Homeostatic regulation of STING protein at the resting state by stabilizer TOLLIP

Abstract

STING (stimulator of interferon genes) is an important innate immune protein, but its homeostatic regulation at the resting state is unknown. Here, we identified TOLLIP as a stabilizer of STING through direct interaction to prevent its degradation. Tollip deficiency results in reduced STING protein in nonhematopoietic cells and tissues, and renders STING protein unstable in immune cells, leading to severely dampened STING signaling capacity. The competing degradation mechanism of resting-state STING requires IRE1α and lysosomes. TOLLIP mediates clearance of Huntington's disease-linked polyQ protein aggregates. Ectopically expressed polyQ proteins in vitro or endogenous polyQ proteins in Huntington's disease mouse striatum sequester TOLLIP away from STING, leading to reduced STING protein and dampened immune signaling. Tollip^-/- also ameliorates STING-mediated autoimmune disease in Trex1^-/- mice. Together, our findings reveal that resting-state STING protein level is strictly regulated by a constant tug-of-war between 'stabilizer' TOLLIP and 'degrader' IRE1α-lysosome that together maintain tissue immune homeostasis.

Figure 1:. Tollip-deficiency impairs STING-mediated IFN signaling.

(A and B) Arrayed siRNA screen of 21 different gene targets in MEFs. Quantitative RT-PCR (qRT-PCR) expression of Ifnb mRNA following cGAMP stimulation (2 μg/ml, transfected by Lipofectamine, same below) for 5 h (A) or intracellular polyI:C (1 μg/ml, transfected by Lipofectamine, same below) for 5 h (B). A representative experiment with 2 biological replicates is showing (same below). Experiments were repeated for at least three times (same below). (C) qRT-PCR analysis of Ifnb mRNA following cGAMP stimulation for 5 h. Wild type MEFs were treated with control siRNA (siCtrl) with 4 distinct siRNA sequences targeting Tollip for 48 h before cGAMP stimulation. Right panel shows that cells with greater Tollip KD had less Ifnb mRNA expression. (D) qRT-PCR analysis of Ifnb mRNA in Tollip KD MEFs stimulated with cGAMP over a 24 h time course. (E) qRT-PCR analysis of Ifnb mRNA in Tollip KD MEFs stimulated with increasing amount of cGAMP (0, 2, 3, 5 μg/ml) for 5 h. (F) qRT-PCR analysis of Ifnb, Il6 mRNA and several ISGs in Tollip KD MEFs stimulated with STING agonist DMXAA (50 μg/ml) for 2 h. Heat map summarizes qRT-PCR data on ISG expression. (G) qRT-PCR analysis of Ifnb in Tollip KD cells stably expressing an empty vector or siRNA-resistant Flag-TOLLIP. Cells were stimulated with DMXAA (50 μg/ml) for 2 h. (H and I) Tollip^+/+ or Tollip^−/− MEFs were stimulated with increasing amount of DMXAA (0, 1, 10, 100 μg/ml) for 2 h (H) or intracellular polyI:C (0, 0.01, 0.1, 1.0, 5.0, 10.0 ng/ml) for 5 h (I). Ifnb mRNA was analyzed by qRT-PCR (J) Bone marrow-derived macrophages (BMDMs) from Tollip^+/+ or Tollip^−/− mice were stimulated with dsDNA (1 μg/ml), cGAMP (2 μg/ml) or DMXAA (50 μg/ml) and Ifnb mRNA was measured by qRT-PCR. For panel G, ***, p < 0.001; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided). Data are representative of at least three independent experiments.

Figure 2:. TOLLIP prevents lysosomal-mediated degradation of STING at the resting state.

