STING orchestrates the crosstalk between polyunsaturated fatty acid metabolism and inflammatory responses

Abstract

Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.

Keywords: FADS2; STING; cGAS; cytosolic DNA; delta-6 Desaturase; inflammation; interferon responses; metabolism; nucleic acid immunity; polyunsaturated fatty acids.

Graphical abstract

Figure 1

STING deficiency leads to global metabolic improvement (A) Cxcl10, Tnf-α, and Il6 mRNA levels were measured in liver, muscle, and visceral adipose tissue (VAT) from Sting^+/+ (n = 5) and Sting^−/− (n = 5) mice. (B) Body weight of Sting^+/+ (n = 32) and Sting^−/− (n = 34) mice under normal chow diet. (C) Body composition of Sting^+/+ (n = 6) and Sting^−/− (n = 6) mice was assessed by EchoMRI. (D) Food intake of Sting^+/+ (n = 8) and Sting^−/− (n = 9) mice. (E) Insulin tolerance test (ITT) in Sting^+/+ (n = 14) and Sting^−/− (n = 15) mice. Left: glycemia (mg/dL) over time, following a bolus of insulin. Right: area above the curve. (F) Glucose tolerance test (GTT) was performed in Sting^+/+ (n = 25) and Sting^−/− (n = 24) mice. Left: glycemia (mg/dL) over time, following a bolus of glucose. Right: area under the curve (AUC). (G) [18F]-FDG clearance rates were measure over time in Sting^+/+ (n = 5) and Sting^−/− (n = 5) mice and are expressed as percent (%) injected dose (ID) per mL. p = 0.0003. (H) Oxygen consumption of Sting^+/+ (n = 6) and Sting^−/− (n = 6) mice as determined in metabolic chambers. (I) Energy expenditure during day (white) and night (gray) was determined as in (H). p value was determined by one-way ANOVA. (J) Daily profile of voluntary running-wheel activity of Sting^+/+ (n = 11) and Sting^−/− (n = 10) mice. (K) [18F]-FDG concentration in the subcutaneous (SAT), visceral (VAT), and brown adipose tissue (BAT) from Sting^+/+ (n = 7) and Sting^−/− (n = 7) mice was measured at 45 min post-injection of [18F]-FDG. Results are expressed as %ID/g of tissue. (L) Pgc1α, Cidea, Dio2, and Ucp1 mRNA levels in the VAT and BAT of Sting^+/+ (n = 4-6) and Sting^−/− (n = 5) in mice. (M) Rectal temperature of Sting^+/+ (n = 13) and Sting^−/− (n = 14) mice under normal chow diet. (N) GTT was performed in Sting^+/+ (n = 7) and Sting^−/− (n = 9) mice under high-fat diet (HFD 60%) for 6 (6w) and 20 weeks (20w). Left: glycemia (mg/dL) over time, following a bolus of glucose. Right: AUC. (O) Rectal temperature of Sting^+/+ (n = 7) and Sting^−/− (n = 9) mice after 6 or 20 weeks of HFD. (P) Survival curve of Sting^+/+ (n = 6) and Sting^−/− (n = 7) mice under HFD. All graphs present means ± SEM. p values were determined by Student’s t test, unless otherwise stated. ns, not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Related to Figure S1.

