| The innate immunity is the first line of defense against invading pathogens, which functions to respond to infection directly and relays signals for the activation of the adaptive immunity. During viral infection, multiple signaling pathways in the innate immune system are triggered to promote the production of cytokines to suppress viral replication. Central to the host antiviral response is the production of type I interferons (IFNs), which include IFN-a and IFN-(3. Several classes of termline-encoded pattern-recognition receptors (PRRs) have been linked to the production of type Ⅰ interferons during viral infection. These PRRs include Toll-like receptors (TLRs), retinoic acid-inducible gene I (RIG-I) like receptor (RLRs) and intracellular DNA sensors. Type I IFNs interact with its receptors then activate the JAK-STAT signal transduction pathways, leading to the induction of multiple downstream IFN-stimulated genes (ISGs).Osteopontin (OPN), also known as early T lymphocyte activation 1 (Eta-1), is a secreted glycoprotein which regulates diverse biological processes including differentiation, adhesion, bone remodeling, malignancy and immune response. OPN is considered to be a potential pro-inflammatory cytokines associated with the promotion of macrophages to secrete IFN-y and IL-12 for a long time. In recent years, with the discovery of intracellular form OPN (iOPN), different function of OPN in diverse immune cells and different stages of immune response gradually attracted people’s attention. But in innate immunity system, especially innate antiviral response, the function of iOPN still remains elusive.Objectives:Investigating the function of OPN, especially iOPN, in regulating the production of type I interferons upon viral infection. Furthermore, finding out the underlying mechanisms.Methods and Results:1. OPN expression is induced upon virus infection, and OPN especially iOPN, positively regulates IFN-β production.The expression of OPN protein and mRNA was increased after infection with SeV. Similarly, infection of VSV also increased OPN protein and mRNA expression in peritoneal macrophages. ELISA analysis showed that sOPN was also increased upon virus infection. These data demonstrated that OPN expression is induced by virus infection in murine peritoneal macrophages. To investigate the function of OPN in innate antiviral immune response, peritoneal macrophages were prepared from WT and OPN-deficient (Spp1-/-) mice and infected with SeV for various times. Then, the expression of IFN-(3 was measured. SeV infection induced the expression of IFN-P mRNA in WT macrophages. However, SeV-induced IFN-P mRNA expression was greatly decreased in OPN-deficient macrophages compared to that in WT macrophages. Consistently, OPN-deficient macrophages secreted less IFN-(3 protein than WT macrophages after SeV infection. The expression of CXCL10, Mx1 and CCL5, which are downstream genes of IFN-β signaling pathway, also decreased in SeV-infected OPN-deficient macrophages. We further investigated the function of OPN on IFN-β expression using overexpression experiments. Transfection of iOPN and full length OPN expression plasmids into HEK293 cells increased SeV-and VSV-induced IFN-β expression. Similar to IFN-β mRNA expression, transfection of iOPN and full length OPN expression plasmids also increased SeV-induced IFN-β promoter activation in a dose-dependent manner. Further, we found that addition of OPN antibody into the culture medium could not inhibit full length OPN transfection-mediated IFN-(3 activation induced by SeV infection. Overexpression of iOPN also increased RIG-I-, melanoma differentiation-associated gene 5 (MDA5)-, TRIF- and cGAS+STING-induced IFN-β promoter activation in a dose-dependent manner. In all circumstances, intracellular OPN seemed more potent to induce IFN-β expression than the full length OPN. Taken together, these data indicated that OPN, especially iOPN, positively regulates IFN-β production downstream of various innate immune signaling pathways including TLR3/4, RLRs and intracellular DNA receptor signaling2 OPN is an important positive regulator of IFN-β production and antiviral immune responses in vivo.IFN-β plays an essential role in antiviral immune response. To investigate the role of OPN in antiviral response, VSV was used to infect cells. Plaque assays showed that VSV replication greatly increased in peritoneal macrophages prepared from OPN-deficient mice compared to that from WT mice in the presence or absence of poly(l:C). Consistently, VSV RNA was also increased in OPN-deficient macrophages compared to that in WT macrophages. In