(A) Immunoblot analysis of control or Tollip-knockdown MEFs stimulated with DMXAA (50 μg/ml) for indicated amount of time (top). Relative intensity of STING protein bands was quantified and presented on the right. Red arrows denote reduced STING protein in Tollip-knockdown cells at time 0. (B) qRT-PCR analysis of basal Tmem173 mRNA in control or Tollip KD MEfs. n=4 biological replicates. (C) Immunoblot analysis of STING and other ER proteins in Tollip^+/+ and Tollip^−/− MEFs. (D) qRT-PCR analysis of Tmem173 mRNA in Tollip^+/+ and Tollip^−/− MEFs. Bar graph shows basal expression level. Line graph shows mRNA turnover after Actinomycin D treatment. n=3 biological replicates. (E) Immunoblot analysis Tollip^+/+ and Tollip^−/− MEFs stably expressing an empty vector or Flag-TOLLIP. Relative protein band intensity from at least two independent experiments were quantified and presented on the bottom. A representative experiment with 2 biological replicates is showing. Experiments were repeated twice. (F) Immunoblot analysis of Tollip^+/+ and Tollip^−/− MEFs treated overnight with 3-MA (5 mM), MG-132(5 μM) and BafA1(0.5 μM). (G) qRT-PCR analysis of basal ISG mRNA in Tollip^+/+ and Tollip^−/− MEFs. n=2 biological replicates. For panel B, D, G, *, P < 0.05; **, P < 0.01; ***, P < 0.005; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided). Data are representative of at least two independent experiments.

Figure 3:. TOLLIP interacts with STING

(A) Co-immunoprecipitation of STING and TOLLIP in HEK293T cells. HEK293T cells were transfected with indicated plasmids (top), and 24 h later anti-HA antibody was used to pull down HA-STING. FLAG-TOLLIP or FLAG-TOM1 (a negative control) co-IP was analyzed by immunoblot. *IgL indicates IgG light chain. (B) Co-immunoprecipitation of endogenous STING and TOLLIP in MEFs. Wild type MEFs were untreated or stimulated with DMXAA for 1 h. Anti-STING antibody was used to pull down endogenous STING. Both IP and lysate were blotted for endogenous TBK1, STING and TOLLIP. (C) Fluorescent microscopy analysis of TOLLIP and STING localization. Scale bar, 10 μm. (D, E) Domain mapping studies. HEK293T cells were transfected with full-length TOLLIP plus various truncations of STING (D) or full-length STING plus various truncations of TOLLIP (E). co-IP analysis was performed as in A. Diagrams of each constructs used are showing on the top. Data are representative of at least three independent experiments.

Figure 4:. PolyQ protein disrupts TOLLIP:STING interaction and results in STING protein turnover in vitro and in vivo.

(A) Immunoblot analysis of HEK293T cells transfected with the same amount HTTq74 plasmid and increasing amount of FLAG-TOLLIP plasmids (as indicated on top). (B) Immunoblot analysis of HEK293T cells transfected with the same amount Flag-STING plasmid and increasing amount of HTTq74 plasmids (as indicated on top). (C) Immunoblot analysis of STING and HTTq74 co-expression treated overnight with MG-132 (5 μM) and BafA1 (0.5 μM). (D) IFNβ-Luciferase assay of cGAS-STING activation after dose titration of HTTq74 protein. A representative experiment with 2 biological replicates is showing. Experiments were repeated twice. (E) Co-immunoprecipitation of STING and TOLLIP in HEK293T cells. HEK293T cells were transfected with indicated plasmids (top) in the presence of BafA1 (0.5 μM), and 24 h later anti-FLAG antibody was used to pull down FLAG-TOLLIP. HA-STING co-IP was analyzed by immunoblot. Note, BafA1 was used to prevent STING degradation caused by polyQ protein. (F) STING protein and mRNA analysis in zQ175 knock-in mouse brain tissues. Wild type, zQ175/+ and zQ175/zQ175 mouse brain striatum and cortex were freshly dissected and processed for immunoblot and qRT-PCR analysis. n=3 mice per group. STING protein blots are showing on the left, densitometry quantitation of STING/TUBULIN band ratio is showing in the middle, and STING mRNA level (normalized to GAPDH) is showing on the right. For panel F, **, P < 0.01; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided). Data are representative of at least two independent experiments.

