The IL-12/STAT4 pathway associates with critical functions in neutrophils

Abstract Signal transducer and activator of transcription 4 (STAT4) becomes activated via phosphorylation by JAK/Tyk kinases in response to IL-12 and other cytokines, which results in downstream transcription of pro-inflammatory genes. Our preliminary data are the first to demonstrate that IL-12 induces phospho-STAT4 in neutrophils. STAT4, JAK2, and p38 MAPK, but not Tyk2, become rapidly phosphorylated in response to IL-12 treatment in a time dependent manner. Pharmacologic inhibition of JAK1/2, but not p38, resulted in a two-fold reduction in phospho-STAT4, confirming its role as a signalling intermediate during STAT4 activation. Although IL-12 does not induce ROS formation in the short term (<3 hours), we were surprised to discover a 4-fold increase in ROS production in response to long term IL-12 treatment (14 hrs), compared to only 1.5-fold increase in STAT4-deficient neutrophils. Additionally, we observed a dramatic 15-fold decrease in in vitro migration of STAT4-deficient neutrophils towards the chemoattractant GM-CSF compared to wild-type. Preliminary in vivo studies have revealed deficient migration to the peritoneal cavity of STAT4-deficient mice following intraperitoneal injection of GM-CSF. Finally, we demonstrate that in vitro NET formation in response to PMA treatment is impaired 5-fold in STAT4-deficient neutrophils compared to wild-type. These findings highlight the novel IL-12/STAT4 pro-inflammatory signalling pathway in murine neutrophils. Our data suggest a major contributory role of STAT4 in mediating neutrophil functions that have been previously associated with the progression of atherosclerosis. Future in vivo studies will determine the neutrophil-specific role of STAT4 in atherosclerotic mice.