F. M. Davis1, A. Kimball1, A. Joshi1, A. DenDekker1, A. Obi1, K. Singer2, B. Moore3, S. Kunkel4, K. Gallagher1 2University Of Michigan,Pediatric Endocrinology,Ann Arbor, MI, USA 3University Of Michigan,Microbiology And Immunology,Ann Arbor, MI, USA 4University of Michigan,Pathology,Ann Arbor, MI, USA 1University of Michigan,Surgery,Ann Arbor, MI, USA
Introduction: Macrophages (MΦ) are critical for both the initiation and resolution of the inflammatory phase of wound repair. Toll-like receptors (TLRs), particularly TLR4, play a critical role in regulating macrophage-mediated inflammation and tissue regeneration. Since an initial inflammatory phase is vital for tissue repair, we hypothesized that TLR4 regulates macrophage-mediated inflammation in wound repair.
Methods: Using a murine wound healing model, percent wound healing was analyzed between control and TLR4 deficient (TLR4-/-) mice (n= 10/group). Bone marrow-derived macrophages (BMDMs) were isolated and cultured in standard fashion. Expression of inflammatory genes (IL1β, IL12, TNFα) were determined by qPCR. Chromatin immunoprecipitation was used to analyze H3K4 methylation. Adoptive transfer was performed with intravenous injection of CD11b+ cells isolated from spleens of control and TLR4-/- mice. Statistical significance was determined using Student t-tests or ANOVA.
Results: TLR4 expression in wound MΦs was significantly increased throughout the wound healing course (p<0.05). We identified that changes in MΦ TLR4 during the course of wound healing correlated with increased expression of the histone methyltransferase Mix-lineage leukemia-1 (MLL1) and its activating epigenetic marker, histone 3 lysine 4 tri-methylation (H3K4me3), on the TLR4 promoter. Mice deficient in TLR4 demonstrated delayed wound healing at all time points post-injury. Furthermore, in vitro BMDMs and in vivo wound macrophages isolated from TLR4-/- wounds had decreased inflammatory cytokine production (p<0.05). Importantly, adoptive transfer of monocyte/macrophages from wild-type mice restored normal healing in the TLR4-/- mice.
Conclusion: These results define a role for macrophage specific TLR4 in the inflammatory response following cutaneous tissue injury and suggest that MLL1 regulates TLR4 expression in wound MΦs.
