Bruton's tyrosine kinase control of cytokine production in Toll‐like receptor signalling

Abstract

Inappropriate and un-regulated inflammation is the hallmark of a variety of inflammatory diseases including Rheumatoid Arthritis (RA). Central to the pathogenesis of this disease is the cytokine Tumour Necrosis Factor α (TNF), a product of activated macrophages. Macrophages are activated by numerous exogenous and endogenous ligands through Toll like receptors (TLR) leading to the activation of numerous protein tyrosine kinases (PTK). These studies have mainly described the LPS pathway and have not focused on endosomal TLRs, which are being increasingly implicated in the pathology of RA. The work presented in this thesis describes the role of Bruton's Tyrosine Kinase (Btk) in TLR8 signalling in human primary macrophages. Using an adenovirus overexpression system and RNAi technology, Btk has been shown to control the production of TNF in R848-stimulated cells. This regulation occurred on a transcriptional level as Btk downregulation decreased the level of the primary TNF transcript at 2h as well as the recruitment of RNA Polymerase II to the promoter of the TNF gene at 1h. This rapid effect of Btk depletion on gene transcription resulted in significantly less TNF protein production at 2h following R848 stimulation. Subsequent analysis of transcription factors potentially involved in the Btk-dependent regulation of TNF transcription revealed that Btk affects both NFkB and AP-1 in TLR8 signalling. In particular, Btk downregulation decreased the binding of NFkB to its consensus site as shown by EMSA as well as the recruitment of p65 to the TNF promoter or 3' enhancer region as shown by ChIP. The regulation of p65 by Btk occurred at the level of p65 phosphorylation on Serine 536 and did not affect the degradation of IkBα. AP-1 activity in EMSA was also decreased under the Btk knockdown conditions, with cJun and ATF-2 but not cFos being involved in Btk signalling. At the same time, Btk did not alter p38, ERK and JNK MAPK activity ruling out their involvement in the Btk-dependent control of AP-1 in R848-challenged macrophages. These data extend current knowledge regarding the involvement of Tec kinases in the regulation of cytokine production under inflammatory conditions by providing a potential mechanism of transcriptional control.