Porcine reproductive and respiratory syndrome virus (PRRSV) induces IL-12p40 production through JNK-AP-1 and NF-κB signaling pathways

Virus Research 225 (2016) 73–81

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Porcine reproductive and respiratory syndrome virus (PRRSV) induces IL-12p40 production through JNK-AP-1 and NF-␬B signaling pathways Zhibin Yu a,b , Chen Huang a,b , Qiong Zhang a,b , Dr. Wen-hai Feng a,b,∗ a b

State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing 100193, China Department of Microbiology and Immunology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

a r t i c l e

i n f o

Article history: Received 3 February 2016 Received in revised form 17 September 2016 Accepted 19 September 2016 Available online 20 September 2016

a b s t r a c t Porcine reproductive and respiratory syndrome virus (PRRSV) mainly infects monocyte/macrophage cells and modulates cytokine production to regulate host immune response. IL-12p40 is the basic subunit of IL-12, a heterodimeric cytokine, which plays key roles in the cell-mediated immune response. In the present study, we demonstrated that PRRSV infection induced IL-12p40 production in vitro and in vivo. Subsequently, we showed that inhibitors of p38 MAPK, JNK, and NF-␬B dramatically reduced PRRSVinduced IL-12p40 expression. To further characterize the molecular mechanism of IL-12p40 production induced by PRRSV infection, we cloned and analyzed the porcine IL-12p40 promoter, in which AP-1 and NF-␬B motifs were found. In addition, both JNK-AP-1 and NF-␬B signaling pathways were activated by PRRSV infection. Taken together, these data indicate that PRRSV induces IL-12p40 expression through the JNK-AP-1 and NF-␬B signaling pathways. Our findings might facilitate our understanding of the molecular mechanisms of IL-12 production induced by PRRSV infection. © 2016 Elsevier B.V. All rights reserved.

1. Background Porcine reproductive and respiratory syndrome (PRRS), characterized by respiratory disorders and reproductive failure in sows, is one of the most economically important infectious disease in swine industry (Albina, 1997; Collins et al., 1992; Li et al., 2007). Porcine reproductive and respiratory syndrome virus (PRRSV), the causative pathogen of PRRS, is an enveloped single-stranded RNA virus that belongs to the genus Arterivirus, family Arteriviridae, order Nidovirales (Balasuriya and MacLachlan, 2004). PRRSV includes type I (European-like) and type II (North Americanlike) genotypes with approximately 60% nucleotide identity at the genomic level (Allende et al., 1999). In 2006, an atypical PRRS featured with high fever, high morbidity, and high mortality broke out in China and a highly pathogenic PRRSV (HP-PRRSV) was then isolated (Tian et al., 2007). PRRSV primarily infects porcine alveolar macrophages (PAMs) and has a highly restricted host cell tropism for monocyte/macrophage and dendritic lineages that play

∗ Corresponding author at: State Key Laboratory of Agrobiotechnology, Department of Microbiology and Immunology College of Biological Science Chin Agricultural University Beijing 100193, China. E-mail address: [email protected] (W.-h. Feng). http://dx.doi.org/10.1016/j.virusres.2016.09.009 0168-1702/© 2016 Elsevier B.V. All rights reserved.

key roles in the immune responses such as phagocytosis, antigen presentation, and cytokine production (Duan et al., 1997). IL-12p40 is the basic subunit shared by heterodimeric cytokine interleukin-12 (IL-12) and interleukin-23 (IL-23). IL-12, also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is vital in the connection of the innate and adaptive immunity (D’Andrea et al., 1992; Kobayashi et al., 1989; Vignali and Kuchroo, 2012). The major function of IL12 is to facilitate the differentiation of Th1 cells from naive T cells and promote cell medicated immune response which is required for the effective elimination of virus and other intracellular pathogens (Hsieh et al., 1993; Manetti et al., 1994, 1993; Park et al., 2000; Wu et al., 1993). In addition, IL-12 is able to stimulate NK cells to produce IFN-␥, which enhances the activity of macrophages, providing a mechanism of T cell independent macrophage activation (Kobayashi et al., 1989; Trinchieri, 2003). The simultaneous expression of both subunits, IL-12p40 and IL-12p35, in the same cell is required to produce the biologically active heterodimer (Wolf et al., 1991). Compared to IL-12p35, IL-12p40 plays a more dominant role in the production of IL-12 (Trinchieri, 2003). IL-12p40 is expressed exclusively in monocytes/macrophages, dendritic cells, and B cells after stimulation, while IL-12p35 shows a basic and constitutive expression level in many cell types (Du and Sriram, 1998; Yamaguchi et al., 2016). Furthermore, IL-12p35 cannot be

