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

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

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

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

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

Table_1_MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.docx

BackgroundGastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development.MethodsThe expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining.ResultsMiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly.ConclusionsIn conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.</p

Image_2_MiR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.tif

BackgroundGastric cancer (GC) is one of the most malignant and lethal cancers worldwide. Multiple microRNAs (miRNAs) have been identified as key regulators in the progression of GC. However, the underlying pathogenesis that miRNAs govern GC malignancy remains uncertain. Here, we identified a novel miR-585-5p as a key regulator in GC development.MethodsThe expression of miR-585-5p in the context of GC tissue was detected by in situ hybridization for GC tissue microarray and assessed by H-scoring. The gain- and loss-of-function analyses comprised of Cell Counting Kit-8 assay and Transwell invasion and migration assay. The expression of downstream microphthalmia-associated transcription factor (MITF), cyclic AMP-responsive element-binding protein 1 (CREB1) and mitogen-activated protein kinase 1 (MAPK1) were examined by Immunohistochemistry, quantitative real-time PCR and western blot. The direct regulation between miR-585-5p and MITF/CREB1/MAPK1 were predicted by bioinformatic analysis and screened by luciferase reporter assay. The direct transcriptional activation of CREB1 on MITF was verified by luciferase reporter assay, chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSAs). The interaction between MAPK1 and MITF was confirmed by co-immunoprecipitation (Co-IP) and immunofluorescent double-labelled staining.ResultsMiR-585-5p is progressively downregulated in GC tissues and low miR-585-5p levels were strongly associated with poor clinical outcomes. Further gain- and loss-of-function analyses showed that miR-585-5p possesses strong anti-proliferative and anti-metastatic capacities in GC. Follow-up studies indicated that miR-585-5p targets the downstream molecules CREB1 and MAPK1 to regulate the transcriptional and post-translational regulation of MITF, respectively, thus controlling its expression and cancer-promoting activity. MiR-585-5p directly and negatively regulates MITF together with CREB1 and MAPK1. According to bioinformatic analysis, promotor reporter gene assays, ChIP and EMSAs, CREB1 binds to the promotor region to enhance transcriptional expression of MITF. Co-IP and immunofluorescent double-labelled staining confirmed interaction between MAPK1 and MITF. Protein immunoprecipitation revealed that MAPK1 enhances MITF activity via phosphorylation (Ser73). MiR-585-5p can not only inhibit MITF expression directly, but also hinder MITF expression and pro-cancerous activity in a CREB1-/MAPK1-dependent manner indirectly.ConclusionsIn conclusion, this study uncovered miR-585-5p impedes gastric cancer proliferation and metastasis by orchestrating the interactions among CREB1, MAPK1 and MITF.</p