Phosphatase PHLPP Selectively Promotes TLR- And RIG-I-triggered Production Of Type Ⅰ Interferon

Innate immune cells recognize pathogen-associated molecular patterns (PAMPs) conserved in microbes by pattern recognition receptors (PRRs) to trigger innate immune responses. As one kind of key PRRs, TLRs comprise a large family consisting of at least 12 members and are mainly expressed in antigen-presenting cells (APCs) including macrophages and dendritic cells (DCs). Upon recognition of PAMPs, TLRs trigger myeloid differentiation factor 88 (MyD88)- and/or Toll/IL-1 receptor (TIR)-domain containing adaptor protein inducing IFN-β(TRIF)-dependent signaling pathway to activate mitogen-activated protein kinases (MAPKs), the transcription factors nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3) and/or IRF7, leading to the production of proinflammatory cytokines and typeⅠinterferon (IFN), and defense against invading pathogens.Besides TLRs, retinoid acid-inducible gene I-like receptors (RLRs) are another important class of PRR families, which are composed of RNA helicase domain-containing proteins retinoic acid-inducible geneⅠ(RIG-Ⅰ), melanoma differentiation associated gene 5 (MDA5) and LGP2. They are localized in the cytoplasm and recognize the genomic RNA of dsRNA viruses and dsRNA generated as the replication intermediate of ssRNA viruses. Vesicular stomatitis virus (VSV) and Sendai virus (SeV) are the representative viruses which can activate RIG-Ⅰsignal pathway. RIG-Ⅰand MDA5 interact with adaptor protein IPS-1 (Interferon-beta promoter stimulator 1, also known as MAVS/Cardif/VISA), which mediates the gene expression of typeⅠinterferon and proinflammatory cytokines by activating IRF3/7 and NF-κB.TLRs and RLRs play important roles in linking innate and adaptive immune responses. It is now well accepted that full activation of TLRs and RLRs is essential for initiating the innate immune response and enhancing adaptive immunity to eliminate invading pathogens. Less efficient activation of TLRs and RLRs or excessive activation of TLRs and RLRs may induce immune disorders and even immunopathological process. Up to now, various signal pathways are known to be involved in the tight regulation of TLR and RLR signaling to maintain the immunological balance. However, the underlying molecular mechanisms for the regulation of TLR and RLR signaling remain to be fully elucidated.Phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase) is a serine/threonine phosphatase, which consist of a pleckstrin homology (PH) domain, a leucine-rich repeat (LRR) region, a PP2C (protein phosphatase 2C) domain, and a PDZ-binding motif. Previous studies showed that PHLPP can directly interact with K-Ras through its LRR domain to negatively regulate Ras-MAPK signal pathway in rat brain. PHLPP also functions as a negative regulator of MAPK and CREB-mediated transcription in hippocampal neurons to regulate rat memory formation. PHLPP is low expressed in many human tumor cells such as breast cancer, colon cancer and glioblastoma cell lines. PHLPP can specifically dephosphorylate the Ser473 of Akt depending on its PP2C and PDZ binding motif to suppress tumor growth and promote apoptosis of tumor cells. In addition, PHLPP controls the cellular levels of PKC by specifically dephosphorylating the hydrophobic motif, thus destabilizing the enzyme and promoting its degradation. All these reports suggest that PHLPP may show different functions depending on its different domain to involve in many cell preceess.Nowadays, the important roles of protein phosphotase in regulating immune response draw more and more attention. In innate immunity, several protein phosphotases, such as SHP-1 (Src homology region 2 domain-containing phosphatase 1), SHP-2 (Src homology region 2 domain-containing phosphatase 2), SHIP-1 (Src homology 2 domain containing inositol-5'-phosphatase-1) and MKP-1 (MAPK phosphatase-1), have been reported to regulate TLR-and RIG-Ⅰ-triggered production of proinflammatory cytokines and/or typeⅠinterferon through different mechanism. However, whether phosphotase PHLPP can regulate TLR and RIG-Ⅰ-triggered immune response remains unknown.The primary aim of this study is to investigate the regulation of TLR-and RIG-Ⅰ-triggered innate immune response by PHLPP in macrophages as well as the underlying mechanisms. PartⅠ. PHLPP selectively promotes TLR-and RIG-Ⅰ-triggered typeⅠinterferon production in macrophagesWe first examined the expression pattern of PHLPP. RT-PCR assay showed that the mRNA of PHLPP is ubiquitously expressed in various mouse tissues and immune cells, with highest levels in brain and spleen. LPS, poly(I:C) or VSV challenge upregulated PHLPP expression in mouse primary peritoneal macrophages.Next, the effects of PHLPP silence or deficiency on the production of TLR- and RIG-Ⅰ-triggered