Figure 5:. TOLLIP stabilizes STING protein in cells.

(A) Immunoblot analysis of STING protein in Tollip^+/+ and Tollip^−/− MEFs treated with cycloheximide (CHX). IkBa is a control protein with short half-life that is unaffected by TOLLIP. Densitometry quantitation of protein bands are showing on the right. (B) Immunoblot analysis of STING protein in Tollip^+/+ and Tollip^−/− MEFs treated with cycloheximide (CHX) alone or in combination of proteasome inhibitor BTZ or lysosome inhibitor BafA. Data are representative of at least two independent experiments.

Figure 6:. Tollip-deficiency selectively activates IRE1α, which facilitate resting-state STING protein turnover.

(A) qRT-PCR analysis of basal expression of UPR genes in Tollip^+/+ and Tollip^−/− MEFs stably expressing an empty vector or Flag-TOLLIP. A representative experiment with 2 biological replicates is showing. Experiments were repeated three times. (B) Immunoblot analysis of UPR proteins in Tollip^+/+ and Tollip^−/− MEFs treated with thapsigargin (Tg, 500 nM) for indicated amount of time (top). Red arrows denote increased IRE1α in Tollip^−/− MEFs. (C) Immunoblot analysis of Ern1^+/+ and Ern1^−/− MEFs stimulated with DMXAA (10 μg/ml) for indicated amount of time (top). (D) qRT-PCR analysis of basal Tmem173 mRNA in Ern1^+/+ and Ern1α^−/− MEFs. A representative experiment with 2 biological replicates is showing. Experiments were repeated three times. (E) Immunoblot analysis of Ern1^+/+ and Ern1^−/− MEFs after treatment with control siRNA or siTollip. (F) Immunoblot analysis of Tollip^+/+ and Tollip^−/− MEFs treated with DMSO or IRE1α inhibitor 4μ8C (100 μM) overnight. (G) Immunoblot analysis of Xbp1^+/+ and Xbp1^−/− MEFs treated with DMSO or 4μ8C (100 μM) overnight. Data are representative of at least three independent experiments.

Figure 7:. Tollip^−/− ameliorates autoinflammatory disease of Trex1^−/− mice

(A-C) Body weight (A), heart H&E staining (B) and spleen (C) from 16-week-old mice of indicated genotypes. n=8 mice. Scale bar, 100 μm. (D) A heat map of ISGs expression in BMDMs of indicated genotypes. Each ISG mRNA expression level was measured by qRT-PCR. Two representative ISGs, Oas3 and Ifit2, are showing on the right. A representative experiment with 2 biological replicates is showing. Experiments were repeated three times. (E) Serum cytokine measurement. n=2 mice. For panel A, C, D, *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant. Error bars represent the SEM. Unpaired Student’s t test (2-sided).

Figure 8.. STING protein level is reduced in Tollip^−/− mouse heart tissue.

(A) H&E staining of fixed mouse heart from indicated genotypes (top). Top images showing the whole heart and bottom images showing enlarged view of cardiomyocytes. Scale bars, 500 μm (upper panels) and 10 μm (lower panels). (B) Fluorescent immunohistochemistry staining with anti-STING (top, and enlarged view at the middle) or Isotype antibody (bottom). STING in green and DAPI in blue. Scale bars, 60 μm (upper panels) and 10 μm (middle and lower panels). (C) A representative enlarged view of cardiomyocytes in wild type heart stained for H&E (left) anti-STING (right) using adjacent tissue slides cut from the same paraffin-embedded block. Dotted lines indicate the same three cardiomyocytes from both images. Scale bars, 10 μm (both panels). (D) Quantification of STING protein fluorescent intensity from B. Four separate areas were chosen from each heart and 25 region-of-interests (ROIs) were randomly chosen from each area (see Supplementary Figure 3). STING fluorescent signal intensity from 100 ROIs are showing for each genotype. ****, P < 0.001. Error bars represent the SEM. Unpaired Student’s t test (2-sided). Experiments were repeated three times using different mouse of each genotype.