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secreted without the covalently binding of IL-12p40 (Wolf et al., 1991). In general, the p40-deficient mice are found to be more suppressed than the p35-deficient mice in terms of their ability to promote Th1 cell development (Becher et al., 2002; Piccotti et al., 1998). IL-12 is important for the control of PRRSV. Recombinant porcine IL-12 (rpIL-12) has been shown to significantly increase the expression of IFN-␥ and reduce viral titers during PRRSV infection both in vivo and in vitro (Carter and Curiel, 2005). The efficacy of porcine IL-12 as a PRRSV vaccine adjuvant has also been well documented (Charerntantanakul, 2009; Charerntantanakul et al., 2006; Wee et al., 2001). Due to the regulatory role of IL-12p40 in IL-12 production, IL-12p40 is often used to show the expression of IL-12 in PRRSV infection (Dwivedi et al., 2012a; García-Nicolás et al., 2014; Thanawongnuwech et al., 2004). IL-12p40-positive cells in PRRSV infected lungs are detected at 1 day post-infection (dpi) by in-situ hybridization, and the hybridization signal is always associated with inflammation (Chung and Chae, 2003). Increased IL-12p40 mRNA levels in PAMs from pigs at 10, 28, and 42 days post infection with PRRSV and/or M. hyopneumoniae are also reported (Thanawongnuwech et al., 2004). Furthermore, an enhanced secretion of IL-12p40 is observed in pigs during early stage of PRRSV infection under farm conditions (Dwivedi et al., 2012a). In contrast, a recent study reveals an inefficient IL-12p40 expression in lung and lymphoid organs during PRRSV infection (García-Nicolás et al., 2014). In addition, it is reported that PRRSV induces a substantially weaker peripheral blood IFN-␥ response than other viruses (Meier et al., 2003; Royaee et al., 2004; Xiao et al., 2004). And the cytotoxic function of CD8+ T and NK cells have been shown to be downregulated after PRRSV infection (Albina et al., 1998; Weesendorp et al., 2013). Thus, whether PRRSV infection could increase the production of IL-12p40 needs to be further clarified. In the present study, we demonstrated that the expression of IL12p40 was induced during PRRSV infection both in vivo and in vitro. Inhibitors of p38, JNK and NF-␬B significantly reduced IL-12p40 production in PAMs infected with PRRSV. The promoter region of porcine IL-12p40 was cloned, in which the binding motifs of NF␬B and AP-1 were found. In addition, both JNK-AP-1 and NF-␬B pathways were activated during PRRSV infection. These findings will help us better understand the molecular mechanisms of IL-12 production induced by PRRSV.

2. Materials and methods 2.1. Cells and virus Porcine alveolar macrophages (PAMs) were obtained by postmortem lung lavage of 8-week-old specific-pathogen-free (SPF) pigs and maintained in RPMI 1640 medium with 10% heatinactivated fetal bovine serum (FBS) and penicillin-streptomycin. PAMs were cultured and maintained at 37 ◦ C with 5% CO2 . JXwn06, a highly pathogenic PRRSV (HP-PRRSV) strain (GenBank accession No., EF641008.1), and CH-1a, a classical type II PRRSV strain (GenBank accession No., DQ217415), were used. HPPRRSV JXwn06 and PRRSV CH-1a were propagated and titrated on PAMs. Briefly, PAMs was prepared in a 96-well plate and then infected with serially diluted PRRSV (10−1 to 10−10 ). PRRSV infection was determined at 48 h post infection (hpi) using immunofluorescent staining for the PRRSV N protein. The viral titer was determined by the Reed-Muench method and expressed as the 50% tissue culture infective dose (TCID50 ). Virus preparations were stored at −80 ◦ C until use. Inactivated viruses were prepared by UV irradiation at 120 mJ cm−2 for 1 h using a Bio-Linkcross-linker (VilberLourmat).

