K63-Linked Polyubiquitination on TRAF6 Regulates LPS-Mediated MAPK Activation, Cytokine Production, and Bacterial Clearance in Toll-Like Receptor 7/8 Primed Murine Macrophages.

Jaya Talreja,Lobelia Samavati

doi:10.3389/fimmu.2018.00279

Abstract

Post viral infection bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness. Despite much efforts put into the discovery of mechanisms of post viral–bacterial infections and their complications in recent years, the molecular mechanisms underlying the increased susceptibility to bacterial infection remain poorly understood. In this study, we focused on the pathways regulating immune responses in murine macrophages and modeled post viral–bacterial infections through pretreatment of bone marrow-derived macrophages (BMDMs) with a toll-like receptor (TLR) 7/8 ligand (R848) and subsequent challenge with TLR2/4 agonists to mimic bacterial infection. We found R848-primed BMDMs upon subsequent exposure to TLR2/4 ligands respond with enhanced inflammatory cytokine production, especially IL-6 and TNF-α. The enhanced cytokine production in R848-primed BMDMs in response to TLR2/4 was due to increased TGF-β-activated kinase (TAK) 1 phosphorylation with subsequent activation of ERK and p38 MAPKs. Furthermore, we identified that R848 priming leads to increased K63-linked polyubiquitination on TRAF6. K63-linked polyubiquitination on TRAF6 is a signal leading to enhanced activation of downstream pathways including TAK1. Importantly, R848-primed BMDMs infected with live bacteria exhibited decreased bacterial clearance. Small-molecule enhancer of rapamycin 3, an ubiquitin ligase inhibitor reversed the K63-linked polyubiquitination on TRAF6 in R848-primed BMDMs and subsequently decreased TAK1 and MAPK phosphorylation, and cytokine production as well as reversed the decreased bacterial clearance capacity of BMDMs. Our study may provide a novel molecular target to alleviate post viral–bacterial infections.

Highlights

Viral infections predispose the host to secondary bacterial infections [1]
An increase in TNF-α with LPS and PAM was observed. These results show that pre-exposure of macrophages to a TLR7/8 ligand (R848) leads to a significantly higher production of pro-inflammatory cytokines after subsequent challenge with either TLR2 or 4
As our results show that R848-primed bone marrow-derived macrophages (BMDMs) leads to an increased expression of K63-linked ubiquitinated proteins especially K63-linked ubiquitinated TRAF6, we determined the effect of SMER3, an E3 Ubiquitin ligase inhibitor (ULI), on TRAF6, the phosphorylation of TAK1, and the activation of MAPKs

Summary

Introduction

Viral infections predispose the host to secondary bacterial infections [1]. Post-influenza bacterial pneumonia is a major cause of morbidity and mortality associated with both seasonal and pandemic influenza virus illness [2]. Epidemiologic evidence suggests that mortality during the 1918–19 pandemic was mostly due to bacterial pneumonia [3]. R848 Priming and TRAF6 K63-Linked Polyubiquitination for the immunopathology of post viral–bacterial pneumonia. These mechanisms include bacterial colonization due to loss of mucociliary clearance, inhibition of the Th17 pathway, increased IL-10 production, suppression of macrophage function due to inhibition of NADPH oxidase dependent decreased phagocytic bacterial clearance [4, 5]. The molecular mechanisms for such increased susceptibility to bacterial infections after a viral infection are not well understood. The cytokine storm triggered by post viral–bacterial infections may contribute to the mortality seen in viral infections, especially influenza infection [6]

Methods

Results

Conclusion