Regulator of calcineurin 1 differentially regulates TLR-dependent MyD88 and TRIF signaling pathways.

Zheng Pang,Robert D Junkins,Adam J Macneil,Tong-Jun Lin,Zhenyu Cheng,Renee Raudonis,Craig Mccormick,Kui Li

doi:10.1371/journal.pone.0197491

Abstract

Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-β (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNβ, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.

Highlights

Toll like receptors (TLRs) are a family of transmembrane receptors that recognize diverse molecular patterns derived from microbes [1]
To assess the effect of Regulator of calcineurin-1 (RCAN1) on the cytokine production regulated through the myeloid differentiation primary response 88 (MyD88)-NF-κB pathway, we stimulated wild-type and RCAN1-deficient bone marrow-derived macrophages (BMMs) with 200 ng/ml of P. aeruginosa LPS for 3 h, 6 h, 12 h, 24 h
We found that the P. aeruginosa LPS-induced production of IL-6 (Fig 1A), TNF (Fig 1B) and macrophage inflammatory protein (MIP)-2 (Fig 1C) was significantly enhanced in RCAN1deficient BMMs compared to wild-type BMMs, suggesting that RCAN1 negatively regulates MyD88-NF-κB-mediated cytokine and chemokine production

Summary

Introduction

Toll like receptors (TLRs) are a family of transmembrane receptors that recognize diverse molecular patterns derived from microbes [1]. TLR4 binding to lipopolysaccharide (LPS) activates two distinct signalling pathways; the myeloid differentiation primary response 88 (MyD88) pathway, and the Toll/IL-1R domain—containing adapter inducing IFN-β (TRIF) pathway [3]. Activated IRF3 and IRF7 drive transcription of interferon-α (IFNα) and interferon-β (IFNβ), and the chemokines RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and IP-10 (Interferon γ-inducible Protein 10) [7, 8]. Both MyD88- and TRIF-dependent pathways have been found to contribute to host defense against the microbial infection [9,10,11,12]

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Results

Conclusion