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Macrophage RNA Binding Protein Pcbp2 Interacts With Pre-Mir-21 In Keratinocyte-Derived Exosome For Resolution Of Inflammation
Anita Yadav1, Anu Sharma1, Chandan K. Sen1, Sashwati Roy1, Subhadip Ghatak1
1Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, United States

Background. Resolution of inflammation is contingent on successful crosstalk between keratinocytes and wound-edge (WE) macrophages (m?). We tested the hypothesis that in m?, RNA binding adaptor poly(C)-binding protein 2 (PCBP2) interacts with pre-miR21 in keratinocyte-derived exosome (Exok) for timely resolution of inflammation.
Methods. Murine WE Exok were genetically labeled with GFP reporter, isolated, and characterized per MISEV 2018 guidelines. This method, reported in EV track, received an EV-metric score of 100%. dSTORM imaging (res:20nm) was performed in conjunction with sequence-specific molecular beacons (MB) to quantify pre-and mature miR21 abundance in Exo? at single exosome resolution. Resolution of inflammation was studied using laser captured microdissection of WE m?, RT-qPCR, and ELISA of inflammatory cytokines and immunostaining of iNOS, ARG1, MPO, and annexinV. Day 5 pro-inflammatory m? (m?pro) from murine WE tissue was isolated from single cell suspension using immunomagnetic sorting of anti-F4/80 and CD11b+ microbeads.
Results. Automated electrophoresis of RNA isolated from WE Exo? showed predominance of small RNA (<200 bp). In silico study identified pre-miR21 with an exomotif sequence in its stem-loop structure. Using MB specific to pre-miR21 and dSTORM imaging, significant high abundance of pre-miR21 was observed in WE Exo? compared to skin (n=6, p<0.01). PCBP2, reported to stabilize mRNA transcript of pro-inflammatory cytokines, was also found in significantly high abundance in m?pro (n=7, p<0.001). Uptake of Exo? by m?pro resulted in significant downregulation of PCBP2 with significant low transcript levels of TNF-?, IL-2, INF-g, GM-CSF (n=4-6, p<0.01). The transcript abundance of pro-resolution Cl3 and Arg1 was also increased. Immunoproximity ligation assay demonstrated binding of pre-miR21 with PCBP2 in m?. Immunoprecipitation of PCBP2 from murine WE tissue indicated presence of miR21. PCBP2-pre-miR21 interactions in day 5 WE m?pro resulted in degradation of the mRNA transcript of pro-inflammatory cytokines. In diabetic mice, WE m?pro demonstrated significant low abundance of pre-miR21 (n=4, p<0.001), suggesting the notion that uptake of pre-miR21 loaded Exok was compromised. To test our hypothesis and block Exok uptake by m?pro, K14 promoter-driven tetraspanins plasmid connected via IRES element with ?eat me not?-CD47 sequence with ?in-frame? GFP reporter was generated. Selective interruption of Exok uptake resulted in the persistence of m?pro with high PCBP2 and iNOS expression (n=4,p<0.01), high annexinV+ dead cells burden, and compromised re-epithelialization.
Conclusion. This work addresses the molecular dynamics of keratinocyte-m? crosstalk in wound microenvironment. Notably, it identifies the pivotal role of WE keratinocytes in directing resolution of inflammation, a process often disrupted in non-healing diabetic wounds.


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