The TIR-domain containing adaptor TRAM is required for TLR7 mediated RANTES production.

Enda Shevlin,Sinéad M Miggin

doi:10.1371/journal.pone.0107141

Enda Shevlin, Sinéad M Miggin

Open Access

https://doi.org/10.1371/journal.pone.0107141

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Journal: PloS one	Publication Date: Sep 11, 2014
Citations: 12	License type: CC BY 4.0

Affiliation: National University of Ireland, Maynooth

Abstract

Toll-like receptor 7 (TLR7) plays a vital role in the immune response to ssRNA viruses such as human rhinovirus (HRV) and Influenza, against which there are currently no treatments or vaccines with long term efficacy available. Clearly, a more comprehensive understanding of the TLR7 signaling axis will contribute to its molecular targeting. TRIF related adaptor molecule (TRAM) plays a vital role in TLR4 signaling by recruiting TRIF to TLR4, followed by endosomal trafficking of the complex and initiation of IRF3 dependent type I interferon production as well as NF-κB dependent pro-inflammatory cytokine production. Towards understanding the molecular mechanisms that regulate TLR7 functionality, we found that TRAM−/− murine macrophages exhibited a transcriptional and translational impairment in TLR7 mediated RANTES, but not TNFα, production. Suppression of TRAM expression in human macrophages also resulted in an impairment in TLR7 mediated CCL5 and IFN-β, but not TNFα, gene induction. Furthermore, suppression of endogenous human TRAM expression in human macrophages significantly impaired RV16 induced CCL5 and IFNβ, but not TNFα gene induction. Additionally, TRAM-G2A dose-dependently inhibited TLR7 mediated activation of CCL5, IFNβ and IFNα reporter genes. TLR7-mediated phosphorylation and nuclear translocation of IRF3 was impaired in TRAM−/− cells. Finally, co-immunoprecipitation studies indicated that TRAM physically interacts with MyD88 upon TLR7 stimulation, but not under basal conditions. Our results clearly demonstrate that TRAM plays a, hitherto unappreciated, role in TLR7 signaling through a novel signaling axis containing, but not limited to, MyD88, TRAM and IRF3 towards the activation of anti-viral immunity.

Highlights

TLRs function by recognizing conserved structural motifs, or pathogen associated molecular patterns (PAMPs) derived from infectious organisms and initiating an intracellular signaling cascade which in turn brings about the appropriate innate and adaptive immune response
Comparable RANTES and TNFa production was evident in TRAM2/2 immortalised bone marrow derived macrophages (iBMDMs) when compared to WT cells following stimulation with Poly(I:C), but not LPS (Fig. 1A, B)
Correlating with ELISA data, real-time PCR data revealed that R848 mediated CCL5 induction was significantly decreased in TRAM2/2 iBMDMs when compared to WT cells (Fig. 1C)

Summary

Introduction

TLRs function by recognizing conserved structural motifs, or pathogen associated molecular patterns (PAMPs) derived from infectious organisms and initiating an intracellular signaling cascade which in turn brings about the appropriate innate and adaptive immune response. Many aspects of their expression, localisation, activation and downstream signaling are tightly regulated by an ever expanding panel of both positive and negative regulators [1]. MyD88 is required for all TLR signaling, except TLR3, and causes pro-inflammatory cytokine production via activation of NFkB and the mitogen activated protein kinases (MAPKs). SARM negatively regulates TLR3 and TLR4 signaling by inhibiting TRIF recruitment [4]. TRAM has been shown to be required for maximal IL-18R signaling [9]

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Conclusion