IL-10 is a regulatory cytokine which is known to inhibit production of several pro-inflammatory cytokines such as IL-1 and TNF-α. In this study, we showed that IL-10 was up-regulated in PRRSV CH-1a-infected PAMs, BMDMs, and MDMs both at mRNA and protein levels in vitro. A recent study focused on the genome-wide host transcriptional responses to CH-1a infection also showed increased expression of IL-10 in an in vivo infection model .
Our study showed that PRRSV infection significantly stimulated p38 phosphorylation (Figure 4a). And IκB was also gradually degraded in PRRSV-infected BMDMs (data not shown), which is consistent with a previous study . In that report, the authors demonstrated that NF-κB pathway was activated in PRRSV-infected MARC-145 and PAMs through IκB degradation. Moreover, through the use of signal transduction pathway specific inhibitors, we further demonstrated that p38 MAPK and NF-κB pathways were involved in the up-regulation of IL-10 production in PRRSV-infected BMDMs (Figure 3). These results suggested that p38 MAPK and NF-κB signal transduction pathways played important roles in the induction of IL-10 during PRRSV infection. This is in accordance with previous reports, in which the authors showed that p38 MAPK pathway was essential for IL-10 production induced by LPS [26, 33]. Another study also reported that IL-10 production stimulated by apoptotic cells was regulated at the transcription level in a p38 MAPK dependent manner . Transcription factor NF-κB is also likely to be a candidate for the transactivation of the IL-10 gene, since the IL-10 promoter has nine putative NF-κB binding sites . We also showed that p38 MAPK and STAT3 pathways were activated in GP5-transfected macrophages (Figure 7). It seems that p38 MAPK pathway is very important in IL-10 induction, since other viral proteins, for instance, extracellular HIV-Tat also induced IL-10 transcription in primary human monocytes through the activation of calmodulin/CaMK-II-dependent p38 MAPK [35, 36]. The STAT3 transcription factor may also bind to an element in the IL-10 promoter and the use of a dominant negative form of STAT3 was able to decrease IL-10 transcription .
MAPK activation has also been shown to be required for the optimal replication of some viruses . In this study, viral replication was not obviously affected when cells were treated with inhibitors of p38 (SB203580), ERK1/2 (PD98059), PI3K (LY294002), and NF-κB (BAY11-7082) pathways (Figure 2). A previous study showed that rhesus rotavirus replication was not significantly altered by the presence of p38 inhibitor, SB203580 at 10 μM in HT-29 cells or at 20 μM in MA104 cells . Lee et al. (2012) showed that SB202190, another p38 inhibitor, down-regulated the viral gene expression only at high concentration (10 μM) . Different p38 inhibitors might cause the differences in the effects of inhibitors on PRRSV replication in treated cells. Lee et al. (2010) also demonstrated that PD98059 had no effect on PRRSV replication in PAMs . In contrast to our results, a recent report indicated that inhibition of PI3K/Akt by treatment with LY294002 at 25 μM prior to PRRSV infection reduced virus replication . The different effects of PI3K inhibitor LY294002 on PRRSV replication might be due to the different concentrations used in their and our studies. In our report, we used no more than 10 μM.
GP5 is the most variable structural protein , and plays an important role in PRRSV pathogenesis. For instance, the existing of a decoy epitope in GP5 and glycan-shielding of the neutralization epitopes contributed to delayed and weak neutralizing antibody response [44, 45]. GP5 was also suggested to be the apoptotic factor mediating the induction of apoptosis of uninfected bystander cells [46, 47]. Recently, a study using chimeric viruses of a highly virulent strain vFL12 and an attenuated vaccine strain showed that NSP3-8 and ORF5 were the location of major virulence determinants . Here, we showd that GP5 induced IL-10 production. Taken together, these results suggested that GP5 might play important roles in the pathogenesis of PRRSV infection. By using constructs truncated at different locations in GP5, the full-length of GP5 structure seems to be essential for IL-10 induction. Truncation of proteins will compromise their overall structure. Thus, more subtle changes could be introduced to map residues involved in specific functions (e.g. mutations) in the future.
Collectively, our results demonstrated that PRRSV strain CH-1a did up-regulate IL-10 production in macrophages. However, studies using vFL12, a virulent PRRSV strain, showed that there was no detectable level of IL-10 in the supernatant of PRRSV-infected macrophages and dendritic cells , and TNF-α was also poorly induced . The authors suggested that the induction of IL-10 by several other PRRSV strains may be associated with their ability to induce pro-inflammatory cytokines during the acute phase of infection. However, IL-10 gene expression was up-regulated (1.9-fold) whereas TNF-α gene was only slightly up-regulated (1.5-fold) in Lelystad PRRSV strain-infected PAMs . Another report demonstrated that exposure of BM-imDCs to PRRSV resulted in a significantly increased secretion of IL-10 but not TNF-α . Interestingly, our lab found that Chinese highly pathogenic PRRSV (HP-PRRSV) infection failed to induce detectable IL-10 (unpublished data), but induced lower levels of TNF-α in PAMs . Further studies need to be done to figure out whether IL-10 up-regulation is associated with virus induced immunosuppression as proposed or just associated with their ability to induce pro-inflammatory cytokines.