Abstract
SummaryDrosophila provides a powerful model in which to study inflammation in vivo, and previous studies have revealed many of the key signaling events critical for recruitment of immune cells to tissue damage. In the fly, wounding stimulates the rapid production of hydrogen peroxide (H2O2).1,2 This then acts as an activation signal by triggering a signaling pathway within responding macrophages by directly activating the Src family kinase (SFK) Src42A,3 which in turn phosphorylates the damage receptor Draper. Activated Draper then guides macrophages to the wound through the detection of an as-yet unidentified chemoattractant.3, 4, 5 Similar H2O2-activated signaling pathways are also critical for leukocyte recruitment following wounding in larval zebrafish,6, 7, 8, 9 where H2O2 activates the SFK Lyn to drive neutrophil chemotaxis. In this study, we combine proteomics, live imaging, and genetics in the fly to identify a novel regulator of inflammation in vivo; the PTP-type phosphatase Pez. Pez is expressed in macrophages and is critical for their efficient migration to wounds. Pez functions within activated macrophages downstream of damage-induced H2O2 and operates, via its band 4.1 ezrin, radixin, and moesin (FERM) domain, together with Src42A and Draper to ensure effective inflammatory cell recruitment to wounds. We show that this key role is conserved in vertebrates, because “crispant” zebrafish larvae of the Draper ortholog (MEGF10) or the Pez ortholog (PTPN21) exhibit a failure in leukocyte recruitment to wounds. This study demonstrates evolutionary conservation of inflammatory signaling and identifies MEGF10 and PTPN21 as potential therapeutic targets for the treatment of inflammatory disorders.
Highlights
To determine whether Pez is expressed in embryonic macrophages, we used Pez-Gal[4] (P{GawB}PezNP4748) to drive UASGFP and investigated GFP expression by immunofluorescence
We found no defect in macrophage recruitment to wounds made in heterozygous src42A[E1]/+, draperD5/+, or PezCB/+ embryos
We found an increase in neutrophil number in PTPN21 crispants—akin to the macrophage phenotype we identify in Drosophila—and a 20% reduction in macrophage numbers in MEGF10 crispant fish (Figure 4B)
Summary
To determine whether Pez is expressed in embryonic macrophages, we used Pez-Gal[4] (P{GawB}PezNP4748) to drive UASGFP and investigated GFP expression by immunofluorescence. We sought to determine whether Pez plays a role in normal macrophage behavior using two independent Pez mutant lines (Figure 1A). Following their specification from the head mesoderm, macrophages follow a stereotypical migration pattern to become evenly distributed by the end of embryogenesis.[14,15,16] This characteristic developmental dispersal of macrophages in Pez mutant embryos occurred normally, with macrophages following the expected dispersal routes at identical migratory speeds to controls (Figures S1H and S1I; Video S1)
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