Post-Transcriptional Inflammatory Response to Intracellular Bacterial c-di-AMP

Linah Mahmoud,Alaa S Abdulkarim,Shaima Kutbi,Khalid S A Khabar,Edward G Hitti,Walid Moghrabi,Sulaiman Altwijri

doi:10.3389/fimmu.2019.03050

Linah Mahmoud, Alaa S Abdulkarim + Show 5 more

Open Access

https://doi.org/10.3389/fimmu.2019.03050

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Abstract

Cyclic-di-AMP (c-di-AMP) is a bacterial second messenger that is produced by intracellular bacterial pathogens in mammalian host macrophages. Previous reports have shown that c-di-AMP is recognized by intracellular pattern recognition receptors of the innate immune system and stimulate type I interferon response. Here we report that the response to c-di-AMP includes a post-transcriptional component that is involved in the induction of additional inflammatory cytokines including IL-6, CXCL2, CCL3, and CCL4. Their mRNAs contain AU-rich elements (AREs) in their 3′ UTR that promote decay and repress translation. We show that c-di-AMP leads to the phosphorylation of p38 MAPK as well as the induction of the ARE-binding protein TTP, both of which are components of a signaling pathway that modulate the expression of ARE-containing mRNAs at the post-transcriptional level. Pharmacological inhibition of p38 reduces the c-di-AMP-dependent release of induced cytokines, while TTP knockdown increases their release and mRNA stability. C-di-AMP can specifically increase the expression of a nano-Luciferase reporter that contains AREs. We propose a non-canonical intracellular mode of activation of the p38 MAPK pathway with the subsequent enhancement in the expression of inflammatory cytokines. C-di-AMP is widely distributed in bacteria, including infectious intracellular pathogens; hence, understanding of its post-transcriptional gene regulatory effect on the host response may provide novel approaches for therapy.

Highlights

Cyclic dimeric AMP (c-di-AMP) or bis-(3′,5′)-cyclic dimeric adenosine monophosphate is a bacterial second messenger that is involved in a number of bacterial metabolic events such as DNA damage-dependent cell cycle control, establishment of infection, cell size control, osmotic homeostasis, and regulation of metabolic enzyme function [1,2,3,4,5,6,7,8]
RAW 264.7 cell lines were purchased from American Type Culture Collection (ATCC; Rockville, MD) and grown in DMEM medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (Invitrogen, Carlsbad, CA)
The inhibition of p38 MAPK with SB203580 in c-di-AMP-treated cells led to a reproducible ∼10% reduction in the expression of nLuc+Ccl3 3′untranslated regions (3′UTR) compared to cells treated with vehicle (Figure 6C)

Summary

INTRODUCTION

Cyclic dimeric AMP (c-di-AMP) or bis-(3′,5′)-cyclic dimeric adenosine monophosphate is a bacterial second messenger that is involved in a number of bacterial metabolic events such as DNA damage-dependent cell cycle control, establishment of infection, cell size control, osmotic homeostasis, and regulation of metabolic enzyme function [1,2,3,4,5,6,7,8]. The canonical activation of the post-transcriptional signaling pathway of p38 MAPK in inflammation starts by the activation of cell surface receptors like cytokine and Toll-like receptors that activate TNF-receptor-associated factors [16, 17]. This is followed by the phosphorylation-dependent activation of a MAP Kinase Kinase Kinase (MAP3K) such as TAK1 (MAP3K7). We report that intracellular c-di-AMP can induce the expression of a subset of inflammatory ARE-containing mRNAs at the transcriptional and post-transcriptional levels

MATERIALS AND METHODS

RESULTS

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DISCUSSION