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. 2018 Jan 2;13(1):e0190597.
doi: 10.1371/journal.pone.0190597. eCollection 2018.

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

Ebony A Monson  1 , Keaton M Crosse  1 , Mithun Das  1 , Karla J Helbig  1
Affiliations

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

Ebony A Monson et al. PLoS One. .

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Lipid droplet mass is reduced by using lowered serum media, with little effect on their biogenesis.
A. Reduced serum (10% to 2% FCS) was applied to Huh-7 and HeLa cells for 48 hrs. Bodipy 505/515 (i) and Oil Red O (ii) were used to stain neutral lipid in both Huh-7 and HeLa cells. Images of fixed cells were captured using a Nikon Eclipse Ti-E microscope at 20X and 40X magnification respectively. DAPI counterstaining was also used to visualise cell nuclei. B. Following visualisation of bodipy stained cells, quantification of fluorescence intensity was performed using NIS Elements AR v.3.22. ***p < 0.001 C. Cells were grown in low serum conditions (2% FCS) for 72 hrs. Rapamycin (RAPA) and Chloroquine (CQ) were used as a positive control for the induction of autophagy. Membranes were probed with anti-LC3 specific antibody and anti-rabbit HRP. Membranes scanned using Amersham 600 chemiluminescence imager (i); Densitometry was performed using Image J analysis (ii) D. LD number was reduced in Huh-7 and HeLa cells by reducing FCS in media to 2% for 48 hrs prior to experiment, representative by time 0. Cells were then returned to 10% FCS media at commencement for experiment, and fixed at the indicated time points. Cells were stained with Bodipy 505/515 and DAPI prior to imaging using a Nikon Eclipse Ti-E microscope at 20X magnification (i), and subsequent image analysis using NIS elements (ii).
Fig 2
Fig 2. LD content alters the regulation of ISGs in a cell-type dependent manner.
A. Huh-7 and HeLa cells were transiently transfected 24 hrs prior to stimulation with 500 ng/well of ISRE-Luc and 5 ng/well of pRL-TK in 12-well tissue culture plates. Luciferase measurements were taken 5 hrs post stimulation with IFN-β (ns = not statistically significant). B. HeLa and C. Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to stimulation with 1000 units/ml of. IFN-β for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFIT-1 (ii) OAS-1 and (iii) Viperin (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).
Fig 3
Fig 3. Lipid droplet content does not impact nucleic entry into Huh-7 cells.
A. A. Huh-7 cells were either incubated in control media or 2% FCS media, 48 hrs prior to transfection with rhodamine conjugated poly I:C. B. Cells were imaged, using a Nikon Ti-E inverted microscope and quantification of fluorescence intensity performed using NIS Elements AR v.3.22.
Fig 4
Fig 4. Reduced lipid droplet content in cells reduces and delays the host response to dsDNA.
A. HeLa and B. Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to transfection with 0.5 μg poly dA:dT per well for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFN-β and (ii) IFN- λ or C. Viperin mRNA (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).
Fig 5
Fig 5. Reduced lipid droplet content in cells reduces and delays the host response to dsRNA.
A. HeLa and B. Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to transfection with 0.5 μg poly I:C per well for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFN-β and (ii) IFN- λ or C. Viperin mRNA (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).
Fig 6
Fig 6. Lipid droplet content alters the host cell response to sendai virus.
Hela cells were pre-incubated in either low serum or control media for 48 hrs prior to infection with 50 HAU/ ml SeV. Following 8 and 24 hrs infections of SeV, RTq-PCR was performed to quantitate mRNA expression of A. IFN-β and B. IFN-λ. (*, p< 0.05; **, p< 0.01; ***, p< 0.001).
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Grants and funding

This work was funded by an internal La Trobe University Grant.