Previous studies have indicated that

Previous studies have indicated that maximal IL-8 protein mg115 requires NF-κB activation as well as the activation of the MAP kinases ERK, JNK, and p38 (Li et al., 2002). In our study, NF-κB inhibitor can reduce the up-regulated IL-8 production by PRRSV infection, indicating that PRRSV-induced IL-8 expression is partially dependent on NF-κB pathway. This is in agreement with the report regarding the significance of NF-κB pathway in IL-8 expression induced by other stimulators (Ohkuni et al., 2011). Moreover, our results suggest that JNK pathway is more essential for PRRSV induced IL-8 expression as JNK and c-Jun inhibitor and siRNA knockdown of c-Jun and c-Fos significantly reduced IL-8 expression. Similarly, under stimulation of HIV-1, HCV and Papillomavirus, the expression of IL-8 is also JNK dependent (Chen et al., 2015, Gangwani and Kumar, 2015, Saeed et al., 2015, Zhang et al., 2014). JNK isoforms are strongly activated in response to various cellular stresses (Cargnello and Roux, 2011). And indeed, we found that PRRSV infection activated JNK, which is in agreement with other reports (Huo et al., 2013, Jing et al., 2014, Lee and Lee, 2012, Yin et al., 2012). Here, we need to indicate that even though JNK inhibitor SP and NF-κB inhibitor BAY significantly inhibit IL-8 mRNA expression induced by PRRSV infection, they do not affect the protein level of IL-8 at the same level, suggesting that there may exist post transcription level regulations such as translation repress, which might be not affected by JNK and NF-kB inhibitors. Interestingly, it has been reported that ARE in IL-8 3′ UTR can recruit TTP, which can repress RNA translation (Anderson and Kedersha, 2009, Eulalio et al., 2007, Franks and Lykke-Andersen, 2008, Winzen et al., 2007). However, this needs to be investigated in the future. The transcription factor c-Jun is a well-described substrate for JNKs. The phosphorylation of c-Jun by JNK has been reported to potentiate the transcriptional capacity of c-Jun, thus enhancing its ability to engage in gene transcription as well as its own expression (Hazzalin and Mahadevan, 2002). Our results also show significant phosphorylation and expression of c-Jun upon PRRSV infection, which is dependent on JNK activation. It is reported that stabilization of IL-8 mRNA is modulated by the p38 mitogen-activated protein kinase pathway (Hoffmann et al., 2002, Winzen et al., 2007). Exposure to the inflammatory cytokine IL-1 has been shown to stabilize IL-8 mRNA through p38 mitogen-activated protein (MAP) kinase and MK2 (Winzen et al., 2007). Interestingly, we found that p38 inhibitor also reduced IL-8 mRNA expression. In human and mouse IL-8 promoters, a sequence from nt +1 to −133 within the 5′flanking region of the IL-8 gene is essential and sufficient for the transcriptional regulation of the gene (Mukaida and Murayama, 1998). Analysis demonstrates that the promoter elements contain NF-κB, AP-1, and C/EBP β binding sites (Matsusaka et al., 1993, Mukaida and Murayama, 1998, Nourbakhsh et al., 2001). The promoter is regulated in a cell type-specific fashion, requiring a NF-κB element plus either an AP-1 or a C/EBP β element (Chang et al., 2004; Wu et al., 1997). We cloned the 2751bp-sequence in the 5′ flanking region of the porcine IL-8 gene. Truncation mutations indicated that the region from −187 to +123bp was essential for the porcine IL-8 promoter activity. However, the longer promoter was not stimulated by PRRSV. The reason could be that there might exist some negative regulation elements in the upstream of the −187 to +123bp. Analysis showed that AP-1, C/EBP β, and NF-κB elements existed in the region from −187 to −76bp. Deletion of these three elements suggested that AP-1 element was the most significant one in PRRSV induced IL-8 expression. Similar result was reported with SARS-CoV (Chang et al., 2004). TAK1 is a key regulator of the innate immunity and the pro-inflammatory signaling pathway. Many viruses have been reported to modulate the activity of TAK1 and thereby the NF-κB and AP-1 pathways. For example, the X protein of hepatitis B virus, LMP1 of Epstein-Barr virus, vGPCR of KHSV, ICP0 of herpes simplex virus type 1, RSV, and Tax of human T cell leukemia virus type 1 can activate NF-κB or AP-1 signaling by targeting TAK1 (Bottero et al., 2011, Diao et al., 2005; Liu et al., 2014; Soni et al., 2007; Zhou et al., 2010). TAK1 regulates JNK, p38 MAPKs and IκB kinase (IKK) signaling pathways, leading to the activation of transcription factors AP-1 and NF-κB (Sakurai, 2002). Consistent with this, we found that PRRSV significantly induced TAK-1 activation and PRRSV-induced IL-8 expression was TAK-1 dependent. Similarly, TAK-1 participates in IL-8 expression under stimulation of other virus (Pera et al., 2012).