Fig. 2
Jmjd3 increases late in diabetic wound Mφs. A Cluster analysis UMAP of single-cell RNA sequencing from human T2D and non-T2D wounds showed 10 unique cell clusters (representative). Dot plot demonstrating JMJD3 expression within the Mφ population in human T2D and non-T2D wound samples (N = 42). The dot size corresponds to the proportion of cells within the group expressing each transcript, while the dot color corresponds to the expression level. B DIO and ND wound Mφs (CD3-/CD19-/NK1.1-/Ly6G-/CD11b+) were harvested on Days 1–10 and analyzed for Jmjd3 expression by RT‒PCR (N = 4/group, repeated once). C Human bulk RNA sequencing heatmap reflecting the expression profiles for selective genes (rows) across different samples (columns; stratified by T2D status) from acute inflammatory response Gene Ontology pathway analysis with upregulation of IL-6 in T2D wounds compared to control wounds (N = 42). D DIO wound Mφs were isolated on Day 5, treated ex vivo for 6 h with recombinant IL-6 (rIL-6; 20 nM) with and without IL-6 receptor inhibition (LMT-28; 200 nM) and analyzed for Jmjd3 expression (N = 3–5/group, pooled, repeated in triplicate). E ChIP analysis for H3K27me3 on the Tnfa and Il1b promoters from diabetic wound Mφs with and without IL-6 receptor inhibition (LMT-28; 200 nM; N = 3–5/group, pooled and repeated in triplicate). F Human single-cell RNA sequencing dot plot demonstrating Jak/Stat gene expression within the Mφ population in human T2D and non-T2D wound samples. (Cluster analysis UMAP shown above in (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. G Jmjd3 expression following rIL-6 stimulation (20 nM) and Jak1/3 inhibition (tofacitinib; 100 μM) in diabetic wound Mφs isolated from Day 5 wounds and treated ex vivo for 4 h. (N = 5/group, pooled and repeated in triplicate). H ChIP analysis for H3K27me3 on the Tnfa and Il1b promoters from diabetic wound Mφs following ex vivo rIL-6 stimulation (20 nM) with and without JAK1/3 inhibition (tofacitinib; 100 μM) for 4 h (N = 5/group, pooled, repeated in triplicate). I Jmjd3 expression in DIO Stat3f/fLyz2Cre+ and DIO littermate control wound Mφs following ex vivo rIL-6 stimulation (20 nM) for 6 h. (N = 3/group, pooled, repeated in triplicate). J ChIP analysis for H3K27me3 on Tnfa and Il1b promoters from DIO Stat3f/fLyz2Cre+ and DIO littermate control wound Mφs following rIL-6 stimulation (20 nM) for 6 h (N = 4/group, pooled, repeated in triplicate). Data are presented as the mean ± SEM. All data are representative of 2–4 independent experiments