2.2. Reagents and antibodies NF-␬B inhibitor (BAY11-7082), JNK inhibitor (SP600125), and MAPK (p38) inhibitor (SB203580) were purchased from Enzo Life Sciences. PKC inhibitor (GF-109203X), PI3K inhibitor (LY294002), and MEK (Erk) inhibitor (PD98059) were purchased from Cell Signaling Technology. AP-1 inhibitor (SR11302) was purchased from Tocris Bioscience. Antibodies against JNK, p-JNK, c-Jun, and p-c-Jun were purchased from Cell signaling Technology, Inc. Anti␤-actin antibody was purchased from Sigma. Goat anti-mouse and anti-rabbit secondary antibodies were purchased from Santa Cruz Biotechnology. 2.3. Animal experiment Six four-week-old SPF piglets were obtained from the Beijing Center for SPF Swine Breeding and Management and randomly divided into two groups (3 piglets per group). Three piglets were intranasally inoculated with 2 ml of HP-PRRSV JXwn06 (105 TCID50 /ml), and three piglets were intranasally inoculated with PBS as controls. At 4 days post infection, sera was collected and postmortem samples, including lung, spleen, liver, and kidney tissues, bronchoalveolar lavage fluid (BALF) and PAMs were prepared for IL-12p40 analysis. 2.4. Inhibition of signal transduction pathways PAMs were pretreated with DMSO, the PKC inhibitor GF109203X (2 ␮M), the p38 mitogen activated protein kinase (MAPK) inhibitor SB203580 (10 ␮M), the MEK inhibitor PD98059 (10 ␮M), the JNK inhibitor SP600125 (10 ␮M), the PI3 K inhibitor LY294002 (5 ␮M), the NF-␬B inhibitor BAY11-7082 (1 ␮M), or the AP-1 inhibitor SR11302 (10 ␮M) for 1 h, and then infected with PRRSV at a multiplicity of infection (MOI) of 1 in the presence of inhibitors. Twenty-four hours later, supernatants were harvested for IL-12p40 analysis by ELISA, and cells were harvested for IL-12p40 mRNA analysis by real-time PCR. 2.5. Western blotting Whole-cell extracts were prepared by lysing cells in radio immunoprecipitation assay lysis buffer with 100 U proteinase inhibitor (Promega) and 20 M NaF (Beyotime). The amount of protein in each sample was quantified with a bicinchoninic acid assay kit (Pierce Biotechnology, Inc.). A similar amount of protein from each sample was run on a 12% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes (Millipore). After blocking with a 5% no-fat milk solution in Tris-buffered saline with 0.1% Tween 20, the membranes were incubated for 2 h at room temperature with the antibodies at a suitable dilution (antiJNK, p-JNK, c-Jun, p-c-Jun at 1:1000; anti-␤-actin at 1:5000). The membranes were then incubated with horseradish peroxidaseconjugated goat anti-mouse IgG or goat anti-rabbit IgG as secondary antibodies for 1 h at a dilution of 1:5000. The antibodies were visualized using the ECL reagent (Cwbiotech) following the manufacturer’s instructions. 2.6. Cloning and characterization of porcine IL-12p40 promoter Genomic DNA of porcine alveolar macrophage was extracted following the instructions of MiniBEST Universal Genomic DNA Extraction Kit (Takara). The unknown 5 -terminal sequence of the porcine IL-12p40 gene was obtained using PCR with the forward primer (5 -GCAACTACGGTTTCAGACCCGACGA-3 ) and reverse primer (5 -CCAGCCAAACCAGGGAAAACCAGGA-3 ) designed according to the porcine IL-12p40 mRNA, and then

