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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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Absence of CCL20 impacts interleukin (IL)-17 priming and IgA secretion. (a) Levels of IL-23, IL-6, and IL-10 in the Peyer's patch of TLR1−/− or anti-CCL20-treated mice infected orally with Y. enterocolitica. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (b) Levels of IL-17 from isolated LP CD4 T cells re-stimulated with irradiated antigen-presenting cell and Y. enterocolitica lysate. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (c) Fecal anti-Yersinia IgA levels from mice collected at day 14 post infection. Data are pooled from two independent experiments (n=6 mice per group). (d) Messenger RNA (mRNA) levels of IL-6 and IL-23 from CCR6+ and CCR6- CD11c+ sorted Peyer's patch cells 3 days post infection. Fold induction is compared with uninfected controls. Data are pooled from two independent experiments (n=5–6 mice per group). (e) Purified CCR6+ and CCR6- dendritic cells (DCs) were sorted from wild-type mice infected with Y. enterocolitica. The purified DCs were co-cultured with CD4+ ovalbumin-specific T cells (OT2) and ovalbumin. Supernatants were analyzed after 3 days for levels of IL-17 (left). CD4 T cells were sorted from the co-cultures and mRNA level of the transcription factor for rorgt was determined. (f) Levels of IL-17 in co-cultures similar to those in 5e with the addition of neutralizing antibodies to IL-6 and IL-23. Data are the average±s.e.m. pooled from three independent experiments. *P<0.01, **P<0.01 (Student's unpaired t-test).
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fig5: Absence of CCL20 impacts interleukin (IL)-17 priming and IgA secretion. (a) Levels of IL-23, IL-6, and IL-10 in the Peyer's patch of TLR1−/− or anti-CCL20-treated mice infected orally with Y. enterocolitica. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (b) Levels of IL-17 from isolated LP CD4 T cells re-stimulated with irradiated antigen-presenting cell and Y. enterocolitica lysate. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (c) Fecal anti-Yersinia IgA levels from mice collected at day 14 post infection. Data are pooled from two independent experiments (n=6 mice per group). (d) Messenger RNA (mRNA) levels of IL-6 and IL-23 from CCR6+ and CCR6- CD11c+ sorted Peyer's patch cells 3 days post infection. Fold induction is compared with uninfected controls. Data are pooled from two independent experiments (n=5–6 mice per group). (e) Purified CCR6+ and CCR6- dendritic cells (DCs) were sorted from wild-type mice infected with Y. enterocolitica. The purified DCs were co-cultured with CD4+ ovalbumin-specific T cells (OT2) and ovalbumin. Supernatants were analyzed after 3 days for levels of IL-17 (left). CD4 T cells were sorted from the co-cultures and mRNA level of the transcription factor for rorgt was determined. (f) Levels of IL-17 in co-cultures similar to those in 5e with the addition of neutralizing antibodies to IL-6 and IL-23. Data are the average±s.e.m. pooled from three independent experiments. *P<0.01, **P<0.01 (Student's unpaired t-test).

Mentions: In order to demonstrate that CCL20 expression has an impact on TH17 responses, we examined the PP of anti-CCL20-treated or TLR1−/− mice 3 days after oral infection with Y. enterocolitica for cytokines important in the induction and maintenance of TH17 cells. TLR1-deficient mice and mice treated with anti-CCL20 had significantly less IL-6 and IL-23 levels, but similar levels of IL-10, when compared with littermate control-treated mice (Figure 5a). In addition, we observed a significant decrease in Yersinia-specific TH17 cells in the LP 7 days following infection in both TLR1-deficient and anti-CCL20-treated mice (Figure 5b), consistent with the hypothesis that the absence of CCL20 and CCR6+ DCs would lead to an inefficient priming of TH17 cells. Of note, levels of IL-17 are more reduced in TLR1−/− mice compared with anti-CCL20-treated mice. This is likely due to the fact that in the absence of TLR1 there is both a reduction in the trafficking of CCR6+ DC that contribute to TH17 priming and loss of IL-6 and IL-23 production due to lack of direct TLR1 signaling on MLN DC. However, anti-CCL20 treatment reduces the trafficking but disseminated Y. enterocolitica would still be able to stimulate IL-6 and IL-23 from DC in the MLN. Our work23 and others39 have shown that IL-17 is important for the induction of mucosal IgA, which is important for the protection against mucosal pathogenic infections. We evaluated Yersinia-specific fecal IgA in our anti-CCL20-treated mice and found a significant reduction compared with control-treated mice (Figure 5c).


