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. 2015 Jan 16:5:7820.
doi: 10.1038/srep07820.

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

Kun Yang  1 , Jinli Wang  1 , Minhao Wu  1 , Meiyu Li  1 , Yi Wang  1 , Xi Huang  1
Affiliations

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

Kun Yang et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. MHV-68 infects MSCs both in vitro and in vivo.
MSCs were infected with MHV-68 (MOI 0.1) for the indicated time, and the cytopathic effect was examined microscopically (a). The replication of viral DNA was detected by real-time PCR (b). The virus titers in the supernatant were determined by plaque assay (c). Data are shown as mean ± SEM of three independent experiments. (d) C57BL/6 mice were intranasally inoculated with MHV-68. Viral DNA in lung, spleen or bone-marrow-derived MSCs was detected with nested PCR of ORF50 gene at the indicated time post-infection. Data are representative of three experiments with similar results.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. Activation of the cytosolic DNA sensing pathway restricts the replication of MHV-68 in MSCs.
MSCs were transfected with poly (dA:dT) (a) or ISD (b) (0.5 μg/ml), and phosphorylation of IRF3 was detected by Western blot. Data are representative of three experiments with similar results. MSCs were transfected with poly (dA:dT) (c), (d) or ISD (e), (f) at the indicated concentration for 6 hr, then infected with MHV-68 (MOI 0.1). The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (c and e). The virus titers in the supernatant were determined by plaque assay at 24 hr post-infection (d), (f). Data are shown as mean ± SD. of three independent experiments.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. Cytosolic DNA sensing pathway mediates both IFN-dependent and -independent antiviral responses.
MSCs were pretreated with JAK inhibitor Ruxolitinib (Rux) for 1 hr, then transfected with poly(dA:dT) (0.5 μg/ml), followed by MHV-68 infection (MOI 0.1). The phosphorylation of STAT1 was examined by Western blot at 2 hr post-tranfection (a). The replication of viral DNA was detected by real-time PCR at 6 hr post-infection (b). The virus titers in supernatant were determined by plaque assay at 24 hr post-infection (c). The mRNA expressions of selected ISGs were analyzed by real-time PCR at 6 hr post-tranfection (d). Data are shown as mean ± SEM of three independent experiments. **, p < 0.01; ***, p < 0.001.
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