Lactobacillus acidophilus and Lactobacillus reuteri modulate cytokine responses in gnotobiotic pigs infected with human rotavirus

Abstract

Probiotic lactic acid bacteria (LAB) have been shown to alleviate inflammation, enhance the immunogenicity of rotavirus vaccines, or reduce the severity of rotavirus diarrhoea. Although the mechanisms are not clear, the differential Th1/Th2/Th3-driving capacities and modulating effects on cytokine production of different LAB strains may be the key. Our goal was to delineate the influence of combining two probiotic strains of Lactobacillus acidophilus and Lactobacillus reuteri on the development of cytokine responses in neonatal gnotobiotic pigs infected with human rotavirus (HRV). We demonstrated that HRV alone, or HRV plus LAB, but not LAB alone, initiated serum cytokine responses, as indicated by significantly higher concentrations of IFN-α, IFN-γ, IL-12, and IL-10 at postinoculation day (PID) 2 in the HRV only and LAB+HRV+ pigs compared to LAB only and LAB-HRV- pigs. Peak cytokine responses coincided with the peak of HRV replication. LAB further enhanced the Th1 and Th2 cytokine responses to HRV infection as indicated by significantly higher concentrations of IL-12, IFN-γ, IL-4 and IL-10 in the LAB+HRV+ pigs compared to the LAB-HRV+ pigs. The LAB+HRV+ pigs maintained relatively constant concentrations of TGF-β compared to the HRV only group which had a significant increase at PID 2 and decrease at PID 7, suggesting a regulatory role of LAB in maintaining gut homeostasis. At PID 28, cytokine secreting cell (CSC) responses, measured by ELISpot, showed increased Th1 (IL-12, IFN-γ) CSC numbers in the LAB+HRV+ and LAB-HRV+ groups compared to LAB only and LAB-HRV- pigs, with significantly increased IL-12 CSCs in spleen and PBMCs and IFN-γ CSCs in spleen of the LAB+HRV+ group. Thus, HRV infection alone, but not LAB alone was effective in inducing cytokine responses but LAB significantly enhanced both Th1 and Th2 cytokines in HRV-infected pigs. LAB may also help to maintain immunological homeostasis during HRV infection by regulating TGF-β production.

Figure 1. Cytokine mean concentration in serum of pigs with or without LAB and inoculated with virulent Wa HRV. Numbers with different letters differ significantly at the same time for the same cytokine among groups (Kruskal Wallis Test, p<0.05, n=4–10)

◇ - LAB+HRV+; □ - LAB-HRV+; ▲ - LAB+HRV−; - LAB-HRV−

Figure 2. Cytokine mean concentration in intestinal contents of pigs with or without LAB and inoculated with virulent Wa HRV. Numbers with different capital letters on top differ significantly at the same time for the same cytokine among groups; numbers with shared letters or without letters do not differ significantly (Kruskal Wallis Test, p<0.05, n=4–10)

■ - LAB+HRV+; - LAB-HRV+; - LAB+HRV−; □ - LAB-HRV−

Figure 3

Cytokine secreting cells (CSC) in intestinal and systemic lymphoid tissues of pigs with or without LAB and infected Wa HRV or mock-infected at PID 28. Data are presented as CSC/ 5×10⁵ MNC. Numbers with different letters differ significantly among groups within the same tissue for the same cytokine; numbers with shared letters or without letters do not differ significantly. (Kruskal-Wallis Test, p<0.05, n=4–10). Legend: ■- LAB+HRV+; - LAB-HRV+; - LAB+HRV−; □ - LAB-HRV−