Src-mediated IRF7 nuclear translocation was required for IFN-β production in poly(I:C)-treated macrophages

Poly(I:C)-mediated IFN-β production required the participation of Src in macrophages (Figure 3). Since IRF3 and IRF7 were reported to be pivotal in ifn-b gene transcription, thereby we wondered whether Src affected nuclear translocation of IRF3 and IRF7. As exhibited in Figure 7A, poly(I:C)-mediated nuclear translocation of IRF3 and IRF7 was PP2-sensitive. To study the role of Src in poly(I:C)-mediated nuclear translocation of IRF3 and IRF7, nuclear extracts derived from RAW264.7, Ctrl-1, siRNA-1, siRNA-1/Src6 and siRNA-1/Src15 cells following

poly(I:C) stimulation were analyzed. As shown in Figure 7B, that poly(I:C)-mediated increase of nuclear IRF7 was significantly suppressed in Src attenuated (siRNA-1) cells as compared to Ctrl-1 cells following 48 hours poly(I:C) treatment. Notably, ectopic Src rescue these events.

Due to the amount of poly(I:C)-induced nuclear IRF3 was not suppressed by Src attenuation. Since, IRF3 was preceded of IRF7, so we chose 12 hour instead of 48 hour poly(I:C) treated cells to analyze the translocation of IRF3 and IRF7 to nucleus. As demonstrated in Figure 7C, the amount of poly(I:C)-enhanced nuclear IRF7 was suppressed in Src attenuated (siRNA-1) cells, but the amount of nuclear IRF3 was not changed in siRNA-1 cells. These results indicated that by augmenting the amount of IRF7, Src was required for poly(I:C)-mediated IFN-β production in macrophages.

Discussion

In this study, we found that iNOS and Src play critical roles in poly(I:C)-mediated IFN-β production in macrophages in vitro (Figure 6) and in vivo (Figure 5, Supplementary Figure 4). The function of type I IFNs is well characterized and they are known to be essential for a powerful host response against viral infection. There are four effector pathways of the IFN-mediated antiviral responses: the ISG15 ubiquitin-like pathway, the Mx GTPase pathway, the 2’, 5’-oligoadenylate-synthetase-directed ribonuclease L pathway, and the protein kinase R pathway. These pathways degrade viral RNA, block viral transcription, inhibit translation and modify protein function to control viral replication.

ISG15 functions to prevent virus-mediated degradation of IRF3, and increases the induction of IFN-β to against viral infection [27]. ISG15 target antiviral effector proteins such as Mx, PKR and RNaseL [28].

Following viral infection, Mx proteins produce from an IFN-stimulated response element (ISRE) to block viral gene transcription [29]. Following activation by viral double-stranded RNA (dsRNA), 2’, 5’-oligoadenylate-synthetase expression is upregulated by type I IFNs [30], and activated the constitutively expressed inactive ribonuclease L (RNaseL). The binding of 2’, 5’-oligoadenylate-synthetase to RNaseL enables RNaseL activation and be capable to cleave virals RNA. Protein kinase R (PKR) expression is also upregulated by type I IFNs. PKR is

activated directly by viral RNA, and can suppress viral proteins translation ability [31] by phosphorylation of eukaryotic translation initiation factor 2α.

IFN-β production is one of the means of innate immune system to against viral infection. Innate immune cells include dendritic cells (DCs), macrophages, neutrophils, and others. In host defense, the innate immune cells recognize microbial pathogens through distinct pattern recognition receptors (PRRs). Through induction of costimulatory molecules and cytokines to activation and differentiation of T lymphocytes, TLRs can trigger adaptive immune responses. TLRs detect microbial nucleic acids, such as double-stranded RNA (dsRNA) (TLR3), single-stranded RNA (ssRNA) (TLR7), and dsDNA (TLR9). TLR3 mainly detect viruses like reovirus, rotavirus in the gastrointestianl system, and Influenza A virus from infected cells but not in the direct viruses themselves [32]. TLR3 can activate its downstream signaling pathways to produce type I IFNs.

To mimic viral infection, we use poly(I:C), which is a synthetic analog of double-stranded RNA (dsRNA), a molecular pattern associated with viral infection and virus replication by-product recognized by TLR3 [33].

