The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides - PubMed (original) (raw)

The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides

John-Demian Sauer et al. Infect Immun. 2011 Feb.

Abstract

Type I interferons (IFNs) are central regulators of the innate and adaptive immune responses to viral and bacterial infections. Type I IFNs are induced upon cytosolic detection of microbial nucleic acids, including DNA, RNA, and the bacterial second messenger cyclic-di-GMP (c-di-GMP). In addition, a recent study demonstrated that the intracellular bacterial pathogen Listeria monocytogenes stimulates a type I IFN response due to cytosolic detection of bacterially secreted c-di-AMP. The transmembrane signaling adaptor Sting (Tmem173, Mita, Mpys, Eris) has recently been implicated in the induction of type I IFNs in response to cytosolic DNA and/or RNA. However, the role of Sting in response to purified cyclic dinucleotides or during in vivo L. monocytogenes infection has not been addressed. In order to identify genes important in the innate immune response, we have been conducting a forward genetic mutagenesis screen in C57BL/6 mice using the mutagen N-ethyl-N-nitrosourea (ENU). Here we describe a novel mutant mouse strain, Goldenticket (Gt), that fails to produce type I IFNs upon L. monocytogenes infection. By genetic mapping and complementation experiments, we found that Gt mice harbor a single nucleotide variant (T596A) of Sting that functions as a null allele and fails to produce detectable protein. Analysis of macrophages isolated from Gt mice revealed that Sting is absolutely required for the type I interferon response to both c-di-GMP and c-di-AMP. Additionally, Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. Our results provide new functions for Sting in the innate interferon response to pathogens.

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Figures

FIG. 1.

FIG. 1.

ENU mutagenesis identifies T596A mutation in Sting. (A) Thioglycolate-elicited peritoneal macrophages harvested from G3 ENU-mutagenized mice were infected with wild-type or Δ_hly L. monocytogenes_ at an MOI of 1. IFN-β was measured by bioassay at 6 h postinfection. (B) Sequencing of the Sting allele in wild-type, heterozygous, and homozygous mutant F2 Gt mice. (C) Sequence alignment showing the conserved amino acid mutated in the Goldenticket mouse. (D) Schematic showing the location of the mutation in the Sting locus and on a predicted topology diagram (

http://proteinformatics.charite.de/rhythm/

). (E) Bone marrow-derived macrophages harvested from F2 Gt mice were infected with wild-type L. monocytogenes at an MOI of 1 for 6 h. The amount of IFN-β in the culture supernatant was measured by bioassay.

FIG. 2.

FIG. 2.

T596A mutation abrogates the function of Sting. (A) Luciferase production was measured 24 h after cotransfection of HEK293T cells with an IFN-β luciferase reporter and increasing concentrations (50 ng, 100 ng, 200 ng per well) of wild-type or Gt Sting. pGL3 is a positive-control luciferase expression vector. (B) Following infection with wild-type L. monocytogenes (L.m.; MOI, 1), infection with Sendai virus (SeV; 150 HAU/ml), or transfection of 500 μg/ml of poly(dAT:dTA) or poly(I:C) for 4 h, RNA was harvested from bone marrow-derived macrophages and the amounts of IFN-β transcripts were measured relative to those of β-actin transcripts. (C) Whole-cell lysates were collected and analyzed for Sting expression by Western blotting following 4 h of treatment with either 4 μg/ml of c-di-GMP or 100 ng/ml of LPS. (D) Whole-cell lysates were collected from HEK293T cells 24 h after transfection with 200 ng/ml of wild-type or Gt Sting. Sting was detected using anti-HA antibody, and β-actin was used as a loading control. (E and F) Sting +/+ or Sting Gt/Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting, or Gt Sting. (E) Transduced cells were transfected with 500 μg/ml poly(dAT:dTA) or 500 μg/ml purified bacterial (Legionella pneumophila) genomic DNA for 4 h, and then RNA was harvested and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. (F) Transduced cells were transfected with 500 μg/ml poly(I:C), infected with Sendai virus at 150 HAU/ml, or treated with 100 ng/ml of LPS for 4 h; RNA was harvested; and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P < 0.05 by Student's t test; un and Unstim, unstimulated; UN, untransfected.

FIG. 3.

FIG. 3.

Sting is required for cyclic dinucleotide-induced IFN-β production. Sting +/+, Sting Gt/Gt, or Sting_−/_− bone marrow-derived macrophages were treated with 4 μg/ml c-di-GMP (A) or c-di-AMP (B) for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 (A) or β-actin (B) transcripts. (C) Sting +/+ or Sting Gt/Gt immortalized bone marrow-derived macrophages were transduced with either empty MSCV2.2 vector, WT Sting, or Gt Sting. Transduced cells were transfected with 4 μg/ml of c-di-GMP or c-di-AMP for 4 h. RNA was harvested, and the amounts of IFN-β transcripts were measured relative to those of rps17 transcripts. Data are representative of those from at least three independent experiments. *, P < 0.05 by Student's t test.

FIG. 4.

FIG. 4.

Sting is required for the response to c-di-GMP and L. monocytogenes in vivo. (A) A total of 200 nmol c-di-GMP was injected intraperitoneally into wild-type C57BL/6 (B6) or isogenic Sting Gt/Gt mice. Serum was collected at 12 h postinjection, and the amount of IFN-β was analyzed by bioassay. (B and C) A total of 1 × 105 wild-type or Δ_hly L. monocytogenes_ cells were injected intravenously into C57BL/6 or Sting Gt/Gt mice, and serum was collected at 4 and 24 h postinfection. The amount of IFN-β in the serum was measured by bioassay at 4 and 24 h postinfection (B), and the amount of TNF-α in the serum was measured by ELISA at 24 h postinfection (C). (D) Wild-type or Sting Gt/Gt mice were infected intravenously with 1 × 104 wild-type L. monocytogenes cells. At 48 h postinfection, spleens were harvested and the bacterial burden was quantified. Data are representative of those from at least two independent experiments. *, P < 0.05 by Mann-Whitney test.

References

    1. Auerbuch, V., D. G. Brockstedt, N. Meyer-Morse, M. O'Riordan, and D. A. Portnoy. 2004. Mice lacking the type I interferon receptor are resistant to Listeria monocytogenes. J. Exp. Med. 200:527-533. - PMC - PubMed
    1. Blasi, E., et al. 1985. Selective immortalization of murine macrophages from fresh bone marrow by a raf/myc recombinant murine retrovirus. Nature 318:667-670. - PubMed
    1. Fitzgerald, K. A., et al. 2003. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4:491-496. - PubMed
    1. Galperin, M. Y. 2005. A census of membrane-bound and intracellular signal transduction proteins in bacteria: bacterial IQ, extroverts and introverts. BMC Microbiol. 5:35. - PMC - PubMed
    1. Hoebe, K., et al. 2003. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424:743-748. - PubMed

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