The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum

Abstract

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca²⁺ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Figure 1.. STIM1 deficiency spontaneously induces type I IFN response in murine and human cells.

a, Representative immunoblot showing expression of STIM1 in wild type (WT) and Stim1^−/− MEFs (left). qPCR analysis of indicated cytokines and ISGs in unstimulated indicated MEFs (right). qPCR data show pooled technical replicates from two independent experiments (Ifnb1 and Il6) and one representative triplicate from two independent experiments (other genes). b, Levels of secreted IFN-β from culture supernatants of unstimulated WT or Stim1^−/− MEFs. c, Representative traces showing averaged SOCE from WT (31 cells), Orai1^−/− (30 cells) and Stim1^−/− (29 cells) MEFs after passive depletion of intracellular Ca²⁺ stores with 1 μM thapsigargin (TG) in the presence of external solution containing 20 mM Ca²⁺ (left). Bar graph (middle) shows averaged baseline subtracted SOCE (± s.e.m.) from four independent experiments. right: qPCR analysis of Ifnb1 mRNA in indicated MEFs. d, Representative immunoblot showing expression of STIM1 in BMDMs (left). qPCR analysis of Ifnb1 and Il6 mRNA in unstimulated WT and Stim1^−/− BMDMs (right). e, Immunoblot showing expression of STIM1 in wild type (WT) and STIM1^−/− THP1 cells generated using two independent sgRNAs (sg#2) and 3 (sg#3). qPCR analysis of IFNB1 and IL6 mRNA in unstimulated WT, STIM1^−/− THP1 cells and those reconstituted for expression of STIM1 (right two panels). f, Secreted IFN-β levels from culture supernatants of untreated or PMA-differentiated WT or STIM1^−/− THP1 cells. Data show representative triplicate from two independent experiments (panels b, e and f) or pooled technical replicates from two (c) or three (d) independent experiments. All immunoblot data (panels a, d and e) are representative of three independent experiments with similar results. Data are shown as mean ± s.e.m. *p < 0.005, and **p < 0.0005 (unpaired/two-tailed t test – a, b, d; One-way ANOVA – c; and Two-way ANOVA – e).

Figure 2.. STING-TBK1-IRF3 pathway links loss of STIM1 expression to Ifnb1 transcription.

a, qPCR analysis of Ifnb1 mRNA in indicated MEFs under resting conditions or after stimulation with 2’,3’-cGAMP for 2 or 4 h (left). Numbers on top indicate average fold change relative to WT MEFs. Secreted IFN-β levels from culture supernatants of indicated MEFs after stimulation with 2’,3’-cGAMP (right). Data show pooled technical replicates from two independent experiments (qPCR) or one representative triplicate from two independent experiments (ELISA) with similar results. b, qPCR analysis of Ifnb1 transcripts in indicated MEFs transfected with interferon stimulatory DNA (ISD), poly(dA:dT) or poly (I:C) for indicated time (left). qPCR analysis of IFNB1 mRNA from untreated or indicated nucleic acid-transfected THP1 cells. c, Representative confocal images showing localization of GFP-IRF3 in indicated MEFs. Bar graph below depicts quantification from indicated number of cells. Scale bars, 5 μm. d, Representative immunoblot for detection of IRF3 under non-reducing conditions in DSP-crosslinked indicated MEFs, (left). Bar graph (right) shows densitometry analysis of IRF3 ratio (dimer/monomer) from three independent experiments. e, Representative immunoblots showing expression of phospho-TBK1 (P-TBK1), total TBK1, and 𝛽-actin from indicated cells. Numbers below indicate normalized fold change in ratio of P-TBK1/total TBK1. f, Representative immunoblots showing expression of STIM1 and STING in WT, Stim1^−/−, or Stim1^−/− and Tmem173^−/− double knock out (DKO) MEFs (left). Expression of Ifnb1 and Il6 transcripts in indicated MEFs under resting conditions (left two panels) or 4 h after stimulation with 2’,3’-cGAMP (right two panels). g, Secreted IFN-β levels from culture supernatants of indicated MEFs after stimulation with indicated nucleic acids. h, Representative immunoblots showing expression of STIM1 and STING in WT, STIM1^−/−, TMEM173^−/− or STIM1^−/− and TMEM173^−/− double knock out (DKO) THP1 cells (left). Representative traces of averaged SOCE from WT (33 cells), STIM1^−/−, (30 cells), TMEM173^−/− (31 cells) and DKO (31 cells) THP1 cells after passive depletion of intracellular Ca²⁺ stores with 1 μM thapsigargin (TG) in the presence of external solution containing 2 mM Ca²⁺ (middle). Bar graph shows averaged baseline subtracted SOCE (± s.e.m.) from three independent experiments. Right panels show qPCR analysis of IFNB1 or IL6 mRNA in indicated THP1 cells. Data show representative triplicates from two independent experiments with similar results (b, d, f, g and h) unless indicated. All immunoblots are representative of at least three independent experiments with similar results. Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.005, ***p < 0.0005 [Two-way ANOVA – a (left panel); unpaired/two-tailed t test – a (right panel), b; Chi-square test – c; and One-way ANOVA – d, f, g, h].

