(WHS-L2.02) SELECTIVE AGONISM OF HISTAMINE RECEPTORS AUGMENTS TISSUE REPAIR
Thursday, May 16, 2024
10:30 AM – 11:30 AM East Coast USA Time
Purpose: Histamine is produced by mast cells and non-canonically by other cells during tissue injury. Four G-protein coupled receptors for histamine (HRH1-4) regulate diverse vascular, fibrotic, and immune signaling. Histamine signaling is critical to healthy wound healing and is dysregulated in diabetic wounds. To understand the role of specific histamine receptors during tissue repair, we performed a systematic investigation of HRH agonism during wound closure in vitro and biomaterial-assisted incisional wound repair in vivo. Methodology: Incisions were made in Balb/c mice and sealed with silk fibroin-based laser-activated sealants (LASE) with codelivery of saline, histamine, or selective agonists for HRH1 (2-pyridylethylamine), HRH2 (dimaprit), HRH3 (immethridine), or HRH4 (4-methylhistamine), hereafter referred to as HRHn-ag (where n is 1-4). Trans-epidermal water loss (TEWL) was measured daily for 3 days. Ultimate tensile strength (UTS) was measured after histamine treatment. IHC was performed for HRH1-4 post-wounding, and for M1/M2 macrophage and neutrophil infiltration, angiogenesis, proliferation, and EMT. Expression of HRH1-4 in HaCaT keratinocytes was evaluated by immunofluorescence and western blot. Migration in response to histamine or agonists was performed by scratch assay.
Results: Histamine treatment of LASE-sealed incisional wounds results in a significant early increase in UTS without alteration of TEWL vs. saline-treated wounds. In contrast, selective agonism of HRHs results in diverse TEWL responses: HRH1-ag (N=3) increased TEWL (p < 0.05), HRH2-ag (N=3) and HRH4-ag (N=3) decreased TEWL (p < 0.05), and HRH3-ag (N=4) causing an initial increase in TEWL (p < 0.001) with a return to physiologic levels after day 1. We found that HaCaT cells express all HRHs. Interestingly, while histamine (p < 0.01) and HRH1-ag (p < 0.0001) accelerate scratch closure, HRH2-ag almost completely prevents migration (p < 0.0001). IHC for tissue responses during repair indicated a pro-resolution macrophage environment (high Arg1, low CD86) with HRH1-ag and HRH4-ag (p < 0.01), and a milder response by HRH2-ag (p < 0.05) and no response by HRH3-ag. HRH2-ag and HRH4-ag robustly suppressed neutrophil presence (p < 0.0001), with a milder response by HRH1-ag (p < 0.01) and no response by HRH3-ag. Angiogenesis (CD31+ vessels) was stimulated by HRH1-ag (p < 0.01) and HRH4-ag (p < 0.0001), with no response by HRH2-ag and HRH3-ag. Epidermal EMT (loss of E-cadherin integrity) was promoted by HRH1-ag (p < 0.05) and suppressed by HRH2-ag (p < 0.01), with no response by HRH3-ag and HRH4-ag. Keratinocyte proliferation was unaffected by all receptors.
Conclusion: HRH1 and HRH4 activation drive a pro-resolution immune response with enhanced angiogenesis, while HRH1 also stimulates increased epidermal EMT. HRH2 activation slows epidermal healing, and HRH3 activation has minimal effect on healing.