Lipid droplet density alters the early innate immune response to viral infection.

Ebony A Monson,Karla J Helbig,Mithun Das,Keaton M Crosse,Luwen Zhang

doi:10.1371/journal.pone.0190597

Abstract

The cellular localisation of many innate signalling events following viral infection has yet to be elucidated, however there has been a few cases in which membranes of certain cellular organelles have acted as platforms to these events. Of these, lipid droplets (LDs) have recently been identified as signalling platforms for innate TLR7 and 9 signalling. Despite their wide range of similar roles in various metabolic pathways, LDs have been overlooked as potential platforms for antiviral innate signalling events. This study established an in vitro model to evaluate the efficiency of the early innate immune response in cells with reduced LD content to the viral mimics, dsDNA and dsRNA, and Sendai viral infection. Using RT-qPCR, the expression of IFN-β and IFN-λ was quantified following stimulation along with the expression of specific ISGs. Luciferase based assays evaluated the combined expression of ISRE-promoter driven ISGs under IFN-β stimulation. Cellular LD content did not alter the entry of fluorescently labelled viral mimics into cells, but significantly decreased the ability of both Huh-7 and HeLa cells to produce type I and III IFN, as well as downstream ISG expression, indicative of an impeded innate immune response. This observation was also seen during Sendai virus infection of HeLa cells, where both control and LD reduced cells replicated the virus to the same level, but a significantly impaired type I and III IFN response was observed in the LD reduced cells. In addition to altered IFN production, cells with reduced LD content exhibited decreased expression of specific antiviral ISGs: Viperin, IFIT-1 and OAS-1 under IFN-β stimulation; However the overall induction of the ISRE-promoter was not effected. This study implicates a role for LDs in an efficient early innate host response to viral infection and future work will endeavour to determine the precise role these important organelles play in induction of an antiviral response.

Highlights

The innate immune response constitutes the first line of host defence to invading viruses; as such, viral infection of a mammalian cell triggers the activation of a number of pattern recognition receptors (PRRs), with subsequent pathway activation resulting in the production of interferon (IFN)
Quantitative real-time PCR was performed in a CFX Connect RealTime Detection System (Bioline) to quantitate the relative levels of IFN and interferon stimulated gene (ISG) mRNA in comparison to the house keeping gene RPLPO
Cells were initially plated in reduced FCS media for 24, 48 and 72 hours, and as can be seen in Fig 1A, a 48 h incubation with 2% FCS media, displayed a marked reduction in Lipid droplet (LD) content as evidenced by staining of neutral lipids with either Bodipy or Oil Red O (Fig 1A), and quantification of Bodipy fluorescent intensity (Fig 1B)

Summary

Introduction

The innate immune response constitutes the first line of host defence to invading viruses; as such, viral infection of a mammalian cell triggers the activation of a number of pattern recognition receptors (PRRs), with subsequent pathway activation resulting in the production of interferon (IFN). Lipid droplet density alters the early innate immune response to viral infection of infected and uninfected cells [1]. The activation of a secondary signalling pathway, the JAK/ STAT pathway, initiates the expression of hundreds of interferon stimulated genes (ISGs). It is these ISGs which promote an antiviral state, decreasing the susceptibility of uninfected cells to subsequent infection by impeding viral proliferation [1]. The germline-encoded innate immune system is able to detect and neutralise incoming foreign pathogens but it primes and shapes the adaptive immune response [2]

Methods

Results

Conclusion