Heterogeneous Neutrophil Influx and Osteopontin Signaling Promote Repair During Skin Infection

Abstract

Skin infections are among the most common types of infections in the United States and pose a serious health threat. Effective resolution requires both bacterial clearance and tissue repair. While most therapeutics for skin infection are aimed at clearing bacteria, repairing the skin barrier is a critical arm of resolution that remains underappreciated. During normal skin wound repair, inflammation is resolved prior to re-epithelialization, however, during skin infection neutrophils remain, prolonging inflammation. Understanding the cell signaling responses in infected tissue is critical for identifying new therapeutic targets to promote tissue repair. In our studies, we combined transcriptomic and genetic knockout approaches in a murine model of S. aureus dermonecrotic skin infection to identify how cellular responses change over the course of skin infection. We selected single cell RNA sequencing for our transcriptomic studies, so that we could determine how individual cell types and subtypes were responding and contributing during skin infection. We identified a major influx of neutrophils during infection including multiple heterogeneous subtypes that were enriched for genes related to non-classical neutrophil functions including antigen presentation and tissue repair. Among these neutrophils, we identified a distinct subtype expressing high levels of Spp1, the gene encoding osteopontin (OPN). We performed cell-cell interaction analysis and identified Spp1 signaling as the most enriched pathway in infected tissue. We determined that this induction of Spp1 occurred once the neutrophils were in the skin and was also present in a population of skin neutrophils during sterile wounding. In vivo, OPN knockout (KO) mice had significantly larger lesions than their wild type (WT) counterpart despite no difference in bacterial load suggesting a role for OPN in tissue repair. Transcriptomic profiling of OPN KO mice revealed a lower proportion of fibroblast and keratinocyte cells at the infection site, and a reduction in associated repair signaling pathways compared to WT mice. We sought to determine if recombinant OPN (rOPN) could be an effective therapeutic for promoting tissue repair during skin infection. We showed that infected WT mice treated with rOPN had accelerated healing relative to PBS treated controls. Together, these findings highlight the importance of specific neutrophil subtypes and OPN signaling as critical mediators in infected tissue repair. Further our study suggests that modulating OPN may represent a promising therapeutic strategy to accelerate repair during skin infection.