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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 production is dependent upon Toll-like receptor 1 (TLR1) signaling and not invasion or type III secretion system (T3SS). (a) Number of migrated cells toward supernatants collected from Caco-2 epithelial cells transfected with wild-type (WT) TLR1 (I602I) and stimulated with wild-type Y. enterocolitica (8081), Y. enterocolitica lacking LcrV (ΔlcrV), T3SS (Δysc), or invasin (ΔinvA). Recombinant human CCL20 (rCCL20) and neutralization of CCL20 (aCCL20) were used as controls. Data are the mean±s.e.m. of pooled from three independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test). (b) In vivo production of CCL20 from IECs of TLR1−/− or wild-type littermate control mice infected with 1 × 106Y. enterocolitica or mutants after 72 h. Data are the mean±s.e.m. of six individual mice pooled from two independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test).
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fig2: CCL20 production is dependent upon Toll-like receptor 1 (TLR1) signaling and not invasion or type III secretion system (T3SS). (a) Number of migrated cells toward supernatants collected from Caco-2 epithelial cells transfected with wild-type (WT) TLR1 (I602I) and stimulated with wild-type Y. enterocolitica (8081), Y. enterocolitica lacking LcrV (ΔlcrV), T3SS (Δysc), or invasin (ΔinvA). Recombinant human CCL20 (rCCL20) and neutralization of CCL20 (aCCL20) were used as controls. Data are the mean±s.e.m. of pooled from three independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test). (b) In vivo production of CCL20 from IECs of TLR1−/− or wild-type littermate control mice infected with 1 × 106Y. enterocolitica or mutants after 72 h. Data are the mean±s.e.m. of six individual mice pooled from two independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test).

Mentions: Y. enterocolitica harbors genes required for cellular invasion, such as invA and yadA, or genes encoding two different type III secretion systems (T3SSs; ysa and ysc). These T3SSs inject effector proteins, called Ysps (Yersinia-secreted proteins) and Yops (Yersinia outer proteins), into target cells.32, 33 These effectors have multiple and diverse functions such as impairing cell signaling, inhibiting actin re-arrangement, or inhibiting nuclear factor-κB activation.34 Yops have also been shown to activate intracellular immune pathways.35 A specific effector protein of the Ysc T3SS, LcrV, has been shown to be essential for immune-evasion via IL-10 production36 and is also critical for the formation of the Ysc T3SS needle.37 In order to determine whether invasion or the T3SS are important for the induction of CCL20, Caco-2 cells were transfected with wild-type TLR1 (I602I) and the production of CCL20 was measured after stimulation with Y. enterocolitica or deletion mutants. The supernatants were collected and used in a chemotaxis assay with differentiated human CD34+ DC to determine whether chemo-attraction occurred in a CCL20-dependent manner. Supernatants from TLR1 I602I-expressing IECs stimulated with wild-type Y. enterocolitica (8081) were able to induce the migration of cells similar to that of recombinant human CCL20 (rCCL20). However, the addition of neutralizing CCL20 antibody to the supernatant almost completely inhibited the migration of DCs, confirming a specific role for CCL20 in this migration (Figure 2a). Supernatants from IECs expressing wild-type TLR1 induced a CCL20-dependent migration that proved to be independent of invasion, type III secretion, and LcrV, as each deletion mutant was able to stimulate CCL20 expression equal to wild-type Y. enterocolitica (Figure 2a). TLR5 signaling by flagellin has been shown to induce CCL20 expression11, 29 and may account for the further reduction seen in I602S-transfected Caco-2 cells treated with anti-CCL20. To determine whether invasion or type III secretion was necessary for the in vivo expression of CCL20, we isolated IECs from TLR1−/− or littermate control mice infected with Y. enterocolitica (8081) or the deletion mutants. CCL20 was equally expressed in the IEC from wild-type Y. enterocolitica as well as the ΔlcrV, Δysc, and ΔinvA mutants (Figure 2b). This was not due to the attenuation of the strains as increasing the dose by 10- and 100-fold still had no effect on CCL20 production (data not shown). In accordance with our data in Figure 1a, TLR1-deficient mice had a defect in CCL20 production from IEC and this was not altered by infection with the various mutants (Figure 2b). These data demonstrate that while invasion and type III secretion effector proteins do not contribute to the production of CCL20, TLR1 is critical for its induction.


