Induction of interleukin-10 is dependent on p38 mitogen-activated protein kinase pathway in macrophages infected with porcine reproductive and respiratory syndrome virus

Abstract

Background: Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure and respiratory illness in pigs and usually establishes a persistent infection. Previous studies suggested that interleukin-10 (IL-10) could play a critical role in PRRSV-induced immunosuppression. However, the ability of PRRSV to induce IL-10 in infected cells is controversial. In this study, we further investigated this issue using PRRSV strain CH-1a, which is the first North American genotype strain isolated in China.

Results: PRRSV strain CH-1a could significantly up-regulate IL-10 production both at mRNA and protein levels in porcine alveolar macrophages (PAMs), bone marrow-derived macrophages (BMDMs), and monocyte-derived macrophages (MDMs). However, up-regulation of IL-10 by PRRSV was retarded by specific inhibitors of p38 mitogen-activated protein kinase (MAPK) (SB203580) and NF-κB (BAY11-7082). Additionally, p38 MAPK and NF-κB pathways but not ERK1/2 MAPK were actually activated in PRRSV-infected BMDMs as demonstrated by western blot analysis, suggesting that p38 MAPK and NF-κB pathways are involved in the induction of IL-10 by PRRSV infection. Transfection of PAMs and PAM cell line 3D4/21 (CRL-2843) with viral structural genes showed that glycoprotein5 (GP5) could significantly up-regulate IL-10 production, which was dependent on p38 MAPK and signal transducer and activator of transcription-3 (STAT3) activation. We also demonstrated that a full-length glycoprotein was essential for GP5 to induce IL-10 production.

Conclusions: PRRSV strain CH-1a could significantly up-regulate IL-10 production through p38 MAPK activation.

Figure 1

IL-10 was up-regulated in PRRSV-infected macrophages. PAMs and BMDMs were infected with PRRSV or UV-inactivated virus at an MOI of 1. Cells stimulated with LPS (1 μg ml^-1) were used as positive control. (a), (c), (e) Cells were harvested at the indicated time points and real-time PCR was performed to evaluate IL-10 mRNA levels. (b), (d), (f) Measurement of secreted IL-10 protein levels in the supernatant of PRRSV-infected PAMs and BMDMs at indicated time points by ELISA. The results represent means ± SD of three independent experiments. *Significant difference (P < 0.05) from media control using Student’s t-test.

Figure 2

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. (a) BMDMs were pretreated with DMSO or PI3K inhibitor LY294002, ERK MAPK inhibitor PD98059, p38 inhibitor SB203580, and NF-κB inhibitor BAY11-7082 at the indicated concentrations for 2 h. Cells were then infected with PRRSV (MOI = 1) and harvested at 24 h.p.i.. Virus titers in the cell supernatants were measured by a standard 50% tissue culture infective doses (TCID₅₀) assay. (b) BMDMs were treated with DMSO or PI3K inhibitor LY294002, ERK MAPK inhibitor PD98059, p38 inhibitor SB203580, and NF-κB inhibitor BAY11-7082 at the indicated concentrations for 24 h. Then the cytotoxicity of the inhibitors on BMDMs was determined by trypan blue exclusion dye staining. Data represent means ± SD of three independent experiments.

Figure 3

PRRSV-stimulated IL-10 production was selectively inhibited by inhibitors of p38 MAPK and NF-κB signaling pathways. BMDMs were pretreated with DMSO or PI3K inhibitor LY294002, ERK MAPK inhibitor PD98059, p38 inhibitor SB203580, and NF-κB inhibitor BAY11-7082 at the indicated concentrations for 2 h. Cells were then infected with PRRSV (MOI = 1) and harvested at 24 h.p.i.. (a) Total RNA isolated from cells was reverse transcribed and then analyzed using real-time PCR. (b) IL-10 production in cell supernatants was analyzed by ELISA. Data represent means ± SD of three independent experiments. *Significant difference (P < 0.05) from PRRSV control using Student’s t-test.

Figure 4

PRRSV infection activated p38 MAPK signaling pathway in BMDMs. Whole-cell extracts of BMDMs infected with (a) PRRSV at an MOI of 1 or (b) equal amounts of UV-inactivated virus were prepared at indicated time points and analyzed by immunoblot for the phosphorylation status of p38 (p-p38) and ERK1/2 (p-ERK1/2). The blot was also probed for tubulin as loading control.

Figure 5

GP5 induced IL-10 mRNA and protein expression. (a) PAMs were transfected with PRRSV structural and non-structural proteins (2 μg) or stimulated with LPS (1 μg ml^-1) as positive control. IL-10 mRNA expression at 24 h post transfection was evaluated by real-time PCR. (b) Cell supernatants collected from viral structural protein transfected PAMs were analyzed for IL-10 production by ELISA. (c) CRL-2843 cells were cotransfected with a mixture of pIL-10-luc plasmid, pRL-TK plasmid, and viral structural protein-encoding plasmids or empty vector. At 24 h post transfection, luciferase activities were measured with the Dual-Luciferase Reporter Assay System. (d) Increasing amounts (0.1, 0.5 and 2 μg) of GP5-encoding plasmid were transfected into PAMs. IL-10 mRNA expression at 24 h post transfection was examined by real-time PCR. The results represent means ± SD of three independent transfections. * Significant difference (P < 0.05) from empty vector control using Student’s t-test.

Figure 6

The full length of GP5 was required for IL-10 induction. (a) Schematic representation of truncated GP5 (numbers indicate the amino acids positions). The black squares represent the translation termination. (b) PAMs were transfected with the truncated GP5 expression plasmids (2 μg) or empty vector. Cells were harvested at 24 h post transfection. Total RNA isolated from cells was reverse transcribed and real-time PCR was performed to detect porcine IL-10 mRNA. Results are shown as means ± SD of three independent transfections. *Significant difference (P < 0.05) from wild-type GP5 using Student’s t-test.

Figure 7

GP5 expression activated p38 MAPK and STAT3 pathways. At 24 h post transfection of the vector control or GP5-encoding plasmid into CRL-2843 cells, the phosphorylation status of p38 (p-p38), ERK1/2 (p-ERK1/2) and STAT3 (p-STAT3) were evaluated by western blot.