Expression of Th1, Th17, and Th2 cytokines in the gastric mucosa after <i>H</i>. <i>pylori</i> infection.

<p>(A) The expression of Th1 and Th17 with TLR4 blocked; (B) The expression of Th1 and Th17 with CD25 blocked; (C) The expression of Th2 with TLR4 blocked; (D) The expression of Th2 with CD25 blocked. ^a<i>P</i> < 0.05–0.001 <i>vs</i>. the control or TLR4 blocked control groups; ^b<i>P</i> < 0.001–0.05 between TLR4 blocked <i>H</i>. <i>pylori</i> and <i>H</i>. <i>pylori</i> groups (Fig 3A); ^a<i>P</i> < 0.001 <i>vs</i>. the control and CD25 blocked control groups; ^b<i>P</i> < 0.001–0.05 between CD25 blocked <i>H</i>. <i>pylori</i> and <i>H</i>. <i>pylori</i> groups (Fig 3B). ^a<i>P</i> < 0.01 <i>vs</i>. control and TLR4 blocked control groups (Fig 3C); ^a<i>P</i> < 0.01–0.001 <i>vs</i>. control and CD25 blocked control groups; ^b<i>P</i> < 0.05 between CD25 blocked and non-blocked <i>H</i>. <i>pylori</i> groups (Fig 3D).</p

Association of TLR4 and Treg in <i>Helicobacter pylori</i> Colonization and Inflammation in Mice

<div><p>The host immune response plays an important role in the pathogenesis of <i>Helicobacter pylori</i> infection. The aim of this study was to clarify the immune pathogenic mechanism of <i>Helicobacter pylori</i> infection via TLR signaling and gastric mucosal Treg cells in mice. To discover the underlying mechanism, we selectively blocked the TLR signaling pathway and subpopulations of regulatory T cells in the gastric mucosa of mice, and examined the consequences on <i>H</i>. <i>pylori</i> infection and inflammatory response as measured by MyD88, NF-κB p65, and Foxp3 protein expression levels and the levels of Th1, Th17 and Th2 cytokines in the gastric mucosa. We determined that blocking TLR4 signaling in <i>H</i>. <i>pylori</i> infected mice decreased the numbers of Th1 and Th17 Treg cells compared to controls (P < 0.001–0.05), depressed the immune response as measured by inflammatory grade (P < 0.05), and enhanced <i>H</i>. <i>pylori</i> colonization (P < 0.05). In contrast, blocking CD25 had the opposite effects, wherein the Th1 and Th17 cell numbers were increased (P < 0.001–0.05), immune response was enhanced (P < 0.05), and <i>H</i>. <i>pylori</i> colonization was inhibited (P < 0.05) compared to the non-blocked group. In both blocked groups, the Th2 cytokine IL-4 remained unchanged, although IL-10 in the CD25 blocked group was significantly decreased (P < 0.05). Furthermore, MyD88, NF-κB p65, and Foxp3 in the non-blocked group were significantly lower than those in the TLR4 blocked group (P < 0.05), but significantly higher than those of the CD25 blocked group (P < 0.05). Together, these results suggest that there might be an interaction between TLR signaling and Treg cells that is important for limiting <i>H</i>. <i>pylori</i> colonization and suppressing the inflammatory response of infected mice.</p></div

Grade of gastritis after <i>H</i>. <i>pylori</i> infection.

<p>(A) The grade of gastritis with TLR4 blocked; (B) The grade of gastritis with CD25 blocked; (C) HE staining of the gastric mucosa of the control group; (D) HE staining of the gastric mucosa of the <i>H</i>. <i>pylori</i> infection group; (E) HE staining of the gastric mucosa of the TLR4 blocked control group; (F) HE staining of the gastric mucosa of the TLR4 blocked <i>H</i>. <i>pylori</i> infection group; (G) HE staining of the gastric mucosa of the CD25 blocked control group; (H) HE staining of the gastric mucosa of the CD25 blocked <i>H</i>. <i>pylori</i> infection group. ^a<i>P</i> < 0.001 between <i>H</i>. <i>pylori</i> and control or TLR4 blocked control groups; ^b<i>P</i> < 0.01 between TLR4 blocked <i>H</i>. <i>pylori</i> and control or TLR4 blocked control groups; ^c<i>P</i> < 0.05 between <i>H</i>. <i>pylori</i> and TLR4 blocked <i>H</i>. <i>pylori</i> groups (Fig 2A). ^a<i>P</i> < 0.001 <i>vs</i>. control and CD25 blocked control groups; ^b<i>P</i> < 0.05 between <i>H</i>. <i>pylori</i> and CD25 blocked <i>H</i>. <i>pylori</i> groups (Fig 2B).</p

Expression of MyD88 and Foxp3 in the gastric mucosa after <i>H</i>. <i>pylori</i> infection by western blot.

