(WHS-I.08) The histone methyltransferase Whsc1 regulates TGFβ-driven macrophage to myofibroblast transition during wound healing
Wednesday, May 15, 2024
1:45 PM – 4:00 PM East Coast USA Time
Introduction: Diabetes contributes to significant morbidity and mortality in the United States, and one of the major features includes poor wound healilng, which leads to high rates of amputation due to ineffective treatment options. In diabetes, macrophages exhibit a pro-inflammatory phenotype that prevents normal wound healing. During normal tissue repair, macrophages undergo transition to myofibroblasts, which aid in late wound repair by promoting wound contraction and closure. Our lab and others have shown that macrophages express key fibrotic genes (e.g., SMA, Col1a1, Col3a1), which increase throughout wound healing. However, this fibrotic gene program is disrupted in diabetic macrophages, and fibroblast numbers are reduced in diabetic wounds. Here, we identify Whsc1 as a TGFβ-dependent histone methyltransferase that is essential for macrophage to myofibroblast transition (MMT), and we show that Whsc1 is disrupted in diabetic macrophages.
Methods: The goal of this study was to identify TGFβ-dependent transcription mechanisms that regulate macrophage phenotype during wound healing. Bone marrow derived macrophages (BMDMs) and wound macrophages (CD11b+CD3-CD19-NK1.1-Ly6G-) were isolated for experiments from a diet-induced obese (DIO) diabetic mouse model and compared with those from normal diet (ND) mice. We performed an epigenetic superarray to identify key chromatin modifying enzymes in macrophages from murine wounds treated ex vivo with TGFβ. siRNA knockdown of Whsc1 and NFkβ p65 (RelA) and downstream chromatin immunoprecipitation (ChIP) and qPCR were also used.
Results: Whsc1 was significantly decreased in DIO macrophages with TGFβ treatment compared to ND macrophages (p < 0.05). siRNA knockdown of Whsc1 in BMDMs decreased TGFβ-dependent expression of fibrotic (SMA, Col1a1, Col3a1) genes (p < 0.05). In ChIP experiments, Whsc1 and its mark H3K36me2 were enriched at fibrotic promoters in ND macrohages in response to TGFβ but decreased in DIO BMDMs. The Whsc1 inhibitor LEM-14 significantly increased wound size and reduced fibrotic gene expression in BMDMs. Interestingly, the pro-inflammatory transcription factor RelA bound more strongly to Whsc1 in DIO BMDMs. Finally, siRNA knockdown of RelA reversed the dysregulated effect of TGFβ on fibrotic gene expression in DIO BMDMs by increasing Whsc1 enrichment at fibrotic promoters.
Conclusion: The current study delineates the TGFβ-Whsc1 signaling axis, which is altered in the diabetic macrophage, thereby leading to defective MMT. Specifically, we show that in ND macrohages Whsc1 controls expression of the fibrotic gene program; however, in DIO macrophages RelA inhibits Whsc1 enrichment at fibrotic gene promoters. Future studies will test whether macrophage-specific targeting of the Whsc1-RelA interaction improves diabetic wound healing in vivo.