TLR3 is activated by extracellular dsRNA and endogenous mRNA [34], which escaping from damaged tissue or in endocytosed cells. TLR3 can not only detect exogenous danger signals, but also sense distress from damaged or stressed tissue. TLR3-mediated type I IFN signaling is only one of TLR3-mediated signaling pathway. All of TLR family membranes,

only TLR3 use MyD88-independent adaptor protein TRIF/

Toll-interleukin 1 receptor domain (TIR)-containing adaptor molecule TICAM-1 [13, 35]. Through TRIF/ TICAM-1-mediated activation of transcription factors (i.e. NF-κB, AP-1 and IRF3/7), transcription of ifn-b and proinflammatory cytokine encoding genes can be induced [36]. In this study, we demonstrated the non-recetpor tyrosine kinase Src participated in TLR3-mediated IFN-β production in macrophage.

Interestingly, c-Src is required for TLR3-mediated activation in dentritic cells [21]. Previously, Src Family Kinases (SFKs) were documented to be involved in inflammatory cytokines production in human macrophages response to HIV-1 envelope protein SFKs were involved [37]. Recently mounting evidence indicated encephalomyocarditis virus-mediated COX2 expression in macrophages is regulated by SFKs [38]. The ability of PP2 and Src attenuation to reduce poly(I:C)-induced IFN-β expression (Figure 2B, Figure 3) and nuclear translocation of IRF7 (Figure 7), suggests that Src participates in poly(I:C)-mediated downstream signaling in the innate immune cells. Furthermore, we also found Src was involved in poly(I:C)-induced proinflammtory cytokines TNFα production in macrophages (data not shown).

Previously, we have demonstrated that LPS-induced Src induction and macrophage migration are required for NO/cGMP [18]. Expression of iNOS and NO production is well known to be an important effector in macrophages in response to viral infection [39, 40]. It is known that iNOS and NO inhibit viral replication and play a role in the host defense against

viruses [41, 42]. Using poly(I:C) to mimic viral infection, we also demonstrated suppressed reduced migration in iNOS null PEMs as compared to WT PEMs (Appendix 2), indicating iNOS expression plays a critical role in macrophages activation including migration, cytokines production. In this study, we use pharmacological blockade, AG (iNOS inhibitor) and ODQ (sGC inhibitor), suppressed poly(I:C)-evoked increment of ifn-b mRNA (Figure 4A), and SNAP (a NO donor) or 8-br-cGMP (a cGMP analogue) could increase the abundance of ifn-b transcript (Figure 4B). With these findings, we conclude that iNOS also plays a critical role in antiviral cytokines production. Since exogenous IFN-β can accelerate the induction of iNOS [43], and iNOS is involved in IFN-β production, therefore there might exist a loop to amplify poly(I:C)-mediated effects. At the same time, we also found Src induction is dependent on iNOS (Supplementary Figure 4).

According to our results, we demonstrate that poly(I:C)-mediated IFN-β production, which is the requirement of iNOS in the innate immune cells macrophages. We suggest that Src seemed to be one of the downstream targets in this process and induction by iNOS. In the immune system, macrophages are important for our body to against infection agents. We clear up iNOS and Src playing important roles in poly(I:C)-mediated IFN-β production in macrophages .

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Figure

Figure 1. Poly(I:C) treatment induces IFN-β production in murine RAW264.7 macrophages.

RAW264.7 cells were treated with poly(I:C) (20 μg/ml) for various times points as indicated. The concentration of IFN-β in culture medium of each sample was determined by ELISA.

(A) (B)

Figure 2. Poly(I:C)-induced IFN-β was PP2 sensitive.

(A) RAW264.7 cells were incubated with PP2 (10 μM) after 30 minutes poly(I:C) (20 μg/ml) stimulation. The concentration of IFN-β in culture medium of each group was determined by ELISA. *** and ^### , P<0.001.

(B) RAW264.7 cells were incubated with PP2 (10 μM) after 30 minutes poly(I:C) (20 μg/ml) stimulation. The amount of ifn-β transcript was analysis by RT-PCR. gapdh was utilized as an internal control for amplification efficiency.

