Fig. 3.
STING-mediated inflammation is regulated by JMDJ3 in wound Mφs. A Bulk RNA sequencing analysis of wound Mφs (CD3-/CD19-/NK1.1-/Ly6G-/CD11b+) isolated on Day 5 from Jmjd3f/fLyz2Cre+ mice and littermate controls with Tmem173 gene expression (N = 4/group). B, C Tmem173 expression and ChIP analysis of H3K27me3 at the Tmem173 promoter in Jmjd3f/fLyz2Cre+ and littermate control wound Mφs (N = 4/group, pooled, repeated in triplicate). D Human single-cell RNA sequencing dot plot demonstrating Tmem173 gene expression within the Mφ population in human T2D and non-T2D wound samples. Cluster analysis UMAP shown above in (2 A). The dot size corresponds to the proportion of cells within the group expressing each transcript, while the dot color corresponds to the expression level. E Tmem173 gene expression from murine diabetic wound Mφs harvested on Days 1–10 compared with that of the nondiabetic controls (N = 4/group, repeated once). F Immunofluorescence against phospho-STING antibody (FITC) in ND and DIO wound Mφs. G Quantification of the immunofluorescence intensity of phospho-STING (N = 3–5 cells/group). H Wound healing curve for DIO STING knockout (DIO Tmem173−/−) mice and littermate controls (DIO Tmem173+/+), with representative healing images on Days 0 and 5 post-injury (6 mm punch wounds; 3–4 mice/group; repeated once). Wounds were harvested on Day 5, paraffin embedded, and stained with Masson’s trichrome stain (N = 3 mice/group). Representative images are shown at ×2 magnification. The black horizontal bar above the wound represents the entire wound distance, the epithelial tongues are denoted by arrowheads, and the asterisk (*) denotes wound debris. The scale bar represents 500 μm. I Collagen quantification of trichrome staining in DIO Tmem173+/+ and DIO Tmem173−/− mice (N = 4 wounds/strain; repeated once). J Il6 mRNA expression in Day 5 wound Mφs from DIO Tmem173+/+ and DIO Tmem173-/- mice. (N = 4 mice/group, pooled, repeated in triplicate). K Il1b mRNA expression in Day 5 wound Mφs from DIO Tmem173+/+ and DIO Tmem173−/− mice. (N = 4 mice/group, pooled, repeated in triplicate). L Tnfa mRNA expression in Day 5 wound Mφs from DIO STING+/+ and DIO STING−/− mice. (N = 4 mice/group, pooled, repeated in triplicate). M Mrc1 mRNA expression in Day 5 wound Mφs from DIO Tmem173+/+ and DIO Tmem173−/− mice. (N = 4 mice/group, pooled, repeated in triplicate). N ChIP analysis of H3K27me3 at the Tmem173 gene promoter in ND and DIO wound Mφs (N = 4/group, pooled, repeated in triplicate). O Tmem173 expression in wound Mφs following rIL-6 stimulation (20 nM) for 4 h (N = 5/group, pooled, repeated in triplicate). P Immunofluorescence against phospho-STING antibody (FITC) in ND wound Mφs ± rIL-6 stimulation (20 nM; 1 hr) and in Jmjd3f/fLyz2Cre+ wound Mφs treated with rIL-6 (20 nM; 1 h). Q Quantification of the immunofluorescence intensity of phospho-STING (FITC) by ImageJ (NIH) (N = 3–5 cells/group). R Tmem173 expression in wound Mφs following rIL-6 stimulation (20 nM) for 6 h with and without JAK1/3 inhibition (tofacitinib; 100 μM; N = 3/group, pooled, repeated in triplicate). S ChIP analysis of H3K27me3 on the Tmem173 promoter in wound Mφs following rIL-6 stimulation (20 nM) for 4 h with and without Jak1/3 inhibition (tofacitinib; 100 μM, N = 4/group, pooled, repeated in triplicate). T Tmem173 expression from Stat3f/fLyz2Cre+ and littermate control wound Mφ following rIL-6 stimulation (20 nM) for 4 h (N = 3/group, pooled, repeated in triplicate). U ChIP analysis of H3K27me3 on the Tmem173 gene promoter from Stat3f/fLyz2Cre+ and littermate control wound Mφs following rIL-6 stimulation (20 nM) for 4 h (N = 6/group, pooled, repeated in triplicate). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as the mean ± SEM