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. 2024 Feb 26;15(1):1750.
doi: 10.1038/s41467-024-46212-z.

S-nitrosothiol homeostasis maintained by ADH5 facilitates STING-dependent host defense against pathogens

Mutian Jia #  1   2 , Li Chai #  1   2 , Jie Wang  1   2 , Mengge Wang  1   2 , Danhui Qin  1   2 , Hui Song  1   2 , Yue Fu  1   3 , Chunyuan Zhao  4 , Chengjiang Gao  1   2 , Jihui Jia  1 , Wei Zhao  5   6
Affiliations

S-nitrosothiol homeostasis maintained by ADH5 facilitates STING-dependent host defense against pathogens

Mutian Jia et al. Nat Commun. .

Abstract

Oxidative (or respiratory) burst confers host defense against pathogens by generating reactive species, including reactive nitrogen species (RNS). The microbial infection-induced excessive RNS damages many biological molecules via S-nitrosothiol (SNO) accumulation. However, the mechanism by which the host enables innate immunity activation during oxidative burst remains largely unknown. Here, we demonstrate that S-nitrosoglutathione (GSNO), the main endogenous SNO, attenuates innate immune responses against herpes simplex virus-1 (HSV-1) and Listeria monocytogenes infections. Mechanistically, GSNO induces the S-nitrosylation of stimulator of interferon genes (STING) at Cys257, inhibiting its binding to the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). Alcohol dehydrogenase 5 (ADH5), the key enzyme that metabolizes GSNO to decrease cellular SNOs, facilitates STING activation by inhibiting S-nitrosylation. Concordantly, Adh5 deficiency show defective STING-dependent immune responses upon microbial challenge and facilitates viral replication. Thus, cellular oxidative burst-induced RNS attenuates the STING-mediated innate immune responses to microbial infection, while ADH5 licenses STING activation by maintaining cellular SNO homeostasis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. GSNO inhibits cGAS-STING activation.
Quantitative polymerase chain reaction (qPCR) analysis of Ifnb mRNA expression in mouse peritoneal macrophages (PMs) pretreated with increasing concentrations of GSNO (0, 100, 250, and 500 μM) (a) or SNAP (0, 250, and 500 μM) (b), following herpes simplex virus-1 (HSV-1) infection. c Enzyme-linked immunosorbent assay (ELISA) analysis of interferon (IFN)-β secretion in mouse PMs pretreated with solvent (Ctrl), GSNO, or SNAP, plus stimulation as indicated. d–f Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1, IFN-stimulating DNA (ISD), or cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP). g qPCR analysis of Isg15, Isg54, Isg56, and Mx1 mRNA expression in PMs pretreated with solvent (Ctrl) or GSNO and then stimulated with cGAMP. h qPCR analysis of IFNB mRNA expression in THP1 cells pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1 or cGAMP. i Immunoblot assays of p-IRF3 and p-STAT1 in THP1 cells pretreated with solvent (Ctrl) or GSNO and then stimulated with ISD. j Luciferase activity assays of IFN-β activation in 293-Dual hSTING-A162 cells pretreated with solvent (Ctrl), GSNO, or SNAP, following stimulation by DMXAA. k qPCR analysis of Ifnb mRNA expression in PMs from Sting+/+ or Sting-/- mice pretreated with solvent (Ctrl) or GSNO and then infected with L. monocytogenes. l Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs pretreated with solvent (Ctrl) or GSNO and then infected with L. monocytogenes. Data represent mean ± standard deviation (SD) or one representative image from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.
Fig. 2
Fig. 2. GSNO attenuates host defenses against HSV-1 infection.
