Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Immunology

First Advisor

Edward M. Behrens

Second Advisor

Gary A. Koretzky

Abstract

Upon invasion of microbial pathogens, cells of the innate immune system respond through the activation of pattern recognition receptors (PRRs) that recognize pattern associated molecular patterns (PAMPs). Once these receptors bind ligand, they initiate a signaling cascade culminating in the expression of proinflammatory and antiviral cytokines, costimulatory molecules, and antimicrobial agents, all of which contribute to pathogen clearance and host defense. However, excessive signaling through these receptors can lead to inflammatory conditions resulting in damage to the host. Therefore, understanding the signaling events downstream of PRR activation is critical for gaining insight into targeting specific mediators for therapeutic intervention to combat infection and to limit host pathology. Canonically, Toll-like receptors (TLRs), one class of PRR, have been thought to signal through serine threonine kinases following ligand recognition. However, there is emerging evidence for the role of protein tyrosine kinases regulating TLR function, but their role is not entirely clear. We sought to understand how TLR9 function is affected by a conserved tyrosine residue in its cytoplasmic domain and by activation of the protein tyrosine kinase Syk. We initially hypothesized that Syk might be participating in tyrosine phosphorylation of TLR9 to induce downstream signaling following receptor activation with CpG DNA. Utilizing genetic deletion of Syk in dendritic cells in vivo and genetic knockdown in a macrophage cell line, here we demonstrate that Syk is important for the intracellular trafficking and exocytosis of the proinflammatory cytokine TNFa, but not IL-6, following CpG stimulation. This secretion event involved activation of calcium signaling and calcium calmodulin kinase II (CaMKII) downstream of Syk. Syk-deficient cells exhibited normal CpG-induced activation of the canonical TLR9 signaling machinery, suggesting that Syk mediates a signaling cascade to promote cytokine exocytosis independent of cytokine transcription and translation, a role unexpected based on its function downstream of ITAM-bearing receptors. These data implicate this signaling pathway in a novel role of cytokine sorting and may have broader implications for release of other cytokines downstream of various pattern recognition receptors.

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