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)

Absence of CCL20 impacts interleukin (IL)-17 priming and IgA secretion. (a) Levels of IL-23, IL-6, and IL-10 in the Peyer's patch of TLR1−/− or anti-CCL20-treated mice infected orally with Y. enterocolitica. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (b) Levels of IL-17 from isolated LP CD4 T cells re-stimulated with irradiated antigen-presenting cell and Y. enterocolitica lysate. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (c) Fecal anti-Yersinia IgA levels from mice collected at day 14 post infection. Data are pooled from two independent experiments (n=6 mice per group). (d) Messenger RNA (mRNA) levels of IL-6 and IL-23 from CCR6+ and CCR6- CD11c+ sorted Peyer's patch cells 3 days post infection. Fold induction is compared with uninfected controls. Data are pooled from two independent experiments (n=5–6 mice per group). (e) Purified CCR6+ and CCR6- dendritic cells (DCs) were sorted from wild-type mice infected with Y. enterocolitica. The purified DCs were co-cultured with CD4+ ovalbumin-specific T cells (OT2) and ovalbumin. Supernatants were analyzed after 3 days for levels of IL-17 (left). CD4 T cells were sorted from the co-cultures and mRNA level of the transcription factor for rorgt was determined. (f) Levels of IL-17 in co-cultures similar to those in 5e with the addition of neutralizing antibodies to IL-6 and IL-23. Data are the average±s.e.m. pooled from three independent experiments. *P<0.01, **P<0.01 (Student's unpaired t-test).
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fig5: Absence of CCL20 impacts interleukin (IL)-17 priming and IgA secretion. (a) Levels of IL-23, IL-6, and IL-10 in the Peyer's patch of TLR1−/− or anti-CCL20-treated mice infected orally with Y. enterocolitica. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (b) Levels of IL-17 from isolated LP CD4 T cells re-stimulated with irradiated antigen-presenting cell and Y. enterocolitica lysate. Data are the mean±s.e.m. pooled from two independent experiments (n=6). (c) Fecal anti-Yersinia IgA levels from mice collected at day 14 post infection. Data are pooled from two independent experiments (n=6 mice per group). (d) Messenger RNA (mRNA) levels of IL-6 and IL-23 from CCR6+ and CCR6- CD11c+ sorted Peyer's patch cells 3 days post infection. Fold induction is compared with uninfected controls. Data are pooled from two independent experiments (n=5–6 mice per group). (e) Purified CCR6+ and CCR6- dendritic cells (DCs) were sorted from wild-type mice infected with Y. enterocolitica. The purified DCs were co-cultured with CD4+ ovalbumin-specific T cells (OT2) and ovalbumin. Supernatants were analyzed after 3 days for levels of IL-17 (left). CD4 T cells were sorted from the co-cultures and mRNA level of the transcription factor for rorgt was determined. (f) Levels of IL-17 in co-cultures similar to those in 5e with the addition of neutralizing antibodies to IL-6 and IL-23. Data are the average±s.e.m. pooled from three independent experiments. *P<0.01, **P<0.01 (Student's unpaired t-test).
Mentions: In order to demonstrate that CCL20 expression has an impact on TH17 responses, we examined the PP of anti-CCL20-treated or TLR1−/− mice 3 days after oral infection with Y. enterocolitica for cytokines important in the induction and maintenance of TH17 cells. TLR1-deficient mice and mice treated with anti-CCL20 had significantly less IL-6 and IL-23 levels, but similar levels of IL-10, when compared with littermate control-treated mice (Figure 5a). In addition, we observed a significant decrease in Yersinia-specific TH17 cells in the LP 7 days following infection in both TLR1-deficient and anti-CCL20-treated mice (Figure 5b), consistent with the hypothesis that the absence of CCL20 and CCR6+ DCs would lead to an inefficient priming of TH17 cells. Of note, levels of IL-17 are more reduced in TLR1−/− mice compared with anti-CCL20-treated mice. This is likely due to the fact that in the absence of TLR1 there is both a reduction in the trafficking of CCR6+ DC that contribute to TH17 priming and loss of IL-6 and IL-23 production due to lack of direct TLR1 signaling on MLN DC. However, anti-CCL20 treatment reduces the trafficking but disseminated Y. enterocolitica would still be able to stimulate IL-6 and IL-23 from DC in the MLN. Our work23 and others39 have shown that IL-17 is important for the induction of mucosal IgA, which is important for the protection against mucosal pathogenic infections. We evaluated Yersinia-specific fecal IgA in our anti-CCL20-treated mice and found a significant reduction compared with control-treated mice (Figure 5c).

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