Role of cyclin-dependent kinase 9 in the resolution of innate inflammation in a zebrafish tailfin injury model
Hoodless, Laura Jane
Neutrophils are an important cell in host defence and migrate rapidly to sites of inflammation when the host is compromised (e.g., in infection or wounding). There, they produce and/or release inflammatory mediators (e.g., LTB4, TNF, IL-8) and ingest and degrade pathogens (e.g., by release of granule proteins and reactive oxygen species). Neutrophils then undergo apoptosis and are cleared by phagocytes such as macrophages, to allow efficient resolution of inflammation. Inducing neutrophil apoptosis by pharmacological means could be a therapeutic strategy to dampen inflammation in diseases where neutrophils are prevalent, e.g., acute respiratory distress syndrome (ARDS) and rheumatoid arthritis (RA). Inhibition of cyclin-dependent kinases (CDKs) using CDK inhibitor (CDKi) compounds induces mammalian neutrophil apoptosis in vitro, and can drive resolution of inflammation in vivo in mouse models. Evidence indicated that this is due to inhibition of CDK9 and CDK7-mediated transcription of the anti-apoptotic protein Mcl-1. The hypothesis of this project was that CDK9, CDK7 and Mcl-1 are pivotal regulators of resolution of inflammation in vivo. The model selected to test this hypothesis was tailfin injury of embryonic zebrafish (Danio rerio). Zebrafish are optically transparent and reporter transgenic lines with neutrophils labelled by enhanced GFP (EGFP - Tg[mpx:EGFP]i114) and macrophages (Tg[MPEG1:mCherry]) have been created, permitting the imaging of the behaviour of these cells in vivo. The model of tailfin transection was chosen to cause an inflammatory response in these animals, with neutrophil and macrophage recruitment to the tailfin. This response was manipulated using CDKi compounds and specific gene knockdowns (using morpholino and CRISPR/cas9 technologies). It was shown that CDKi compounds could reduce neutrophil numbers at 24 h post-injury at the transected tailfin, but did not affect macrophage numbers. The CDKi AT7519 increased neutrophil apoptosis at 12 h post-injury. Specific CDK9 knockdown using morpholinos or CRISPR/cas9 also reduced neutrophilic inflammation at the tailfin 24 h after transection, accompanied by increased apoptosis levels at 8 h in the morpholino-treated group. Inhibition of an endogenous CDK9 inhibitor, LaRP7, had the opposite effect and increased neutrophil numbers; and could oppose the neutrophil- reducing effect of AT7519 and CDK9 morpholino knockdown. Preliminary genetic knockdown studies into the roles of CDK7 and Mcl-1 have been carried out. Taken together, the results demonstrate CDK9 is important in the resolution of neutrophilic inflammation, indicating that manipulation of CDK9 activity could be a good target for therapeutic intervention in inflammatory disease.