Figure 3. Poly(I:C)-mediated IFN-β secretion was inhibited by src-specific siRNA, which could be reversed by ectopic Src.

RAW264.7 cell and its derived control (ctrl-1, -2), Src attenuated cells (siRNA-1, -2) and Src attenuated cells harboring plasmid encoding avian Src (siRNA/Src6, siRNA/Src15) were stimulated without or with poly(I:C) (20 μg/ml) for 48 hours. Equal amounts of lysates (80 μg) from each sample were resolved by SDS-PAGE and probed with antibodies as indicated. The concentration of IFN-β in culture medium of each group was determined by ELISA. The arrow indicates the position of Src. ***, P<0.001.

(A)

(B)

Figure 4. Poly(I:C)-induced ifn-β transcript was sensitive to inhibitors of inducible nitric oxide synthase (iNOS) and soluble guanylyl cyclase (sGC).

(A) RAW264.7 cells were pretreated without or with AG (2 mM), ODQ (100 μM) for 30 mins , and then cells were stimulated without and with poly(I:C). (B) RAW264.7 cells were stimulated without and with SNAP (100 μM), 8-br-cGMP (cGMP analogue, 100 μM) for 48 hours. The amount of ifn-β transcript was analysis by RT-PCR. gapdh was utilized as an internal control for amplification efficiency.

Figure 5. iNOS was involved in poly(I:C)-mediated IFN-β production.

Primary peritoneal macrophages from wild type (WT) and iNOS^-/- mice were treated without and with poly(I:C) (20 μg/ml) for 48 hours. The concentration of IFN-β in culture medium of each sample was determined by ELISA. ***, P<0.001.

Figure 6. SNAP-mediated IFN-β secretion was inhibited by src-specific siRNA, which could be reversed by ectopic Src.

RAW264.7 cell and its derived control (ctrl-1, -2), Src attenuated cells (siRNA-1, -2) and Src attenuated cells harboring plasmid encoding avian Src (siRNA/Src6, siRNA/Src15) were stimulated without or with SNAP for 48 hours. Equal amounts of lysates (80 μg) from each sample were resolved by SDS-PAGE and probed with antibodies as indicated. The concentration of IFN-β in culture medium of each group was determined by ELISA. The arrow indicates the position of Src. ***, P<0.001.

(A)

(B) (C)

Figure 7. Poly(I:C)-mediated IRF7 nuclear translocation was inhibited by src-specific siRNA, which could be reversed by ectopic Src.

(A) RAW 264.7 cells were incubated with PP2 (10 μM) after 30 minutes poly(I:C) (20 μg/ml) stimulation. The amount of nuclear IRF3 and IRF7 were analysis by nuclear extraction. (B)(C)The RAW264.7 expressing nonspecific siRNA (Ctrl-1), Src attenuated cells (siRNA-1) and Src attenuated cells harboring plasmid encoding avian Src (siRNA/Src6, siRNA/Src15) were stimulated without or with poly(I:C) (20 μg/ml) for 48 hours and 12 hours. The amount of nuclear IRF3 and IRF7 were analysis by nuclear extraction.

Figure 8. The signaling pathway for IFN-β secretion in poly(I:C)-stimulated macrophages.

Following poly(I:C) treatment, the signal is transduced into macrophages via Toll-like receptor 3, increases expression of iNOS. NO produced by iNOS contribures to Src induction. And Src activation effect the amount of nuclear IRF7 cause IFN-β production.

Appendix

Supplementary Figure 1. The inhibitory effect of PP2 on poly(I:C)-induced protein tyrosine phosphorylation. (provide for 妙 瑩學姐和青昭學姐)

Supplementary Figure 2. iNOS is involved in poly(I:C)-mediated macrophage migration. (provide for 妙瑩學姐和青昭學姐)

Supplementary Figure 3. Poly(I:C) induced Src expression in murine RAW264.7 macrophages and rat peritoneal macrophages (PEMs).

(provide for 妙瑩學姐和青昭學姐)

Supplementary Figure 4. Poly(I:C) induced Src expression is dependent on iNOS. (provide for 妙瑩學姐和青昭學姐)