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Epidermal Nrf2 Orchestrates Tissue Regeneration Through Regulation Of Ccl2
Alvaro Villarreal-Ponce, PhD1, Melat Tiruneh-Worku, MS2, Jasmine Lee, BA1, Kristen Dammeyer, BS1, Christian Guerrero-Juarez, PhD3, Joe Kuhn, MD1, Jennifer Kwong, BA1, Piul Rabbani, PhD1, Daniel J. Ceradini, MD1.
1NYU Langone Health, New York, NY, USA, 2Rutgers Robert Wood Johnson School of Medicine, New Jersey, NJ, USA, 3UC Irvine, Irvine, CA, USA.

PURPOSE:
Type 2 diabetic (T2D) chronic wounds affect more than 4.5M people in the US and remain the leading cause of non-traumatic amputations. While many signaling pathways are implicated as responders to tissue damage, our work has demonstrated that poor diabetic wound healing is partly due to mismanagement of intracellular oxidative stress that results from dysfunction in the Nrf2/Keap1 signaling pathway. This study focuses on uncovering the molecular mechanisms orchestrated by epidermal Nrf2 that are critical for initiating a regenerative response, and are defective in diabetic wounds.
METHODS:
T2D mice (Leprdb/db) were used to describe dysfunctional cellular and molecular programs that impair wound healing. To investigate epidermal Nrf2 function in the context of tissue regeneration in vivo, we generated inducible transgenic mice to conditionally ablate Nrf2 expression in K14+ basal keratinocytes (K14 CreER; Nrf2fl/fl; cKO) upon administration of tamoxifen. Ten-millimeter diameter, stented, full-thickness excisional wounds were created on the dorsum of 6-8-week-old WT, diabetic, and cKO mice to examine wound closure kinetics and to analyze histologically. To mechanistically understand epidermal Nrf2 function, we isolated WT and cKO wound-associated keratinocytes by flow cytometry and performed RNA-seq, further coupling with chromatin immunoprecipitation (ChIP), immunohistochemistry, qPCR/protein expression analysis, and functional assays.
RESULTS:
Nrf2 expression analysis in full-excisional wounds reveals nuclear translocation of Nrf2 is defective in wound edge-associated keratinocytes of diabetic mice (WT 97.03.6% vs. diabetic 22.516.4%; p<0.05). We demonstrate the functional importance of epidermal Nrf2 in wound regeneration, as its induced deletion severely delays wound closure to levels observed in diabetic mice (WT 15.61.2days vs. cKO 341.7days vs. diabetic 300days; p<0.0001). Histologic assessment of the gross wound reveals not only impaired re-epithelialization (p<0.0001), but also reduced neovascularization (p<0.05) and collagen maturation, suggesting its governing role of both keratinocyte and non-keratinocyte autologous regenerative programs.
Transcriptome-wide analysis (2-fold differential expression; q<0.05) coupled with ChIP enrichment analysis corroborates our finding, showing epidermal Nrf2 directly regulates expression of not only oxidoreductase-related genes, but also those affecting many paracrine signaling-mediated regenerative responses, including inflammation, immune-cell guidance, extracellular structure, and angiogenesis (p<0.05). The significance of epidermal Nrf2-mediated intercellular communication is demonstrated through a prominent defect in monocyte/macrophage trafficking, observed during early (130.315.0cells/area vs. 352.6cells/area; p<0.05) and later phases of repair (p<0.05). This defect results from downregulated expression of chemokine Ccl2 in cKO wounds, which we find to possess a Nrf2-binding motif that exhibits dynamic Nrf2 binding upon wounding. Induced expression of Nrf2 in primary keratinocytes results in Ccl2 upregulation, and its application is sufficient for restoring physiologic wound regeneration in cKO wounds (Vehicle: 29.71.73days vs +Ccl2: 17.30.6days; p<0.0001).
CONCLUSION:
In-depth analysis of Nrf2 in the wound environment uncovers an indispensable role of epidermal Nrf2 in regulating initiation of the regenerative response that is critical for physiologic wound repair. We find epidermal Nrf2 is necessary for mediating paracrine crosstalk between keratinocytes and monocytes/macrophages, specifically through the direct regulation of Ccl2 which promotes immune cell guidance to the wound edge. Together, our findings provide the basis for continued investigation on the therapeutic value of Nrf2 in restoring diabetic wound regeneration.


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