Fig 1: Activation of the TLR4 signaling pathway after H. suis infection(A) Tlr4 mRNA levels in the stomachs of H. suis-infected WT, Myd88 KO, Trif KO, and Myd88/Trif DKO mice 6 months after infection were determined with qRT-PCR.(B) Immunohistology of TLR4-expressing cells analyzed with confocal microscopy. Magnification, 100×.(C) Immunohistology of EpCAM-positive cells in the stomachs of H. suis-infected WT mice. Magnification, 100×. Formation of gastric lymphoid follicles is indicated by dotted lines. Representative images are shown.(D–H) C57BL/6J WT mice were infected with H. suis for 6 months and gastric cells were analyzed with flow cytometry. (D) Staining with B220 and CD19 antibodies, followed by staining for TLR4. Right panels show TLR4 staining among B220 + CD19 + cells. (E) Staining with CD4 and TCRβ antibodies, followed by staining for TLR4. Right panels show TLR4 staining among CD4+ TCRβ+ cells. (F) Staining for CD11c and MHC class antibodies, followed by staining for TLR4. Right panels show TLR4 staining among CD11c + MHC class + cells.(G) Staining for FDC M1, followed by staining for TLR4. Right panels show TLR4 staining among FDC M1+ cells. (H) Staining for EpCAM, followed by staining for TLR4. Right panels show TLR4 staining among EpCAM + cells.Ifna (I) and Ifnb (J) mRNA levels in the stomach after H. suis infection were determined with qRT-PCR. (A, I, and J) Data were normalized to β-actin levels.Data are shown as the mean ± SD (n = 5) of three independent experiments. ∗∗p < 0.01, N.S., no significant difference (ANOVA). Ab, antibody; DKO, double knockout. Scale bar, 100 μm.
Fig 2: Activation of TLR4 signaling in gastric epithelial cells after treatment with monophosphoryl lipid A (MPLA)Gastric epithelial cell lines (AGS cells) were treated with 1 μg/mL MPLA for 24 and 48 hr with or without the TLR4 inhibitor peptide (30 μM) 2 hr before MPLA treatment.(A and B) qRT-PCR analysis of TLR4 mRNA levels in AGS cells after treatment with MPLA at 24 and 48 hr (A) and with or without the TLR4 inhibitor at 48 hr (B).(C–E) (C) qRT-PCR analysis of TRIF mRNA levels in AGS cells after treatment with MPLA, with or without the TLR4 inhibitor, after 48 hr qRT-PCR analysis of IFNA (D) and IFNB (E) expression levels in AGS cells at 24 and 48 hr after MPLA treatment.ELISA of IFN-α (F) and IFN-β (G) production in AGS cells at 24 and 48 hr after MPLA treatment.qRT-PCR analysis of IFNA (H) and IFNB (I) mRNA levels at 48 hr after MPLA treatment with or without the TLR4 inhibitor.Data are shown as the mean ± SD (n = 5) of three independent experiments. qRT-PCR data were normalized to β-actin levels ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001, N.S., no significant difference (ANOVA).
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