Delayed Activation of Innate Immunity in Murine Model of Diabetic Wounds: Initiating An Aberrant Inflammatory Response

Abstract

Introduction: Diabetic wounds are a costly and morbid condition requiring continuous wound care, frequent debridement, and in some cases, amputation. Aberrant immune responses, characterized by delayed infiltration of inflammatory cells, paucity of growth factors and persistence of the inflammatory phase of wound healing, are well-documented manifestations in chronic diabetic wounds. the relationship of these findings has not been well described. Skin resident immune cells function as a first line of defense in the activation of the inflammatory wound-healing cascade. Skin resident γδ T cells, also known as Dendritic Epidermal T cells (DETC), recognize damage-associated molecular patterns. Upon activation, DETC recruit neutrophils and monocytes to the wound bed and stimulate fibroblast and keratinocyte proliferation. Abnormal activation and signaling from these cells would result in poor clearance of pathogens and debris, faulty ECM formation, and delayed keratinocyte migration. We hypothesize that wounds in leptin receptor deficient, diabetic mice will exhibit decreased activation of DETC resulting in atypical macrophage phenotypes. Methods: An excisional wound-healing model was implemented with leptin receptor deficient (db/db) mice and their wild type (WT) littermates. on post-operative days (POD) 3 and 7, wounds were photographed, and skin was harvested. Wound samples were dispersed in an enzyme cocktail to single cell suspension. Cells were stained with two antibody panels for 12-color Flow Cytometry (FC) to identify surface markers for leukocyte subtypes, skin homing, and activation states. Results: the leptin receptor deficient model of Type II Diabetes was validated with serial blood glucose and body mass readings (p<0.05) (n>5; 2-tailed Ttest). Delayed wound healing was observed in the db/db group with widened epithelial gap on photomicroscopy (p<0.05). DETC in wounds of db/db animals had lower levels of surface activation markers CD25 (p=0.03) and CD44 (p=0.02) on POD3. Activation of these cells in skin was comparable to WT on POD7. on POD3, Ly6G+ neutrophils were increased (p=0.006) and activated macrophages (F480+, MHCII+, CD11b high) (p=0.003) were decreased in db/db wounds. Activated macrophages were also decreased in the db/db wound on POD7 (p=0.02). Total wound macrophages were equivocal in both groups on POD3 and POD7. Conclusions: Diabetic mice show an impaired ability to activate DETC upon wounding. Blunted activation of DETC likely leads to delayed recruitment of neutrophils and inflammatory monocytes in the early phases of wound healing. the result is persistent neutrophils and less activated macrophages in the wound on POD3 and 7. Ineffective macrophage activation could lead to poor clearance of wound debris and pathogens as well as altered signaling within the microenvironment of the wound. These findings could explain the improper regulation of the inflammatory wound-healing cascade and the clinical manifestations of chronic diabetic wounds.