| ObjectiveHand,foot and mouth disease(HFMD)is a disease caused by enterovirus infection,which usually causes fever,ulcers or rashes in mouth,hands,feet and other parts of the patient,generally with self limitation.But serious infection can cause various complications,such as aseptic meningoencephalitis,myocarditis and pulmonary edema,and even death.In 2008,an outbreak of enterovirus 71(EV71)infection in mainland China resulted in 490,000 infections and 126 deaths.Since then,EV71 infection has repeatedly occurred throughout the Asia-Pacific region and has become a major kind of viral infectious diseases that threatens the health of infants and young children.However,the current treatment of EV71 mainly uses hormone and symptomatic treatment,there is no effective antiviral drugs.Therefore,it is necessary to further study the pathogenesis of EV71 and accelerate the development of preventive drugs and therapeutic methods.EV71 belongs to the genus enterovirus in the family of picornaviruses,which is a 20～30 nm diameter non enveloped icosahedral virus particle.Its genome length is about 7.5 kb,including structural proteins(VP1,VP2,VP3 and VP4)and nonstructural proteins(2A,2B,2C,3A,3B,3C and 3D).Structural protein VP1 promotes virus infection by binding to specific receptors on the surface of host cells.There are many types of receptors,including h-SCARB2,PSGL-1,annexin Ⅱ,etc.Among these receptors,h-SCARB2 can more effectively promote virus infection.Because EV71 has different susceptibility to different species of hosts,infections can be aggrated by overexpression of h-SCARB2 in cells or mice that are not susceptible to infection.Innate immunity is the first line of defense to recognize and resist virus infection.It can directly recognize various invasive pathogens and effectively limit virus infection.At the same time,it plays an important role in the activation of adaptive immunity.PRRs are widely expressed in or on the surface of host cells,can monitor and recognize PAMPs,which can activate host innate immune response,and induce the production of IFNs and a variety of cytokines,such as inflammatory factors.IFNs mainly include three families:type Ⅰ IFN,type Ⅱ IFN and type Ⅲ IFN.Type Ⅰ IFN is widely expressed in mammalian cells and plays a key role against various types of virus infection.TypeⅡ IFN is IFN-y,which is produced by activated T cells or NK cells and has immunomodulatory and antiviral activities.Type Ⅲ IFN contains IFN-λ1,IFN-λ2 and IFN-λ3.It is similar to IFN-α/β and also has antiviral function.Once the EV71 invades the body,TLRs,RIG-I,MDA5 can quickly recognize the dsRNA component of EV71,start downstream signaling pathways,activate IRF-3,IRF-7 and NF-κB,and induce the transcription levels of IFNs.The IFNs can play an antiviral effect through autocrine or paracrine binding with specific receptors on the cell membrane,which can induce the cells to produce a large number of ISGs and other antiviral proteins.IFNs also have immunomodulatory functions,such as promoting the expression level of MHCI on various types of cells,promoting the expression levels of costimulatory molecules on the surface of DCs,promoting the proliferation of T cells,and stimulating the antiviral function of adaptive immunity.However,in the process of the game between EV71 and the host,the virus has evolved many different mechanisms to escape the innate immune surveillance and antiviral function of the organism.For example,the nonstructural proteins 2A and 3C of EV71 can escape the antiviral function of IFNs by inhibiting many adaptor molecules in the production and response stages of IFNs.The 2A protein can cleave MDA5,the 3C protein can block the interaction between RIG-I and MAVS to prevent PRRs from recognizing invading viruses.The 2A protein can cleave adaptor molecule MAVS in the downstream of MDA5 and RIG-I,and TRIF in the downstream of TLR3.The 3C protein can cleave IRF7 and IRF9 to block the transcription of IFNs and ISGs,and it also can cleave the TAK1 complex to interrupt the downstream signaling process.The 2A protein can inhibit the transcription of ISGs by down-regulating the expression level of IFNAR1.These are the main reasons that EV71 can escape the body’s innate immune response.As EV71 belongs to enterovirus,intestinal tract is its main invasion portal.However,until now,how EV71 escapes and passes through the critical gut mucosal innate immune barrier to cause systemic infection has not been explored.The intestinal mucosal immune barrier consists of densely arranged monolayer IECs and IELs,which constitute the first line of intestinal mucosa against infection of