a Microscopy analysis of HSV-1 replication in PMs pretreated with solvent (Ctrl) or GSNO, and then infected with HSV-GFP for 12 h. Scale bars, 100 μm. qPCR analysis of HSV-1 UL30 mRNA level (left) and HSV-1 DNA level (right) in PMs pretreated with solvent (Ctrl), GSNO (b), or SNAP (c), and then infected with HSV-1. d–l C57BL/6 J mice were pretreated with solvent (Ctrl) or GSNO and then infected with HSV-1. Serum levels of IFN-β (d) and IL-6 (e) were analyzed by ELISA (PBS group, n = 2; HSV-1 group, n = 8). qPCR analysis of Ifnb mRNA expression in the spleen (f), brain (g), and lung (h) tissues (PBS group, n = 2; HSV-1 group, n = 8). The HSV-1 viral burden was determined by measurement of HSV-1 UL30 mRNA levels in lung tissues (i) (PBS group, n = 2; HSV-1 group, n = 7). Plaque analysis of homogenizes of lung (j) from C57BL/6 J mice were pretreated with solvent (Ctrl) or GSNO (n = 4), and then infected with HSV-1 for 2 days. Hematoxylin and eosin staining of lung tissue sections. Scale bar: 200 μm (k). The Kaplan-Meier method was used to evaluate survival curves (n = 13 per group) (l). Statistical significance was determined by unpaired two-sided multiple Student’s t tests in (a–k) or the log-rank Mantel-Cox test in (l). Data represent mean ± SD or one representative image from three independent experiments.
Fig. 3
Fig. 3. GSNO targets STING.
ELISA analysis of cGAMP production in HSV-1-infected (a) and ISD-stimulated (b) PMs pretreated with solvent (Ctrl) or GSNO. c Co-immunoprecipitation analysis of cGAMP-biotin binding to STING in HSV-1-infected PMs pretreated with solvent (Ctrl) or GSNO. d Immunoblot assays of dimer-STING in cGAMP-stimulated PMs pretreated with solvent (Ctrl) or GSNO. Immunoblot assays of Oligo-STING in HSV-1-infected (e) and cGAMP-stimulated (f) PMs pretreated with solvent (Ctrl) or GSNO. g Confocal microscopy analysis of the co-localization of STING (green) and cis-Golgi (GM130, red) in BJ cells pretreated with solvent (Ctrl) or GSNO and then stimulated with cGAMP. The intensity profiles of each line were quantified by Image J software. h Colocalization analysis of STING and cis-Golgi (GM130) by Manders’ Colocalization Coefficients (MCC). i ELISA analysis of interferon (IFN)-β secretion in PMs from Sting+/+ or Sting-/- mice pretreated with solvent (Ctrl) or GSNO, plus stimulation as indicated. j qPCR analysis of Ifnb mRNA expression in PMs from Sting+/+ or Sting-/- mice pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1, LPS or poly(I:C). k qPCR analysis of IFNB mRNA expression in THP1 cells pretreated with solvent (Ctrl) or GSNO and then stimulated with HSV-1, cGAMP, poly(I:C) or VSV. Data represent mean ± SD or one representative image from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.
Fig. 4
Fig. 4. GSNO induces S-nitrosylation of STING to inhibit cGAMP binding.
a, b Immunoblot assays of STING S-nitrosylation in PMs infected with HSV-1 or L. monocytogenes using the irreversible biotinylation procedure (IBP). c Immunoblot assays of STING S-nitrosylation in HEK293T cells transfected with STING-FLAG and treated with solvent (Ctrl) or GSNO. d Immunoblot assays of in vitro S-nitrosylation of recombinant human STING protein treated with GSNO. e Liquid chromatography-mass spectrometry (LC-MS) spectra of STING S-nitrosylation at residue C257. f Immunoblot assays of S-nitrosylation in HEK293T cells transfected with empty vector, wild-type (WT) STING, or STING mutants (C257S or C309S) using the IBP. g Co-immunoprecipitation analysis of cGAMP-biotin binding to STING in HEK293T cells transfected with empty vector, WT, or C257S STING mutant. h, i The binding pattern of cGAMP with WT STING or S-nitrosylation of STING at C257 (STING-C257-NO) after an 80–100 ns simulation. The dashed green lines represent hydrogen bonds, and the red gear represents hydrophobicity. j Immunoblot assays of S-nitrosylation in HEK293T cells transfected with empty vector, wild-type (WT) STING, or STING mutants (R238A/Y240A) using the IBP. Data are shown as one representative image from three independent experiments.
