Short communicationIRF9 as a negative regulator involved in TRIF-mediated NF-κB pathway in a teleost fish, Miichthys miiuy
Introduction
The immune defense system has evolved in vertebrates to eliminate infective pathogens when the body is constantly threatened by invasion of microorganisms; this system consists of innate and acquired immunity (Akira et al., 2006). The innate immune response as the first line of body could protect the host from invading microbial pathogens, and its activation may be as a precondition to trigger the acquired immunity (Akira et al., 2001). The innate immune response against invading pathogens depends on pattern-recognition receptors (PRRs) to sensing of pathogen-associated molecular patterns (PAMPs) (Thompson et al., 2011, Kawai and Akira, 2010). These PRRs mainly include TLRs, NLRs, RLRs, and CLRs (Kawai and Akira, 2010, Kingeter and Lin, 2012, Eisenacher and Krug, 2011, Elinav et al., 2011). Then, each PRR recruits downstream adaptor molecule to transmit signals and activates distinct transcription factors, leading to the production of type I interferons (IFNs) and cytokines (Werts et al., 2006, O’Neill and Bowie, 2007). Therefore, the innate immune response is indispensable for controlling microbial infection. However, excessive immune response could lead to many inflammatory and autoimmune diseases, so varieties of regulatory factors are need to tightly regulate the TLR signaling pathway to maintain immune balance.
TLRs, as an important PRR, play a critical role in host defense against pathogenic microbes by recognizing PAMPs (Medzhitov, 2007). Until now, there are at the least 26 members of TLRs with various names identified from different species, containing fish, birds, amphibians, reptiles, and mammals. However, fish TLRs reveal distinct and high variety characteristics; TLR18-20 and TLR23-28 were identified only exist in fish, and certain nonmammalian TLRs have been detected in fish (Wang et al., 2016). Signaling through TLRs can be broadly separated into two pathways that are associated with downstream activation of NF-κB, MAPKs, and IFN regulatory factors (IRFs) (O’Neill and Bowie, 2007). All TLRs have a Toll/IL-1R domain through which interacte with TLR adaptor proteins. To date, five of adaptor proteins have been identified, including MyD88, MAL/TRIAP, TRIF, TRAM, and SARM respectively (O’Neill and Bowie, 2007). And these adaptors promote the activation of the MyD88-dependent and MyD88-independent pathways. Almost all TLRs use MyD88 adaptor to transmit signals and activate the NF-κB and other transcription factors that facilitate expression of classic proinflammatory cytokines (Lin et al., 2010). However, TRIF is only used by both TLR3 and TLR4 to mediate a MyD88-independent pathway which drives the expression of type I IFN and proinflammatory cytokines (Gay et al., 2014, Alexopoulou et al., 2001), thereby leading the body to producing inflammatory response. However, excessive inflammatory response can cause damage on body. In order to maintain immune balance, signal pathway must be tight regulated in the immune response. Recent work has revealed complex regulation of TLR signal transduction at numbers of different levels including phosphorylation, degradation, and sequestration of signaling molecules (Komuro et al., 2008). For example, MyD88s, have been reported to be an endogenous negative regulator in MyD88-dependent singaling pathway (Burns et al., 2003). In addition, SOCS-1 as a critical regulator in the immune systems and has been shown to negatively regulate the IFN signaling pathways (Nie et al., 2014).
TRIF facilitates TLR3 and TLR4 signaling and activate the transcription factors, NF-κB and IRF3 leading to the production of proinflammatory cytokine and type I IFN. Previous studies have shown many cases of negative regulation of TRIF involved in above signaling pathway. For instance, SARM could negatively regulate the TRIF-mediated signaling pathway (Carty et al., 2006). And ADAM15 is a negative regulator of TLR3 and TLR4 signaling and involved in the mechanism of TRIF degradation (Ahmed et al., 2013). Although some reports have confirmed about the regulation mechanism of TRIF, but there is no report that IRF family could regulate TRIF.
IRF9 belongs to IRF family, and it is first recognized from a component called ISGF3 (Veals et al., 1992). And in the fish genome, alike to the mammals, it has the total IRF family members exhibiting a clear orthologous relationship with mammalian the counterparts (Stein et al., 2007). The previous research demonstrated that, in IRF9-deleted of the mouse cells, the activation of ISGs by IFNα or IFNγ established the host antiviral state are all showed certain impaired, and the corresponding ISRE-binding activities by the IFNs are absent, thus the result indicating that IRF9 is a vital type I IFN and type II IFN response (Kimura et al., 1996). In this paper, we have firstly reported that IRF9 is a negative regulator of TRIF-mediated NF-κB pathway. In addition, we discovered that inhibitory effect of IRF9 primary depends on DBD domain. This study provides an evidence for the regulatory mechanisms of TRIF signaling by IRFs and enriches the content of the negative regulation of TLR signaling pathway.
Section snippets
Plasmids construction
The full-length CDS region of miiuy croaker (Miichthys miiuy) IRF9 was amplified by PCR with primers including restriction enzyme cutting sites Kpn I and Xba I with HA tag, the product was digested by corresponding enzymes and ligated into the pcDNA3.1 vector. The recombinant plasmid of TRIF from miiuy croaker was cloned into the Kpn I and BamH I sites with FLAG tag of pCDNA3.1 (Supplemental Table 1). The recombinant plasmid was confirmed by double enzyme digestion and sequencing. The four
Overexpression of IRF9 attenuates the TRIF-mediated NF-κB pathway
In the previous studies, TRIF possesses the ability which activates not only the NF-κB but also the IFN-β promoter, and associates with both TLR3 and IRF3 (Yamamoto et al., 2002). In addition, IRF9 plays an important role in IFN-inducing JAK/STAT signaling pathway. In order to detect the role of IRF9 in TRIF-mediated signaling pathway, we firstly examined the expression of the TRIF in the protein level used the Western blot, the result exhibited that the molecular weight was in agreement with
Discussion
IRF family offers a basis for host innate and adaptive immune responses in mammals and fish (Zhang and Gui, 2012, Ozato et al., 2007, Tamura et al., 2008). IRFs, as the best basic transcription factor, can also cooperatively regulate the transcription of some cytokine genes by associating with NF-κB family members (Ikushima et al., 2013). Moreover, recent reports also indicated that IRF could negatively regulate other adaptor proteins or signaling pathway, such as, IRF4 functions as a negative
Author contributions
Conceived and designed the experiments: XZ TX. Performed the experiments: XZ RH TX. Analyzed the data: XZ TX. Contributed reagents/materials/analysis tools: XZ TX. Wrote the paper: XZ QC JC RH TX.
Acknowledgments
This study was supported by National Natural Science Foundation of China (31370049, 31672682) and Natural Science Foundation of Zhejiang Province (LR14C040001).
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