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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

The cGAS-STING cytosolic DNA sensing pathway mediates recognition of MHV-68 in MSCs.MSCs were infected with MHV-68 (MOI 0.1) (a) or transfected with MHV-68 DNA (0.5 μg/ml) (b) for the indicated time, and then analyzed for IFN-β expression by real-time PCR. The expressions of TLR9 and MyD88 in MSCs or BMDM were detected with RT-PCR (c). MSCs and RAW264.7 cells were stimulated with CpG DNA (2 μM) for the indicated time, and then analyzed for IFN-β mRNA expression (d). The expressions of cytosolic DNA sensors and adaptor STING in MSCs or BMDM were detected with RT-PCR (e). MSCs were transfected with indicated siRNA for 48 hr, and then stimulated with MHV-68 DNA (0.5 μg/ml) for 6 hr (f)–(i). The knockdown efficacy was confirmed by real-time PCR (f) or Western blot (h), and the expressions of IFN-β were detected by real-time PCR (g), (i). RT-PCR and Western blot data are representative of three experiments with similar results. Real-time PCR data are shown as mean ± SEM of three independent experiments. *, p < 0.05; ***, p < 0.001.
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f2: The cGAS-STING cytosolic DNA sensing pathway mediates recognition of MHV-68 in MSCs.MSCs were infected with MHV-68 (MOI 0.1) (a) or transfected with MHV-68 DNA (0.5 μg/ml) (b) for the indicated time, and then analyzed for IFN-β expression by real-time PCR. The expressions of TLR9 and MyD88 in MSCs or BMDM were detected with RT-PCR (c). MSCs and RAW264.7 cells were stimulated with CpG DNA (2 μM) for the indicated time, and then analyzed for IFN-β mRNA expression (d). The expressions of cytosolic DNA sensors and adaptor STING in MSCs or BMDM were detected with RT-PCR (e). MSCs were transfected with indicated siRNA for 48 hr, and then stimulated with MHV-68 DNA (0.5 μg/ml) for 6 hr (f)–(i). The knockdown efficacy was confirmed by real-time PCR (f) or Western blot (h), and the expressions of IFN-β were detected by real-time PCR (g), (i). RT-PCR and Western blot data are representative of three experiments with similar results. Real-time PCR data are shown as mean ± SEM of three independent experiments. *, p < 0.05; ***, p < 0.001.

Mentions: To investigate the innate immune response to MHV-68 in MSCs, cells were infected with MHV-68, and induction of the downstream gene IFN-β was examined. We found that IFN-β was not induced in MSCs after infection (Fig. 2a), suggesting that MHV-68 may inhibit the IFN response. To explore how MSCs detected foreign nucleic acid of invading MHV-68, we stimulated cells with viral DNA by transfection. Real-time PCR data showed that viral DNA induced the expression of IFN-β (Fig. 2b), indicating activation of the innate immune response. Next, we examined which receptors detected viral DNA in MSCs. Since MHV-68 is a dsDNA virus, we first tested whether TLR9 was involved in detection of MHV-68. The expression of TLR9 was examined by RT-PCR in MSCs. While BMDM expressed TLR9, mRNA of TLR9 was not detectable in MSCs (Fig. 2c). We further stimulated MSCs with TLR9 ligand CpG DNA, and examined the expression of the downstream gene IFN-β. While CpG DNA induced the expression of IFN-β in murine macrophage-like RAW264.7 cells, MSCs failed to respond to CpG DNA (Fig. 2d). These results indicated that mouse MSCs did not express functional TLR9, thus ruling out the possibility of TLR9-mediated recognition of MHV-68 in MSCs. In addition to endosomal TLR9, foreign dsDNA can also be detected by cytosolic DNA sensors. Therefore, we tested the hypothesis that cytosolic DNA sensors may recognize MHV-68 in MSCs. The expressions of well-characterized cytosolic DNA sensors including cGAS, DDX41, p204, AIM2, DAI and the adaptor STING were detected with RT-PCR. BMDM were used as positive control, which expressed the receptors above (Fig. 2e). Similarly, all of the receptors were expressed in MSCs, though at different levels (Fig. 2e). Furthermore, we knocked down each DNA sensor with a small interfering RNA (siRNA) to determine which sensor was responsible for detection of MHV-68 (Fig. 2f). Notably, MHV-68 DNA-induced IFN-β expression in MSCs was impaired by cGAS-specific siRNA, whereas siRNAs targeting other DNA sensors did not reduce IFN-β expression (Fig. 2g). During cytosolic DNA sensing, STING is a central adaptor protein. Thus, we knocked down STING to examine whether STING mediated recognition of MHV-68 in MSC. The knockdown efficacy was confirmed by Western blot (Fig. 2h). Real-time data showed that knockdown of STING attenuated the expression of IFN-β in MSCs stimulated with viral DNA (Fig. 2i). These results suggest that the cGAS-STING cytosolic DNA sensing pathway recognized MHV-68 in MSCs.


