Resveratrol Attenuates the Mitochondrial RNA-Mediated Cellular Response to Immunogenic Stress

Abstract

Human mitochondria contain a circular genome that encodes 13 subunits of the oxidative phosphorylation system. In addition to their role as powerhouses of the cells, mitochondria are also involved in innate immunity as the mitochondrial genome generates long double-stranded RNAs (dsRNAs) that can activate the dsRNA-sensing pattern recognition receptors. Recent evidence shows that these mitochondrial dsRNAs (mt-dsRNAs) are closely associated with the pathogenesis of human diseases that accompany inflammation and aberrant immune activation, such as Huntington's disease, osteoarthritis, and autoimmune Sjögren's syndrome. Yet, small chemicals that can protect cells from a mt-dsRNA-mediated immune response remain largely unexplored. Here, we investigate the potential of resveratrol (RES), a plant-derived polyphenol with antioxidant properties, on suppressing mt-dsRNA-mediated immune activation. We show that RES can revert the downstream response to immunogenic stressors that elevate mitochondrial RNA expressions, such as stimulation by exogenous dsRNAs or inhibition of ATP synthase. Through high-throughput sequencing, we find that RES can regulate mt-dsRNA expression, interferon response, and other cellular responses induced by these stressors. Notably, RES treatment fails to counter the effect of an endoplasmic reticulum stressor that does not affect the expression of mitochondrial RNAs. Overall, our study demonstrates the potential usage of RES to alleviate the mt-dsRNA-mediated immunogenic stress response.

Keywords: Sjögren’s syndrome; dsRNA stress; immunogenic stress; innate immunity; mitochondrial double-stranded RNAs; oligomycin A; resveratrol; tunicamycin.

Figure 1

RES alleviated the IFN response by poly I:C. (A) The expression of mtRNAs in NS-SV-AC cells upon poly I:C transfection. (B) Heatmap of mRNA-seq results upon poly I:C transfection with or without RES pretreatment for type I IFN genes. The two columns represent log₂ fold changes of two biological replicates. (C) The ratios of ISG induction upon poly I:C transfection between RES-pretreated and control samples. (D) The APH assay was used to measure the effect of RES pretreatment on cell viability. n = 4 and error bars are s.e.m. (E) The ratios of mtRNAs induction upon poly I:C transfection between RES-pretreated and control samples. For ratios, Cq values were first normalized to that of ACTB mRNA. For untreated samples, the values were normalized to RNAs from control cells without poly I:C transfection. Similarly, RNAs from RES-pretreated cells with poly I:C transfection were normalized separately from those from RES-treated cells without poly I:C. Unless mentioned otherwise, three independent experiments were carried out, and error bars denote s.e.m. All statistical significances were calculated using one-tailed Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 2

RES pretreatment rescued SS-related protein expressions. (A) Representative images of ZO-1 and occludin upon poly I:C transfection with or without RES pretreatment. The quantification of ZO-1 and occludin expressions is presented as the mean fluorescence intensity per spheroid. Intensities were normalized to the data from each experimental group cultured without poly I:C. n = 3 and error bars are s.e.m. (B) Visualization of GFP-tagged AQP5 expression upon poly I:C transfection. The scale bar represents 100 μm. The mean fluorescence intensities were normalized to the data from each experimental group cultured without poly I:C. In the bar graphs, the “–“ sign indicates RES-untreated/Mock-transfected, whereas the “+” sign indicates RES-treated/poly I:C-transfected. n = 3 and error bars are s.e.m. All statistical significances were calculated using one-tailed Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

OA elevated mtRNA and ISG expressions. The expression of mtRNAs in HCT116 cells upon poly I:C transfection (A), OA treatment (B), or TN treatment (C). All Cq values are relative to that of ACTB mRNA. The values of poly I:C-transfected samples were normalized to the mock-transfected samples, and values of OA- or TN-treated samples were normalized to the DMSO-treated samples. (D,E) Heatmap of differentially expressed genes represents mtRNAs (D) and ISGs (E) upon OA or TN treatment. Each column represents the log₂ fold change of three biological replicates with OA or TN treatment normalized by the control average. Unless mentioned otherwise, three independent experiments were carried out, and error bars denote s.e.m. All statistical significances were calculated using one-tailed Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 4

RES treatment alleviated cell death and IFN response to OA. (A) Cell viability upon OA treatment with or without RES treatment measured using a SRB assay. (B) The ratios of mtRNA induction upon OA treatment between RES-treated and control samples. (C) The ratios of ISG induction upon OA treatment between RES-treated and control samples. For ratios, Cq values were first normalized to that of ACTB mRNA. For untreated samples, the values were normalized to RNAs from control cells without OA treatment. Similarly, RNAs from cells co-treated with RES and OA were normalized separately from those from cells treated only with RES without OA. Unless mentioned otherwise, three independent experiments were carried out, and error bars denote s.e.m. All statistical significances were calculated using one-tailed Student’s t-tests; * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.

Figure 5

The rescue effect of RES on cellular response to immunogenic stresses. Two immunogenic stressors, poly I:C stimulation and OA, exert their downstream effects partly via mtRNAs. By promoting mitochondrial function and preventing mtRNA induction, an antioxidant RES can counter the molecular and cellular responses to these stressors.