Cell type specific gene expression profiling reveals a role for complement component C3 in neutrophil responses to tissue damage

Abstract

Tissue damage induces rapid recruitment of leukocytes and changes in the transcriptional landscape that influence wound healing. However, the cell-type specific transcriptional changes that influence leukocyte function and tissue repair have not been well characterized. Here, we employed translating ribosome affinity purification (TRAP) and RNA sequencing, TRAP-seq, in larval zebrafish to identify genes differentially expressed in neutrophils, macrophages, and epithelial cells in response to wounding. We identified the complement pathway and c3a.1, homologous to the C3 component of human complement, as significantly increased in neutrophils in response to wounds. c3a.1−/− zebrafish larvae have impaired neutrophil directed migration to tail wounds with an initial lag in recruitment early after wounding. Moreover, c3a.1−/− zebrafish larvae have impaired recruitment to localized bacterial infections and reduced survival that is, at least in part, neutrophil mediated. Together, our findings support the power of TRAP-seq to identify cell type specific changes in gene expression that influence neutrophil behavior in response to tissue damage.

Trap-TRAP, a versatile tool for tissue-specific translatomics in zebrafish

“…Previous studies in zebrafish have reported the use of TRAP methods for translational profiling in zebrafish using a few tissue-specific promoters 14,15,16,17 . Here we applied the Trap-TRAP strategy to generate random insertions of the eGFP-rpl10a cassette into the genome.…”

“…In zebrafish, different laboratories have successfully implemented the TRAP approach using tissuespecific drivers. They include the promoters of the genes tyrp1 for melanocytes 14 , cmlc2 for cardiomyocytes 15 , actc1b for skeletal myocytes 16 , or lyz for neutrophils 17 . One of the main disadvantages of the TRAP methodology is precisely the need to create a customized transgenic line for each cell type to be analysed.…”

Zebrafish Embryo Infection Model to Investigate Pseudomonas aeruginosa Interaction With Innate Immunity and Validate New Therapeutics

“…The recruitment of neutrophils at the site of infection has also been observed ( Diaz-Pascual et al., 2017 ; McCarthy et al., 2017 ). The effect of neutrophil depletion in the outcome of P. aeruginosa in zebrafish embryos has not been investigated, but defects in neutrophil migration greatly sensitize embryos to P. aeruginosa infections ( Rosowski et al., 2016 ; Houseright et al., 2020 ). More precisely, embryos defective for Rac2 function, which have neutrophils that are unable to migrate, are highly susceptible to P. aeruginosa ( Rosowski et al., 2016 ).…”

“…Upon localized otic infection with P. aeruginosa , systemic activation and mobilization of neutrophils from hematopoietic tissues is also mediated by Cxcr2 signaling, a receptor enabling neutrophils to sense the IL8 chemokine ( Deng et al., 2013 ). In addition, the increased susceptibility to P. aeruginosa infection observed in zebrafish deficient for c3a.1 , which is homologous to the C3 component of human complement, is likely due to a neutrophil-intrinsic function of C3, possibly its ability to recruit neutrophils at the infection site ( Houseright et al., 2020 ).…”

A Novel Function of TLR2 and MyD88 in the Regulation of Leukocyte Cell Migration Behavior During Wounding in Zebrafish Larvae

“…Moreover, the expression of Myd88 and Tlr2 is significantly increased in diabetic wounded mice ( Dasu et al, 2010 ). In addition, some evidence indicate that Myd88 is involved in the modulation of wound healing ( Macedo et al, 2007 ; Houseright et al, 2020 ), but the underlying mechanism is still unclear. Although TLR signaling is important for chemokine production, little is known about the role of MyD88 in leukocyte migratory responses to tissue injuries in the absence of pathogenic infections.…”