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TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica.

Sugiura Y, Kamdar K, Khakpour S, Young G, Karpus WJ, DePaolo RW - Mucosal Immunol (2013)

Bottom Line: Intestinal epithelial cells are a central player in barrier function and also in communicating signals from invading pathogens to the underlying immune tissue.Here we demonstrate that activation of Toll-like receptor 1 (TLR1) in the epithelium leads to the upregulation of the chemokine CCL20 during oral infection with Yersinia enterocolitica.These data demonstrate a novel role for TLR1 signaling in the intestinal epithelium and demonstrate that together TLR1 and CCL20 are critical mediators of TH17 immunity through the activation and recruitment of DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Midwestern University, Downers Grove, Illinois, USA.

ABSTRACT
Our gastrointestinal tract is a portal of entry for a number of bacteria and viruses. Thus, this tissue must develop ways to induce antigen-specific T cell and antibody responses quickly. Intestinal epithelial cells are a central player in barrier function and also in communicating signals from invading pathogens to the underlying immune tissue. Here we demonstrate that activation of Toll-like receptor 1 (TLR1) in the epithelium leads to the upregulation of the chemokine CCL20 during oral infection with Yersinia enterocolitica. Further, both neutralization of CCL20 using polyclonal antibody treatment and deletion of TLR1 resulted in a defect in CCR6+ dendritic cells (DCs), which produce innate cytokines that help to induce anti-Yersinia-specific T helper 17 (TH17) cells and IgA production. These data demonstrate a novel role for TLR1 signaling in the intestinal epithelium and demonstrate that together TLR1 and CCL20 are critical mediators of TH17 immunity through the activation and recruitment of DCs.

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CCL20 is secreted in a Toll-like receptor 1 (TLR1)-dependent manner from intestinal cells. Levels of CCL20 protein (a) and messenger RNA (mRNA; b) from various mucosal tissues 3 days after oral Y. enterocolitica infection. Data are pooled from two independent experiments (n=4–6 mice per group). (c) Levels of CCL20 mRNA in intestinal epithelial cells (IECs; CD13+CD45-) and lamina propria immune cells (LPCs; CD13-CD45+) sorted from wild-type (WT) and TLR1−/− mice. Purified cells were stimulated with TLR2/1 ligand, TLR2/6 ligand, and Y. enterocolitica (Ye). Data are the average from three to four individual mice. (d) Level of CCL20 from Caco-2 cells transfected with control vector (control), WT TLR1 (I602I), or TLR1 containing a single-nucleotide polymorphism (I602S) 18 h after stimulation with TLR ligands or Y. enterocolitica lysate (Ye). Data are pooled from three independent experiments. (n=6). *P<0.05, **P<0.01, ***P<0.001. Student's unpaired t-test. MLN, mesenteric lymph node.
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fig1: CCL20 is secreted in a Toll-like receptor 1 (TLR1)-dependent manner from intestinal cells. Levels of CCL20 protein (a) and messenger RNA (mRNA; b) from various mucosal tissues 3 days after oral Y. enterocolitica infection. Data are pooled from two independent experiments (n=4–6 mice per group). (c) Levels of CCL20 mRNA in intestinal epithelial cells (IECs; CD13+CD45-) and lamina propria immune cells (LPCs; CD13-CD45+) sorted from wild-type (WT) and TLR1−/− mice. Purified cells were stimulated with TLR2/1 ligand, TLR2/6 ligand, and Y. enterocolitica (Ye). Data are the average from three to four individual mice. (d) Level of CCL20 from Caco-2 cells transfected with control vector (control), WT TLR1 (I602I), or TLR1 containing a single-nucleotide polymorphism (I602S) 18 h after stimulation with TLR ligands or Y. enterocolitica lysate (Ye). Data are pooled from three independent experiments. (n=6). *P<0.05, **P<0.01, ***P<0.001. Student's unpaired t-test. MLN, mesenteric lymph node.

