USP8-Governed MDA5 Homeostasis Promotes Innate Immunity and Autoimmunity

Abstract

The essential cytoplasmic RNA sensor Melanoma Differentiation-Associated protein 5 (MDA5) initiates type I interferons (IFNs) signaling and subsequent immune responses. However, aberrant activation of MDA5 by viral infections or gain-of-function mutations leads to severe autoimmune diseases, for most of which effective treatment is limited. Here, it is shown that inactivation of ubiquitin-specific protease 8 (USP8/UBPy) degrades the MDA5 protein, suppressing antiviral signaling and autoimmunity. It is found that viral infection modulates the AKT-dependent phosphorylation of USP8 at serine 718, which not only promotes the activation of USP8 but also enhances the association between USP8 and MDA5 and the consequent deubiquitination and stabilization of MDA5. Inactivation of USP8 specifically degrades the MDA5 protein regardless of the mutation pattern. Genetic deletion of Usp8 in mice contributes to decreased levels of type I interferons and proinflammatory cytokines. Importantly, inhibition of USP8 or AKT can effectively suppress MDA5-induced autoimmunity in Aicardi-Goutières syndrome (AGS) mice and anti-MDA5-positive dermatomyositis (DM)/systemic lupus erythematosus (SLE) patient cells. Therefore, these results highlight the critical roles of USP8 in innate antiviral immunity against RNA viruses and autoimmunity and provide a potential therapy for treating autoimmune diseases associated with MDA5.

Keywords: AKT; MDA5; USP8; autoimmunity; deubiquitination; innate immunity.

Figure 1

USP8 interacts with and upregulates the MDA5 protein. A) Immunoblot analysis of MDA5 and GAPDH (up panel) and quantification of the intensities of MDA5 (relative to GAPDH) (bottom panel) in THP1 cells infected with EMCV for 5 h followed by treatment with CHX for 0–8 h. B) Luciferase assay in HEK293T cells co‐transfected with the NF‐κB luciferase reporter (NF‐κB pro.), pRL‐TK, MDA5 CARD, or empty vector or DUBs for 24 h. Luciferase activities of DUBs on the NF‐κB luciferase reporter (Renilla luciferase as an internal control) were normalized to empty vector control activity. C) Luciferase assay in HEK293T cells co‐transfected with the IFNβ luciferase reporter (IFNβ pro.), pRL‐TK, MDA5, or empty vector or DUBs for 24 h. Luciferase activities of DUBs on the IFNβ luciferase reporter (Renilla luciferase as an internal control) were normalized to empty vector control activity. D) Venn diagram showing the overlapping DUBs that enhanced promoter's activity in the two screening assays described in (B) and (C). E) Immunoblot analysis of the MDA5, RIG‐I, VISA, TBK1, IRF3, IRF7 and SFB‐GFP in HEK293T cells transfected with empty vector or SFB‐USP8. F) Pull‐down analysis of the direct interaction between exogenous USP8 and MDA5 purified from BL21 E. coli. G) Pull‐down analysis of the interaction between endogenous USP8 and MDA5 in THP1 cells stably overexpressing MDA5 and infected with EMCV as indicated. H) Pull‐down analysis (with anti‐S protein) of the interaction between USP8 and wild‐type MDA5 or deletion mutants of MDA5 in HEK293T cells. I) Pull‐down analysis (with anti‐S protein) of the interaction between MDA5 and wild‐type USP8 (Myc‐USP8) or deletion mutants of USP8 in HEK293T cells. J) Immunoblot analysis of exogenous MDA5 in HEK293T cells transfected with full‐length SFB‐USP8 or truncated mutants. K) Immunoblot analysis of full‐length MDA5 or truncated mutants in HEK293T cells transfected with empty vector or SFB‐USP8. The data from the indicated wells per group are presented as the means ± SEMs. Each blot data is representative of three independent experiments. (B and C) n = 3 biologically independent experiments. P values were determined by t tests (B and C).

