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. 2015 Apr 1;194(7):3236-45.
doi: 10.4049/jimmunol.1402764. Epub 2015 Feb 20.

"VSports注册入口" cGAS and Ifi204 cooperate to produce type I IFNs in response to Francisella infection

Kelly M Storek  1 , Nina A Gertsvolf  1 , Maikke B Ohlson  2 , Denise M Monack  3
Affiliations

cGAS and Ifi204 cooperate to produce type I IFNs in response to Francisella infection

Kelly M Storek et al. J Immunol. .

Abstract

Type I IFN production is an important host immune response against viral and bacterial infections. However, little is known about the ligands and corresponding host receptors that trigger type I IFN production during bacterial infections. We used a model intracellular pathogen, Francisella novicida, to begin characterizing the type I IFN response to bacterial pathogens. F. novicida replicates in the cytosol of host cells and elicits a robust type I IFN response that is largely TLR independent, but is dependent on the adapter molecule STING, suggesting that the type I IFN stimulus during F. novicida infection is cytosolic. In this study, we report that the cytosolic DNA sensors, cyclic GMP-AMP synthase (cGAS) and Ifi204, are both required for the STING-dependent type I IFN response to F. novicida infection in both primary and immortalized murine macrophages. We created cGAS, Ifi204, and Sting functional knockouts in RAW264. 7 macrophages and demonstrated that cGAS and Ifi204 cooperate to sense dsDNA and activate the STING-dependent type I IFN pathway. In addition, we show that dsDNA from F. novicida is an important type I IFN stimulating ligand. One outcome of cGAS-STING signaling is the activation of the absent in melanoma 2 inflammasome in response to F VSports手机版. novicida infection. Whereas the absent in melanoma 2 inflammasome is beneficial to the host during F. novicida infection, type I IFN signaling by STING and IFN regulatory factor 3 is detrimental to the host during F. novicida infection. Collectively, our studies indicate that cGAS and Ifi204 cooperate to sense cytosolic dsDNA and F. novicida infection to produce a strong type I IFN response. .

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Figures

FIGURE 1.
FIGURE 1.
cGAS and Ifi204 are required for type I IFN production in response to cytosolic F. novicida in BMMs. siRNA targeting known cytosolic sensor genes or an NT control was transfected into BMMs for 36 h. (A) Quantitative RT-PCR measured mRNA levels for each targeted gene in uninfected BMMs. Gene expression was normalized to GAPDH and the NT control. Type I IFN levels were measured (B) 9 h postinfection with the indicated F. novicida strain at an MOI of 10 or (C) 4 h poststimulation with LPS. Results are presented as RLUs. Data presented as normalized RLUs are type I IFN levels normalized to the uninfected, NT control. (D) Type I IFN levels were measured from unstimulated primary C57BL/6 (WT), Stinggt/gt, and cGAS−/− BMMs infected with F. novicida strains at an MOI of 10 for 12 h. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 2.
FIGURE 2.
The type I IFN response to F. novicida is similar in BMMs and RAW264.7 macrophages. BMMs and RAW264.7 macrophages were infected with the indicated F. novicida strains at an MOI of 10. (A) Immunofluorescence microscopy of BMMs and RAW264.7 macrophages stained for F. novicida (green), LAMP-1 (red), and DAPI (blue) 8 h postinfection. Scale bars, 10 μm. (B) Intracellular survival was assessed by CFU plating. Type I IFN levels from (C) RAW264.7 macrophages or (D) C57BL/6 BMMs were measured 24 h and 12 h postinfection, respectively. (E) Cytotoxicity was determined by measuring LDH release 24 h postinfection. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 3.
FIGURE 3.
The type I IFN response to cytosolic F. novicida requires cGAS, Ifi204, and STING in RAW264.7 macrophages. Type I IFN levels were measured from RAW264.7 (WT) macrophages and the indicated genotypes (A, C, and E) infected with F. novicida strains at an MOI of 10 for 24 h or not infected (NI). Type I IFN levels were measured from deficient macrophages stably expressing Sting (B), cGAS (D), or Ifi204 (F) infected with U112 at an MOI of 10 for 24 h. Type I IFN levels were measured from RAW264.7 (WT) macrophages and indicated genotypes infected with F. novicida for 12 (G) and 24 h (H) or infected with WT L. monocytogenes for 8 h at an MOI of 20 (I). mRNA expression levels of IFN-β1 (J) and TNF-α (K) from uninfected and U112-infected macrophage cell lines at an MOI of 10 for 8 h. mRNA levels were normalized to GAPDH and the WT RAW264.7 macrophages. (L) Type I IFN levels were measured from macrophages stimulated with LPS for 8 h. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. NS, not stimulated.
FIGURE 4.
FIGURE 4.
Both cGAS and Ifi204 are required for the full type I IFN response to F. novicida infection. mRNA expression levels were measured in uninfected RAW264.7 (WT) cell lines stably expressing Ifi204 (A) or cGAS (C). mRNA expression was normalized to GAPDH and WT RAW264.7 macrophages. Macrophages were infected with WT F. novicida at an MOI of 10 for 24 h and type I IFN levels measured (B and D, respectively). Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 5.
FIGURE 5.
cGAS and Ifi204 cooperate to sense cytosolic dsDNA. (A) Type I IFN levels were measured from WT RAW264.7 macrophages transfected for 24 h with U112 lysates either untreated (UT) or treated with DNase I, heat-killed (HK) DNase I or RNase A. Type I IFN levels were measured from RAW264.7 macrophages transfected for 24 h with (B) endo-free plasmid DNA (pCherry), (C) 1 μg/ml polyinosinic-polycytidylic acid [poly(I:C)], or (D) 10 μg/ml c-di-GMP NT, not transfected. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. ***p < 0.001.
FIGURE 6.
FIGURE 6.
cGAS increases inflammasome activity in BMMs. (A) Cytotoxicity, (B) IL-1β secretion, and (C) intracellular survival were measured from unstimulated primary C57BL/6 (WT), Stinggt/gt, cGAS−/−, Aim2−/−, and ASC−/− BMMs infected with F. novicida strains at an MOI of 10 for 12 h unless otherwise indicated. (D) Release of processed caspase-1 (casp-1 p10 and p20) into the supernatants (SN) was measured by Western blot from cells either not infected (NI) or infected with WT F. novicida at an MOI of 100 for 12 h. Corresponding cell lysates were probed for procaspase-1 and β-actin. Graphs show the mean ± SD of triplicate wells and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
FIGURE 7.
FIGURE 7.
STING and IRF3 are detrimental to host survival during F. novicida infection. WT and Stinggt/gt mice were infected s.c. with 105 CFU U112. (A) The spleen and liver from five infected WT and Stinggt/gt mice were harvested and plated for CFU/g 3 d postinfection; geometric mean is shown. (B) Ten WT and 10 Stinggt/gt mice were monitored twice daily over 15 d for survival. (C) Nine WT and 10 IRF3−/− mice were infected s.c. with 2 × 105 CFU U112 and were monitored twice daily over 15 d for survival. *p < 0.05, **p < 0.01, ****p < 0.0001.
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