Mentions: Our previous study demonstrated that TLR1−/− mice were unable to effectively mount a protective mucosal TH17 response during mucosal infection by Y. enterocolitica. This was due to a defect in the activation of TLR1-deficient DCs through direct contact with Y. enterocolitica, resulting in decreased interleukin (IL)-6 and ultimately decreased TH17 priming.23 Interestingly, TLR1−/− mice also had fewer DCs in the mesenteric lymph node (MLN) 3 days following infection. IECs come in contact with bacteria during infection and secrete CCL20 to attract CCR6+ DC to the site of infection.9 After activation, CCR6+CD11c+ cells downregulate CCR6 and traffic to draining lymph nodes, where they present antigens to naive T cells. We hypothesized that the reduction of DCs observed in the MLN of TLR1−/− mice may be due to a defect in recruitment of DCs to the site of infection. To address the mechanism of DC trafficking in TLR1-deficient mice, CCL20 expression was examined in mucosal tissues 72 h after oral infection with Y. enterocolitica. Y. enterocolitica-induced CCL20 expression was reduced in the IECs and PPs of TLR1−/− mice compared with wild-type littermate controls by both protein (Figure 1a) and messenger RNA (mRNA; Figure 1b). CCL20 expression was not induced in either the lamina propria (LP) or MLN of littermate controls or TLR1-deficient mice (Figure 1a, b). Interestingly, the absence of TLR6, which also forms a heterodimer with TLR2,24, 25 showed no effect on CCL20 production (data not shown), suggesting a specific role for TLR1 in the IEC for CCL20 expression. To show directly that CCL20 was being produced by IECs in a TLR1-dependent manner, IECs and lamina propria immune cells (LPCs) were isolated using expression of CD13 and CD45. IECs (CD13+CD45-) and LPCs (CD13-CD45+) were purified from the ileum of the small intestine from TLR1−/− and wild-type littermate control mice. CCL20 mRNA was measured after stimulation with TLR ligands or Y. enterocolitica. CCL20 transcript was induced by TLR2/1 ligand and Y. enterocolitica stimulation of IEC (Figure 1c, left) but not LPC (Figure 1c, right). These data indicate a TLR1 dependence for CCL20 production in IECs but not LPCs.


TLR1-induced chemokine production is critical for mucosal immunity against Yersinia enterocolitica.

Sugiura Y, Kamdar K, Khakpour S, Young G, Karpus WJ, DePaolo RW - Mucosal Immunol (2013)

CCL20 is secreted in a Toll-like receptor 1 (TLR1)-dependent manner from intestinal cells. Levels of CCL20 protein (a) and messenger RNA (mRNA; b) from various mucosal tissues 3 days after oral Y. enterocolitica infection. Data are pooled from two independent experiments (n=4–6 mice per group). (c) Levels of CCL20 mRNA in intestinal epithelial cells (IECs; CD13+CD45-) and lamina propria immune cells (LPCs; CD13-CD45+) sorted from wild-type (WT) and TLR1−/− mice. Purified cells were stimulated with TLR2/1 ligand, TLR2/6 ligand, and Y. enterocolitica (Ye). Data are the average from three to four individual mice. (d) Level of CCL20 from Caco-2 cells transfected with control vector (control), WT TLR1 (I602I), or TLR1 containing a single-nucleotide polymorphism (I602S) 18 h after stimulation with TLR ligands or Y. enterocolitica lysate (Ye). Data are pooled from three independent experiments. (n=6). *P<0.05, **P<0.01, ***P<0.001. Student's unpaired t-test. MLN, mesenteric lymph node.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3760963&req=5

