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Regulation of immune responsiveness in vivo by disrupting an early T-cell signaling event using a cell-permeable peptide.

Guimond DM, Cam NR, Hirve N, Duan W, Lambris JD, Croft M, Tsoukas CD - PLoS ONE (2013)

Bottom Line: We found that mice treated with R9-QQP and sensitized and challenged with the surrogate allergen ovalbumin (OVA) display significant inhibition of lung inflammation in a peptide-specific manner.Thus, parameters of the allergic response, such as airway hyper-responsiveness, suppression of inflammatory cell infiltration, reduction of bronchial mucus accumulation, and production of relevant cytokines from draining lymph nodes were significantly suppressed.Furthermore, the experimental approach of intracellular peptide-mediated inhibition might be applicable to the study of other important intracellular interactions thus providing a paradigm for dissecting signal transduction pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.

ABSTRACT
The inducible T cell kinase (ITK) regulates type 2 (Th2) cytokines that provide defense against certain parasitic and bacterial infections and are involved in the pathogenesis of lung inflammation such as allergic asthma. Activation of ITK requires the interaction of its SH3 domain with the poly-proline region of its signaling partner, the SH2 domain containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). The specific disruption of the ITK-SH3/SLP-76 poly-proline interaction in vitro by a cell-permeable competitive inhibitor peptide (R9-QQP) interferes with the activation of ITK and the transduction of its cellular functions in T lymphocytes. In the present investigation, we assessed the effects of R9-QQP treatment on the induction of an in vivo immune response as represented by lung inflammation in a murine model of allergic asthma. We found that mice treated with R9-QQP and sensitized and challenged with the surrogate allergen ovalbumin (OVA) display significant inhibition of lung inflammation in a peptide-specific manner. Thus, parameters of the allergic response, such as airway hyper-responsiveness, suppression of inflammatory cell infiltration, reduction of bronchial mucus accumulation, and production of relevant cytokines from draining lymph nodes were significantly suppressed. These findings represent the first demonstration of the biological significance of the interaction between ITK and SLP-76 in the induction of an immune response in a whole animal model and specifically underscore the significance of the ITK-SH3 domain interaction with the poly-proline region of SLP-76 in the development of an inflammatory response. Furthermore, the experimental approach of intracellular peptide-mediated inhibition might be applicable to the study of other important intracellular interactions thus providing a paradigm for dissecting signal transduction pathways.

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R9-QQP specifically inhibits eosinophilia.(A) Representative flow cytometric density plots obtained by specific antibody staining of BAL fluid cells from mice treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box at bottom of graph outlines staining and analysis strategy: CD45+ cells gated and analyzed for Siglec-F and CD11c expression as described in Materials and Methods. Gates depict eosinophils and macrophages as percentages of total CD45+ cells. (B,C) Average (± SEM) number of eosinophils and macrophages respectively, calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (A). (D) Separate aliquots of BAL fluid cells from the experiment depicted in panel (A) analyzed as indicated in the inset box at the bottom of the graph: CD45+ cells gated by scatter and analyzed for CD3ε+ cells as described in Materials and Methods. Gates depict T lymphocytes as percentage of total CD45+ cells. (E) Average (± SEM) number of T cells calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (D). Axes in all histograms are displayed on a log scale for all fluorescence channels and on a linear scale for FSC. **, p < 0.0001; #, p>0.05; *, p<0.005.
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pone-0063645-g005: R9-QQP specifically inhibits eosinophilia.(A) Representative flow cytometric density plots obtained by specific antibody staining of BAL fluid cells from mice treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box at bottom of graph outlines staining and analysis strategy: CD45+ cells gated and analyzed for Siglec-F and CD11c expression as described in Materials and Methods. Gates depict eosinophils and macrophages as percentages of total CD45+ cells. (B,C) Average (± SEM) number of eosinophils and macrophages respectively, calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (A). (D) Separate aliquots of BAL fluid cells from the experiment depicted in panel (A) analyzed as indicated in the inset box at the bottom of the graph: CD45+ cells gated by scatter and analyzed for CD3ε+ cells as described in Materials and Methods. Gates depict T lymphocytes as percentage of total CD45+ cells. (E) Average (± SEM) number of T cells calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (D). Axes in all histograms are displayed on a log scale for all fluorescence channels and on a linear scale for FSC. **, p < 0.0001; #, p>0.05; *, p<0.005.

Mentions: In order to more precisely characterize as well as quantitate the cellular phenotype and frequency of infiltrating cells, respectively, we analyzed BAL fluid and lung cells by multi-color flow cytometry utilizing a staining approach previously reported by Stevens et al [14]. In these experiments, CD45-positive leukocytes that were stained for Siglec-F and CD11c allowed for the identification of eosinophils as defined by SiglecF-positive and CD11c-negative expression of these cell surface markers (Figure 5A). Asthmatic mice treated with R9-QQP displayed an almost two-fold decrease in the relative percentage of eosinophils found in BAL fluids (Figure 5A, compare PC and R9-QQP panels). For a more quantitative assessment of this decrease, we calculated the total numbers of eosinophils for multiple experiments using the indicated numbers of mice (Figure 5B). Compared to non-peptide treated animals (PC bar), R9-QQP treatment resulted in a 73% decrease in total eosinophils (Figure 5B). This significant reduction (p<0.05) was peptide specific, as it was not seen when mice were treated with the control peptide R9-QQA (Figure 5B). Furthermore, the same effect on eosinophilia was observed when cells isolated from lung tissue were analyzed with the same markers as above (data not shown). In contrast, the total numbers of alveolar macrophages were not affected upon R9-QQP treatment (Figure 5C). Using a similar multicolor FACS-based approach, the percentage of T cells was measured from BAL cells that were stained for expression of CD45 and the TCR/CD3ε (Figure 5D). The effect of R9-QQP treatment on infiltrating T cells mirrored that of eosinophils. That is, enumeration of total T cell numbers in BAL fluids showed a reduction of 63% upon R9-QQP treatment that was significant at p<0.05 (Figure 5E). This effect was specific for R9-QQP, as treatment with the control peptide R9-QQA had no significant effect on T cell infiltration (Figure 5E).


