Exosomes in wound healing

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ABSTRACT Chronic wounds represent a major healthcare burden. One of the common complications of non- healing chronic diabetic wounds is stalled inflammation. Mechanisms underlying such derailed immune responses are therefore of extraordinary interest. This proposal rests on our novel observation that keratinocytes (κ) of the wound-edge tissue cross-talk with visiting macrophages via κ-originating exosomes which carry genetic information to wound-site macrophages in a highly organized and directed manner. EXOmotif in genes selects them for exosomal packaging. We propose that miR-21 is one such non-coding gene the precursor of which, pre-miR-21, is packaged in κ-originating exosomes and delivered to wound macrophages to resolve inflammation in a timely manner. This process of cross-talk is compromised in diabetes. We observed that surface protein chemistry of a specific κ-originating exosomal subset, i.e. presence of mannose favors uptake by wound macrophages. Diabetes modifies this surface protein chemistry causing mannose modifications thus compromising the uptake of these exosomes by wound macrophages. The following three specific aims are thus proposed: Aim 1. Determine whether exosomal miR-21 transfer from keratinocyte to macrophage is compromised in diabetic wound-edge tissue 1.1 Uptake of keratinocyte-originating miR-21 (pre and mature) loaded exosomes by wound-site macrophages (wmφ) is compromised under conditions of diabetes. 1.2 Compromised keratinocyte-macrophage cross-talk in diabetic wound impairs resolution of inflammation. Aim 2. Test whether surface glycoproteins of keratinocyte-derived exosomes from the wound site are glycosylated under conditions of diabetes impairing macrophage targeting. 2.1 Mannose, on the surface of κ-GFPEXO at the wound site, is glycosylated under conditions of diabetes such that the uptake by macrophages is impaired 2.2 Wound-edge κ-GFPEXO undergoes mannose isomerization under conditions of diabetes. Aim 3. Isolate keratinocyte- originating exosomes (HuK14EXO) from human chronic wound fluid (cWF) from patients with diabetes to determine whether these exosomes contain lower miR-21 (pre and mature) compared to those from patients without diabetes. Aim 3 tests the significance of the findings of Aim 1 in a patient setting. This resubmitted proposal from an early-stage investigator is aimed at laying the foundation of a laboratory dedicated to studying the cell-specific origin of exosomal subsets in bodily fluids, their targeting/homing in other cells/tissues, and functional significance. The premise of such work is that malfunction of such processes is implicated in diabetic complications and that rescue strategies can only be developed if underlying processes are understood.