Macrophage-Specific Nanotherapy Targeting Histone Demethylase JMJD3 Decreases Pathologic Inflammation and STING1 in Diabetic Wounds

Objectives: Macrophage (Mφ) mediated inflammation is controlled by epigenetic modifications, which play an important role in normal and diabetic wound healing. JMJD3, an epigenetic enzyme that demethylates the trimethylated lysine 27 residue on histone 3 (H3K27me3), is upregulated in chronic inflammation. However, its upstream regulation and its role in inflammatory pathways important to wound healing remains unclear. Herein, we show that JMJD3 regulation uses the JAK/STAT3 axis and plays a role in the Nod-like Receptor inflammasome (NLRP3) and the Stimulator of Interferon Genes (STING) inflammatory cascade. In addition to inflammation, these pathways are involved in foreign DNA sensing and cell apoptosis. Finally, we demonstrate that macrophage specific nanoparticle inhibition of JMJD3 in diabetic wounds leads to improved wound healing.

Methods: 4mm punch wounds were created on the dorsum of C57BL/6J mice (n=3-5 per group). Wound Mφs (CD3-,CD19-,Ly6G-,CD11b+) were harvested on day 3 by magnetic sorting (MACS) and levels of Jmjd3 expression and activity were assessed by RT-qPCR and chromatin immunoprecipitation (ChIP). Wound healing curves were obtained by measuring wound surface areas over 7-10 days. Jmjd3 Lyz2Cre+ floxed mice were generated and their wound Mφs analyzed. Diet-induced obesity (DIO) mice were obtained by maintaining male C57BL/6 mice on a standard high fat diet (60% kcal saturated fat, 20% proteins, 20% carbohydrate; Research Diets, Inc) for 12-18 weeks. Levels of NLRP3 inflammasome and STING expression were obtained by a combination of RT-qPCR, ChIP (H3K27me3 levels on the respective promoters) and Western blot. A wound healing curve was obtained following daily wound injection of macrophage specific nanoparticles laden with a JMJD3 inhibitor.

Results: JMJD3 expression is upregulated late in diabetic wound macrophages, with concomitant increase in inflammatory cytokines. Specific inhibition or myeloid depletion of the Jak/Stat3 pathway led to decreased inflammation, decreased Jmjd3 expression and increased H3K27me3 levels. Wound Mφs from interferon receptor knockout (IFNAR-/-) mice prevented Jmjd3 expression and activity. Depletion of JMJD3 from myeloid lineage conferred improved wound healing in the chronic phase of wound healing. Additionally, macrophage specific inhibition of Jmjd3 in diabetic wounds led to significantly improved wound healing in diabetic mice with decreased inflammatory markers for the STING, NLRP3 and NFkB pathways.

Conclusions: Histone demethylase, JMJD3, plays an important role in multiple pathways that sustain chronic Mφ-mediated inflammation in diabetes, and its resulting poor tissue repair. Targeting JMJD3 activity in a Mφ-specific manner is a translationally viable method to improve diabetic wound healing.