foreign pathogens.IECs express many kinds of PRRs,such as TLR,which can directly identify the pathogens,activate the related signal pathway.Then IECs can secrete IFNs,inflammatory chemokines and many kinds of cytokines to resist the infection of foreign pathogens.IELs contain cytoplasmic particles and express a variety of NKRs,which patrol between IECs.They can identify infected IECs in time,and exert NK-like killing activity and ADCC effect.Intestinal NK cells are also important members of the intestinal mucosal immune system,which express high levels of CD56,NKRs,CD 16 and TLRs.They can identify the pathogenic bacteria and viruses in time,and produce proinflammatory factors such as IFN-y and TNF-α,to kill the infected cells and quickly clear the infected pathogens.Intestinal NK cells play an essential role in the fight against intestinal bacterial and viral infection.It has been reported that they can inhibit HIV infection in intestinal mucosa for long-term.Therefore,IECs,IELs and NK cells constitute the first defense line to maintain the intestinal immune homeostasis and resist the foreign pathogens.However,the current researches focus on the effects of EV71 on various innate immune signaling pathways and their signaling proteins,little attention has been paid to the mechanism of how EV71 breaks through the intestinal mucosal immune barrier and enters the whole body to cause nervous system diseases,and what role the intestinal mucosal immune barrier plays in the process of resistance to EV71 infection.From the perspective of intestinal mucosa,this paper discusses how EV71 escapes from the recognition and elimination of IECs,NK cells and IELs in intestinal mucosal immune barrier.Firstly,the h-SCARB2 vector was successfully constructed and h-SCARB2-MC38 cell lines were established.It was verified that h-SCARB2 could significantly promote the susceptibility of mouse derived cells to EV71.Then,based on the establishment of EV71 virus infection system,we confirmed that EV71(especially 2A and 3C proteins)could inhibit the key molecules of innate immune signaling pathway,and could significantly inhibit the expression levels of PRRs such as RIG-1,TLR3 and MDA5 induced by polyI:C and the production level of IFN-β.In order to further observed the innate immune response of intestinal epithelial cells after EV71 infection,we used EV71 to infect colon cancer cell lines such as HT29,CaCo2 and HCT116.On this basis,we first found that the production levels of IFN-β and IFN-λ were significantly different after IECs infected by EV71,and the response ability of IECs to IFN-β and IFN-λ,was also different.The levels of IFN-λ produced by IECs after EV71 infection were significantly higher than that of IFN-β,and the response of IECs to IFN-λ was more intense.These phenomena suggested that IFN-λ played a more important role in the resistance of IECs to EV71 infection.Furthermore,we found that the 2A and 3C proteins of EV71 inhibited the production and response of IFN-λ,suggesting that this is one of the main mechanisms of EV71 escaping from intestinal mucosal immune barrier.At the same time,we also found that EV71 2A and 3C proteins could significantly inhibit the expression levels of NKG2DL on IECs and promoted infected IECs to escape from the recognition and killing of NK cells or IELs.Methods1.The h-SCARB2 vector was constructed and the h-SCARB2-MC38 cell lines were established through the sleeping beauty transposons system.After MC38 and h-SCARB2-MC38 cells infected by EV71-Mouse-Adapted,the virus titers in supernatant,the viral load and the expression levels of IFNs were detected by plaque experiment and Q-PCR,respectively.2.A variety of colon cancer cell lines,such as HT29,CaCo2 and HCT116,were infected by EV71-Human-BrCr to establish model of intestinal epithelial cell lines infected by EV71.3.After HT29 cells infected by EV71-Human-BrCr,we observed the changes of cell morphology by microscope,detected the virus titers in the supernatant by plaque experiment,and detected the cell viral load and the expression levels of type Ⅰ IFN,type Ⅲ IFN and ISGs by Q-PCR at different MOI and time points.4.After polyI:C was used to activate 293T cells,EV71 2A and 3C plasmids were transfected into 293T cells,and the effects of 2A and 3C proteins on the expression levels of PRRs,IFNs,and ISGs were detected by Q-PCR.5.The basic expression levels of IFN-λR and IFN-αR on intestinal epithelial cells CaCo2,HCT116 and HT29 and bone marrow-derived NK cells and T cells were detected by flow cytometry and Q-PCR,and how EV71,EV71 2A and 3C proteins affected the expression level of IFN-λR was