM2+ macrophages in the CAIA model with administration of AS1517499 or DMSO for 10 days. I) Quantitative analysis of TREM2+ macrophages in (H). J,K) µCT images and quantitative analysis of pathological new bone formation in CAIA model with administration of DMSO or AS1517499 for 4 weeks. n = 6 per group. Scale bar: 1.5 mm. L) H&E staining and SOFG staining in the hind paws of the CAIA model. Scale bar: 200 µm. Data is shown as mean±SD. ** p < 0.01; ns, not significant (P > 0.05) determined by unpaired, two‐tailed Student's t‐test. AS, ankylosing spondylitis; CAIA, collagen antibody‐induced arthritis; Ctrl, control; Ab, Rat Anti‐Mouse ST2/IL‐33R Monoclonal Antibody; DMSO, dimethyl sulfoxide; AS1517499, STAT6 inhibitor; BV, bone volume; CB, cortical bone; NB, new bone.

Fig 5: TREM2+ macrophages promote pathological new bone formation by secreted CREG1 A) Dynamic network venn diagram of secretory protein upregulated genes form the bulk RNA‐seq of spinal ligament tissue for patients with patients of AS and non‐AS and from the TREM2+ macrophage subsets of the scRNA‐seq dataset. B) Feature plots displaying the single‐cell expression of Creg1. C) Immunoblot analysis of expression of CREG1 in hind paws of control and CAIA model D) Immunoblot analysis of expression of CREG1 in spinal ligament tissues from patients with AS and non‐AS patients. E,F) SOFG staining, IHC analysis, and quantitative analysis of CREG1 in hind paws of CAIA model. n = 6 per group. Scale bar: 200 µm. G,H) SOFG staining, IHC analysis, and quantitative analysis of CREG1 in spinal ligament tissues from patients with AS and non‐AS patients. n = 12 per group. Scale bar: 200 µm. I,J) Double IF staining and quantitative analysis in hind paws of CAIA model and control, including staining for TREM2 and CREG1. Scale bar: 200 µm. K,L) Double IF staining and quantitative analysis in spinal ligament tissues from patients with AS and non‐AS, including staining for TREM2 and CREG1. Scale bar: 200 µm. M) Alizarin Red staining of LDPCs treatment with CREG1 for 14 days. Scale bar: 100 µm. N,O) RT‐qPCR analysis and immunoblot analysis of the level of Runx2, Osx of LDPCs treatment with creg1. P,Q) µCT images and quantitative analysis of pathological new bone formation in CAIA model with the administration of shCtrl or shILcreg1 for 4 weeks. n = 6 per group. Scale bar: 1.5 mm. R) H&E staining and SOFG staining in hind paws of shCtrl or shCreg1 treated CAIA model. Scale bar: 200 µm. Data is shown as mean±SD. * p < 0.05; ** p < 0.01 determined by unpaired, two‐tailed Student's t‐test. AS, ankylosing spondylitis; CAIA, collagen antibody‐induced arthritis; BV, bone volume; CB, cortical bone; NB, new bone.