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Fig. 1. IL-12p40 production is significantly up-regulated by PRRSV infection in PAMs. (A) PAMs were inoculated with medium alone, HP-PRRSV (MOI = 1) or UV-inactivated HP-PRRSV. IL-12p40 expression was analyzed by real-time PCR at 6, 12, 24 and 36 hpi. (B) Culture supernatants of PAMs inoculated with or without HP-PRRSV were collected at 24 and 36 hpi, and the protein levels of IL-12p40 were analyzed using ELISA. (C) PAMs were infected with HP-PRRSV at an MOI of 0.01, 0.1 or 1.0, and IL-12p40 expression was analyzed by real-time PCR at 36 hpi. (D) PAMs were inoculated with medium alone, classical PRRSV strain CH-1a (MOI = 1) or UV-inactivated CH-1a. IL-12p40 mRNA expression was analyzed at 6, 12, 24 and 36 hpi. (E) Culture supernatants of PAMs inoculated with or without CH1a were collected at 24 and 36 hpi, and the protein levels of IL-12p40 were analyzed. Data are means ± SD from three independent experiments. Statistical analysis was performed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

sequenced. Then, the 5 -flanking region of the porcine IL-12p40 gene was cloned using a genome walking kit (Takara) according to the manufacturer’s instructions. The AP2 degenerate primer used in the asymmetry PCR was provided by the kit and the three specific primers for SP1 (5 -AGTTCGTCGGGTCTGAAACCGTAGT-3 ), SP2 (5 -AAGTAATGCTTTCAGGAGTCCACGC-3 ), and SP3 (5 TATGCGTTTGTCCACAGACCTTAGT-3 ) were designed according to the 5 terminal gene sequence of the porcine IL-12p40. The resulting fragment was inserted into pMD18-T vector and sequenced. Transcriptional regulating elements were predicted using TFSERCH (http://www.cbrc.jp/research/db/TFSEARCH.html). And the comparison of porcine IL-12p40 promoter with other species was done using DNAMAN software. 2.7. ELISA for IL-12p40 and IFN- The levels of IL-12p40 in BALF, serum, and cell culture supernatants were analyzed using a porcine IL-12/IL-23 p40 quantitative ELISA kit (R&D Systems) according to the instruction manual. IFN␥ proteins in BALF and serum were assessed by a porcine IFN-␥ quantitative ELISA kit (R&D Systems). Cell culture supernatants and BALF were centrifuged at 720g for 5 min at 4 ◦ C, and plasma was centrifuged at 1000 g for 15 min at 4 ◦ C before ELISA was performed. 2.8. Quantitative real-time PCR Total RNAs of PAMs or tissues were extracted with TRIzol (Invitrogen) following the manufacturer’s protocol. cDNA was obtained using moloney leukemia virus (M-MLV) reverse transcriptase

according to the manufacturer’s instructions (Takara). Quantitative real-time PCR analysis was performed using a FastSYBR mixture (Cwbiotech) on a ViiA7 real-time PCR system (Applied Biosystems). Primers used in the reaction included: GAPDH forward: 5 -CCTTCCGTGTCCCTACTGCCAAC-3 , GAPDH reverse: 5 -GACGCCTGCTTCACCACCTTCT-3 , IL-12p40 forward: 5 -CATTGAGGTCGTGCTGGAAGCTGTT-3 IL-12p40 reverse: 5 -GGTCTGGTTTGATGATGTCCCT-3 IFN-␥ forward: 5 -CTTTGCGTGACTTTGTGTTTTTCTG-3 IFN-␥ reverse: 5 -TTTTTTTGTCACTCTCCTCTTTCCA −3 2.9. Statistical analysis Our data were analyzed by GraphPad Prism (GraphPad Software, San Diego, CA). Differences were analyzed using Student’s t-test and considered to be statistically significant if the P value was less than 0.05. *P < 0.05; **P < 0.01; ***P < 0.001. 3. Results 3.1. PRRSV induced IL-12p40 expression in vitro To investigate whether PRRSV is able to induce IL-12p40 production, PAMs were inoculated with HP-PRRSV JXwn06 or UVinactivated virus at an MOI of 1, and then harvested for IL-12p40 analysis at 6, 12, 24, and 36 hpi. As shown in Fig. 1A, HP-PRRSV infection significantly up-regulated IL-12p40 mRNA expression with 5.5- and 23.5- fold increase at 24 and 36 hpi, respectively. Consistent with this, an increased IL-12p40 protein level in the