Fig. 5
Fig. 5. ADH5 attenuates S-nitrosylation of STING to facilitate its activation.
a Immunoblot assays of S-nitrosylation of STING in PMs from Adh5+/+ or Adh5-/- mice infected with HSV-1 using the IBP. b Immunoblot assays of dimer-STING in cGAMP-stimulated PMs from Adh5+/+ or Adh5-/- mice. Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs from Adh5+/+ or Adh5-/- mice stimulated with HSV-1 (c) or cGAMP (d). e ELISA analysis of IFN-β secretion in PMs from Adh5+/+ or Adh5-/- mice, plus stimulation as indicated. f Immunoblot assays of p-TBK1, p-IRF3, and p-STAT1 in PMs transfected with negative control siRNA (siNC) or Adh5 siRNA (siAdh5-2), followed by DMXAA stimulation. g qPCR analysis of Ifnb expression in PMs transfected with siNC or siAdh5-2, plus stimulation as indicated. h Immunoblot assays of p-IRF3 and p-STAT1 in PMs pretreated with increasing concentrations of N6022, followed by HSV-1 infection. i ELISA analysis of IFN-β secretion in PMs pretreated with N6022 and then stimulated with HSV-1 or DMXAA. j Luciferase activity analysis of IFN-β activation in HEK293T cells transfected with empty vector or cGAS plus STING plasmids, together with ADH5 plasmid or empty vector plasmid. Adh5+/+ or Adh5-/- mice were stimulated with HSV-1 by intraperitoneal injection. Serum levels of IFN-β (k) and IL-6 (l) were analyzed by ELISA (PBS group, n = 1; HSV-1 group, n = 4). m, n Adh5+/+ or Adh5-/- mice were stimulated with DMXAA by intraperitoneal injection. Serum levels of IFN-β were analyzed by ELISA (PBS group, n = 1; DMXAA group, n = 4) (m). qPCR analysis of Ifnb mRNA expression in spleen and lung tissues (n). Data represent mean ± SD or one representative from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.
Fig. 6
Fig. 6. ADH5 is downregulated during pathogens infection by promoting DNA methylation.
Immunoblot assays of ADH5 in PMs during HSV-1 (a), L. monocytogenes (b) infection or IFN-β (c) stimulation. qPCR analysis of Adh5 mRNA expression during HSV-1 (d) or L. monocytogenes (e) infection. f qPCR analysis of Adh5 mRNA expression in HSV-1-infected PMs from Sting1+/+ or Sting1-/- mice. qPCR analysis of Adh5 mRNA expression in PMs pretreated with solvent (DMSO) or Azacitidine and then infected with HSV-1 (g) or L. monocytogenes (h). Data represent mean ± SD or one representative from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.
Fig. 7
Fig. 7. ADH5 deficiency attenuates HSV-1- and L. monocytogenes-induced innate responses.
a–c qPCR analysis of Isg15, Isg54, Isg56, Mx1, HSV-1 UL30 mRNA expression and HSV-1 DNA level in HSV-1-infected PMs from Adh5+/+ or Adh5-/- mice. d Immunoblot assays of STING, p-TBK1, p-IRF3, and p-STAT1 in L. monocytogenes-infected PMs from Adh5+/+ or Adh5-/- mice. e ELISA analysis of IFN-β secretion in L. monocytogenes-infected PMs from Adh5+/+ or Adh5-/- mice. f–h qPCR analysis of Ifnb, Cxcl10, Isg15, Isg54, Isg56, and Mx1 mRNA expression in L. monocytogenes-infected PMs from Adh5+/+ or Adh5-/- mice. Data represent mean ± SD or one representative from three independent experiments. The p values were calculated using unpaired two-sided t test and adjustments were made for multiple comparisons.
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