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Mesenchymal stem cells detect and defend against gammaherpesvirus infection via the cGAS-STING pathway.

Yang K, Wang J, Wu M, Li M, Wang Y, Huang X - Sci Rep (2015)

Bottom Line: Cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) was identified as the sensor of MHV-68 in MSCs for the first time.Moreover, the cytosolic DNA sensing pathway mediated a potent anti-herpesviral effect through the adaptor STING and downstream kinase TBK1.Furthermore, blockade of IFN signaling suggested that cytosolic DNA sensing triggered both IFN-dependent and -independent anti-herpesviral responses.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China [2] Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China.

ABSTRACT
Mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders due to their multipotentiality and immunomodulation. Long-term observations reveal several complications after MSCs infusion, especially herpesviral infection. However, the mechanism of host defense against herpesviruses in MSCs remains largely unknown. Here we showed that murine gammaherpesvirus-68 (MHV-68), which is genetically and biologically related to human gammaherpesviruses, efficiently infected MSCs both in vitro and in vivo. Cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) was identified as the sensor of MHV-68 in MSCs for the first time. Moreover, the cytosolic DNA sensing pathway mediated a potent anti-herpesviral effect through the adaptor STING and downstream kinase TBK1. Furthermore, blockade of IFN signaling suggested that cytosolic DNA sensing triggered both IFN-dependent and -independent anti-herpesviral responses. Our findings demonstrate that cGAS-STING mediates innate immunity to gammaherpesvirus infection in MSCs, which may provide a clue to develop therapeutic strategy.

No MeSH data available.


Related in: MedlinePlus

STING adaptor and TBK1 kinase are required for the antiviral response of cytosolic DNA sensing pathway.MSCs were transfected with poly(dA:dT) (0.5 μg/ml) for 1 hr, and the subcellular distribution of STING and phosphorylated TBK1 were analyzed by immunofluorescence microscopy (a). Phosphorylation of TBK1 kinase was detected by Western blot (b). MSCs were transfected with siSTING for 48 hr (c)-(e) or pretreated with BX795 for 1 hr (f)-(h), and then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). Protein levels of STING and phosphorylated IRF3 were analyzed by Western blot (c), (f). Data are representative of three experiments with similar results. The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (d), (g). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (e), (h). Real-time PCR data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.
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f4: STING adaptor and TBK1 kinase are required for the antiviral response of cytosolic DNA sensing pathway.MSCs were transfected with poly(dA:dT) (0.5 μg/ml) for 1 hr, and the subcellular distribution of STING and phosphorylated TBK1 were analyzed by immunofluorescence microscopy (a). Phosphorylation of TBK1 kinase was detected by Western blot (b). MSCs were transfected with siSTING for 48 hr (c)-(e) or pretreated with BX795 for 1 hr (f)-(h), and then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). Protein levels of STING and phosphorylated IRF3 were analyzed by Western blot (c), (f). Data are representative of three experiments with similar results. The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (d), (g). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (e), (h). Real-time PCR data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.

Mentions: During cytosolic DNA sensing, the adaptor STING recruits and phosphorylates TBK1 kinase to activate downstream signaling. To examine STING-TBK1 signaling, MSCs were stimulated with poly(dA:dT), and the subcellular distribution of STING and phosphorylated TBK1 was visualized with immunofluorescence microscopy. In mock-treated MSCs, STING distributed diffusely in the cytosol, and phosphorylation of TBK1 was not observed (Fig. 4a). However, in response to dsDNA transfection, STING aggregated perinuclearly, and phosphorylated TBK1 was found to co-localize with STING (Fig. 4a). Moreover, Western blot showed that phosphorylation of TBK1 in MSCs after poly(dA:dT) transfection was time-dependent (Fig. 4b). These observations indicated activation of the STING-TBK1 signaling in dsDNA-stimulated MSCs. To elucidate whether the adaptor STING mediated the cytosolic DNA sensing-induced antiviral response, we silenced endogenous STING with siRNA in MSCs, and stimulated the cells with poly(dA:dT). Western blot showed that phosphorylation of downstream transcription factor IRF3 was dramatically attenuated in poly(dA:dT)-stimulated MSCs in which STING was knocked down (Fig. 4c), suggesting a critical role of STING in cytosolic DNA sensing. Furthermore, viral DNA replication and virion yield were examined in STING-silenced MSCs. Both real-time PCR (Fig. 4d) and plaque assay (Fig. 4e) data showed that dsDNA stimulation decreased viral replication in siNC-treated MSCs, while the antiviral effect was abolished in STING-silenced MSCs (Fig. 4d, 4e). These results indicated that the adaptor STING mediated the antiviral response of cytosolic DNA sensing pathway.


Mesenchymal stem cells detect and defend against gammaherpesvirus infection via the cGAS-STING pathway.

