Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing

Abstract

Bidirectional cell-cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte-macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exo_κ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exo_κ-GFP were also characterized. Unlike skin exosome, wound-edge Exo_κ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. <i>In vitro</i> addition of wound-edge Exo_κ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exo_κ-GFP<i>in vivo</i>, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNP_κ) were designed with 94.3% encapsulation efficiency. Application of TLNP_{κ/si-hnRNPA2B1} to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNP_{κ/si-control} group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNP_{κ/si-hnRNPA2B1} treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNP_{κ/si-control} group. Furthermore, TLNP_{κ/si-hnRNPA2B1} treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exo_κ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.