Figure 3.. STIM1 deficiency causes enhanced type I IFN response in patient cells.

a, Representative immunoblot showing expression of STIM1 in WT, Stim1^−/− MEFs or those expressing either WT STIM1 (+STIM1) or STIM1^E136X (+E136X) mutant. b, qPCR analysis of Ifnb1 and Il6 mRNA in indicated MEFs under resting conditions or 2 h after stimulation with 2’,3’-cGAMP. Data show representative triplicate from two independent experiments. c, Representative immunoblot showing expression of STIM1 and GAPDH in PBMCs isolated from a healthy control (HC) and patient (Pat.). d, Levels of indicated cytokines in serum samples from healthy controls (three independent donors) and STIM1-deficient patient. Data show one representative triplicate from two independent experiments (n=9 for three HCs). e, Taqman qPCR analysis of indicated ISGs from peripheral blood mononuclear cells (PBMCs, top) or purified monocytes (below) from two independent healthy controls and STIM1-deficient patient. Patient data (normalized to those of healthy controls) are derived from two independent experiments performed in duplicates. Data are shown as mean ± s.e.m. *p < 0.05, **p < 0.005, ***p < 0.0005 (One-way ANOVA – b; and unpaired/two-tailed t test – d, e).

Figure 4.. STIM1 interacts with STING for its retention in the endoplasmic reticulum.

a, Representative confocal microscopy image of STING-GFP and STIM1 in a MEF cell. Scale bar, 5 µm, Inset – 1 µm. Pearson’s r = 0.67 ± 0.08 from 9 cells. b, FLAG-immunoprecipitates (IP) from lysates of HEK293T cells overexpressing FLAG-tagged STING and His-tagged STIM1 were immunoblotted for detection of STIM1. Arrow, monomeric STING or STIM1; *, STING multimers. c, Immunoprecipitates of endogenous STING from HEK293 cells were immunoblotted for detection of indicated proteins. d, FLAG-immunoprecipitates (IP) from lysates of HEK293T cells expressing FLAG-tagged STING and His-tagged STIM1 with or without treatment with thapsigargin (1 μM, 10 min; left) or 2’, 3’-cGAMP (1 μM, 30 min and further incubation in media for 1 h) were immunoblotted for detection of the indicated proteins. Bar graphs show densitometry analysis of normalized fold changes (mean ± s.e.m.) in STIM1 and STING band intensity from three (left) and four (right) independent experiments. e, Schematic showing domain structure of STING and STIM1 as indicated in the text. Amino acid residues of STING and STIM1 fragments used in this study are indicated. f, Left – FLAG-immunoprecipitates (IP) from lysates of HEK293T cells expressing FLAG-tagged full-length STING (FL), NTD (a.a. 1–140), and CTD (a.a. 140–379) were immunoblotted for detection of STIM1. Right – Purified recombinant GST-fused indicated fragments of STIM1 incubated with lysates of HEK293T cells expressing FLAG-tagged, FL, NTD or CTD of STING were immunoblotted with anti-FLAG antibody. Immunoblots in panels b, c, and f are representative of four independent experiments. *p < 0.005 (unpaired/two-tailed t test - d).