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Fig. 2. PRRSV induces IL-12p40 production in vivo. Piglets were infected intranasally with HP-PRRV at a dose of 2 ml (105 TCID50 /ml) for 4 days or inoculated with PBS as controls. After the infection, tissues of lung, spleen, liver and kidney (A) or PAMs (B) were collected for IL-12p40 mRNA expression analysis by real-time PCR. IL-12p40 protein in serum (C) and BALF (D) was analyzed using an ELISA kit. IFN-␥ mRNA expression in PAMs (E) and lung tissues (F) from controls or HP-PRRSV infected pigs were analyzed by real-time PCR. (H) IFN-␥ production in BALF was assessed by an ELISA kit. Data are means ± SD from three independent experiments. Statistical analysis was performed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

supernatants was observed at 24 and 36 hpi (Fig. 1B). The upregulation of IL-12p40 by HP-PRRSV infection was dose-dependent (Fig. 1C). CH-1a, the conventional type II PRRSV strain, showed a similar kinetic of IL-12p40 production (Fig. 1D and 1E). Collectively, these data suggest that PRRSV induces IL-12p40 production in PAMs in vitro.

pigs was also significantly increased (Fig. 2H). However, IFN-␥ in sera from HP-PRRSV infected pigs was undetectable (data not shown).

3.2. IL-12p40 production was up-regulated by PRRSV infection in vivo

To dissect the signaling pathways involved in PRRSV-induced IL12p40 production, PAMs were pretreated with DMSO or inhibitors of the key signaling pathways, including MEK, MAPK p38, JNK, PI3 K, PKC, and NF-␬B for 1 h before PRRSV infection. At 36 h later, the expression levels of IL-12p40 were analyzed by real-time PCR and ELISA. As shown in Fig. 3A and B, SB203580 (a MAPK p38 inhibitor), SP600125 (a JNK inhibitor), and BAY11-7082 (a NF-␬B inhibitor) significantly inhibited IL-12p40 production induced by PRRSV infection both at mRNA and protein levels. To confirm the inhibitory effects of these inhibitors on IL-12p40 production, we pretreated PAMs with the inhibitor of MAPK p38, JNK, or NF-␬B at increasing doses for 1 h before PRRSV infection. At 36 h later, cells were harvested for IL-12p40 analysis by real-time PCR. As shown in Fig. 3C and D, the inhibitions of IL-12p40 expression by SB203580, SP600125 and BAY11-7082 were in a dose dependent manner. These results indicate that p38, JNK, and NF-␬B signaling pathways might be involved in PRRSV-induced IL-12p40 production.

Next, we sought to determine whether PRRSV infection induces IL-12p40 induction in vivo. Three 4-week old specific pathogen free (SPF) piglets were infected intranasally with HP-PRRV JXwn06 at a dose of 2 × 105 TCID50 /ml, and three age-matched SPF piglets were inoculated with PBS as controls. At 4 days post infection, serum and BALF samples, tissues of lung, spleen, liver and kidney were collected for IL-12p40 analysis. As shown in Fig. 2A, an increased IL-12p40 mRNA expression was observed in lungs after PRRSV infection. However, IL-12p40 mRNA up-regulation was not observed in spleen, liver and kidney. IL-12p40 mRNA expression in PAMs lavaged from the PRRSV infected piglets was also up-regulated (Fig. 2B). Consistently, the protein levels of IL-12p40 in serum and BALF were significantly increased with an average amount of ∼1200 pg/ml (Fig. 2C) and 280 pg/ml (Fig. 2D) after HP-PRRSV infection, respectively. Together, these data suggest that HP-PRRSV JXwn06 infection induces a remarkable IL-12p40 production in vivo. We also analyzed IFN-␥ production. Our results showed that IFN-␥ expression in PAMs (Fig. 2E) and lungs (Fig. 2F) of pigs infected with HP-PRRSV JXwn06 was significantly increased. IFN-␥ production in BALF from HP-PRRSV infected

3.3. PRRSV induced IL-12p40 expression through MAPK p38, JNK and NF-B pathways

3.4. Cloning and characterization of porcine IL-12p40 promoter To further investigate the roles of p38, JNK and NF-␬B in PRRSVinduced IL-12p40 production, we tried to examine whether porcine