cytokines were investigated. RNAi knockdown of PHLPP significantly suppressed TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages, but had no effect on the production of IL-6 and TNF-α. Meanwhile, silencing of PHLPP expression had no influence on TLR9-triggered production of the above cytokines. Coincidently, overexpression of PHLPP significantly enhanced TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/βin macrophages.Overexpression of PHLPP significantly increased TRIF-, constitutively active RIG-Ⅰ-or MDA-5-activated expression of IFN-βreporter gene in a dose-dependent manner, but had no effect on TRIF-or MyD88-activated expression of TNF-αreporter gene, or MyD88-activated expression of IFN-βreporter gene.In macrophages from PHLPP knockout mice generated by PB (PiggyBac) transposon system, PHLPP expression was downregulated over 80% as compared to that in macrophages from wild type mice. PHLPP deficiency significantly inhibited TLR3,4- and RIG-Ⅰ-triggered production of IFN-α/β, while had no effect on the production of IL-6 and TNF-αin macrophages. The production of above cytokines induced by CpG ODN remain unchanged in PHLPP-/- and PHLPP+/+ macrophages. PHLPP-/- mice and PHLPP+/+ mice were challenged by intraperitoneal injection with LPS, poly(I:C), or infection with VSV. After these challenges, the mRNA levels of IFN-α/βin primary peritoneal macrophages and IFN-p level in serum of PHLPP-/- mice were much lower than that in PHLPP+/+ mice, while there was no significant difference in the levels of proinflammatory cytokines.Taken together, these results indicated that PHLPP selectively promotes TLR-triggered TRIF-dependent and RIG-Ⅰ-triggered production of typeⅠinterferon, but has no effect on the production of proinflammatory cytokines and MyD88-dependent production of cytokines.PartⅡ. The underlying molecular mechanisms for the positive regulation of TLR and RIG-Ⅰ-triggered typeⅠinterferon production by PHLPPTo investigate the function of each domain of PHLPP in the enhancement of typeⅠinterferon production triggered by TLR3,4 and PIG-Ⅰ, various truncated or deleted mutants of PHLPP were constructed according to its known structural and functional domains, and then transfected into HEK293 cells to detect TRIF-activated expression of IFN-(3 reporter gene. We revealed that the mutants of PHLPP deleting LRR region or PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutants of PHLPP deleting PH domain or PDZ-binding motif significantly increased TRIF-dependent activation of IFN-βreporter gene as full-length PHLPP did. The mutant of PHLPP only expressing PP2C domain couldn't increase TRIF-activated expression of IFN-βreporter gene, while the mutant of PHLPP expressing LRR region and PP2C domain significantly could did so. Meanwhile, overexpression of the mutants of PHLPP deleting LRR region or PP2C domain failed to enhance poly(I:C)-induced production of IFN-(3 in macrophages. So, the results indicated that the LRR and PP2C domain were responsible for the positive regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon by PHLPP.Then, we tested whether PHLPP regulates TLR-and RIG-Ⅰ-triggered activation of MAPKs and NF-κB pathways. PHLPP deficiency had no significant effect on poly(I:C)-induced phosphorylation of ERK, JNK, p38 and IKKα/βin macrophages. Consistently, PHLPP overexpression did not affect MyD88-, TRIF- or constitutively active RIG-Ⅰ-activated expression of NF-κB reporter gene. So, PHLPP can't enhance TLR- and RIG-Ⅰ-triggered activation of MAPKs and NF-κB, which contributes to the unchanged production of inflammatory cytokines.Next, we investigated which molecule in TLR and RIG-Ⅰsignal pathway PHLPP could interact with. Luciferase report gene assay showed that overexpression of PHLPP significantly increased TBK1- and IRF3-activated expression of IFN-βreporter gene, and TRIF-activated expression of IRF3 reporter gene in a dose-dependent manner. These results indicated IRF3 may be the potential target PHLPP can interact with. Immunoprecipitation experiment and GST pull-down assay confirmed PHLPP could directly interact with the C-terminal of IRF3 through its LRR region.As we known, IRF3 resides in the cytoplasm in resting cells, and, upon stimulation, becomes activated leading to nuclear translocation. The confocal laser scanning microscope observed that PHLPP resided in both cytoplasm and nuclear of resting macrophages. Upon the stimulation of poly(I:C) or SeV, more PHLPP translocated into nuclear, and co-localized with IRF3 in the nuclei of macrophages. Immunoprecipitation experiment using separated extracts of cytoplasm and nuclei revealed that PHLPP could co-precipitate with IRF3 in the nuclei of macrophages.To further confirm the target of PHLPP in regulation of TLR-and RIG-Ⅰ-triggered production of typeⅠinterferon is IRF3, we transduce PHLPP into