The Role And The Underlying Molecular Mechanism Of Riok3 In Regulating Antiviral Innate Immune Responses

The exploration ofinteraction between viral infection and host innate immunity and the underlying mechanisms has long been as hotspot in the fields of virology and immunology.As the first line for host to defense against foreign pathogens' invasion,the innate immune system plays key roles in the processes of recognizing,eliminating or presenting the pathogens to T cells to maintain host's homeostasis.Upon infection of organisms,the pathogen associated molecular patterns(Pathogen-associated molecular,patterns,PAMPs)of these pathogens were immediately recognized by host's pattern recognition receptors(PRRs)such as retinoid acid-inducible gene I-like receptors(RLRs)and Toll like receptors(TLRs),thereby initiating host immune response.RLRs,mainly including the retinoic acid inducible gene I(RIG-I)and melanoma differentiation associated antigen 5(MDA5),apperceived byRNA viruses like vesicular stomatitis virus(VSV)and influenza virus H1N1 PR8(Puerto-Rico/8),may recruitthe adaptor molecule mitochondrial antiviral signaling protein(MAVS),MAVS then recruits TRAF3(tumor necrosis factor receptor-associated factor 3)and TRAF6(tumor necrosis factor receptor-associated factor 6),to activate the TRAF3-TBK1-IRF3 and TRAF6-TAK1-NF-?B/MAPK signaling pathway,respectively.Activation of transcription factors such as IRF3 and NF-?B promotes their nuclear entry to initiate type ? IFNs and inflammatory cytokines' release.Type ? interferon further activates the downstream JAK/STAT signaling pathway,resulting in the production of hundreds of antiviral proteins(interferon stimulated genes,ISGs),thereby inhibiting the replication of viruses and bacteria.Toll like receptor(TLRs)is another important group of PRRs.So far,up to 13 different TLRs have been identified,among which,TLR1-TLR9 were found both in human and mouse,and TLR10 was only found in human while TLR11?TLR13 were detected in mice only.They recognize the specific PAMPs,transduce downstream MyD 8 8 or TRIF dependent signaling pathway,activate transcription factors such as IRF3,NF-?B or AP1,and finally initiate the production of inflammatory factors and type I IFNs.RLRs and TLRs are involved in the innate immune response and acquired immune response,and either over-activation or deficiency would lead to immune-related diseases.In addition,there are PRRs to recognize DNA PAMPs in cells like TLR9,DNA-dependent activator of IFN-regulatory factor(DAI),stimulator of interferon gene(STING),RNA III polymerase(Pol III),leucine-rich repeat flightless-interacting protein 1(LRRFIP1)and absent in melanoma protein 2(AIM2),etc.They sense the exogenous or intracellular DNA molecules,then mediate the prduction of type I interferon and inflammatory factors.Taking the cGAS-STING-TBK1 pathway found in recent years for example:cGAS recognizes DNA molecules to form cGAS-DNA complex,subsequently cGASbinds and catalytes ATP and GTP to generate cGAMP,cGAMP then binds and activates STING,which may phosphorylate TBK1 through the C-terminal domain,and the STING-TBK1 complex binds and induces the activation of IRF3,thereby initiating antiviral immune response.Protein kinases participate in the modification of protein phosphorylation,thus affect almost all cell life activities like cell growth,differentiation,development,metabolism and cell interaction through modulating the signaling pathways transduction.As an atypical kinase,Riok3(RIO Kinase 3)is widely expressed in eukaryotes.It lacks of typical kinase domain,but retains the kinase activity.The conservative RIO domain of Riok3 is very different from other RIO family members,and few studies have focused on its functions.It is highly expressed in immunocytes,indicating its possible role in the immune responses.Previous studies have reported that Riok3 has the function of regulating type ? type interferon signaling pathway in HEK293T cells,but its specific regulation mechanism is not clear.So far,we have not seen the function of Riok3 in immune cells.The aim of this study is to investigate the role and mechanism of Riok3 in the regulation of type ? interferon and inflammatory cytokines expression in immunological cells(primary macrophages).Firstly,we constructed the myeloid-specific knockout mice LysMcreRiok3F/F,used VSV(Vesicular Stomatitis Virus),PR8(A/PR/8,H1N1),SeV(Sendai Virus),HSV(Herpes Simplex Virus)and MHV68(Murine Gammaherpesvirus 68)to infect Riok3 knockout macrophages in vitro and LysMcreRiok3F/mice in vivo,finding that Riok3 selectively negativelyregulates the type ? IFNs pathway induced by RNA viruses infection,which means that loss of Riok3 inhibits RNA virus replication both in primary macrophages and mice in vivo.Then,we transfected Poly(I:C)into cells to directly activate RIG-I/MDA5 receptors,finding that Riok3 negatively regulates the type ? IFNs signaling pathway.This phenomena was also confirmed in mouse embryonic fibroblasts(MEFs)and HEK293T cells.At the same time,Poly(I:C)and LPS were added directly to stimulate the primary macrophages to detect the regulatory effect of Riok3 on TLR3/TLR4 signaling pathway,and we found that Riok3 positively regulates the release of type ?IFNs and pro-inflammatory cytokines.Western blot assay showed the phosphorylation of TBK1,IRF3 and NF-?B was significantly increased in Riok3 KO macrophages compared to the control groupupon activation of RIG-I/MDA5 pathway,while the NF-kappa B phosphorylation was decreased significantly compared with the control group upon stimulating TLR3/TLR4.Preliminary mechanistic studies indicated that Riok3 could bind with TRAF3 and may influence the different sites of ubiquitination,thus achieving different results on the regulation of downstream pathways.When RIG-I/MDA5 is activated,Riok3 may promote the K48 ubiquitination of TRAF3 for degradation,then inhibit the transduction of the downstream signaling.Our study is the first to explore the Riok3 function of regulating innate immune response in macrophages,and we revealed the following findings:1)Riok3 deficiency selectively inhibits RNA virus replication both in vitro and in vivo;2)Riok3 negatively regulates the immune response triggeredby RIG-I/MDA5 receptors activation;3)Riok3 positively regulatethe immune response induced by TLR3/TLR4 activation;4)Riok3 does not affect the immune response when activating the DNA molecule-related PRRs;5)Riok3 may interact with TRAF3,promoting itsK48 ubiquitination to degradate it,thus leading to the suppression of its downstream signaling pathway.Our experimental results reveal the possible mechanism of Riok3 negatively regulating the RIG-I/MDA5 pathway,indicating that Riok3 may act as a negative regulator of RIG-I/MDA5 signaling pathway.Therefore,it is of great interest to confirm Riok3's role upon RIG-I or MDA5 over-activation in cells,or even in autoimmune diseases.It is not clear how Riok3 positively regulates the TLR3/4 signaling pathway,we'll focus on finding the underlying mechanism in the next step.Our study provides a new sight for understanding the host innate immune response,and may also provide a new target for the prevention and treatment of autoimmune diseases caused by over-activation of RIG/MDA5 pathway upon RNA viruses infection.