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Fig. 3. UP-regulation of IL-12p40 production is impaired by p38, JNK and NF-␬B inhibitors. PAMs were pretreated for 1 h with DMSO or inhibitors of signaling pathways, including SB203580 (SB; a p38 MAPK inhibitor; 10 ␮M), SP600125 (SP; a JNK inhibitor; 10 ␮M), PD98059 (PD; an MEK inhibitor; 10 ␮M), LY294002 (LY; a PI3 K inhibitor; 5 ␮M), GF-109203X (GF; a PCK inhibitor; 2 ␮M) and BAY11-7082 (BAY; an NF-␬B inhibitor; 1 ␮M), and then infected with HP-PRRSV (MOI = 1) for 36 h. IL-12p40 expression level was then examined by real-time PCR (A) and ELISA assay (B). (C and D) PAMs were pretreated for 1 h with SB203580, SP600125 or BAY11-7082 at increasing doses of SB (1, 5, 10 ␮M), SP (2, 10, 20 ␮M) and (BAY, 0.1, 1, 5 ␮M), then infected with HP-PRRSV (MOI = 1) for 36 h. The expression of IL-12p40 mRNA was analyzed by real-time PCR. Data are means ± SD from three independent experiments. Statistical analysis was performed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

IL-12p40 promoter has the binding sites for NF-␬B and transcription factors involved in p38 or JNK pathways. Human and mouse IL-12p40 promoters have been well characterized with regulatory elements such as C/EBP ␤, NF-␬B, AP-1, ISRE and TATA box (Murphy et al., 1995; Plevy et al., 1997; Wang et al., 2000; Zhu et al., 2001). However, the porcine IL-12p40 promoter has not been identified yet. Furthermore, the DNA sequence of porcine IL-12p40 gene is incomplete with an ∼2.0 kb missed sequence at the 5 terminus (Fig. 4A). To obtain the promoter region, we first cloned the 5 -terminal sequence of the porcine IL-12p40 gene based on the IL-12p40 mRNA sequence (Foss and Murtaugh, 1997). Then, the promoter region located next to the 5 -terminus of porcine IL12p40 gene was cloned by genome walking technology. Sequence alignment revealed that the porcine IL-12p40 promoter was highly conserved with mouse and human IL-12p40 promoters, and the nucleotide identity was 75.43%. The putative binding sites for NF␬B and AP-1, a transcription factor in JNK signaling pathway, were found in the porcine IL-12p40 promoter region (Fig. 4B). Other crit-

ical elements such as C/EBP␤ and ISRE also existed in the porcine IL-12p40 promoter region. 3.5. PRRSV induced IL-12p40 expression though JNK-AP-1 signaling pathway AP-1 is a heterodimeric transcriptional factor composed of c-Jun and c-Fos. Since a biding site of AP-1 was found in the core promoter region of porcine IL-12p40, we examined whether AP-1 was required for porcine IL-12p40 expression induced by PRRSV. PAMs were pretreated with SR11302, a specific inhibitor of c-Jun, before PRRSV infection. At 36 hpi, the expression of IL-12p40 was analyzed, and our results showed that SR11032 suppressed IL-12p40 up-regulation upon PRRSV infection at both mRNA (Fig. 5A) and protein levels (Fig. 5B). To determine whether JNK and AP-1 are activated by PRRSV infection, PAMs were harvested at 0, 3, 5, 8, 12 and 24 hpi for western blot analysis using antibodies against JNK, p-JNK, c-Jun, and p-c-Jun As shown in Fig. 5C, the phosphorylation of JNK and

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Fig. 4. Porcine IL-12p40 promoter contains conserved transcriptional regulatory elements including AP-1. (A). Structure model of porcine IL-12p40 gene. Translation starting point (ATG) was denoted, exons were abbreviated as e1 to e8, and the promoter region and 5 -terminus of porcine IL-12p40 gene had not been identified. (B) Porcine IL-12p40 promoter was isolated by a genome walking kit. A highly identity of porcine IL-12p40 promoter with mouse IL-12p40 promoter was revealed by sequence comparison. Transcriptional regulatory elements such as C/EBP ␤, NF-␬B, AP-1, ISRE and TATA-box were found.

c-Jun were increased post infection, indicating that JNK and c-Jun are activated by PRRSV infection. However, when PAMs were pretreated with SP600125, the phosphorylation level of c-Jun induced by PRRSV infection was decreased (Fig. 5D). Together, these results indicate that PRRSV infection induces IL-12p40 expression through JNK-AP-1 signaling pathway.