Yang K, Wang J, Wu M, Li M, Wang Y, Huang X - Sci Rep (2015)

STING adaptor and TBK1 kinase are required for the antiviral response of cytosolic DNA sensing pathway.MSCs were transfected with poly(dA:dT) (0.5 μg/ml) for 1 hr, and the subcellular distribution of STING and phosphorylated TBK1 were analyzed by immunofluorescence microscopy (a). Phosphorylation of TBK1 kinase was detected by Western blot (b). MSCs were transfected with siSTING for 48 hr (c)-(e) or pretreated with BX795 for 1 hr (f)-(h), and then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). Protein levels of STING and phosphorylated IRF3 were analyzed by Western blot (c), (f). Data are representative of three experiments with similar results. The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (d), (g). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (e), (h). Real-time PCR data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4296288&req=5

f4: STING adaptor and TBK1 kinase are required for the antiviral response of cytosolic DNA sensing pathway.MSCs were transfected with poly(dA:dT) (0.5 μg/ml) for 1 hr, and the subcellular distribution of STING and phosphorylated TBK1 were analyzed by immunofluorescence microscopy (a). Phosphorylation of TBK1 kinase was detected by Western blot (b). MSCs were transfected with siSTING for 48 hr (c)-(e) or pretreated with BX795 for 1 hr (f)-(h), and then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). Protein levels of STING and phosphorylated IRF3 were analyzed by Western blot (c), (f). Data are representative of three experiments with similar results. The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (d), (g). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (e), (h). Real-time PCR data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.
Mentions: During cytosolic DNA sensing, the adaptor STING recruits and phosphorylates TBK1 kinase to activate downstream signaling. To examine STING-TBK1 signaling, MSCs were stimulated with poly(dA:dT), and the subcellular distribution of STING and phosphorylated TBK1 was visualized with immunofluorescence microscopy. In mock-treated MSCs, STING distributed diffusely in the cytosol, and phosphorylation of TBK1 was not observed (Fig. 4a). However, in response to dsDNA transfection, STING aggregated perinuclearly, and phosphorylated TBK1 was found to co-localize with STING (Fig. 4a). Moreover, Western blot showed that phosphorylation of TBK1 in MSCs after poly(dA:dT) transfection was time-dependent (Fig. 4b). These observations indicated activation of the STING-TBK1 signaling in dsDNA-stimulated MSCs. To elucidate whether the adaptor STING mediated the cytosolic DNA sensing-induced antiviral response, we silenced endogenous STING with siRNA in MSCs, and stimulated the cells with poly(dA:dT). Western blot showed that phosphorylation of downstream transcription factor IRF3 was dramatically attenuated in poly(dA:dT)-stimulated MSCs in which STING was knocked down (Fig. 4c), suggesting a critical role of STING in cytosolic DNA sensing. Furthermore, viral DNA replication and virion yield were examined in STING-silenced MSCs. Both real-time PCR (Fig. 4d) and plaque assay (Fig. 4e) data showed that dsDNA stimulation decreased viral replication in siNC-treated MSCs, while the antiviral effect was abolished in STING-silenced MSCs (Fig. 4d, 4e). These results indicated that the adaptor STING mediated the antiviral response of cytosolic DNA sensing pathway.

Bottom Line: Cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) was identified as the sensor of MHV-68 in MSCs for the first time.Moreover, the cytosolic DNA sensing pathway mediated a potent anti-herpesviral effect through the adaptor STING and downstream kinase TBK1.Furthermore, blockade of IFN signaling suggested that cytosolic DNA sensing triggered both IFN-dependent and -independent anti-herpesviral responses.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Immunology, Institute of Tuberculosis Control, Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, China [2] Key Laboratory of Tropical Diseases Control (Sun Yat-sen University), Ministry of Education, Guangzhou 510080, China.

ABSTRACT
Mesenchymal stem cells (MSCs) are widely used in clinical settings to treat tissue injuries and autoimmune disorders due to their multipotentiality and immunomodulation. Long-term observations reveal several complications after MSCs infusion, especially herpesviral infection. However, the mechanism of host defense against herpesviruses in MSCs remains largely unknown. Here we showed that murine gammaherpesvirus-68 (MHV-68), which is genetically and biologically related to human gammaherpesviruses, efficiently infected MSCs both in vitro and in vivo. Cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) was identified as the sensor of MHV-68 in MSCs for the first time. Moreover, the cytosolic DNA sensing pathway mediated a potent anti-herpesviral effect through the adaptor STING and downstream kinase TBK1. Furthermore, blockade of IFN signaling suggested that cytosolic DNA sensing triggered both IFN-dependent and -independent anti-herpesviral responses. Our findings demonstrate that cGAS-STING mediates innate immunity to gammaherpesvirus infection in MSCs, which may provide a clue to develop therapeutic strategy.

No MeSH data available.


Related in: MedlinePlus