The Histone Methyltransferase MLL1 Directs Macrophage-Mediated Inflammation in Wound Healing and Is Altered in a Murine Model of Obesity and Type 2 Diabetes

Abstract

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)-mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1^f/fLyz2^Cre+ ) to determine the function of MLL1 in wound healing. Mll1^f/fLyz2^Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1^f/fLyz2^Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

Figure 1

MLL1 is upregulated in macrophages in the early inflammatory phase of wound healing. A: Wounds were created by 4-mm punch biopsy on the back of C57BL/6 mice. Wounds were harvested on days 1–5 postwounding. Wound macrophages CD11b⁺[CD3⁻CD19⁻Ly6G⁻] were isolated by MACS. Mll1 gene expression was quantified by qPCR over time (n = 16, repeated two times) B: Wounds were created by 4-mm punch biopsy on the back of C57BL/6 mice. Wounds were harvested on days 1, 3, 5, and 7 postinjury; cells were isolated; and single-cell suspensions were processed for flow cytometry. Pseudocolor plots and data analysis of MLL1⁺ cells as a percentage of live, lineage⁻, Ly6G⁻, CD11b⁺ cells (n = 20, repeated one time). C: Mll1^+/− heterozygote and Mll1^+/+ wild-type mice were wounded, and Mll1 expression was examined by qPCR (n = 10, repeated one time). D: Wound healing model. Wound closure was monitored daily with photographs of the mice by using an internal scale, and wound areas were measured with ImageJ software. E: Mll1^+/− heterozygote and Mll1^+/+ wild-type mice were wounded and images recorded until day 3 postinjury. Scatter plot of Mll1^+/− heterozygote and Mll1^+/+ wild-type wound area at day 3 and representative photographs of wounds at days 0 and 3 (n = 10, repeated one time). Statistical analysis was by Student t test. Data are mean ± SEM. FSC-A, forward scatter area.

Figure 2

Wound healing is impaired in macrophage-specific MLL1-deficient mice (Mll1^f/fLyz2^Cre+). We generated mice deficient in Mll1 in cells of myeloid lineage with lysosomes (monocytes, macrophages, granulocytes) by using the Cre-lox system. A: Myeloid depletion of Mll1 was examined by qPCR in MACS splenic and wound macrophages CD11b⁺[CD3⁻CD19⁻Ly6G⁻] from Mll1^f/fLyz2^Cre+ mice and littermate controls (Mll1^f/fLyz2^Cre−) (n = 10, repeated one time). B: Depletion of MLL1 was examined by flow cytometry in nonmyeloid Lin⁺ cells (CD3, CD19, NK1.1, Ter-119)⁺ and CD11b⁺, Ly6C^Hi cells from spleens of Mll1^f/fLyz2^Cre+ mice and littermate controls (Mll1^f/fLyz2^Cre−). C: Wounds were created by 4-mm punch biopsy on the backs of Mll1^f/fLyz2^Cre+ mice and littermate control mice. The change in wound area was recorded daily with ImageJ software until complete healing was observed. Representative photographs of the wounds of Mll1^f/fLyz2^Cre+ mice and littermate controls on days 0 and 3 postinjury are shown (n = 20, repeated three times). D: Wounds were created by 4-mm punch biopsy on the backs of Mll1^f/fLyz2^Cre+ mice and littermate control mice. Wounds were harvested on day 2, paraffin embedded, and sectioned. Sections (5 μmol/L) were stained with hematoxylin-eosin (H&E) and Masson’s trichrome. Percent reepithelialization was calculated by measuring the distance traveled by epithelial tongues on both sides of the wound divided by total distance for full reepithelialization. Representative images are shown (n = 10, repeated one time). E: CD3⁻CD11c⁻CD19⁻Ly6G⁻NK1.1⁻CD11b⁺ single-cell suspensions were isolated from Mll1^f/fLyz2^Cre+ and Mll1^f/fLyz2^Cre− spleens by MACS. Cells (1 × 10⁶) were injected intravenously in wounded (day 1) Mll1^f/fLyz2^Cre− mice, and wound closure was measured daily with ImageJ software (n = 15). Statistical analysis was by Student t test. Data are mean ± SEM. FSC-A, forward scatter area.

Figure 3

BMDMs from mice insufficient for the histone methyltransferase MLL1 demonstrated decreased inflammatory cytokine expression in vitro. Cultured BMDMs harvested from Mll1^f/fLyz2^Cre+ mice and littermate controls were stimulated with LPS (100 ng/mL) for 6 h and then collected for analysis. A: IL1β, TNFα, and NOS2 gene expression was quantified by qPCR (n = 9, repeated two times in triplicate). B: NO assay in BMDM supernatants (n = 16, repeated one time in triplicate). Statistical analysis was by Student t test. Data are mean ± SEM.

Figure 4

Macrophages isolated from wounds of Mll1^f/fLyz2^Cre+ mice display decreased inflammatory cytokine gene expression and protein levels. A: Wound macrophages were isolated from Mll1^f/fLyz2^Cre+ mice and littermate controls at day 2 postinjury by MACS for CD11b⁺[CD3⁻CD19⁻Ly6G⁻] cells. TNFα and IL1β gene expression in isolated macrophages was measured by qPCR (n = 12, repeated two times). B: Mll1^f/fLyz2^Cre+ and littermate control wound cell isolates were processed for ex vivo intracellular flow cytometry after stimulation with LPS (100 ng/mL) for 2 h. The gating strategy used for ex vivo intracellular flow cytometry selecting live, lineage⁻, Ly6G⁻, CD11b⁺, Ly6C^Hi cells is shown. Flow cytometry quantification of IL-1β and TNF-α in wounds (n = 10, repeated one time). C: IL-1β protein levels in wound cell lysates from Mll1^f/fLyz2^Cre+ mice and littermate controls by Western blot (n = 20, repeated one time). Statistical analysis was by Student t test. Data are mean ± SEM. FMO, fluorescence minus one; FSC, forward scatter; FSC-A, forward scatter area; FSC-H, forward scatter height; MΦ, macrophage; SSC, side scatter.