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 production is dependent upon Toll-like receptor 1 (TLR1) signaling and not invasion or type III secretion system (T3SS). (a) Number of migrated cells toward supernatants collected from Caco-2 epithelial cells transfected with wild-type (WT) TLR1 (I602I) and stimulated with wild-type Y. enterocolitica (8081), Y. enterocolitica lacking LcrV (ΔlcrV), T3SS (Δysc), or invasin (ΔinvA). Recombinant human CCL20 (rCCL20) and neutralization of CCL20 (aCCL20) were used as controls. Data are the mean±s.e.m. of pooled from three independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test). (b) In vivo production of CCL20 from IECs of TLR1−/− or wild-type littermate control mice infected with 1 × 106Y. enterocolitica or mutants after 72 h. Data are the mean±s.e.m. of six individual mice pooled from two independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test).
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fig2: CCL20 production is dependent upon Toll-like receptor 1 (TLR1) signaling and not invasion or type III secretion system (T3SS). (a) Number of migrated cells toward supernatants collected from Caco-2 epithelial cells transfected with wild-type (WT) TLR1 (I602I) and stimulated with wild-type Y. enterocolitica (8081), Y. enterocolitica lacking LcrV (ΔlcrV), T3SS (Δysc), or invasin (ΔinvA). Recombinant human CCL20 (rCCL20) and neutralization of CCL20 (aCCL20) were used as controls. Data are the mean±s.e.m. of pooled from three independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test). (b) In vivo production of CCL20 from IECs of TLR1−/− or wild-type littermate control mice infected with 1 × 106Y. enterocolitica or mutants after 72 h. Data are the mean±s.e.m. of six individual mice pooled from two independent experiments. *P<0.01, **P<0.001 (Student's unpaired t-test).
Mentions: Y. enterocolitica harbors genes required for cellular invasion, such as invA and yadA, or genes encoding two different type III secretion systems (T3SSs; ysa and ysc). These T3SSs inject effector proteins, called Ysps (Yersinia-secreted proteins) and Yops (Yersinia outer proteins), into target cells.32, 33 These effectors have multiple and diverse functions such as impairing cell signaling, inhibiting actin re-arrangement, or inhibiting nuclear factor-κB activation.34 Yops have also been shown to activate intracellular immune pathways.35 A specific effector protein of the Ysc T3SS, LcrV, has been shown to be essential for immune-evasion via IL-10 production36 and is also critical for the formation of the Ysc T3SS needle.37 In order to determine whether invasion or the T3SS are important for the induction of CCL20, Caco-2 cells were transfected with wild-type TLR1 (I602I) and the production of CCL20 was measured after stimulation with Y. enterocolitica or deletion mutants. The supernatants were collected and used in a chemotaxis assay with differentiated human CD34+ DC to determine whether chemo-attraction occurred in a CCL20-dependent manner. Supernatants from TLR1 I602I-expressing IECs stimulated with wild-type Y. enterocolitica (8081) were able to induce the migration of cells similar to that of recombinant human CCL20 (rCCL20). However, the addition of neutralizing CCL20 antibody to the supernatant almost completely inhibited the migration of DCs, confirming a specific role for CCL20 in this migration (Figure 2a). Supernatants from IECs expressing wild-type TLR1 induced a CCL20-dependent migration that proved to be independent of invasion, type III secretion, and LcrV, as each deletion mutant was able to stimulate CCL20 expression equal to wild-type Y. enterocolitica (Figure 2a). TLR5 signaling by flagellin has been shown to induce CCL20 expression11, 29 and may account for the further reduction seen in I602S-transfected Caco-2 cells treated with anti-CCL20. To determine whether invasion or type III secretion was necessary for the in vivo expression of CCL20, we isolated IECs from TLR1−/− or littermate control mice infected with Y. enterocolitica (8081) or the deletion mutants. CCL20 was equally expressed in the IEC from wild-type Y. enterocolitica as well as the ΔlcrV, Δysc, and ΔinvA mutants (Figure 2b). This was not due to the attenuation of the strains as increasing the dose by 10- and 100-fold still had no effect on CCL20 production (data not shown). In accordance with our data in Figure 1a, TLR1-deficient mice had a defect in CCL20 production from IEC and this was not altered by infection with the various mutants (Figure 2b). These data demonstrate that while invasion and type III secretion effector proteins do not contribute to the production of CCL20, TLR1 is critical for its induction.

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