<p>(A) The expression of MyD88 and Foxp3 with TLR4 blocked; (B) The expression of MyD88 and Foxp3 with CD25 blocked; (C, D) The expression of MyD88 and Foxp3 by western blotting. ^a<i>P</i> < 0.001 <i>vs</i>. control and TLR4 blocked control groups; ^b<i>P</i> < 0.001–0.05 <i>vs</i>. control and TLR4 blocked control groups and the <i>H</i>. <i>pylori</i> group (Fig 5A). ^a<i>P</i> < 0.01–0.001 <i>vs</i>. control and CD25 blocked control groups; ^b<i>P</i> < 0.05 between CD25 blocked <i>H</i>. <i>pylori</i> and <i>H</i>. <i>pylori</i> groups (Fig 5B).</p

Western blot detection conditions for MyD88 and Foxp3.

<p>Western blot detection conditions for MyD88 and Foxp3.</p

Expression of MyD88, NF-κB p65, and Foxp3 in the gastric mucosa by immunohistochemistry after <i>H</i>. <i>pylori</i> infection.

<p>Expression of MyD88, NF-κB p65, and Foxp3 with TLR4 (A) or CD25 (B) blocked; (C-T) Representative images of immunohistochemical staining. MyD88 staining in the gastric mucosa of the untreated (C, D), TLR4 blocked (E, F), or CD25 blocked (G, H) control and <i>H</i>. <i>pylori</i> infection groups; NF-κB p65 staining of the gastric mucosa of the untreated (I, J), TLR4 blocked (K, L), or CD25 blocked (M, N) control and <i>H</i>. <i>pylori</i> infection groups; Foxp3 staining of the gastric mucosa of the untreated (O, P), TLR4 blocked (Q, R), or CD25 blocked (S, T) control and <i>H</i>. <i>pylori</i> infection groups. ^a<i>P</i> < 0.001 <i>vs</i>. control or TLR4 blocked control groups; ^b<i>P</i> < 0.001–0.05 <i>vs</i>. control and TLR4 blocked control groups and the <i>H</i>. <i>pylori</i> group (Fig 4A). ^a<i>P</i> < 0.001–0.01 <i>vs</i>. control and CD25 blocked control groups; ^b<i>P</i> < 0.01–0.05 between CD25 blocked <i>H</i>. <i>pylori</i> and <i>H</i>. <i>pylori</i> groups (Fig 4B).</p

<i>H</i>. <i>pylori</i> colonization score in the gastric mucosa after infection.

<p>(A) <i>H</i>. <i>pylori</i> colonization score in the gastric mucosa with TLR4 blocked; (B) <i>H</i>. <i>pylori</i> colonization score in the gastric mucosa with CD25 blocked; (C) Giemsa staining of the gastric mucosa of the control group; (D) Giemsa staining of the gastric mucosa of the <i>H</i>. <i>pylori</i> infection group; (E) Giemsa staining of the gastric mucosa of the TLR4 blocked control group; (F) Giemsa staining of the gastric mucosa of the TLR4 blocked <i>H</i>. <i>pylori</i> infection group; (G) Giemsa staining of the gastric mucosa of the CD25 blocked control group; (H) Giemsa staining of the gastric mucosa of the CD25 blocked <i>H</i>. <i>pylori</i> infection group. ^a<i>P</i> < 0.001 between <i>H</i>. <i>pylori</i> and control or TLR4 blocked control groups; ^b<i>P</i>< 0.05 between <i>H</i>. <i>pylori</i> and TLR4 blocked <i>H</i>. <i>pylori</i> groups (Fig 1A). ^a<i>P</i> < 0.001 <i>vs</i>. control or CD25 blocked control groups; ^b<i>P</i> < 0.05 between <i>H</i>. <i>pylori</i> and CD25 blocked <i>H</i>. <i>pylori</i> groups (Fig 1B).</p