Age-dependent variation in innate immune responses to porcine epidemic diarrhea virus infection in suckling versus weaned pigs

Abstract

Porcine epidemic diarrhea (PED) is an enteric coronaviral infection that causes severe morbidity and mortality in suckling pigs, but less severe disease in older pigs. Consequently, it causes significant economic losses to the pork industry. There are limited studies on the innate immune responses to PED virus (PEDV) in pigs. The aims of our study were to investigate differences in innate immune responses to PEDV infection in suckling and weaned pigs and to examine if disease severity coincides with reduced innate immune responses. Weaned 26-day-old pigs (n=20) and 9-day-old nursing pigs (n=20) were assigned to PEDV inoculated or uninoculated control groups. The pigs were observed daily for clinical signs, virus shedding and were euthanized at post-inoculation days (PIDs) 1 and 5 to assay immune responses. Blood samples were collected at PIDs 1, 3 and 5. The natural killer (NK) cell frequencies, NK cell activities (lysis of target K562 tumor cells in vitro), CD3+CD4+ T cell and CD3+CD8+ T cell frequencies were measured in blood and ileum at PIDs 1 and 5. The PEDV infected suckling pigs showed severe diarrhea and vomiting at PID 1, whereas the PEDV infected weaned pigs showed milder clinical signs starting at PID 3. PEDV infected suckling pigs had significantly higher diarrhea scores, earlier fecal PEDV RNA shedding and significantly higher viremia (viral RNA in serum) compared to weaned pigs. There was no mortality in either infected suckling or infected weaned pigs. The control pigs not inoculated with PEDV did not show any clinical signs and no detectable fecal or serum PEDV RNA. Strikingly, PEDV infected suckling pigs had significantly lower NK cell frequencies, undetectable NK cell activity and lower IFNγ producing NK cells in blood and ileum compared to PEDV infected weaned pigs. Pro-inflammatory cytokine profiles of PEDV infected suckling pigs differed from those of PEDV infected weaned pigs and coincided with onset of fecal PEDV RNA shedding and serum PEDV RNA titers. The infected suckling pigs have higher and earlier increases in serum IFNα, but lower serum IL-8 and TNFα levels compared to infected weaned pigs. CD3+CD4+ T cell frequencies were significantly higher in ileum of suckling pigs than in weaned pigs, whereas there was no difference in CD3+CD8+ T cell frequencies. In conclusion, the observations of impaired lytic activity and IFN-γ production by NK cells in suckling pigs coincided with the increased severity of PEDV infection in the suckling pigs compared with the weaned pigs.

Keywords: Cytokine; Innate immunity; NK cells; PEDV; Porcine epidemic diarrhea virus.

Fig. 1

Suckling piglets had lower NK cell activity (% lysis of K562 cells) compared to weaned piglets in blood and ileal mononuclear cells at PID 1 (A and B) and PID 5 (C and D). Blood and ileal mononuclear cells were co-cultured with CFSE stained K562 cells at indicated ratios overnight. The dead cells were stained by incubating with 7-AAD. The cells were observed by flow cytometry to obtain % of dead K562 cells. The groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. The bar graphs labeled with different alphabetical letters are significantly different (P < 0.01).

Fig. 2

Weaned infected piglets had higher NK cell frequencies compared to suckling infected piglets in blood (A) and ileum (B) at PID 5. Blood and ileal MNCs were stained with anti-porcine CD3-FITC, CD4-PE and CD8-SPRD. The NK cell (CD3-CD4-CD8+) frequency was expressed as percentage of lymphocytes. The groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. The different time points were also compared within an age group and tissue type. Bar graphs labeled with no common alphabetical letter are significantly different (P < 0.05).

Fig. 3

IFNγ producing CD3-CD4-CD8+ NK cell frequencies were higher in weaned pigs compared to suckling pigs at PID 1 and 5 in blood (A) and at PID 5 in ileum (B). Blood and ileal MNCs were cultured in E-RPMI with protein transporter inhibitor, Brefeldin A added. The cells were stained with CD3-FITC, CD8-SPRD, and CD4-biotin followed by streptavidin APC as secondary antibody. Samples were stained intracellularly with anti-porcine IFNγ–PE. Flow cytometric analysis was done and the percentage of CD3-CD4-CD8+ NK cells that were also IFNγ positive was calculated. The groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. The different time points were also compared within an age group and tissue type. Bar graphs labeled with no common alphabetical letter are significantly different (P < 0.05).

Fig. 4

Inflammatory cytokine profile of PEDV infected suckling piglets differed from PEDV infected weaned pigs and coincided with fecal and serum PEDV RNA. The graphs summarize the serum IFNα (A), IL-12 (B), IL-8 (C), TNFα (D) and IL-17 (E) in suckling and weaned pigs at PIDs 1, 3 and 5. Blood samples were collected from the pigs at the specified time points and sera separated by centrifugation and frozen at −20 °C until use. The cytokine assays were done using standard assays. The groups that were compared were, infected suckling versus infected weaned at different time points, uninfected suckling or weaned versus infected suckling or weaned at different time points. The different time points within infected groups of suckling or weaned pigs were also compared. Bar graphs labeled with no common alphabetical letter are significantly different (P < 0.01).

Fig. 5

CD3+CD4+ cells were higher in ileum of suckling pigs than weaned pigs whereas there was no difference in CD3+CD8+ cells in ileum of suckling and weaned pigs. The graphs show the percentage of CD3+CD4+ cells in blood (A) and ileum (B) of suckling and infected pigs at PIDs 1 and 5, and the CD3+CD8+ cells in blood (C) and ileum (D) of suckling and infected pigs at PIDs 1 and 5. Blood and ileal mononuclear cells were stained with CD3-FITC, CD4-PE and CD8-SPRD for 15 min at 4˚C. The frequency of T cells or NK cells was expressed as percentage of lymphocytes. The groups that were compared were, suckling uninfected versus weaned uninfected, suckling infected versus weaned infected, suckling infected versus suckling uninfected and weaned infected versus weaned uninfected within a time point. The different time points were also compared within an age group and tissue type. Bar graphs labeled with no common alphabetical letter are significantly different (P < 0.05).