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. 2009 Feb 15;127(3-4):304-15.
doi: 10.1016/j.vetimm.2008.10.322. Epub 2008 Oct 31.

Toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs

Ke Wen  1 , Marli S P Azevedo, Ana Gonzalez, Wei Zhang, Linda J Saif, Guohua Li, Ahmed Yousef, Lijuan Yuan
Affiliations

Toll-like receptor and innate cytokine responses induced by lactobacilli colonization and human rotavirus infection in gnotobiotic pigs

Ke Wen et al. Vet Immunol Immunopathol. .

Abstract

Toll-like receptors (TLR) play an important role in the recognition of microbes by host sentinel cells that leads to the subsequent innate and adaptive immune responses. In this study, we evaluated the patterns of TLR2-, TLR3- and TLR9-expressing antigen presenting cells (APCs) in spleen and blood of gnotobiotic (Gn) pigs after colonization with a mixture of two strains of lactic acid bacteria (LAB), Lactobacillus acidophilus and Lactobacillus reuteri or infection with the virulent human rotavirus (HRV) Wa strain. We also assessed the influence of LAB on TLR and serum innate cytokine responses induced by HRV. Distributions of subpopulations of APCs [CD14+/-SWC3+CD11R1- monocytes/macrophages and CD14+/-SWC3+CD11R1+ conventional dendritic cells (cDCs)] were described in our previous report (Zhang, W., Wen, K., Azevedo, M.S., Gonzalez, A.M., Saif, L.J., Li, G., Yousef, A.E., Yuan, L., 2008. Lactic acid bacterial colonization and human rotavirus infection influence distribution and frequencies of monocytes/macrophages and dendritic cells in neonatal gnotobiotic pigs. Vet. Immunol. Immunopathol. 121, pp. 222-231). We demonstrated that LAB induced strong TLR2-expressing APC responses in blood and spleen, HRV induced a TLR3 response in spleen, and TLR9 responses were induced by either HRV (in spleen) or LAB (in blood). LAB and HRV have an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of LAB. Overall, the frequencies of TLR-expressing CD14+ APCs were higher than CD14- APCs. LAB enhanced the IFN-gamma and IL-4 responses in serum, but it had a suppressive effect on the TLR3- and TLR9-expressing CD14- APC responses in spleen and the serum IFN-alpha response induced by HRV. These results elucidated the systemic TLR2-, TLR3-, and TLR9-expressing monocyte/macrophage and cDC responses after HRV infection, LAB colonization, and the two combined. Our findings facilitate the understanding of the mechanism of LAB's adjuvant effect on rotavirus vaccines and the diverse innate and adaptive immune responses induced by commensal LAB colonization versus rotavirus infection and the interactions between them.

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Figures

Fig. 1
Fig. 1
Comparisons between anti-human and anti-pig TLR antibodies for detection of TLR2 and TLR9 expression in pig blood monocytes. The frequencies of SWC3+TLR2+ and SWC3+TLR9+ monocytes detected by the different TLR antibodies are labeled on each dot plot. Dot plots show the representative of results from three pigs. Blood MNCs isolated from three normal conventional pigs were stimulated in vitro with CpG OND D19 (1 μM) (Watarai et al., 2008) or Bacillus subtilis peptidoglycan (10 μg/ml) (Yamamoto et al., 2003) for 48 h before staining. The MNCs were stained with SWC3a-biotin and streptavidin-PerCP and followed by the PE conjugated anti-human TLR2 and TLR9, respectively (see Section 2) or followed by the rabbit anti-pig TLR2 and TLR9 polyclonal antibodies (Cosmo Bio Co., Japan) respectively, and then a PE conjugated goat anti-rabbit IgG (Open Biosystems, Huntsville, AL).
Fig. 2
Fig. 2
Example of frequencies of TLR3-expressing splenic monocytes/macrophages of Gn pigs from each treatment group. Treatment group is labeled on each dot plot: LAB+HRV+, pigs were inoculated with LAB and virulent Wa strain HRV; LAB−HRV+, pigs were inoculated with HRV only; LAB+HRV−, pigs were inoculated with LAB only; and LAB−HRV−, pigs were mock inoculated. Monocytes/macrophages were defined as CD14+/−SWC3+CD11R1− (Zhang et al., 2008c). CD14/TLR3 dot plots were performed within the SWC3+CD11R1− subpopulation. The dot plot at lower left corner shows a TLR3 isotype control staining which included antibodies to all the cell markers (SWC3, CD11R1 and CD14), except that the antibodies to TLR3 were replaced by the isotype-matched antigen-irrelevant control antibodies.
Fig. 3
Fig. 3
Frequencies of TLR2-, TLR3-, and TLR9-expressing CD14+ and CD14− APCs in spleen of Gn pigs at PID 5. Gn pigs were inoculated with LAB and virulent Wa strain HRV (LAB+HRV+), HRV only (LAB−HRV+), LAB only (LAB+HRV−) or mock (LAB−HRV−). The y-axis is the mean frequencies (%) of CD14+ or CD14− TLR-expressing cells among monocytes/macrophages or cDCs. Note the difference in y-axis scales between CD14+ and CD14− APCs. The error bars represent standard error of the mean (n = 4). The letters A, B, and C indicate the results of significance testing for difference between treatments. Unshared letters indicate significant difference between treatment groups on frequencies of the TLR-expressing APCs (Kruskal–Wallis rank sum test, p < 0.05), while shared letters indicate no significant difference.
Fig. 4
Fig. 4
Frequencies of TLR2-, TLR3-, and TLR9-expressing CD14+ and CD14− APCs in blood of Gn pigs at PID 5. Gn pigs were inoculated with LAB and virulent Wa strain HRV (LAB+HRV+), HRV only (LAB−HRV+), LAB only (LAB+HRV−) or mock (LAB−HRV−). The y-axis is the mean frequencies (%) of CD14+ or CD14− TLR-expressing cells among monocytes/macrophages or cDCs. Note the difference in y-axis scales between CD14+ and CD14− TLR2-expressing APCs. The error bars represent standard error of the mean (n = 4). For letters A, B, C, see figure legend for Fig. 3.
Fig. 5
Fig. 5
Cytokine levels in serum of Gn pigs. Gn pigs were inoculated with LAB and virulent Wa strain HRV (LAB+HRV+), HRV only (LAB−HRV+), LAB only (LAB+HRV−) or mock (LAB−HRV−). Cytokine concentrations were measured by ELISA on PID 0, PID 2, and PID 5. The error bars represent standard error of the mean. For letters A, B, C, see figure legend for Fig. 3.
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