Fig. 1
Inhibition of SIKs promotes macrophage efferocytosis and regulates CD4+ T cell immune responses. a Gene expression patterns of SIKs and efferocytosis-related transcripts in synovial tissue during collagen-induced arthritis (CIA) progression (GSE13071) and clinical scores of female CIA mice injected with vehicle (n = 9), a pan SIK inhibitor YKL-05-099 (20 mg/kg, n = 10), or a SIK3-selective inhibitor Pterosin B (20 mg/kg, n = 10). Hindpaws of normal DBA1/J or CIA mice were fixed and scanned with μCT, followed by immunohistochemistry to identify cartilage damage (Safranin O, violet indicates cartilage) and inflammation score (cell infiltration). Arrows indicate the hollow region due to osteolysis. Immunofluorescence analyses for the presence of apoptotic cells (TUNEL+) and F4/80+ macrophages among the infiltrated cells (DAPI+). b Expression of SIKs in non-efferocytic and efferocytic BMDMs analyzed using scRNAseq data (GSE180638), and experimental comparisons of efferocytic efficiency (DIO engulfment) over 24 h in BMDMs treated with YKL-05-099 (1 μM). An ingenuity pathway analysis (QIAGEN, IPA) on the bulk mRNAseq dataset obtained from efferocytic BMDMs treated with YKL-05-099. NaN indicates non-available number. Luciferase reporter activities of nuclear peroxisome proliferator-response elements (PPREs) and efferocytic efficacy in p300-knockdowned BMDMs. c Relative average expression of SIKs, MERTK, and TIMD4, and the frequency of expressing cells according to disease status. MTX, methotrexate. The relationship between SIK3 expression and MERTK or TIMD4 expression in RA patients STMs. Percentages of STM subsets, classified based on SIK3 and MERTK expressions, in RA patients. Gene ontology (GO) analysis related to biological process (BP) of the STM subsets. d SIK3 expression in BMDMs challenged with each cytokine (10 ng/ml) or LPS (100 ng/ml) for 24 h. SIK3 levels in BMDMs alone or co-cultured with lipophilic dye-labeled apoptotic cells. Frequency of efferocytic macrophages (DIO+) in BMDMs treated with YKL-05-099 (1 μM) or Pterosin B (20 μM) for 4 h. Expression of PD-L1/L2 in BMDMs challenged with vehicle or YKL-05-099 for 24 h and PTEN level of CD4+ T cells in αCD3e-activated splenocytes treated with vehicle or YKL-05-099 for 72–96 h. Number of CD4+ Foxp3+ Treg in macrophage-naïve CD4+ T cell cocultures, and effects of PD-L1 and PD-L2 blocking antibodies on YKL-05-099-mediated Treg generation. The image of (d) in the middle was created with the assistance of BioRender (https://www.biorender.com/). Data are expressed as the mean ± S.D. and p-values were calculated using the unpaired parametric Welch’s corrected t-test (two-tailed). (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)