Figure 5

MLL1 mediates wound macrophage inflammation by increasing H3K4me3 at the NF-κB transcription factor binding site. A: ChIP analysis for H3K4me3 at the NF-κB binding site of the TNFα and IL1β promoters in Mll1^f/fLyz2^Cre+ and littermate control BMDMs (n = 10, repeated two times in triplicate). B: Wound macrophages were isolated from Mll1^f/fLyz2^Cre+ mice and littermate controls at day 2 postinjury by MACS for CD11b⁺[CD3⁻CD19⁻Ly6G⁻] cells. ChIP analysis for H3K4me3 at the NF-κB binding site of the TNF-α and IL-1β promoters in macrophages isolated from the wounds of Mll1^f/fLyz2^Cre+ and littermate controls was performed (n = 10, repeated two times). For all ChIP experiments, isotype control antibody to IgG was run in parallel. Statistical analysis was by Student t test. Data are mean ± SEM. MΦ, macrophage.

Figure 6

Macrophages isolated from DIO mice display decreased early inflammatory cytokine production, MLL1, and H3K4me3 at inflammatory gene promoters. A: DIO and control murine wounds were harvested 2 days after injury, and wound cells were isolated. Single-cell suspensions were processed for flow cytometry. Density plots and associated data for IL-1β and TNF-α staining in live, lineage⁻, Ly6G⁻, CD11b⁺, Ly6C^Hi cells (as gated in Fig. 4) from DIO and control wounds (n = 12, repeated one time). B: DIO and control wounds were harvested 2 days after injury for cell isolation and macrophage selection for CD11b⁺[CD3⁻, CD19⁻, Ly6G⁻] cells through MACS. MLL1 expression as measured by qPCR from wound macrophages (n = 14, repeated one time). C: Pseudocolor plots and data analysis of wound MLL1⁺, CD11b⁺, Ly6C^Hi cells as a percentage of live cells in control and DIO mice at day 2 postinjury. Mll1^f/fLyz2^Cre+ and littermate control spleens were used as staining controls for MLL1 (n = 10; repeated one time). D and E: DIO and control wounds were harvested 2 days after wounding for cell isolation and macrophage selection for CD11b⁺[CD3⁻, CD19⁻, Ly6G⁻] cells through MACS. ChIP analyses of wound macrophages for H3K4me3 at the NF-κB binding sites of the promoters of IL1β (D) and TNFα (E) were performed (n = 12, repeated two times). Statistical analysis was by Student t test. Data are mean ± SEM. FMO, fluorescence minus one; MΦ, macrophage; WT, wild type.

Figure 7

MLL1 upregulation in DIO mice and consequent epigenetic changes are associated with increased inflammation that is reversible with an MLL1 inhibitor. A: DIO and control wounds were harvested 5 days after injury for cell isolation and macrophage selection for CD11b⁺[CD3⁻, CD19⁻, Ly6G⁻] cells through MACS. MLL1 expression as measured by qPCR from wound macrophages (n = 12, replicated two times). B: DIO and control murine wounds were harvested 5 days after injury, and wound cells were isolated and processed for flow cytometry. Pseudocolor plots and data analysis of MLL1⁺, CD11b⁺, Ly6C^Hi cells as a percentage of live cells in DIO and control wounds at day 5 postinjury. Mll1^f/fLyz2^Cre+ and littermate control spleens were used as staining controls for MLL1 (n = 10). C: DIO and control wound cell isolates were collected at day 5 postinjury and processed for ex vivo intracellular flow cytometry after 2 h of LPS (100 ng/mL) stimulation. Density plots and associated data for IL-1β and TNF-α staining in live, lineage⁻, Ly6G⁻, CD11b⁺, Ly6C^Hi cells (as gated in Fig. 4) (n = 10, repeated one time). D and E: DIO and control wounds were harvested 5 days after injury for wound cell isolation and macrophage selection for CD11b⁺[CD3⁻, CD19⁻, Ly6G⁻] cells through MACS. ChIP analyses of wound macrophages for H3K4me3 at the NF-κB binding sites of the promoters of IL1β (D) and TNFα (E) were performed (n = 15, repeated two times). F: BMDMs from DIO mice were cultured in the presence of an MLL1 inhibitor, MI-2, for 24 h followed by stimulation with LPS (100 ng/mL) for 6 h. qPCR was performed for expression of IL1β and TNFα (n = 6, repeated two times in triplicate). Statistical analysis was by Student t test. Data are mean ± SEM. FMO, fluorescence minus one; MΦ, macrophage; WT, wild type.

Figure 8

Human monocytes from patients with T2D display increased Mll1. Peripheral blood (30 mL) was collected from patients with T2D and control subjects without diabetes. No statistical differences were found between groups with respect to sex, age, or comorbid conditions. Peripheral blood mononuclear cells underwent red blood cell lysis followed by Ficoll separation. CD14⁺ monocytes were then positively selected by MACS, and Mll1 gene expression was measured by qPCR (n = 11). Statistical analysis was by Student t test. Data are mean ± SEM.