fig1: CCL20 is secreted in a Toll-like receptor 1 (TLR1)-dependent manner from intestinal cells. Levels of CCL20 protein (a) and messenger RNA (mRNA; b) from various mucosal tissues 3 days after oral Y. enterocolitica infection. Data are pooled from two independent experiments (n=4–6 mice per group). (c) Levels of CCL20 mRNA in intestinal epithelial cells (IECs; CD13+CD45-) and lamina propria immune cells (LPCs; CD13-CD45+) sorted from wild-type (WT) and TLR1−/− mice. Purified cells were stimulated with TLR2/1 ligand, TLR2/6 ligand, and Y. enterocolitica (Ye). Data are the average from three to four individual mice. (d) Level of CCL20 from Caco-2 cells transfected with control vector (control), WT TLR1 (I602I), or TLR1 containing a single-nucleotide polymorphism (I602S) 18 h after stimulation with TLR ligands or Y. enterocolitica lysate (Ye). Data are pooled from three independent experiments. (n=6). *P<0.05, **P<0.01, ***P<0.001. Student's unpaired t-test. MLN, mesenteric lymph node.
Mentions: Our previous study demonstrated that TLR1−/− mice were unable to effectively mount a protective mucosal TH17 response during mucosal infection by Y. enterocolitica. This was due to a defect in the activation of TLR1-deficient DCs through direct contact with Y. enterocolitica, resulting in decreased interleukin (IL)-6 and ultimately decreased TH17 priming.23 Interestingly, TLR1−/− mice also had fewer DCs in the mesenteric lymph node (MLN) 3 days following infection. IECs come in contact with bacteria during infection and secrete CCL20 to attract CCR6+ DC to the site of infection.9 After activation, CCR6+CD11c+ cells downregulate CCR6 and traffic to draining lymph nodes, where they present antigens to naive T cells. We hypothesized that the reduction of DCs observed in the MLN of TLR1−/− mice may be due to a defect in recruitment of DCs to the site of infection. To address the mechanism of DC trafficking in TLR1-deficient mice, CCL20 expression was examined in mucosal tissues 72 h after oral infection with Y. enterocolitica. Y. enterocolitica-induced CCL20 expression was reduced in the IECs and PPs of TLR1−/− mice compared with wild-type littermate controls by both protein (Figure 1a) and messenger RNA (mRNA; Figure 1b). CCL20 expression was not induced in either the lamina propria (LP) or MLN of littermate controls or TLR1-deficient mice (Figure 1a, b). Interestingly, the absence of TLR6, which also forms a heterodimer with TLR2,24, 25 showed no effect on CCL20 production (data not shown), suggesting a specific role for TLR1 in the IEC for CCL20 expression. To show directly that CCL20 was being produced by IECs in a TLR1-dependent manner, IECs and lamina propria immune cells (LPCs) were isolated using expression of CD13 and CD45. IECs (CD13+CD45-) and LPCs (CD13-CD45+) were purified from the ileum of the small intestine from TLR1−/− and wild-type littermate control mice. CCL20 mRNA was measured after stimulation with TLR ligands or Y. enterocolitica. CCL20 transcript was induced by TLR2/1 ligand and Y. enterocolitica stimulation of IEC (Figure 1c, left) but not LPC (Figure 1c, right). These data indicate a TLR1 dependence for CCL20 production in IECs but not LPCs.

Bottom Line: Intestinal epithelial cells are a central player in barrier function and also in communicating signals from invading pathogens to the underlying immune tissue.Here we demonstrate that activation of Toll-like receptor 1 (TLR1) in the epithelium leads to the upregulation of the chemokine CCL20 during oral infection with Yersinia enterocolitica.These data demonstrate a novel role for TLR1 signaling in the intestinal epithelium and demonstrate that together TLR1 and CCL20 are critical mediators of TH17 immunity through the activation and recruitment of DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Midwestern University, Downers Grove, Illinois, USA.

ABSTRACT
Our gastrointestinal tract is a portal of entry for a number of bacteria and viruses. Thus, this tissue must develop ways to induce antigen-specific T cell and antibody responses quickly. Intestinal epithelial cells are a central player in barrier function and also in communicating signals from invading pathogens to the underlying immune tissue. Here we demonstrate that activation of Toll-like receptor 1 (TLR1) in the epithelium leads to the upregulation of the chemokine CCL20 during oral infection with Yersinia enterocolitica. Further, both neutralization of CCL20 using polyclonal antibody treatment and deletion of TLR1 resulted in a defect in CCR6+ dendritic cells (DCs), which produce innate cytokines that help to induce anti-Yersinia-specific T helper 17 (TH17) cells and IgA production. These data demonstrate a novel role for TLR1 signaling in the intestinal epithelium and demonstrate that together TLR1 and CCL20 are critical mediators of TH17 immunity through the activation and recruitment of DCs.

Show MeSH
Related in: MedlinePlus