A CpG triggered Syk-Calcium-CaMKII signal directs the secretion of TNFα by innate immune cells (P6297)

Abstract

Abstract The kinase Syk is reported to regulate cytokine secretion in multiple and conflicted manners. We therefore examined Syk in regulating CpG DNA stimulated TNFα secretion. Secreted cytokines were measured by ELISA and surface cytokines by flow cytometry. Gene deletion was achieved with both Cre-Lox and shRNA methods. Deletion of Syk decreases CpG induced TNFα secretion while TNFα mRNA and intracellular protein levels remain normal. In contrast, IL-6 secretion is unaffected. CpG induced plasma membrane levels of TNFα are decreased in Syk deficiency, suggesting that Syk is important for TNFα intracellular trafficking. A downstream effector of Syk is calcium (Ca) flux mediated by PLCγ activation. Supporting a role for Ca in Syk mediated TNFα release, we show: 1) CpG stimulation triggers calcium flux only in Syk-sufficient cells, 2) silencing PLCγ2 phenocopies the TNFα defect of Syk-deficient cells, 3) forced Ca mobilization using ionomycin rescues the TNFα secretion defect in Syk-deficient cells. CpG-triggered phosphorylation of the calcium dependent kinase CaMKII is defective in Syk-deficient cells. CpG-induced TNFα secretion is defective in CaMKII-deficient cells, and is not rescued by ionomycin, suggesting that CaMKII is downstream in the pathway initiated by Syk signaling. These data suggest that CpG provides active signals for TNFα transcription and translation as well as intracellular trafficking, via a Syk-Ca-CaMKII axis, allowing for the fine-tuning of TNFα secretion.