4. Discussion IL-12p40 is an important subunit which determines the production of heterodimenric cytokine IL-12 and IL-23. It has been demonstrated that IL-12 plays key role in host defense against viral infections, primarily through the promotion of Th1 cell development and NK cell activation, (Del Vecchio et al., 2007; Jankovic et al.,

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Fig. 5. c-Jun activation by PRRSV is required for IL-12p40 induction. (A and B) Effect of c-Jun inhibitor on PRRSV induced IL-12p40 expression. PAMs were pretreated with c-Jun specific inhibitor SR11302 (10 ␮M) for 1 h and then infected with HP-PRRSV (MOI = 1). At 24 hpi, cells were collected for IL-12p40 mRNA (A) analysis by real-time PCR, and supernatants were harvested for IL-12p40 protein (B) analysis using an ELISA kit. (C) Western blotting was performed to detect JNK and c-Jun activation. PAMs inoculated with HP-PRRSV (MOI = 1) were harvested for JNK, p-JNK, c-Jun and p-c-Jun analysis at 0, 3, 5, 8, 12, and 24 hpi. (D) Effect of JNK inhibitor SP600125 on c-Jun phosphorylation in PAMs infected with HP-PRRSV. PAMs were pretreated with SB203580 (10 ␮M) or SP600125 (10 ␮M) for 1 h and then infected with HP-PRRSV (MOI = 1). At 24 hpi, cells were harvested for c-Jun and p-c-Jun analysis by Western blotting using specific antibodies against c-Jun and p-c-Jun. Data are means ± SD from three independent experiments. Statistical analysis was performed by Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001.

2002; Schurich et al., 2013). Many viruses such as influenza virus, herpes simplex virus and vaccinia virus can induce IL-12p40 production (Gherardi et al., 2003; Monteiro et al., 1998; Parker et al., 2000). In the present study, we showed that PRRSV infection upregulated IL-12p40 production both in vitro and in vivo. We revealed a substantial increased level of IL-12p40 in serum and BALF of PRRSV infected pigs. Elevated IL-12p40 mRNA expression in lung rather than other organs (including spleen, liver and kidney) was also detected (Fig. 2A). Together, these results suggest that PRRSV is able to induce IL-12p40 expression in PAMs. Consistent with our findings, previous studies have also reported that type II PRRSV strains (VR-2358, MN 1-18-2, SD23983, SNUVR970501) are able to induce IL-12p40 production in vivo or in vitro (Chung and Chae, 2003; Dwivedi et al., 2012b; Renukaradhya et al., 2010; Thanawongnuwech and Thacker, 2003). However, another study reports that IL-12p40 expression is not increased by the infection of a type I PRRSV strain (PRRSV-2982) (García-Nicolás et al., 2014). Thus, we propose that the ability to induce IL-12p40 by type I and type II PRRSV strains varies. However, this needs to be investigated further.

In agreement with the report that a strong influx of NK cells and cytotoxic T cells in the BALF of pig infected with PRRSV was observed (Samsom et al., 2000), we showed an increased IFN-␥ mRNA expression in PAMs and lungs infected with PRRSV (Fig. 2E and F) and a remarkable production of IFN-␥ protein in BALF (Fig. 2H). As recombinant porcine IL-12p40 can up-regulate IFN␥ expression and reduce viral titers during PRRSV infection (Carter and Curiel, 2005), there might be a correlation between IL-12p40 and IFN-␥ in lungs during PRRSV infection. However, earlier literatures claim that IL-12 and IFN-␥ are under-induced after PRRSV infection (Albina et al., 1998; Weesendorp et al., 2013). Even though we confirmed the up-regulation of IL-12p40 production by PRRSV infection, our further studies showed that PRRSV-induced IL-12p40 was 6 to 26 folds lower compared with other viruses such as PRV and SeV (data not shown), implicating that PRRSV is able to induce a significant but relatively lower expression of IL-12p40. Thus, we suppose that PRRSV-induced IL-12 production may contribute to the IFN-␥ production and inflammation response in lungs, but it is not sufficient to induce a robust cell mediated immune response. To study the underlying mechanism, inhibitors of different signaling pathways were used to treat PAMs before PRRSV infection.