Figure 2

STING interacts with FADS2 in the absence of pro-inflammatory stimulation (A) Left: experimental scheme. Right: silver-staining of immunopurified Flag- and HA-tagged Sting (F/HA-Sting) separated on SDS-PAGE. Numbers on the left: molecular weight in kDa. (B) Simplified schematic representation of the LA (omega-6 [Ω6], yellow) and ALA (omega-3 [Ω3], blue) fatty acid (FA) desaturation pathway, leading to the generation of LC-PUFAs. Where Fads2 is indicated is where the Fads2-dependent delta-6 desaturase (Δ6D) activity is required. Where Fads1 is indicated is where the Fads1-dependent delta-5 desaturase (Δ5D) activity is required. ALA, α-linolenic acid; LA, linoleic acid; DGLA, dihomo-γ-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid. (C) Flag immunoprecipitation was performed on whole-cell extracts (WCEs) from MEF^Sting−/− expressing F/HA-Sting or not. Inputs and eluates from Flag-immunoprecipitated F/HA-Sting were analyzed by western blot (WB) using indicated antibodies. (D) Immunoprecipitation was performed using a Sting-specific antibody on WCEs from MEF and MEF^Sting−/−. Inputs and immunoprecipitated material were analyzed by WB using indicated antibodies. (E) As in (D), except that WCEs from MEF^Sting−/− expressing F/HA-Sting or not were used. (F) Flag immunoprecipitation was performed on WCEs from MEFs expressing F-Fads2 or not. Inputs and eluates from Flag-immunoprecipitated Flag-Fads2 (F-Fads2) were analyzed by WB using indicated antibodies. (G) Immunofluorescence analysis of MEF^Sting−/− stably expressing F/HA-Sting using anti-Fads2 and anti-HA antibodies and DAPI nuclear staining. BF, bright field. Pearson’s correlation coefficient values for co-localization of Fads2 and Sting in MEF cells. Pearson’s correlation coefficients were calculated from four randomly selected areas. (H) As in (C), except that WCEs from MEF^Sting−/−, MEFs expressing F/HA-Sting, or MEFs expressing Flag-tagged STING (STING-F) or HAQ-STING (HAQ-STING-F) were used. (I) As in (C), except that WCEs from MEFs expressing F/HA-WT-Sting or F/HA-StingΔTM were used. (J) As in (G), except that cells were stimulated (DMXAA) or not (DMSO) for 30 min prior to immunofluorescence analysis. Pearson’s correlation coefficient values for co-localization of Fads2 and Sting in MEF cells treated with DMXAA or not. Pearson’s correlation coefficients were calculated from four randomly selected areas in each group. All graphs present means ± SEM. p values were determined by Student’s t test. ^∗∗p < 0.01. Related to Table 1 and Figure S2.

Figure 3

STING modulates PUFA pools (A) Ratio between total omega-6 (Ω6) and omega-3 (Ω3) PUFAs, LC-PUFAs, and derivatives measured in Sting^+/+ (n = 5) and Sting^−/− (n = 5) mice liver using LC-MS. (B) Sum of indicated PUFAs and LC-PUFAs and respective derivatives in samples analyzed as in (A). (C) Fads1- and Fads2-associated enzymatic activities, respectively Δ5D and Δ6D, were estimated by calculating the substrate/product ratio of PUFAs and LC-PUFAs measured in (A). (D) Ratio between total omega-6 and omega-3 PUFAs and derivatives measured in WT-MEF, MEF^Sting−/−, and MEF^cGas−/− (n = 4–5) using LC-MS. (E) Sum of indicated PUFAs, LC-PUFAs, and respective derivatives in samples analyzed as in (D). (F) qRT-PCR analysis of Prdm16 and Pgc1a mRNA levels in WT-MEF, MEF^Sting−/−, and MEF^cGas−/− (n = 4). (G) WCEs from WT-MEF, MEF^Sting−/−, and MEF^Sting−/− stably expressing F/HA-WT-Sting or F/HA-StingΔTM were analyzed by WB using indicated antibodies. (H) Prdm16, Pgc1α, and Ucp1 mRNA levels in WT-MEF, MEF^Sting−/−, and MEF^Sting−/− stably expressing F/HA-WT-Sting or F/HA-StingΔTM (n = 3). (I) Prdm16, Pgc1α, and Ucp1 mRNA levels in WT-MEF and MEF^Sting−/− treated or not with the sc26196 Fads2 inhibitor for 48 h (n = 4). (J) GTT was performed in Sting^+/+ (n = 4) and Sting^−/− mice under standard (STD) (n = 8) or low omega-3 (Ω3^L) diet (n = 7) for 4 weeks. (K) AUC before and after 4 weeks of STD or Ω3^L diet in Sting^−/− mice was calculated from data in (J). (L) Rectal temperature of Sting^+/+ and Sting^−/− mice from (J). (M) Sting inhibits Fads2. Consequently, absence of Sting leads to increased Fads2-dependent desaturation of PUFAs and LC-PUFAs, driving the activation of thermogenic program genes and thermogenesis in vivo. All graphs present means ± SEM. p values were determined by Student’s t test. ns, not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Related to Figure S3.