further detected.6.Before or after HCT116 cells infected by EV71-Human-BrCr,the expression level of MICA/B was detected by flow cytometry and Q-PCR,and how EV71 2A and 3C proteins affected the expression levels of NKG2DL were further detected.7.After HT29 cells activated by polyI:C or transfected by EV71 2A plasmid based on polyI:C,then the killing sensitivity of HT29 cells to NK cells were detected by CFSE/7AAD.Results1.The h-SCARB2 vector was successfully constructed and h-SCARB2-MC38 cell lines were established.Mouse intestinal epithelial cells MC38 that overexpress h-SCARB2 were more sensitive to EV71,showing that the virus titers in supernatant,the viral load and the expression levels of IFN-β and IFN-λ3 were significantly higher than those of MC38 cells.2.PolyI:C obviously induced the expression levels of RIG-I,TLR3,MDA5,IFN-βand ISGs in 293T cells,but EV71 2A and 3C plasmids could significantly weaken the enhancement of polyI:C.3.After HT29 cells infected by EV71-Human-BrCr,we found that the cells damage were aggravated,the virus titers and cell viral load were gradually increasing,meanwhile the expression levels of IFN-β,IFN-λ1,IFN-λ2,and IFN-λ3 were significantly up-regulated,and the expression levels of antiviral proteins MXA and ISG54 also were increased,but these expression levels began to decline after reaching the highest peak at 48 h.4.After HT29 cells infected by EV71-Human-BrCr,the expression level of IFN-λ was significantly higher than that of IFN-β.10 ng/ml IFN-β or 10 ng/ml IFN-λ1 was added before HT29 infection,then it could be seen that the IFN-λ1 could significantly reduced the cells damage and the virus titers than the IFN-β.5.Colon cancer cells were treated with 10 ng/ml IFN-λ1 before or after EV71 infection,then it could be seen that treatment with IFN-λ1 before infection could significantly reduced the cells damage and the virus titers.However,treatment with IFN-λ1 after infection could aggravate the cells damage and increase the virus titers,a large number of cells became round and shed,and there was no significant difference between the EV71 infection without IFN-λ1 treatment group.6.Treatment with IFN-λ1 before EV71 infection significantly increased the expression levels of ISG54,ISG15 and PKR in the intestinal epithelial cells.However,treatment with IFN-λ1 after EV71 infection could not enhance the expression levels of ISG54,ISG15 and PKR.7.The surface of intestinal epithelial cells HCT116,HT29 and CaCo2 highly expressed IFN-λR,while the surface of bone marrow-derived cells such as NK cells and T cells hardly expressed IFN-λR.8.PolyI:C stimulation could significantly up-regulate the expression level of IFN-λR,on the basis of which over expression of 2A protein could significantly inhibit the expression level of IFN-XR.The expression levels of IFN-λ1,IFN-λ2 and IFN-λ3 induced by polyI:C and the expression levels of ISGs induced by IFN-λ1 were significantly inhibited by 2A and 3C proteins.9.EV71 infection significantly inhibited the expression level of MICA/B in intestinal epithelial cells.The 2A and 3C proteins generally inhibited the expression levels of NKG2DL,especially significantly inhibited the expression level of MICA/B.10.The killing experiment showed that the killing sensitivity of HT29 cells stimulated by polyI:C to NK-92 cells was significantly reduced.The killing sensitivity ofHT29 cells stimulated by pcDNA3.1+polyI:C or polyI:C to NK-92 cells was similar.Compared with the pcDNA3.1+polyI:C stimulation group,the pcDNA3,1-2A+polyI:C stimulation group significantly reduced the killing sensitivity of HT29 cells to NK-92 cells.Conclusions1.The h-SCARB2 receptor can significantly promote the susceptibility of mouse derived cells to EV71 infection and increase the expression levels of IFNs.2.The production level of IFN-λ by intestinal epithelial cells after EV71 infection is significantly higher than that of IFN-β,and the response of intestinal epithelial cells to IFN-λ is stronger,indicating that IFN-λ plays an important role in the process of anti-EV71 infection.3.The EV71 virus antagonizes the antiviral function of IFN-λ and its 2A and 3C proteins exert a significant inhibitory effect on the production and response of IFN-λ,which can inhibit the production level of IFN-λ,the membrane expression level of IFN-λR and the transcription levels of ISGs in the downstream of IFN-λ.4.The 2A and 3C proteins of EV71 virus can significantly inhibit the expression levels of NKG2DL on intestinal epithelial cells and promote infected intestinal epithelial cells to escape from the recognition and killing of NK cells. |
PDF Full Text Request