Cytokine profiles in pregnant gilts experimentally infected with porcine reproductive and respiratory syndrome virus and relationships with viral load and fetal outcome

Abstract

In spite of extensive research, immunologic control mechanisms against Porcine Reproductive and Respiratory Syndrome virus (PRRSv) remain poorly understood. Cytokine responses have been exhaustively studied in nursery pigs and show contradictory results. Since no detailed reports on cytokine responses to PRRSv in pregnant females exist, the objectives of this study were to compare host cytokine responses between PRRSv-infected and non-infected pregnant gilts, and to investigate relationships between cytokine levels in infected gilts and viral load or fetal mortality rate. Serum samples and supernatants of peripheral blood mononuclear cells (PBMC) either stimulated with PRRSv or phorbol myristate acetate/Ionomycin (PMA/Iono) were analyzed for cytokines/chemokines: interleukins (IL) 1-beta (IL1β), IL4, IL8, IL10, IL12, chemokine ligand 2 (CCL2), interferon alpha (IFNα) and interferon gamma (IFNγ). Three cytokines (IFNα, CCL2, IFNγ) in gilt serum differed significantly in inoculated versus control gilts over time. In supernatants of PRRSv stimulated PBMC from PRRSv-infected gilts, levels of IFNα were significantly decreased, while IL8 secretion was significantly increased. PRRSv infection altered the secretion of all measured cytokines, with the exception of IFNα, from PBMC after mitogen stimulation, indicating a possible immunomodulatory effect of PRRSv. IFNα, CCL2, and IFNγ in serum, and IFNγ in supernatants of PMA/Iono stimulated PBMC were significantly associated with viral load in tissues, serum or both. However, only IFNα in supernatants of PRRSv stimulated PBMC was significantly associated with fetal mortality rate. We conclude that of the eight cytokines tested in this study IFNα was the best indicator of viral load and severity of reproductive PRRSv infection.

Figure 1

Examples of standard curves obtained by FMIA and ELISA. Examples of standard curves of recombinant protein standards serially diluted in serum (A) or cell culture medium (B) for the seven cytokines tested by FMIA; (C) examples of IFNγ-standard curves (recombinant protein serially diluted) in serum (ser) or cell culture medium (sup) by ELISA.

Figure 2

CCL2, IFNα and IFNγ levels in gilt serum over time. Mean (± SEM) CCL2 (A), IFNα (B) and IFNγ (C) levels in serum from 111 INOC and 19 CTRL gilts across respective study days; P-values indicate significant differences between INOC and CTRL gilts on individual days.

Figure 3

Adjusted IFNα and IL8 levels in supernatants of PRRSv stimulated PBMC over time. Mean (± SEM) adjusted IFNα (A) and IL8 (B) levels in supernatants of PRRSv stimulated PBMC from 111 INOC and 19 CTRL gilts across respective study days. P-values indicate significant differences between INOC and CTRL gilts on individual days. Adjusted cytokine levels were calculated by subtracting values in supernatants of unstimulated cells from PRRSv stimulated cells.

Figure 4

Adjusted CCL2, IL8, IL10, IL12, IL1β, and IL4 levels in supernatants of PMA/Iono stimulated PBMC over time. Mean (± SEM) adjusted CCL2 (A), IL8 (B), IL10 (C), IL12 (D), IL1β (E), and IL4 (F) levels in supernatants of PMA/Iono stimulated PBMC from 111 INOC and 19 CTRL gilts across respective study days. P-values indicate significant differences between INOC and CTRL gilts on individual days. Adjusted cytokine levels were calculated by subtracting values in supernatants of unstimulated cells from PMA/Iono stimulated cells.

Figure 5

Scatter plots of selected cytokines in serum and supernatants of PRRSv stimulated PBMC versus viral load in gilt serum or percent dead fetuses per litter. The levels of IFNα (A) and CCL2 (B) in serum over time are plotted against the viral load in gilt serum over time (area under the curve (AUC) from 0 to 21 dpi) illustrating individual variation. (C) The levels of IFNα in supernatants of PRRSv stimulated PBMC over time (AUC) are plotted against the percentage of dead fetuses per litter. Animals in the upper right quadrant are helping to drive the relationship. No gilts had high cytokine levels and low viral load in serum or low percent of dead fetuses per litter.