contrast, overexpression of iOPN and the full length OPN in HEK293 cells greatly attenuated VSV replication in the presence or absence of poly(l:C). Taken together, these data indicated that OPN positively regulates antiviral immune response. To investigate the physiological role of OPN in antiviral response in vivo, Spp1-/- mice and WT mice were infected with VSV, and the antiviral immune responses were examined. The amount of IFN-β protein induced by VSV infection was much less in sera of VSV-infected Spp1-/- mice than that of WT mice. In accordance with reduced IFN-β production, VSV replication in the livers, spleens, and lungs was much higher in OPN-deficient (Spp1-/-) mice than in WT controls. Importantly, Spp1-/-mice were more susceptible to VSV infection than WT mice. Spp1-/- mice all died, while 50% of WT mice were alive 5 days after infection. These data suggested that OPN is an important oositive regulator of IFN-β production and antiviral immune responses.3 iOPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.IRF3 is the main transcription factor responsible for IFN-β transcription during the early phase of viral infection. To investigate the function of OPN on IRF3 activation, series of experiments were performed. First, IFN-β PRD Ⅰ/Ⅲ reporter, which harbors only IRF3 binding site in IFN-β promoter, was used. RIG-I, TRIF-and cGAS+STING-induced-IFN-p PRD Ⅰ/Ⅲ activation was increased by iOPN overexpression in a dose-dependent manner. IRF3 activation requires the phosphorylation of conserved serine and theronine residues at the c-terminal region. SeV infection induced IRF3 phosphorylation in macrophages from WT mice. While, SeV-induced IRF3 phophorylation was greatly decreased in macrophages from OPN-deficient (Spp1-/-) mice. Similarly, VSV infection-induced IRF3 phosphorylation was also greatly decreased in macrophages from Spp1-/-mice compared to that from WT mice. In contrast, overexpression of iOPN in HEK293 cells substantially increased SeV-and VSV-induced IRF3 phosphorylation. After phosphorylation, IRF3 dimerizes and translocates into nuleus to initiate IFN-(3 transcription. IRF3 dimerization was greatly decreased in OPN-deficient (Spp1-/-) macrophages compared to that in WT macrophages after SeV infection. Western blot analysis of cytoplasmic fraction and nuclear fraction showed that more IRF3 was transfected into nucleus in macrophages from WT mice compared to that from OPN-deficient (Spp1-/-) mice after virus infection. All together, these data demonstrated that OPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.4. iOPN interacts with TRAF3 to positively regulate IFN-β production and antiviral response.OPN positively regulates IFN-P production downstream of TLR3/4, RLR and intracellular DNA receptor signaling pathways, indicating OPN targets common molecules in these signaling pathways. To identify the molecules, IFN-(3 promoter activation induced by various molecules in RLRs signaling pathway was investigated. We found iOPN increased RIG-I-, MDA5-, MAVS-induced IFN-β activation, but not TBK1-and IRF3 5D-induced IFN-β activation. RT-PCR analysis of IFN-β mRNA also confirmed iOPN increased RIG-I-, MDA5-, MAVS-induced IFN-β expression, but not TBK1-and IRF3-5D-induced IFN-β expression. These data indicated that iOPN targets molecules upstream of TBK1 to positively regulate innate signaling. To directly identify iOPN targets, immunoprecipitation (IP) and western blotting (WB) were performed in HEK293 cells transfected with expression plasmids for RIG-I, MAVS, TRAF3, TNF receptor-associated factor 6 (TRAF6), STING, TBK1 and IRF3 together with iOPN. iOPN was shown to interact with TRAF3, but not with RIG-I, MAVS, STING, TBK1 and IRF3. TRAF3 has been shown to be required for the IFN-β expression downstream of TLR3/4 and RLR signaling. Interestingly, iOPN could not interact with TRAF3 homologue TRAF6, which has been shown to activate NF-κB, leading to the production of proinflammatory cytokines. Interaction between endogenous TRAF3 and OPN also detected in macrophages after SeV infection. To confirm iOPN interacts with TRAF3 directly, iOPN and TRAF3 were expressed in an in vitro protein expression system, then mixed together and followed by pull-down assays with anti-OPN antibody. TRAF3 could coimunoprecipitate with OPN, indicating a direct interaction between iOPN and TRAF3. The interactions were further supported by the colocalization studies. iOPN-GFP was found to diffuse in the cytoplasm and nucleus without SeV infection. TRAF3 was present in the cytoplasm exclusively. iOPN-GFP and TRAF3 showed less or no colocalization without SeV infection. SeV