Figure 2

USP8 deficiency impairs the type I IFN antiviral immune response in vitro. A) Immunoblot analysis of the USP8 protein in USP8‐knockdown HEK293T cells. B) Luciferase assay of the NF‐κB, IFNβ, and ISRE luciferase reporter in a scramble or shUSP8‐HEK293T cells transfected with Poly(I:C) HMW or MAD5. C) RT‒qPCR analysis of IFNβ, CXCL10, and ISG56 mRNA in a scramble or shUSP8‐SUM159 cells with or without Poly(I:C) HMW transfection. GAPDH was used as the internal reference. D) Immunoblot analysis of p‐IRF3, p‐TBK1, USP8, and GAPDH protein levels in scramble or shUSP8‐HEK293T SUM159 cells with or without Poly(I:C) HMW transfection at the indicated times. E) RT‒qPCR analysis of IFNβ, CXCL10 and ISG56 mRNA in USP8^+/+ (sgGFP) or USP8^+/‐ (sgUSP8) A549 cells with or without infection with EMCV. GAPDH was used as the internal reference. F) Viral titers of USP8^+/+ or USP8^+/‐ A549 cells infected with EMCV for 24 h were determined via a standard plaque assay. The cells were stained with crystal violet. G) Immunoblot analysis of p‐IRF3, p‐TBK1, USP8 and GAPDH protein levels in Tet‐on pLKO‐shUSP8‐ A549 cells treated ‐/+ Dox for 72 h followed by infecting with EMCV at the indicated times. H,I) RT‒qPCR analysis of Ifnb1, Cxcl10 (H) and Ifih1 (I) mRNA in Usp8 ^fl/fl MEFs that were transduced for 48 h with control or Cre lentivirus and then infected with EMCV for 0–8 h. J) Immunoblot analysis of the MDA5, USP8, p‐TBK1, and p‐IRF3 proteins in Usp8 ^fl/fl MEFs that were transduced and infected as described in (H). K,L) ELISA of IFN‐β K) from the supernatants of Usp8^fl/fl MEFs and RT‒qPCR analysis of EMCV replication L) in Usp8 ^fl/fl MEFs that were transduced for 48 h with control or Cre lentivirus followed by infection with EMCV for 8 h. Data represent the analysis of the indicated n wells per group, means ± SEMs. Each blot data is representative of three independent experiments. (B, C, E, G, H, I and L) n = 3 biologically independent experiments; J) n = 3 independent experiments. P values were determined via two‐way ANOVA (B, C, E, I, and K), Multiple unpaired t‐tests H), Welch's t‐test F), and unpaired t‐tests L). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 3

USP8 deficiency impairs the type I IFN antiviral response in vivo. A,B) Survival of age‐ and sex‐matched Usp8 ^fl/fl and Lyz2‐Cre Usp8 ^fl/fl mice after intranasal instillation of EMCV (1x10¹⁴ TCID₅₀ per mouse) A) or intraperitoneal infection with EMCV (5x10¹² TCID₅₀ per mouse) B). C) Flow diagram of the experiment in (D, E). D) ELISA analysis of IFN‐β in the sera of Usp8 ^fl/fl and Lyz2‐Cre Usp8 ^fl/fl mice infected for 48 h via intraperitoneal injection of phosphate‐buffered saline (PBS) or EMCV (2x10¹⁴ TCID₅₀). E,F) RT‒qPCR analysis of Ifnb1 and Cxcl10 mRNA and EMCV RNA in the brains E) and lungs F) of the mice described in (D). G) Flow diagram of the experiment in (G‐J). H) Immunoblot analysis of the MDA5, USP8, p‐TBK1 and p‐IRF3 proteins in BMDMs (Usp8 ^fl/fl or Lyz2‐Cre Usp8 ^fl/fl) infected with EMCV for the indicated times. I) ELISA analysis of IFN‐β in the supernatants of BMDMs infected with EMCV for 12 h. J) RT‒qPCR analysis of EMCV replication in BMDMs infected with EMCV for 12 h. K) Survival of age‐ and sex‐matched Usp8^fl/fl, Cre‐ER Usp8 ^fl/+ and Cre‐ER Usp8 ^fl/fl mice intraperitoneally injected with tamoxifen (80 µg g⁻¹ dissolved in corn oil) for five consecutive days and intravenously injected with EMCV (5x10¹² TCID₅₀ per mouse) 7 days later. L) Immunoblot analysis of the MDA5, USP8, p‐TBK1, and p‐IRF3 proteins in Usp8 ^fl/fl and Cre‐ER Usp8 ^fl/fl MEFs treated with 4‐OH tamoxifen followed by infection with EMCV for 0–12 h. M) RT‒qPCR analysis of Ifnb1 and Cxcl10 expression as described in (K). The data from the indicated wells per group are presented as the means ± SEMs. Each blot data is representative of three independent experiments. D,E) n = 4 in Mock group, n = 5 of Usp8 ^fl/fl in EMCV group and n = 6 of Lyz2‐Cre Usp8 ^fl/fl in EMCV group; F) n = 5 per group; I) n = 3 independent experiments; J,M) n = 3 biologically independent experiments. A,B, and K) n as shown in the figure. p values were determined via the log‐rank (Mantel‒Cox) test A,B, and K), two‐way ANOVA D,I, and M), Multiple unpaired t tests (E and F), upaired t‐tests J). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.

