| Toll-like receptor(TLR)is a typical pattern recognition receptor(PRR)to recognize pathogen associated molecular patterns(PAMPs)and active downstream signaling pathway,initiating innate immune response.TLR3 is an important member of TLR family and plays a significant role in innate immune response against viral infection.TLR3 recognizes double-stranded RNA(dsRNA)structure produced in viral infection to activate IRF3 and NF-κB through Myd88-indepent pathway,inducing secretion of type I interferon and pro-inflammatory cytokines to restrict viral infection.Many researches demonstrate that TLR3 signaling pathway is indispensable for immune response against herpesvirus infection and tumor.Marek’s disease virus is a a-herpesvirus,leading Marek’s disease with typical T-cell lymphomas and immune suppression clinical symptoms,cause great economic loss for poultry industry.MDV pathogenesis consists of early cytolytic phase,latency phase,,late cytolytic phase and neoplastic phase.According to trancriptome profiling,the transcription level of molecules associated with the TLR3 pathway in chicken immune organs,including spleen,bursa of Fabricius and thymus,was significantly changed at all phases of MDV infection.Moreover,TLR3 pathway is obviously suppressed at late cytolytic phase and neoplastic phase,suggesting that TLR3 pathway is an important element for host immune response against MDV infection.Recent progress demonstrates that activation of TLR3 pathway can effectively inhibit MDV infection in vitro and lymphoma development in vivo.However it is unclear how the TLR3 pathway achieves these functions.In this study,we investigate the function of the TLR3 pathway in MDV-infected chicken embryo fibroblasts and Marek’s disease lymphoma-derived chicken cell line using transcriptomics,immunological and molecular biological methods.Our results provides important data and clues for better understanding the role of TLR3 pathway in host immune response against MDV.1.Mechanism of poly(I:C)inhibiting MDV infectionIn this study,we activate TLR3 pathway in MDV-infected CEFs at different time,in order to investigate whether TLR3 response can inhibit MDV infection.As a result,no matter at any time post infection,activation of TLR3 was able to decrease virus titer and gB transcription.Real-time PCR data showed that the transcription level of molecules involved in TLR3 pathway significantly up-regulated during this process,especially for TLR3,IRF3 and IKKa.At the same time,up-regulation of IFN-β,IL-6 and TNF-a was also found.Using small molecular inhibitors BX795 and Resveratrol,we respectively verified the effect of type I interferon and pro-inflammatory cytokines on MDV replication.In consequence,BX795 at a concentration of 10μM and Resveratrol at a concentration of 100μM can effectively block TLR3-mediated production of type I interferon pro-inflammatory cytokines without cytotoxicity.And both BX795 and Resveratrol were able to weaken rather than abolish the inhibitory effect of TLR3 activation on MDV infection.This data indicated that TLR3 pathway inhibits MDV infection in CEFs through type I interferon response and pro-inflammatory cytokines.2.Poly(I:C)specifically induces apoptosis of Marek’s disease lymphoma cell line MSB1Several reports have showed that dsRNA can trigger tumor cells apoptosis through different mechanisms.MSB1 is a chicken cell line derived from Marek’s disease lymphoma.It is unknown whether dsRNA is able to induce apoptosis in MSB1 cell.In this study,after stimulation with dsRNA analog poly(I:C),the apoptosis of MSB1 cells was verified using methods based on morphology,molecular and cellular biology,and immunology.As a result,poly(I:C)caused MSB1 cells death rather than other avian cells such as DF-1,CEF,CEK,HD11 and DT40,depending on dose and incubation time.MSB1 cells treated with poly(I:C)exhibited disintegration and a low refractivity,which was similar to morphologic changes in apoptotic cells.Compared to cells without treatment,the percentage of Annexin V positive cells was obviously higher in MSB1 cells treated with poly(I:C),indicating the occurrence of apoptosis.Furthermore,the activity of Caspase 3/7,8 and 9 was significantly up-regulated in poly(I:C)-treated cells,which further confirmed the occurrence of apoptosis.And mitochondria membrane potential was almost completely down-regulated in cells treated with poly(I:C),agreeing with the up-regulation of Caspase 9 activity.In genome electrophoresis assay,a distinct laddering effect was noticed,indicating nucleosomal fragmentation of DNA samples obtained from MSB1 cells treated with poly(I:C).At the same time,pan-caspase inhibitor Z-VAD-FMK can abolish cell apoptosis caused by poly(I:C)stimulation.Collectively,TLR3 ligand poly(I:C)can induce apoptosis of Marek’s disease lymphoma-derived chicken cell line MSB1 through caspase-dependent pathway,suggesting a potential application of poly(I:C)for MD prevention and lymphoma treatment.3.Mechanism of poly(I:C)inducing MSB1 cells apoptosisTLR3 pathway has been demonstrated to induce tumor cells apoptosis through different ways.In previous research,it was identified that TLR3 ligand poly(I:C)specifically induces apoptosis of Marek’s disease lymphoma-derived chicken cell line MSB1.In this study,using several methods including small molecular inhibitors,flow cytometry,real-time PCR and so on,we explored the function of TLR3 pathway in MSB1 cells apoptosis induce by poly(I:C).At first,we found that the transcription level of adaptors involved in TLR3 pathway and pro-apoptotic factors was significantly up-regulated during this process.Correspondingly,IFN-β and TNF-αwere also significantly up-regulated,suggesting a relationship between apoptosis and activation of TLR3 pathway.After that,inhibitors Pepinh-TRIF,BX795 and concentration were used to respectively block activation of TRIF,IRF3 and NF-κB.Both Pepinh-TRIF at a concentration of 50μM and Resveratrol at a concentration of 50μM can obviously inhibit MSB 1 cells apoptosis induce by poly(I:C),while BX795 a concentration of 10μM showed no effect on suppression of apoptosis.It indicated that TRIF and NF-κB was indispensable for poly(I:C)-induced apoptosis of MSB1 cells.Our results explained how poly(I:C)induced MSB1 cells apoptosis through TLR3 pathway,which helped us to better understand the function of TLR3 pathway in host immune response against MDV and tumor.These data provided certain reference for development of novel MDV vaccines and anti-tumor drugs. |
