Supplementary Figure S2. Impact of TLR Ligands on Influenza Virus Infectedmice

Supplementary Figure S1. The expression level of MCP-1 mRNA did not differ significantly in the E. faecalis-fed and vehicle groups.

Mice were orally intubated with E. faecalis once daily for 12 consecutive days. MCP-1 gene expression was measured by real-time PCR in peritoneal macrophages.

Supplementary Figure S2. Impact of TLR ligands on influenza virus–infectedmice.

(A) Schematic representation of the experimental setup. After oral intubation of C57BL/6 male mice, PBS (vehicle) or different TLR ligands were administered once daily for 12 consecutive days before experimental infection with influenza A virus (1 × 103plaque-forming units [PFU]). (B) Percent survival was calculated on a daily basis. Survival curves were compared using the Gehan–Breslow–Wilcoxon test. (C) Body weight was measured daily after the experimental infection, and all data were normalized to the initial weight of each mouse. Error bars represent the standard error of the mean (SEM), total number (n) = 5 mice per group, *P0.05, LTAversusvehicle (two-way repeated-measures ANOVA followed by the Bonferroni’s post-hoc test). (D) Lung viral titers were determined at the indicated time points post-infection using the plaque assay (5 mice per group). Error bars represent the SEM. (E, F)Total cell and differential cell counts in bronchoalveolar lavage (BAL)specimens were obtained using standard blood count methods. *P < 0.05, **P < 0.01, ***P < 0.001 (n = 5 mice per group, two-tailedStudent’s t-test). Abbreviations: n.d. = not detected; LTA = lipoteichoic acid.

Supplementary Figure S3. Post-infection oral administration of heat-killed Enterococcus faecalisdoes not protects against enterovirus 71 infection.

(A) Schematic representation of the experimental setup. Ten-day-old ICR mice were orally intubated once daily with PBS (vehicle) or Enterococcus faecalis (E. faecalis) for 7 consecutive days (≥ 5 mice per group) after experimental infection with EV71 (5 × 105plaque-forming units [PFU]). (B and C) Clinical scores and body weight were measured daily after the EV71 infection. Error bars represent the standard error of mean (SEM). Body weight data were normalized to the initial body weight of each mouse. (D) Viral titers in the brain and different tissues were determined in RD cells at the indicated time points after infection by using the plaque assay. Error bars represent the SEM.