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Staphyloccocus aureus Biofilms Impair Reepithelialization and Granulation Tissue Deposition in Cutaneous Wounds via a MyD88-Dependent Mechanism
Eugene Park, MD, Seok J. Hong, PhD, Claudia Chavez-Munoz, MD, PhD, Wei Xu, PhD, Thomas A. Mustoe, MD, Robert D. Galiano, MD.
Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Toll-like receptors (TLRs) have mainly been studied for their role in the innate immune response, but they have also been shown to affect wound healing. The effects of TLR function on wound healing in the presence of bacterial biofilms have not been previously studied. In this study, we investigate the role of MyD88, an adaptor protein critical to the function of multiple TLRs, in the impairment of wound healing caused by Staphylococcus aureus biofilms.
In vivo wound healing was analyzed using a murine model of S. aureus biofilm previously developed by our group. Six mm excisional wounds were created on the backs of MyD88 -/- mice and analyzed over time using a splinted wound model to minimize wound contraction. Wound healing was quantified by photo planimetry and by measuring epithelial gaps and granulation tissue thickness in cross-section using paraffin sections with immunohistochemical staining. In vitro scratch assays to measure cell migration were performed using the HaCat keratinocyte cell line and S. aureus biofilm exudate collected using a flow chamber model of bacterial biofilm formation.
MyD88 -/- mice (n=10) displayed delayed wound healing compared to wild-type C57bl/6 (WT) mice (n=9) as measured by the ratio of final wound size to initial wound size on both post-operative day (POD) 6 (WT: 0.710.16 vs. MyD88 -/-: 0.950.25; p<0.05) and POD 8 (WT: 0.510.23 vs. MyD88 -/-: 0.800.21; p<0.0001). Wound reepithelialization in WT mice (n=5) was delayed in the presence of S.aureus biofilm compared to non-biofilm controls on both POD 7 (p<0.05) and POD 10 (p<0.05). In MyD88 -/- mice (n=5), there was no significant difference in reepithelialization between biofilm and non-bioflim wounds on either POD 7 or POD 10. Granulation tissue deposition in non-biofilm wounds (n=5) was greater in WT mice compared to MyD88 -/- mice (p<0.001). In the presence of biofilm, this difference was no longer seen. Cell migration was delayed in MyD88-knockdown HaCat cells compared to WT (n=5) at both 16 hours (p<0.01) and 32 hours (p<0.05). In the presence of S. aureus biofilm exudate, migration was further impaired in WT (p<0.05) but not MyD88-knockdown cells. On immunofluorescence staining, WT biofilm wounds displayed 4-fold greater macrophage recruitment at POD 3 than MyD88 -/- biofilm wounds (p<0.001). Gross examination of immunofluorescence staining on POD 7 revealed greater M1-phase activation of macrophages in WT biofilm wounds compared to MyD88 -/- biofilm wounds. Transcription analysis revealed a higher upregulation of iNOS (p<0.01) and CCL2 (0<0.05) in WT biofilm wounds compared to MyD88 -/- biofilm wounds. Col1A1a expression was more highly upregulated in MyD88 -/- biofilm wounds (0<0.05).
In the presence of a S. aureus biofilm, MyD88 influences wound healing impairment by affecting keratinocyte migration, granulation tissue deposition, and the regulation of inflammation. By targeting the innate immune system, we may one day be able to develop therapeutics that control the host response to bacterial biofilms and improve biofilm wound healing.
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