Figure 4

USP8 deubiquitinates and stabilizes MDA5 through its catalytic activity. A) Immunoblot analysis of SFB‐MDA5, Myc‐USP8, and SFB‐GFP (upper panel) and quantification of the intensities of SFB‐MDA5 (relative to SFB‐GFP) (lower panel) in HEK293T cells transfected with the control vector, WT Myc‐USP8 or the Myc‐USP8 C786A mutant in the presence of CHX for 0–9 h. B) Immunoblot analysis of HA‐MDA5, SFB‐USP8 and SFB‐GFP (left blots) and quantification of the intensities of HA‐MDA5 (relative to SFB‐GFP) (right graphs) in scramble HEK293T cells or USP8‐knockdown HEK293T cells reconstituted with vector or SFB‐USP8 in the presence of CHX for 0–12 h. C) Denature pull‐down (with anti‐S protein) and immunoblot analysis (with anti‐Flag, anti‐HA or anti‐Myc) of HEK293T cells transfected with SFB‐MDA5, HA‐Ub, and empty vector, Myc‐USP8 WT, or Myc‐USP8 C786A for 24 h. D) In vitro deubiquitination analysis of ubiquitin‐modified SFB‐MDA5 eluted from Streptavidin Sepharose by biotin (2 mg mL⁻¹) incubated with in vitro‐translated USP8 WT or USP8 CA generated from an in vitro transcription and translation kit. E) Ni‐His pull‐down and immunoblot analysis of exogenous MDA5. SFB‐MDA5, Myc‐USP8, and Myc‐USP8 CA were expressed in 6xHis‐Ub‐HEK293T stable cells, and ubiquitin conjugates were recovered on Nickel magnetic beads under denaturing conditions. The cells were treated with MG‐132 for 6 h prior to harvesting. F) Denature pull‐down (with anti‐S protein) and immunoblot analysis (with anti‐Flag, anti‐HA, or anti‐Myc) of USP8^‐deficient HEK293T cells transfected with SFB‐MDA5, HA‐Ub, and empty vector, Myc‐USP8 WT, or Myc‐USP8 C786A for 24 h. G,H) Denature pull‐down (with anti‐S protein) and immunoblot analysis (with anti‐Flag, anti‐HA or anti‐Myc) of HEK293T cells transfected with SFB‐MDA5, HA‐Ub (K6 or K27 only) and empty vector, Myc‐USP8 WT, or Myc‐USP8 CA for 24 h. Each blot data is representative of three independent experiments.

Figure 5

AKT1 phosphorylates and promotes the activity of USP8 and its association with MDA5. A) SFB‐USP8 was purified from cells infected with or without EMCV and incubated with K6‐linked Di‐ubiquitin at 37 °C for 40 min. B) SFB‐USP8 was purified and incubated with K6‐linked Di‐ubiquitin in the presence of DUB‐IN‐2 (USP8 inhibitor, U8IN) at the indicated dosages at 37 °C for 1 h. C) SFB‐USP8 and phosphorylation site mutants infected or not infected with EMCV were purified and incubated with K6‐linked Di‐ubiquitin in the presence of the compounds at 37 °C for 3 h. D) Immunoblot analysis of Myc‐MDA5 and SFB‐GFP in HEK293T cells transfected with empty vector, SFB‐USP8 WT, S718A, or S718D for 24 h. E) Immunoblot analysis of the phosphorylation levels of endogenous USP8 in THP1 cells infected with EMCV for 0–24 h. F) Immunoprecipitation (with Strep) and immunoblot analysis (with anti‐p‐USP8 Ser718) of the phosphorylation levels of USP8 in SFB‐USP8‐overexpressing HEK293T cells transfected with empty vector or HA‐AKT1 prior to treatment with MK2206. G) Purified SFB‐USP8 (co‐transfection with empty vector or HA‐AKT1 with or without MK2206) were incubated with K6‐linked Di‐ubiquitin in the presence of the compounds at 37 °C for 2 h. H) Purified SFB‐USP8 and S718A (co‐transfection with empty vector or HA‐AKT1) were incubated with K6‐linked Di‐ubiquitin in the presence of the compounds at 37 °C for 1 h. () Immunoblot analysis of exogenous protein levels of MDA5 in HEK293T cells overexpressing HA‐AKT1. J) Immunoblot analysis of Myc‐MDA5 and GAPDH protein expression in USP8^‐deficient HEK293T cells transfected with empty vector, SFB‐USP8, SFB‐USP8 S718A or HA‐AKT1 for 24 h. K) Co‐immunoprecipitation analysis of the interaction between USP8 and MDA5 in HEK293T cells transfected with empty vector or the indicated dose of HA‐AKT1. Each blot data is representative of three independent experiments.

