PKCδ-IRAK1 axis regulates oxidized LDL-induced IL-1β production in monocytes

Rajiv Lochan Tiwari,Vishal Singh,Ankita Singh,Minakshi Rana,Anupam Verma,Nikhil Kothari,Monica Kohli,Jaishri Bogra,Madhu Dikshit,Manoj Kumar Barthwal

doi:10.1194/jlr.m045658

Abstract

This study examined the role of interleukin (IL)-1 receptor-associated kinase (IRAK) and protein kinase C (PKC) in oxidized LDL (Ox-LDL)-induced monocyte IL-1β production. In THP1 cells, Ox-LDL induced time-dependent secretory IL-1β and IRAK1 activity; IRAK4, IRAK3, and CD36 protein expression; PKCδ-JNK1 phosphorylation; and AP-1 activation. IRAK1/4 siRNA and inhibitor (INH)-attenuated Ox-LDL induced secreted IL-1β and pro-IL-1β mRNA and pro-IL-1β and mature IL-1β protein expression, respectively. Diphenyleneiodonium chloride (NADPH oxidase INH) and N-acetylcysteine (free radical scavenger) attenuated Ox-LDL-induced reactive oxygen species generation, caspase-1 activity, and pro-IL-1β and mature IL-1β expression. Ox-LDL-induced secretory IL-1β production was abrogated in the presence of JNK INH II, Tanshinone IIa, Ro-31-8220, Go6976, Rottlerin, and PKCδ siRNA. PKCδ siRNA attenuated the Ox-LDL-induced increase in IRAK1 kinase activity, JNK1 phosphorylation, and AP-1 activation. In THP1 macrophages, CD36, toll-like receptor (TLR)2, TLR4, TLR6, and PKCδ siRNA prevented Ox-LDL-induced PKCδ and IRAK1 activation and IL-1β production. Enhanced Ox-LDL and IL-1β in systemic inflammatory response syndrome (SIRS) patient plasma demonstrated positive correlation with each other and with disease severity scores. Ox-LDL-containing plasma induced PKCδ and IRAK1 phosphorylation and IL-1β production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes. Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKCδ-IRAK1-JNK1-AP-1 axis in Ox-LDL-induced IL-1β production.

Highlights

Because it is reported that IRAK1 is downstream to IRAK4 and relays the signal forward [25], we performed IRAK1 kinase assay to ascertain the activation of the IRAK4-IRAK1 signaling pathway
We have evaluated the role of the protein kinase C (PKC) and interleukin-1 receptor-associated kinase (IRAK) kinase families and associated signaling events during Oxidized LDL (Ox-LDL)-induced IL-1␤ production
Because Ox-LDL treatment induces inflammation and subsequent cholesterol accumulation causes cell death [41, 42], we have used an optimal concentration that induces significant IL1␤ production along with minimal cell death; this has been routinely used by other investigators as well [6, 30]

Summary

Introduction

In THP1 macrophages, CD36, toll-like receptor (TLR), TLR4, TLR6, and PKC␦ siRNA prevented Ox-LDLinduced PKC␦ and IRAK1 activation and IL-1␤ production. Ox-LDL-containing plasma induced PKC␦ and IRAK1 phosphorylation and IL-1␤ production in a CD36-, TLR2-, TLR4-, and TLR6-dependent manner in primary human monocytes. Results suggest involvement of CD36, TLR2, TLR4, TLR6, and the PKC␦-IRAK1-JNK1AP-1 axis in Ox-LDL-induced IL-1␤ production.—Tiwari, R. Recent reports suggest that Ox-LDL can induce sterile inflammation by stimulating production of various inflammatory cytokines, including interleukin (IL)-1␤ [6, 7]. Sterile inflammation is characterized by the recruitment of neutrophils and macrophages and production of inflammatory cytokines like IL-1␤ and TNF-␣ [8]. Ox-LDL-induced sterile inflammation was dependent on CD36-induced heterodimerization of toll-like receptor (TLR) and TLR6 [6].

Methods

Results

Conclusion