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We found that inhibitors of p38 MAPK, JNK and NF-␬B remarkably decreased IL-12p40 production by PRRSV both at mRNA and protein levels. Stress-activated MAP kinases (MAPKs), comprised of JNK and p38, play prominent roles in the production of many cytokines in innate and adaptive immune systems (Huang et al., 2009). However, the role of JNK in the production of IL-12p40 is controversial (Cho et al., 2012; Korhonen et al., 2012; Ma et al., 2004; Yang et al., 2010). It has been reported that JNK positively controls IL-12p40 expression, and dexamethasone (DXM), an antiinflammatory glucocorticoid, can inhibit IL-12p40 production in LPS-stimulated monocytes by down-regulating the activation of JNK (Ma et al., 2003). In contrast, artemisinin induces the production of IL-12p40 in LPS-stimulated macrophages by inhibiting JNK activity (Cho et al., 2012). Interestingly, it is reported that the modulation of IL-12p40 expression by JNK intracellular signaling pathways is influenced by IL-12p40 promoter polymorphism (Dobreva et al., 2009). To investigate whether there are any transcription factors in p38, JNK and NF-␬B signaling pathways involved in the PRRSV induced IL-12p40 production, we cloned and analyzed the porcine IL-12p40 promoter. As expected, the porcine IL-12p40 promoter has a high similarity with human and mouse promoters, and the putative binding motifs for NF-␬B (TTGAAATTCCCCC) and AP-1 (AGTCAG) exist. The function of NF-␬B on IL-12p40 regulation is first revealed in murine cells and confirmed by following studies (Murphy et al., 1995; Plevy et al., 1997). While the activation of NF-␬B by PRSSV CH-1a infection has been revealed before (Fu et al., 2012), we showed a classical activation of NF-␬B signaling pathway by HP-PRRSV infection (data not shown). AP-1, a heterodimeric transcription factor composed of c-Jun and c-Fos, is involved in JNK signaling pathway. Both JNK and AP-1 were activated by PRRSV infection (Fig. 5C) and the phosphorylation of c-Jun was suppressed by JNK inhibitor (Fig. 5D). Consistent with the role of JNK in IL-12p40 induction, AP-1 inhibitor also significantly reduced the expression of IL-12p40 after PRRSV infection, suggesting that PAMs are able to produce IL-12p40 triggered by PRRSV through JNK-AP-1 and NF-␬B signaling pathways. The transcription of IL-12p40 is regulated by multiple routes, and other transcription factors such as C/EBP ␤ and IRFs may also be involved in the regulation of PRRSV-induced IL-12p40 production (Murphy et al., 1995; Plevy et al., 1997; Wang et al., 2000; Zhu et al., 2001). Although p38 MAPK positively regulated the expression of IL-12p40, we did not find any transcription factors controlled by p38 in the porcine IL-12p40 promoter. Since p38 is demonstrated to play a role in mRNA stabilization (Dean et al., 2004; Winzen et al., 1999), it might regulate IL-12p40 expression post-transcriptionally. In summary, our results demonstrate that PRRSV infection could induce IL-12p40 production both in vivo and in vitro, and the upregulation of IL-12p40 by PPRSV depends on p38, JNK, and NF-␬B pathways. This work may provide some insights into the molecular mechanisms of IL-12p40 induced by PRRSV. Acknowledgments This work was supported by the Beijing Natural Science Foundation (Grant No. 6151001), China, the National Natural Science Foundation of China (Grant No. 31572516), and the State Key Laboratory of Agrobiotechnology Fund (Grant 2014SKLAB1-3 and 2015SKLAB1-2), China Agricultural University. References Albina, E., Piriou, L., Hutet, E., Cariolet, R., L’Hospitalier, R., 1998. Immune responses in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Vet. Immunol. Immunopathol. 61 (1), 49–66. Albina, E., 1997. Epidemiology of porcine reproductive and respiratory syndrome (PRRS): an overview. Vet. Microbiol. 55 (1–4), 309–316.

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