Porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus in pigs

Abstract

We performed a comprehensive analysis of innate and adaptive immune responses in dual-virus infected pigs to understand whether a pre-existing immunomodulatory respiratory viral infection affects the overall immunity to a subsequent porcine respiratory coronavirus (PRCV) infection in pigs. Pigs were either mock-infected or infected with porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to cause immunosuppressive respiratory disease, and then pigs were co-infected with PRCV, which normally causes subclinical respiratory infection. We collected samples for six independent experiments from 178 pigs that were also used for pathological studies. We detected a significant reduction in innate NK-cell-mediated cytotoxic function in PRRSV-infected pigs, which was synergistically further decreased in pigs co-infected with PRCV. Subsequently, in association with clinical signs we observed elevated levels of proinflammatory (IL-6), Th-1 (IL-12), and regulatory (IL-10 and TGF-β) cytokines. Increased frequencies of CD4CD8 double-positive T lymphocytes and myeloid cells, in addition to the elevated Th-1 and proinflammatory cytokines in dual-infected pigs, contributed to the severity of lung disease in pigs. The results of our study clarify how each virus modulates the host innate and adaptive immune responses, leading to inflammatory reactions and lung pathology. Thus measurements of cytokines and frequencies of immune cells may serve as indicators of the progression of respiratory viral co-infections, and provide more definitive approaches for treatment.

FIG. 1.

Clinical responses in pigs infected with PRRSV, and then infected with PRCV 10 d later. Clinical signs of pigs mock-infected (n = 43), PRRSV-infected (n = 39), PRCV-infected (n = 48), or infected with both PRRSV and PRCV (n = 48) were recorded on the indicated post-inoculation days (PID). (A) Rectal temperature. (B) Body weight gain. Each data point represents the mean ± SEM of 5–48 pigs. A significantly higher incidence of fever was observed in PRCV/PRRSV co-infected pigs than in PRRSV-alone pigs (p = 0.027), and a significantly higher proportion had less body weight gain in the PRCV/PRRSV co-infected pigs than in those infected with PRRSV alone (p = 0.005), as indicated by the asterisks. Statistical analysis was performed using Fischer's exact test to evaluate the proportions of pigs with fever and decreased body weight gain, and p < 0.05 was considered statistically significant.

FIG. 2.

Significant reductions occurred in NK-cell cytotoxicity despite a lack of significant changes in NK-cell populations following PRRSV and PRCV co-infection in pigs. (A) Percentages of CD3⁻CD4⁻CD8α⁺ lymphocytes (NK cells) in PBMCs of pigs were evaluated in mock-infected, PRRSV-infected, PRCV-infected, or dual-infected (PRCV/PRRSV) pigs on the indicated post-inoculation days (PID) by flow cytometric analysis. Each bar represents the mean ± SEM of NK cells from 4–8 pigs, and total numbers of pigs at each PRCV/PRRSV PID: −2/8 (n = 17); 2/12 (n = 18); 4/14 (n = 18); 8/18 (n = 20); 10/20 (n = 20); 14/24 (n = 21); and 21/31 (n = 21). (B) Percentages of NK-specific cytotoxicity were measured using pig PBMCs (effectors) harvested from mock-infected or infected pigs as described above against target cells (K-562). Effectors and targets at the different indicated ratios were cultured together, and the supernatants harvested were analyzed for amounts of LDH released using its substrate at 490 nm. Each line is from one pig, and each data point on the line is the mean of triplicate readings ± SEM at the respective E:T ratios tested. Altogether there were 19 pigs from one independent experimental trial (a total of four PIDs). A similar trend in NK-specific lysis was detected in another two independent experimental trials comprised of the same numbers of pigs. Statistical analyses were performed from the pooled results of all three independent experimental trials, and “a” denotes a statistically significant difference (p < 0.05) between the mock- and dual-infected groups; “b” denotes a statistically significant difference between the mock- and PRRSV-infected groups; “c” denotes a statistically significant difference between the mock- and PRCV-infected groups; and “d” denotes a statistically significant difference between the PRCV- and dual-infected groups, as analyzed by the nonparametric Kruskal-Wallis test.

FIG. 3.

Serum and lung cytokine levels in PRRSV- and PRCV-infected pigs. Pigs were mock infected or infected with the indicated viruses as described in the legend to Fig. 2. Serum samples and lung lysates were assayed for different cytokines by ELISA. (A) IL-12, (B) IL-6, (C) IL-10, and (D) TGF-β. Each data point represents the average cytokine levels of 5–8 pigs from each indicated PID ± SE. “a” denotes a statistically significant difference (p < 0.05) between the mock- and dual-infected groups; “b” denotes a statistically significant difference (p < 0.05) between the PRCV- and dual-infected groups; “c” denotes a statistically significant difference (p < 0.05) between the PRCV- and PRRSV-infected groups; and “d” denotes a statistically significant difference (p < 0.05) between the mock- and PRRSV-infected groups, as analyzed by the nonparametric Kruskal-Wallis test.

FIG. 4.

Serum cytokine levels in PRRSV- and PRCV-infected pigs. Pigs were mock-infected (n = 43), or infected with PRRSV (n = 39), PRCV (n = 48), or PRCV/PRRSV (n = 48), and serum was collected on the indicated post-inoculation days and assayed for different cytokines by ELISA. (A) IL-12, (B) IL-6, (C) IL-10, and (D) TGF-β. Each data point represents the average cytokine levels of 5–48 pig serum samples collected on the indicated post-inoculation days ± SEM. “a” denotes a statistically significant difference (p < 0.05) between the mock- and dual-infected groups; “b” denotes a statistically significant difference (p < 0.05) between the PRCV- and dual-infected groups; “c” denotes a statistically significant difference (p < 0.05) between the PRCV- and PRRSV-infected groups; and “d” denotes a statistically significant difference (p < 0.05) between the mock- and PRRSV-infected groups, as analyzed by the nonparametric Kruskal-Wallis test. Asterisks indicate a statistically significant difference for repeated sera collected from the respective pig groups (those infected with PRRSV alone and those infected with both viruses), as analyzed by repeated-measures ANOVA.

FIG. 5.

Variable frequencies of lymphoid and myeloid cell populations in PBMCs of pigs were detected following PRRSV and PRCV co-infection. Pigs were mock-infected or infected with the indicated viruses as described in the legend to Fig. 2. Percentages of CD3⁺ lymphocyte subpopulations in PBMCs of pigs on the indicated post-inoculation days (PID) were evaluated by flow cytometric analysis, and then further grouped based on CD4- and CD8-specific staining: (A) T-helper cells (CD3⁺CD4⁺CD8α⁻); (B) cytotoxic T lymphocytes (CD3⁺CD4⁻CD8α⁺); and (C) double-positive T cells (CD3⁺CD4⁺CD8α⁺). (D) Total myeloid cell populations (CD172⁺) in PBMCs were also evaluated by flow cytometric analysis. Each bar represents the mean ± SEM from 3–8 pigs, and the total numbers of pigs at each PRCV/PRRSV PID were: −2/8 (n = 17); 2/12 (n = 18); 4/14 (n = 18); 8/18 (n = 20); 10/20 (n = 20); 14/24 (n = 21); and 21/31 (n = 12). On each PID, 4–6 pigs were euthanized, representing all four treatment groups. “a” denotes a statistically significant difference (p < 0.05) between the PRRSV- and mock-infected groups; “b” denotes a statistically significant difference (p < 0.05) between the dual- and mock-infected groups; and “c” denotes a statistically significant difference (p < 0.05) between the PRCV- and mock-infected groups, as analyzed by the nonparametric Kruskal-Wallis test.