Abstract
The inappropriate retention of neutrophils at inflammatory sites is a major driver of the excessive tissue damage characteristic of respiratory inflammatory diseases including COPD, ARDS, and cystic fibrosis. The molecular programmes which orchestrate neutrophil recruitment to inflammatory sites through chemotactic guidance have been well-studied. However, how neutrophil sensitivity to these cues is modulated during inflammation resolution is not understood. The identification of neutrophil reverse migration as a mechanism of inflammation resolution and the ability to modulate this therapeutically has identified a new target to treat inflammatory disease. Here we investigate the role of the CXCL12/CXCR4 signaling axis in modulating neutrophil retention at inflammatory sites. We used an in vivo tissue injury model to study neutrophilic inflammation using transgenic zebrafish larvae. Expression of cxcl12a and cxcr4b during the tissue damage response was assessed using in situ hybridization and analysis of RNA sequencing data. CRISPR/Cas9 was used to knockdown cxcl12a and cxcr4b in zebrafish larvae. The CXCR4 antagonist AMD3100 was used to block the Cxcl12/Cxcr4 signaling axis pharmacologically. We identified that cxcr4b and cxcl12a are expressed at the wound site in zebrafish larvae during the inflammatory response. Following tail-fin transection, removal of neutrophils from inflammatory sites is significantly increased in cxcr4b and cxcl12a CRISPR knockdown larvae. Pharmacological inhibition of the Cxcl12/Cxcr4 signaling axis accelerated resolution of the neutrophil component of inflammation, an effect caused by an increase in neutrophil reverse migration. The findings of this study suggest that CXCR4/CXCL12 signaling may play an important role in neutrophil retention at inflammatory sites, identifying a potential new target for the therapeutic removal of neutrophils from the lung in chronic inflammatory disease.
Highlights
The inappropriate retention of activated innate inflammatory cells at inflammatory sites is major driver of chronic inflammatory diseases including asthma, COPD and rheumatoid arthritis [1]
Aside from generation of neutrophil retention signals in multiple physiological settings [41, 42], neutrophils taken from patients with chronic inflammatory disease have increased CXCR4 expression, and
A specific role for the CXCL12/CXCR4 signaling axis in retaining neutrophils in the caudal haematopoietic tissue (CHT) has recently been suggested following the study of neutrophil behavior in zebrafish Cxcr4b and Cxcl12a mutant larvae [39]
Summary
The inappropriate retention of activated innate inflammatory cells at inflammatory sites is major driver of chronic inflammatory diseases including asthma, COPD and rheumatoid arthritis [1]. Despite the global burden of chronic inflammatory diseases, there are currently no effective therapies to treat the neutrophilic component of these conditions, highlighting a need to identify novel drug targets to promote the successful resolution of inflammation It has been known for 30 years that neutrophils undergo apoptosis followed by efferocytosis by macrophages, and this is the best characterized mechanism by which neutrophils are removed from inflammatory sites [7, 8]. The inhibition of CXCL12 using blocking antibodies prevented the accumulation of neutrophils in the lung during the late stages of LPS induced lung injury [25] Based on this evidence we hypothesized that CXCL12/CXCR4 functions as a retention signal in the context of tissue damage, functioning to maintain active neutrophils at the inflammatory site. We have identified a druggable signaling axis which could be a therapeutic target to remove excessively retained neutrophils at inflammatory sites during disease
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