Figure 5.. STIM1 inhibits STING trafficking to the ER-Golgi intermediate compartment.

a, Representative confocal microscopy images of WT or Stim1^−/− MEFs stably expressing STING-GFP under resting conditions (top two panels) or 4 h after HSV-1 infection (bottom 3 panels) and stained for endogenous p58 (ERGIC). Scale bars, 10 µm. Bar graph shows quantification of indicated number of cells showing STING translocation to the ERGIC under resting conditions or after infection with HSV-1 for indicated times. Data are derived from two independent experiments. b, Representative live cell epifluorescence images of WT (top) or Stim1^−/− (bottom) MEFs after treatment with 1 µM 2’, 3’-cGAMP for the indicated times showing translocation of STING-GFP into the ERGIC (left). Line graph on the right shows normalized rate of translocation of STING in WT (9 cells) and Stim1^−/− (11 cells) MEFs from two independent experiments. Scale bar, 10 µm. c, Reporter assays for Ifnb1 promoter activity in HEK293T cells transfected with STING and increasing amounts of full length STIM1 or its indicated fragments, 6 hours after stimulation with 2’, 3’ cGAMP (top) or poly(I:C) (below). Data show representative triplicate from two independent experiments. *p < 0.005, **p < 0.0005 Chi square test (a) and one-way ANOVA (c); N.S. – not significant.

Figure 6.. Ablation of STIM1 enhances host defense towards DNA viruses and HIV by priming type I IFN responses.

a, qPCR analysis of Ifnb1 and GFP transcripts in uninfected or HSV-1-GFP-infected (MOI 0.1, 24 h) WT or Stim1^−/− MEFs. Data show pooled technical replicates from two independent experiments. b, qPCR analysis of GFP and indicated viral mRNAs in MHV-68-GFP-infected (MOI 0.2, 24 h) WT or Stim1^−/− MEFs. Data show pooled technical replicates from three independent experiments. c, Representative immunoblots showing expression of phospho-IRF3 (P-IRF3), total IRF3, and 𝛽-actin from untreated or HSV-1-infected (MOI 5.0) WT or Stim1^−/− MEFs for indicated time points. d, qPCR analysis of Ifnb1 and Il6 mRNA in untreated or HSV-1-GFP-infected (indicated MOI, 24 h) WT or Stim1^−/− BMDMs. Data shows representative triplicate from two independent experiments. e, Top two panels show representative GFP images in HSV-1-GFP-infected (MOI 10, 24 h) WT, (left) and STIM1^−/− (right) THP-1 cells. Below: qPCR analysis of IFNB1 and GFP transcripts from the same cells. Scale bars, 10 µm. Data shows representative triplicate from two independent experiments. f, Representative flow plots showing frequency of HIV-GFP-infected WT (left) or two different STIM1^−/− (right two panels) THP1 cell lines (MOI 2.0, 24 h). Bar graph shows averaged frequency of HIV-GFP-positive indicated THP1 cell lines in the presence or absence of HIV reverse transcriptase inhibitor azidothymidine (AZT, 5 µM) from four independent experiments. Immunoblots in panel c and epifluorescence images in panel e are representative of three and two independent experiments respectively. *p < 0.005 and **p < 0.0005 [Two-way ANOVA – a (left panel), d, e (right panel), f; unpaired/two-tailed t test – a (right panel), b; One-way ANOVA – e (left panel)].

Figure 7.. STIM1 deficiency enhances host defense against HSV-1 infection in vivo.

a, Kinetics of survival (top) and body weight changes (bottom) of indicated numbers of control (Stim1^fl/fl) and STIM1-deficient (Stim1^fl/flLyz2-cre) mice (6–7-week old) after intravenous injection with HSV-1 (1 × 10⁷ PFU per mouse). b, Kinetics of survival (top) and body weight changes (bottom) of indicated numbers of control (Orai1^fl/fl) and Orai1-deficient (Orai1^fl/flLyz2-cre) mice after intravenous injection with HSV-1 (1 × 10⁷ PFU per mouse). Mice that lost >20% body weight were euthanized. c, Virus load in control (Stim1^fl/fl) and STIM1-deficient (Stim1^fl/flLyz2-cre) mouse brains 3 days after intravenous injection with HSV-1. d, ELISA analyses of the indicated cytokines from the sera of control (Stim1^fl/fl) and Stim1-deficient (Stim1^fl/fl Lyz2-cre) mice after intravenous injection with HSV-1 for indicated times. Data in panels a and b are pooled from two independent experiments. Panels c and d show mean +/− s.e.m. from indicated number of animals (each symbol represents data from individual animal). *p < 0.05, **p < 0.005, ***p < 0.0005 (unpaired/two-tailed t test).