Figure 4

STING activation promotes FADS2-dependent desaturation (A) Ifnβ and Cxcl10 mRNA levels in 293T cells transfected or not with dsDNA for 24 h (n = 3). (B) WCEs of cells treated as in (A) were analyzed by WB using indicated antibodies. (C) Sum of indicated PUFAs, LC-PUFAs, and derivatives in samples prepared as in (A). (D) Ratio between total omega-6 and omega-3 PUFAs and derivatives in samples from (C). (E) Ifnβ and Cxcl10 mRNA levels in WT and Trex1-deficient MEF cells (n = 3). (F) WCEs of WT and Trex1-deficient MEF cells were analyzed by WB using indicated antibodies. (G) Sum of indicated PUFAs, LC-PUFAs, and derivatives in WT and Trex1-deficient MEF cells. (H) Ratio between total omega-6 and omega-3 PUFAs and derivatives in samples from (G). All graphs present means ± SEM. p values were determined by Student’s t test. ns, not significant; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Related to Figure S4 and Table S1.

Figure 5

STING agonists promote FADS2-dependent desaturation (A) WCEs from WT, Sting^−/−, or cGas^−/− MEFs stimulated or not with DMXAA for 2 h were analyzed by WB using indicated antibodies. (B) Sum of omega-6 and omega-3 PUFAs, LC-PUFAs, and derivatives measured in MEFs stimulated or not with DMXAA for 6 h. (C) WCEs from WT, Sting^−/−, or cGas^−/− MEFs stimulated or not with dsDNA for 6 and 24 h were analyzed by WB using indicated antibodies. (D) Molecular docking of cGAMP, DMXAA, and 6 PUFAs to STING (top) or FADS2 (bottom). Color coding for ligand: LA in blue, ALA in green, AA in orange, DHA in turquoise, DGLA in brown, and EPA in magenta. (E) Binding of Flag-purified F-Fads2 or F/HA-Sting to cGAMP was analyzed by WB using anti-Sting or anti-Fads2 antibodies. (F) Prdm16 and Ucp1 mRNA levels in MEFs stimulated or not with DMXAA for 3 h in presence or not of the sc26196 Fads2 inhibitor (n = 3). (G) Ucp1 mRNA levels in the VAT of Sting^−/− mice treated or not with DMXAA for 4 weeks (n = 5). (H) Ucp1 protein levels in the VAT of Sting^−/− mice treated or not with DMXAA for 4 weeks. Representative WB, n = 3. (I) Sting agonist promotes Fads2-dependent desaturation of PUFAs, leading to increased LC-PUFAs and derivatives, thereby driving the activation of thermogenic program genes and thermogenesis in vitro and in vivo. All graphs present means ± SEM. p values were determined by Student’s t test. ^∗∗p < 0.01, ^∗∗∗p < 0.001. Related to Figure S5.

Figure 6

Fads2 and PUFAs regulate Sting activation (A) WCEs from WT-MEFs treated or not with DMXAA, in the presence or absence of 50 μM ALA or LA for 2 or 6 h, were analyzed by WB using indicated antibodies. (B) Ifnβ, Cxcl10, and Isg15 mRNA levels in cells treated as in (A) (n = 3). (C) WT-MEFs were treated or not with DMXAA in the presence or not of 50 μM ALA or OA for 3 h prior to analysis by WB using indicated antibodies. (D) WT-MEFs were transfected or not with dsDNA in the presence or absence of 50 μM ALA for 6 h prior to analysis by WB using indicated antibodies. (E) Flag-immunoprecipitation was performed from MEF^Sting−/− stably expressing F/HA-Sting or not, following treatment with DMXAA or not in combination with ALA for 3 h. Input and eluates were analyzed by WB using indicated antibodies. (F) MEFs expressing stably expressing shScr or shFads2 were treated or not with 50 μM ALA for 6 h prior to analysis by WB using indicated antibodies. (G) WCEs from MEFs expressing shScr or shFads2 transfected or not with dsDNA for 6 h were analyzed by WB using indicated antibodies. (H) Ifnβ and Cxcl10 mRNA levels in cells expressing shScr or shFads2 after stimulation or not with dsDNA for 6 h (n = 4). (I) WT-MEFs were infected with HSV-KOS64 for 16 h prior to analysis of Ifnβ, Cxcl10, and Isg15 mRNA levels. (J) As in (I), except that Prdm16, Pgc1α, and Ucp1 mRNA levels were analyzed. (K) WT-MEFs were infected with HSV-KOS64 in presence or not of a Fads2 inhibitor (sc26196). Infection is presented as mean plaque number per cm². (L) WT-MEFs were infected with HSV-KOS64 in presence or not of 10 μM ALA. Infection is presented as mean plaque number per cm². (M) PUFAs and FADS2 inhibit STING. All graphs are means ± SEM from at least 3 independent experiments. p values were determined by Student’s t test. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001. Related to Figure S6.