Alcohol intoxication prior to traumatic injury delays wound healing and increases the risk for wound infection. Since over half of trauma patients have blood alcohol levels at or above the legal limit, it is imperative to understand how ethanol exposure and the products of its metabolism, including reactive oxygen species (ROS), impair normal wound healing and antimicrobial responses. Although alcohol exposure and wound healing have been extensively studied independently, few reports have directly evaluated the impact of ethanol exposure on wound infection. This highlights a considerable breach in our understanding of how ethanol exposure influences antimicrobial responses during wound infection, and also identifies an important niche for clinical and translational research. Delayed wound closure and reduced antimicrobial responses contribute to bacterial susceptibility in the skin. Antimicrobial peptides (AMPs), such as cathelicidin (human:CAMP;mouse:CRAMP), are key innate immune system elements that have the capacity to combat microbes directly, initiate wound healing processes, and maintain cutaneous barrier function. Toll-like Receptor-2 (TLR2), present on epidermal keratinocytes, detects bacterial membrane components and ROS to stimulate AMP production at the site of injury or infection. In parallel, TLR2 aids microbial resistance by maintaining epidermal barrier integrity. Ethanol can increase barrier permeability in several epithelial tissues, but its effects on skin barrier function are unknown. Furthermore, factors that promote gene transcription through histone modifications, such as histone acetyltransferases (HATs) are decreased by ethanol exposure and may suggest a mechanism for impaired wound healing through AMP transcription suppression. Therefore, we hypothesize that ethanol exposure prior to wound infection inhibits TLR2-mediated AMP production and epithelial barrier integrity to delay wound closure and increase bacterial survival. Our previous work establishes that ethanol exposure delays wound closure and wound inflammation. Our rationale for this work is that there are no published reports investigating alcohol exposure and epidermal TLR2-mediated AMP and barrier responses, particularly in the field of epigenetics. Driven by convincing proof-of-concept data, our hypothesis will be tested by addressing two Specific Aims: 1) To characterize the role of TLR2 and its co-factors in ethanol-mediated reduction of CRAMP and permeability barrier function in infected mouse wounds and primary human keratinocytes. 2) To determine if ethanol-mediated suppression of CAMP is facilitated by epigenetic histone modifications of CAMP in primary human keratinocytes and establish whether topical histone deacetylase inhibitors (HDACi) restores CRAMP expression and decreases bacterial survival in infected wounds from ethanol-treated mice. Our approach is innovative, as these results will provide a fundamental understanding of how ethanol exposure dampens TLR2 responses during wound infection and may indicate HDACi as a novel therapy for chronic or infected wounds in intoxicated patients and in the wound care population as a whole.
The proposed investigations are directly relevant to improving overall human health since we will explore novel pathways for alcohol-mediated delays in wound repair processes and suppression of microbial responses. The results from these studies are likely to identify potential therapeutic regimens for chronic non-healing wounds. The anticipated research is relevant to the NIH's mission to reduce global economic and social hardship by advancing current therapies and preventative measures related to alcohol use disorders, ultimately improving the overall quality of public health.
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