Cytokine responses in gnotobiotic pigs after infection with virulent or attenuated human rotavirus

Abstract

To understand the role of cytokines during rotavirus infection, we assessed the kinetics of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) (proinflammatory), IL-12 (Th1 inducer), gamma interferon (IFN-gamma) (Th1), IL-4 and IL-10 (Th2), and transforming growth factor beta (Th3) cytokine responses by enzyme-linked immunosorbent assay in serum and intestinal contents of neonatal gnotobiotic pigs and IL-12, IFN-gamma, IL-4, and IL-10 cytokine-secreting cell (CSC) responses of mononuclear cells from ileum, spleen, and blood by ELISPOT. Pigs received the virulent Wa P1A[8]G1 strain of human rotavirus (HRV) (VirHRV), attenuated Wa HRV (AttHRV), or mock (controls). The TNF-alpha levels peaked earlier and remained elevated in serum of the VirHRV group but peaked later in the AttHRV group. In serum, IL-6 was significantly elevated at postinoculation day (PID) 1 in the VirHRV group and at PID 3 in both HRV groups. The IL-12 was detected in serum of all pigs including controls with significantly elevated peaks in both HRV-infected groups, indicating a role for IL-12 in the induction of immune responses to rotavirus infection. Only low and transient IFN-gamma responses occurred in serum and intestinal contents of the AttHRV-infected pigs, compared to significantly higher and prolonged IFN-gamma responses in the VirHRV-infected pigs. This observation coincides with the diarrhea and viremia induced by VirHRV. The number of IFN-gamma-secreting cells was significantly higher in the ileum of the VirHRV group than in that of the controls. The number of IL-4 CSCs was significantly higher in ileum of both HRV groups than in that of the controls. Significantly higher levels of IL-10 in the serum occurred early in the VirHRV group, compared to lower levels in the AttHRV group. However, the number of IL-10 CSCs was significantly higher later in ileum and spleen of the AttHRV than in the VirHRV group, suggesting a delayed initiation of a Th2 response induced by AttHRV. A significantly higher percentage of pigs had IFN-gamma and IL-10 responses in serum after VirHRV infection than after AttHRV infection or in controls. These data indicate a balanced Th1/Th2 response during rotavirus infection, with higher cytokine levels early after infection with VirHRV compared to that with AttHRV. Mapping the kinetics and patterns of cytokine responses after rotavirus infection has important implications for induction of protective immunity by HRV vaccines. Higher protection rates may be associated with more balanced Th1- and Th2-type responses, but induction of higher earlier IFN-gamma (Th1) and proinflammatory cytokines triggered by VirHRV may also play an important role in the higher intestinal immunoglobulin A responses and protection rates induced by VirHRV.

FIG. 1.

Cytokine levels in serum of gnotobiotic pigs. Symbols: squares, VirHRV; diamonds, AttHRV; triangles, control. Statistical symbols for P < 0.05: *, higher than AttHRV and controls; #, higher than controls.

FIG. 2.

Cytokine levels in intestinal contents of gnotobiotic pigs. Symbols: ▪, VirHRV; ⋄, AttHRV; ▵, control.

FIG. 3.

(A) IFN-γ, IL-6, and TNF-α and antigenemia in gnotobiotic piglets inoculated with HRV. Symbols: ▪, serum IFN-γ levels in VirHRV group; □, HRV antigen in VirHRV group; ⋄, serum IFN-γ levels in AttHRV group; *, HRV antigen in AttHRV group. Dotted line represents cutoff value (ELISA = 0.08). (B) IFN-γ, IL-6, and TNF-α and diarrhea in gnotobiotic piglets inoculated with HRV. Symbols: ▪, IFN-γ in VirHRV group; □, diarrhea score in VirHRV group; ⋄, IFN-γ in AttHRV group; *, diarrhea score in AttHRV group.

FIG. 4.

Th1 (IFN-γ and IL-12) cytokine-secreting cell responses in ileum, spleen, and PBMCs of gnotobiotic pigs inoculated with VirHRV, AttHRV, or controls. Due to higher IL-12 numbers in PBMCs, the PBMC graph is shown using a different scale. Symbols: ▨, VirHRV; ░⃞, AttHRV; □, controls. Statistical symbols for P < 0.05: *, higher than AttHRV and controls; +, higher than VirHRV and controls; #, higher than controls.

FIG. 5.

Th2 (IL-10 and IL-4) cytokine-secreting cells in ileum, spleen, and blood of gnotobiotic piglets inoculated with HRV. Symbols: ▨, VirHRV; ░⃞, AttHRV; □, controls. Statistical symbols for P < 0.05: *, higher than AttHRV and controls; +, higher than VirHRV and controls; #, higher than controls.