infection induced translocation of large amount of iOPN from nucleus into cytoplasm, where colocalization between iOPN and TRAF3 was greatly increased. Taken together, these data suggested that iOPN interacts with TRAF3 to positively regulate IFN-β production and antiviral response.5 iOPN inhibits K48-linked polyubiquitination and degradation of TRAF3.TRAF3 activation is tightly regulated by protein ubiquitination. K63-linked TRAF3 polyubiquitination is responsible for the activation of downstream signaling. While, K48-linked ubiquitination leads to the degradation of TRAF3 and deactivation of TRAF3-mediated downstream signaling. To investigate the molecular mechanism of iOPN in the regulation of IFN-P production, TRAF3 polyubiquitination was investigated. TRAF3 was transfected into HEK293 cells together with WT HA-ubiquitin plasmid and iOPN expression plasmid. IP and WB showed that TRAF3 polyubiquitination was greatly inhibited by iOPN expression To investigate which form of TRAF3 polyubiquitination was affected by iOPN, HA-ubiquitin mutants K48 and K63, which has only one lysine residue at position 48 and 63 respectively, were transfected into HEK293 cells together with iOPN expression plasmid. Overexpression of iOPN decreased TRAF3 polyubiquitination in HA-K48-transfected cells, whereas, TRAF3 polyubiquitination was not affected by iOPN in HA-K63-transfected cells, indicating iOPN mainly prevents TRAF3 from K48-linked polyubiquitination. K48-linked polyubiquitination leads to protein degradation by 26S proteasome. To investigate the function of iOPN on TRAF3 degradation, Myc-TRAF3 was transfected into HEK293 cells together with iOPN expression plasmid or control plasmid. After SeV infection, the half life of Myc-TRAF3 protein was measured. SeV infection led to the degradation of Myc-TRAF3 in control vector transfected cells with a half life of ～0.8 h. However, the degradation of Myc-TRAF3 was greatly attenuated in iOPN expression vector transfected cells. Similarly, SeV infection induced the degradation of Myc-TRAF3 in A549 cells, whereas, overexpression of iOPN reversed Myc-TRAF3 protein degradation after SeV infection, indicating inhibition of TRAF3 degradation by iOPN is not cell specific. To further confirm OPN stabilizes TRAF3 protein through inhibition of K48-linked ubiquitination in physiological conditions, peritoneal macrophages from WT and Spp1-/-mice were prepared and infected with SeV. IP and WB showed that K48-linked polyubiquitination of TRAF3 was greatly increased in the macrophages from Spp1-/-mice compared to that in macrophages from WT mice after SeV infection. Increased K48-linked TRAF3 polyubiquitination in OPN-deficient macrophages was further confirmed with proteins immunoprecipitated with anti-TRAF3 under stringent conditions. Consistent with more TRAF3 ubiquitination, TRAF3 was degraded more rapidly in OPN-deficient macrophages. All together, these data indicated that OPN prevents TRAF3 from K48-linked polyubiquitination and degradation.6. iOPN inhibits Triad3A-mediated TRAF3 polyubiquitination.iOPN alone does not have the ability to modulate protein ubiquitination. There are two possibilities for iOPN to inhibit TRAF3 polyubiquitination. One is that iOPN may recruit deubiquitinating enzymes (DUB) to cleave K48-linked polyubiquitin chains from TRAF3 to stabilize TRAF3. Recently, USP25 has been reported to cleave K48-linked polyubiquitin chains from TRAF3. To investigate whether iOPN recruit USP25 to stabilize TRAF3, TRAF3 ubiquitination was measured in the presence of USP25 and iOPN expression plasmids. iOPN expression decreased TRAF3 ubiquitination. USP25 expression indeed decreased TRAF3 polyubiquitination. While, cotransfection of USP25 and iOPN could not further decrease USP25-mediated deubiquitination from TRAF3, suggesting iOPN may not recruit USP25 to cleave K48-linked ubiquitin from TRAF3. Another possibility is that iOPN prevents an E3 ligase from binding to TRAF3. Triad3A has been reported to be an E3 ligase involved in TRAF3 ubiquitination and degradation after virus infection. To investigate whether iOPN inhibits Triad3A-mediated TRAF3 ubiquitination, TRAF3 was transfected into HEK293 cells together with Triad3A and iOPN. IP and WB showed that Triad3A promoted TRAF3 ubiquitination. Overexpression of iOPN greatly decreased TRAF3 ubiquitination mediated by Triad3A. In vitro ubiquitination assays with in vitro expressed proteins also confirmed that Triad3A-induced K48-linked TRAF3 polyubiqutination was greatly attenuated by iOPN. Triad3A binding to TRAF3 was also decreased by iOPN in a dose-dependent manner. In vitro pull-down assays