Figure 6

USP8 or AKT1 inhibition promotes the degradation of gain‐of‐function MDA5 mutants. A) Luciferase assay in HEK293T cells co‐transfected with the IFNβ luciferase reporter, pRL‐TK, and MDA5 G821S for 24 h, followed by treatment with U8IN for 9 h. Luciferase values were normalized to Renilla luciferase activity. B) Immunoblot analysis of the SFB‐MDA5 G821S, p‐IRF3, SFB‐MDA5 G821S, and GAPDH proteins in stable‐overexpressing‐SFB‐MDA5 G821S HEK293T cells treated with U8IN for 9 h. C) Immunoblot analysis of SFB‐MDA5 G821S and Hsp90 (top panel) and quantification of the intensities of SFB‐MDA5 G821S (relative to Hsp90) (bottom panel) in stable‐overexpressing‐SFB‐MDA5 G821S HEK293T cells treated with U8IN for 1 h followed by treatment with CHX for 0–8 h. D) Immunoblot analysis of the SFB‐MDA5 G821S, p‐IRF3, and GAPDH proteins in stable‐overexpressing SFB‐MDA5 G821S THP1 cells treated with MK2206 for 9 h. E) Immunoblot analysis of SFB‐MDA5 G821S and Hsp90 (left blots) and quantification of the intensities of SFB‐MDA5 G821S (relative to GAPDH) (right graphs) in stably overexpressing SFB‐MDA5 G821S HEK293T cells treated with MK2206 for 1 h followed by treatment with CHX for 0–8 h. F) Illustration of the IFIH1 mutation sites. G) Denaturing immunoprecipitation (with anti‐S protein) and immunoblot analysis (with anti‐Flag, anti‐HA or anti‐Myc) of HEK293T cells transfected with SFB‐MDA5 (WT/R337G/G821S/M854K/A946T/I956V), HA‐Ub and empty vector or Myc‐USP8 for 24 h. Data represent the analysis of the indicated n wells per group, means ± SEMs. Each blot data is representative of three independent experiments. (A) n = 3 biologically independent experiments. p values were determined via two‐way ANOVA (A). ^*** p < 0.001.

Figure 7

USP8 or AKT1 inhibition alleviates autoimmunity in AGS mice and anti‐MDA5‐positive DM/SLE patient cells. A) Illustration of CRISPR technology editing to generate G821S (gs/+) mutant mice. B) Sequencing identification of gs/+ mice. C) Genotyping analysis of embryos and mice at the indicated ages was performed from the interbreeding of WT and gs/+ mice. D) Body weights (right) and representative images (left) of WT and gs/+ mice. E) Flow diagram of the experiment in (F‐H). F–G) RT‒qPCR analysis of Ifnb1, Tnfα and Il6 mRNA in the liver (F) or kidney (G) of the mice described in (E). H) Survival of age‐matched +/+ and gs/+ mice after intraperitoneal injection of U8IN or MK2206 alone or in combination. I) HE staining analysis of the livers treated as described in (E). Red arrow, increased cytoplasmic eosinophilic acid. Yellow arrow, hepatocyte steatosis, variously sized vacuoles in the cytoplasm. Green arrow, small focal aggregation of lymphocytes. J,K) Immunoblot analysis J) and normalized (K) of MDA5 and p‐TBK1 in PBMCs from patients treated with DMSO or the USP8 inhibitor for 16 h. Ponceau S staining for total protein normalization. Data represent the analysis of the indicated n wells per group, means ± SEMs. Each blot data is representative of three independent experiments. D) n = 7 in +/+ group, n = 3 in gs/+ group; (F) n = 3 in +/+, NC; gs/+, U8IN; gs/+, MK2206 group; n = 4 in gs/+, NC; gs/+, U8IN+MK2206 group; G) n = 4 in +/+, NC; gs/+, NC; gs/+, U8IN+MK2206 group; n = 3 in gs/+, U8IN; gs/+, MK2206 group; C,H) n as shown in the figure. p values were determined via unpaired t tests (D), one‐way ANOVA (F and G), log‐rank (Mantel‒Cox) tests H), and two‐way ANOVA K). ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.