IRF3 deficient (IRF3-/-) macrophages. Overexpression of PHLPP had no effect on poly(I:C)- or VSV-induced production of IFN-βin IRF3-/- macrophages, while PHLPP overexpression promoted poly(I:C) or VSV- triggered IFN-βproduction when rescuing the expression of IRF3 in IRF3-/- macrophages. So, the above results confirmed PHLPP can interact with IRF3 to promote TLR- and RIG-Ⅰ-triggered production of typeⅠinterferon.As one of the key transcription factors in both TLR and RIG-I signal pathway, IRF3, upon stimulation, becomes activated via serine/threonine phosphorylation (between residues 385 and 405) leading to its dimerization, nuclear translocation and association with the coactivator CBP/p300 for inducing the production of typeⅠinterferon, then IRF3 degrades through proteasome-dependent pathway to maintain the balance of the production of typeⅠinterferon. We found that IRF3 of PHLPP-/- macrophages degraded faster and more significantly than that of wild-type macrophages challenged with poly(I:C), VSV or SeV. Considently, overexpression of PHLPP could delay and inhibit SeV-induced degradation of IRF3 in macrophages. MG132 could almost completely inhibit the degradation of IRF3 in PHLPP-/- and PHLPP+/+ macrophages. Meanwhile, PHLPP deficiency enhanced the poly(I:C)-, VSV- or SeV-induced polyubiquitination of IRF3. These results indicated PHLPP directly interacts with IRF3 to maintain the stability of IRF3, preventing it from ubiquitination and degradation.Next, we wanted to know how PHLPP maintains the stability of IRF3. It has been reported that phosphorylation of Ser339 of human IRF3 (IRF3 Ser332 in mice IRF3) led to polyubiquitination of IRF3 and then proteasome-dependent degradation of IRF3. Combination with the above result that PHLPP promoted the production of typeⅠinterferon depending on its phosphatase activity of PP2C domain, we predicted that PHLPP may maintain the stability of IRF3 through dephosphorylating IRF3 Ser332. In orderto confirm the hypothesis, we generated the mutant IRF3 with alanine substituted for serine in residue 332 of IRF3 (IRF3 S332A). Overexpression of PHLPP had no effect on SeV-induced polyubiquitination and degradation of IRF3 S332A, while obviously inhibited polyubiquitination and degradation of wild-type IRF3. Reporter gene assay showed that overexpression of PHLPP couldn't enhance IRF3 S332A-activated expression of IFN-βreporter gene. These results demonstrated that PHLPP could dephosphorylate IRF3 Ser332 to inhibit its phosphorylation-dependent degradation of IRF3.Taken together, these results demonstrated PHLPP directly interacted with the C-terminal of IRF3 through its LRR region in the nuclei of macrophages, and dephosphorylated IRF3 Ser332 through PP2C domain to maintain the stability of IRF3 by inhibiting IRF3 Ser332 phosphorylation-dependent degradation of IRF3, leading to the enhanced production of typeⅠinterferon triggered by TLR3,4 and RIG-Ⅰ.PartⅢ. PHLPP enhances host defense against viral infection by promoting typeⅠinterferon productionBecause PHLPP has been shown to be able to potentiate typeⅠinterferon production in macrophages, we wondered whether PHLPP-mediated enhancement of typeⅠinterferon production could protect the host from virus infection. We tested this with VSV infection model both in vitro and in vivo. Upon infection of VSV, VSV titers (TCID50) in the cultural supernatant of PHLPP-/- acrophages were significantly higher than that in wild type macrophages, and HEK293 cells cultured with the supernatant of PHLPP-/-macrophages quickly showed pathology of virus infection and almost all died. Addition of recombinant mouse IFN-(3 could decrease the titer of VSV in PHLPP-/- macrophages and rescue the death of HEK293 cells cultured with the supernatant of PHLPP-/- macrophages. These results indicated that PHLPP inhibits VSV replication through promoting the production of typeⅠinterferon during viral infection.Next, we investigated the possible role of PHLPP in suppressing VSV infection in vivo. VSV RNA replicates and VSV titer in liver and spleen of PHLPP-/- mice were much higher than that of control mice after VSV challenge. These in vivo data revealed that PHLPP can inhibit VSV replication through promoting RIG-Ⅰ-triggered production of typeⅠinterferon, and protect the host from virus infection.In conclusion, we have demonstrated that PHLPP can selectively promote the TLR3,4-and RIG-Ⅰ-triggered production of typeⅠinterferon in macrophages. PHLPP directly interacts with IRF3 through its LRR domain, dephosphorylates IRF3 Ser332 depending on the phosphatase activity of PP2C domain and subsequently inhibit polyubiquitination and Ser332 phosphorylation-dependent degradation of IRF3, thus maintaining the stability of IRF3. Our results indicate that phosphatase PHLPP may be an essential component of the host antiviral'machinery'.