confirmed the binding Triad3A to TRAF3 was gradually decreased with the increasing binding of iOPN to TRAF3. Consistent with the inhibition of Triad3A-induced TRAF3 ubiqutination, Triad3A-induced degradation of TRAF3 was reversed by iOPN expression. The Y residue and Q residue at position 441 and 443 of TRAF3 have been reported for Triad3A binding. To confirm the importance of these two residues, TRAF3 mutant was constructed by mutating Y441 and Q443 to A. Mutation of YQ to AA ablated Triad3A binding to TRAF3. Notably, iOPN binding to TRAF3 mutant was also ablated, indicating iOPN binding to the same sites in TRAF3 as the Triad3A. All together, these data demonstrated that iOPN competes with Trida3A for the binding to TRAF3, which prevents TRAF3 from K48-linked polyubiquitination and degradation promoted by Triad3A.7. C-terminal fragment of iOPN binds to TRAF3.Endogenous OPN can be cleaved by thrombin at position 168 into two fragments. In order to investigate the OPN fragment involved in the binding and regulation of TRAF3 ubiquitination, two OPN truncations were constructed and expressed in vitro. In vitro pull-down assays demonstrated that full length and the C-terminal fragment of iOPN, but not the N-terminal fragment, bound to TRAF3. Consistent with the C-terminal fragment binding to TRAF3, Trida3A-induced TRAF3 ubiqutination was inhibited by the C-terminal fragment, but not the N-terminal fragment. These data indicated that the C-terminal fragment of OPN is responsible for the binding and inhibition of ubiquitination of TRAF3. To investigate the inhibition of TRAF3 ubiquitination by WT and the C-terminal fragment of iOPN has a physiological role on IFN-P production, lentiviral expression plasmids for WT, N-terminal fragment and C-terminal fragment of iOPN were constructed and used to infect WT and OPN-deficient macrophages. Infection of lentivirus containing WT iOPN plasmid increased OPN expression in WT macrophages and restored iOPN expression in OPN-deficient (Spp1-/-) macrophages. Consistent with positive function of iOPN on IFN-(3 production, lentiviral infection of WT iOPN expression plasmid into WT macrophages further increased SeV-induced expression of IFN-β, CXCL10, Mx1 and CCL5. Lentiviral infection of WT iOPN expression plasmid into OPN-deficient macrophages restored SeV-induced expression of IFN-β, CXCL10, Mx1 and CCL5 to the same level as that in WT macrophages. Consistent with ability to inhibit TRAF3 ubiquitination by the C-terminal fragment, lentiviral infection of the C-terminal fragment of iOPN increased SeV-induced expression of IFN-(3, CXCL10, Mx1 and CCL5 in WT macrophages. SeV-induced expression of IFN-β in OPN-deficient macrophages was also restored upon infection with lentivirus containing the C-terminal fragment of iOPN. But, infection of lentivirus containing the N-terminal fragment of iOPN could not increase or restore SeV-induced expression of IFN-βin WT and OPN-deficient macrophages, respectively Taken together, these data demonstrated that iOPN binds to TRAF3 through the C-terminal fragment, preventing TRAF3 from K48-linked ubiquitination and degradation and leading to increased IFN-β production and innate antiviral response.Conclusions:1. OPN expression is induced upon virus infection, and OPN especially iOPN, positively regulates IFN-β production.2. OPN is an important positive regulator of IFN-(3 production and antiviral immune responses in vivo.3. iOPN positively regulates IRF3 activation to regulate IFN-β production and antiviral response.4. iOPN interacts with TRAF3 to positively regulate IFN-(3 production and antiviral response.5. iOPN inhibits K48-linked polyubiquitination and degradation of TRAF3.6. iOPN compete with Trida3A for the binding to TRAF3, which prevents TRAF3 from K48-linked polyubiquitination and degradation promoted by Triad3A.7. iOPN binds to TRAF3 through the C-terminal fragment, preventing TRAF3 from K48-linked ubiquitination and degradation and leading to increased IFN-β production and innate antiviral response.Innovation and significance:1. To the best of our knowledge, this study is the first to completely prove that iOPN can positively regulate IFN-β production and antiviral innate immune response.2. We first time illustrated that the C terminal of iOPN could inhibit the ubiquitination of any protein through competitive combination.3. These researches improve the function of iOPN in innate immunity, propose new understanding about expression regulation mechanism of type I interferons, and provide the theoretical basis for the relevant drug development. |
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