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)

The cGAS-STING cytosolic DNA sensing pathway mediates recognition of MHV-68 in MSCs.MSCs were infected with MHV-68 (MOI 0.1) (a) or transfected with MHV-68 DNA (0.5 μg/ml) (b) for the indicated time, and then analyzed for IFN-β expression by real-time PCR. The expressions of TLR9 and MyD88 in MSCs or BMDM were detected with RT-PCR (c). MSCs and RAW264.7 cells were stimulated with CpG DNA (2 μM) for the indicated time, and then analyzed for IFN-β mRNA expression (d). The expressions of cytosolic DNA sensors and adaptor STING in MSCs or BMDM were detected with RT-PCR (e). MSCs were transfected with indicated siRNA for 48 hr, and then stimulated with MHV-68 DNA (0.5 μg/ml) for 6 hr (f)–(i). The knockdown efficacy was confirmed by real-time PCR (f) or Western blot (h), and the expressions of IFN-β were detected by real-time PCR (g), (i). RT-PCR and Western blot data are representative of three experiments with similar results. Real-time PCR data are shown as mean ± SEM of three independent experiments. *, p < 0.05; ***, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2: The cGAS-STING cytosolic DNA sensing pathway mediates recognition of MHV-68 in MSCs.MSCs were infected with MHV-68 (MOI 0.1) (a) or transfected with MHV-68 DNA (0.5 μg/ml) (b) for the indicated time, and then analyzed for IFN-β expression by real-time PCR. The expressions of TLR9 and MyD88 in MSCs or BMDM were detected with RT-PCR (c). MSCs and RAW264.7 cells were stimulated with CpG DNA (2 μM) for the indicated time, and then analyzed for IFN-β mRNA expression (d). The expressions of cytosolic DNA sensors and adaptor STING in MSCs or BMDM were detected with RT-PCR (e). MSCs were transfected with indicated siRNA for 48 hr, and then stimulated with MHV-68 DNA (0.5 μg/ml) for 6 hr (f)–(i). The knockdown efficacy was confirmed by real-time PCR (f) or Western blot (h), and the expressions of IFN-β were detected by real-time PCR (g), (i). RT-PCR and Western blot data are representative of three experiments with similar results. Real-time PCR data are shown as mean ± SEM of three independent experiments. *, p < 0.05; ***, p < 0.001.
Mentions: To investigate the innate immune response to MHV-68 in MSCs, cells were infected with MHV-68, and induction of the downstream gene IFN-β was examined. We found that IFN-β was not induced in MSCs after infection (Fig. 2a), suggesting that MHV-68 may inhibit the IFN response. To explore how MSCs detected foreign nucleic acid of invading MHV-68, we stimulated cells with viral DNA by transfection. Real-time PCR data showed that viral DNA induced the expression of IFN-β (Fig. 2b), indicating activation of the innate immune response. Next, we examined which receptors detected viral DNA in MSCs. Since MHV-68 is a dsDNA virus, we first tested whether TLR9 was involved in detection of MHV-68. The expression of TLR9 was examined by RT-PCR in MSCs. While BMDM expressed TLR9, mRNA of TLR9 was not detectable in MSCs (Fig. 2c). We further stimulated MSCs with TLR9 ligand CpG DNA, and examined the expression of the downstream gene IFN-β. While CpG DNA induced the expression of IFN-β in murine macrophage-like RAW264.7 cells, MSCs failed to respond to CpG DNA (Fig. 2d). These results indicated that mouse MSCs did not express functional TLR9, thus ruling out the possibility of TLR9-mediated recognition of MHV-68 in MSCs. In addition to endosomal TLR9, foreign dsDNA can also be detected by cytosolic DNA sensors. Therefore, we tested the hypothesis that cytosolic DNA sensors may recognize MHV-68 in MSCs. The expressions of well-characterized cytosolic DNA sensors including cGAS, DDX41, p204, AIM2, DAI and the adaptor STING were detected with RT-PCR. BMDM were used as positive control, which expressed the receptors above (Fig. 2e). Similarly, all of the receptors were expressed in MSCs, though at different levels (Fig. 2e). Furthermore, we knocked down each DNA sensor with a small interfering RNA (siRNA) to determine which sensor was responsible for detection of MHV-68 (Fig. 2f). Notably, MHV-68 DNA-induced IFN-β expression in MSCs was impaired by cGAS-specific siRNA, whereas siRNAs targeting other DNA sensors did not reduce IFN-β expression (Fig. 2g). During cytosolic DNA sensing, STING is a central adaptor protein. Thus, we knocked down STING to examine whether STING mediated recognition of MHV-68 in MSC. The knockdown efficacy was confirmed by Western blot (Fig. 2h). Real-time data showed that knockdown of STING attenuated the expression of IFN-β in MSCs stimulated with viral DNA (Fig. 2i). These results suggest that the cGAS-STING cytosolic DNA sensing pathway recognized MHV-68 in MSCs.

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