The CXCL12/CXCR4 Signaling Axis Retains Neutrophils at Inflammatory Sites in Zebrafish

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.

ISG15 drives immune pathology and respiratory failure during viral infection

“…Importantly, complete deletion of Isg15 from Usp18 C61A mice reversed the accumulation of CXCR4 + neutrophils and the associated pathological outcomes. CXCR4 expression on neutrophils promotes retention and survival of neutrophils in inflamed tissues (Isles et al, 2019). Elevated levels of neutrophil CXCR4 expression and its ligand CXCL12 has been found in patients with severe lung inflammation in COPD and cystic fibrosis patients (Carevic et al, 2015; Hartl et al, 2008), and CXCR4 + neutrophils are elevated in late stage LPS-induced lung injury (Yamada et al, 2011).…”

Hif-1alpha stabilisation is protective against infection in a zebrafish model of comorbidity

“…Likely candidates for Hif-1α targets include G protein coupled receptors (GPCRs) that are involved in neutrophil migration (many chemokine receptors are GPCRs) and are regulated by Hif-1α in immune cells (eg., CXCR1, CXCR2 or CXCR4) [37–41]. Cxcr1/2 have been implicated in retention of neutrophils at a tailfin wound in zebrafish and we have recently demonstrated that decreasing Cxcr4 signalling causes premature reverse migration away from the tailfin wound [42].…”

CXCR4 signalling, metastasis and immunotherapy: zebrafish xenograft model as translational tool for anti-cancer discovery

“…We found that neutrophils display altered motility and their number fluctuates during embryo development, leading to the conclusion that CXCR4 regulates neutrophil development in zebrafish. Moreover, a link between CXCR4 signalling and neutrophil response during inflammation has been recently described [98] . In our model, the neutrophilic response towards cancer cells was also altered in zebrafish mutants with a non-functional Cxcr4 (Cxcr4b).…”