Regulation of immune responsiveness in vivo by disrupting an early T-cell signaling event using a cell-permeable peptide.

Guimond DM, Cam NR, Hirve N, Duan W, Lambris JD, Croft M, Tsoukas CD - PLoS ONE (2013)

R9-QQP specifically inhibits eosinophilia.(A) Representative flow cytometric density plots obtained by specific antibody staining of BAL fluid cells from mice treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box at bottom of graph outlines staining and analysis strategy: CD45+ cells gated and analyzed for Siglec-F and CD11c expression as described in Materials and Methods. Gates depict eosinophils and macrophages as percentages of total CD45+ cells. (B,C) Average (± SEM) number of eosinophils and macrophages respectively, calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (A). (D) Separate aliquots of BAL fluid cells from the experiment depicted in panel (A) analyzed as indicated in the inset box at the bottom of the graph: CD45+ cells gated by scatter and analyzed for CD3ε+ cells as described in Materials and Methods. Gates depict T lymphocytes as percentage of total CD45+ cells. (E) Average (± SEM) number of T cells calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (D). Axes in all histograms are displayed on a log scale for all fluorescence channels and on a linear scale for FSC. **, p < 0.0001; #, p>0.05; *, p<0.005.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646824&req=5

pone-0063645-g005: R9-QQP specifically inhibits eosinophilia.(A) Representative flow cytometric density plots obtained by specific antibody staining of BAL fluid cells from mice treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box at bottom of graph outlines staining and analysis strategy: CD45+ cells gated and analyzed for Siglec-F and CD11c expression as described in Materials and Methods. Gates depict eosinophils and macrophages as percentages of total CD45+ cells. (B,C) Average (± SEM) number of eosinophils and macrophages respectively, calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (A). (D) Separate aliquots of BAL fluid cells from the experiment depicted in panel (A) analyzed as indicated in the inset box at the bottom of the graph: CD45+ cells gated by scatter and analyzed for CD3ε+ cells as described in Materials and Methods. Gates depict T lymphocytes as percentage of total CD45+ cells. (E) Average (± SEM) number of T cells calculated as described in Materials and Methods for the indicated number (n) of mice that had been treated as in panel (D). Axes in all histograms are displayed on a log scale for all fluorescence channels and on a linear scale for FSC. **, p < 0.0001; #, p>0.05; *, p<0.005.
Mentions: In order to more precisely characterize as well as quantitate the cellular phenotype and frequency of infiltrating cells, respectively, we analyzed BAL fluid and lung cells by multi-color flow cytometry utilizing a staining approach previously reported by Stevens et al [14]. In these experiments, CD45-positive leukocytes that were stained for Siglec-F and CD11c allowed for the identification of eosinophils as defined by SiglecF-positive and CD11c-negative expression of these cell surface markers (Figure 5A). Asthmatic mice treated with R9-QQP displayed an almost two-fold decrease in the relative percentage of eosinophils found in BAL fluids (Figure 5A, compare PC and R9-QQP panels). For a more quantitative assessment of this decrease, we calculated the total numbers of eosinophils for multiple experiments using the indicated numbers of mice (Figure 5B). Compared to non-peptide treated animals (PC bar), R9-QQP treatment resulted in a 73% decrease in total eosinophils (Figure 5B). This significant reduction (p<0.05) was peptide specific, as it was not seen when mice were treated with the control peptide R9-QQA (Figure 5B). Furthermore, the same effect on eosinophilia was observed when cells isolated from lung tissue were analyzed with the same markers as above (data not shown). In contrast, the total numbers of alveolar macrophages were not affected upon R9-QQP treatment (Figure 5C). Using a similar multicolor FACS-based approach, the percentage of T cells was measured from BAL cells that were stained for expression of CD45 and the TCR/CD3ε (Figure 5D). The effect of R9-QQP treatment on infiltrating T cells mirrored that of eosinophils. That is, enumeration of total T cell numbers in BAL fluids showed a reduction of 63% upon R9-QQP treatment that was significant at p<0.05 (Figure 5E). This effect was specific for R9-QQP, as treatment with the control peptide R9-QQA had no significant effect on T cell infiltration (Figure 5E).

Bottom Line: We found that mice treated with R9-QQP and sensitized and challenged with the surrogate allergen ovalbumin (OVA) display significant inhibition of lung inflammation in a peptide-specific manner.Thus, parameters of the allergic response, such as airway hyper-responsiveness, suppression of inflammatory cell infiltration, reduction of bronchial mucus accumulation, and production of relevant cytokines from draining lymph nodes were significantly suppressed.Furthermore, the experimental approach of intracellular peptide-mediated inhibition might be applicable to the study of other important intracellular interactions thus providing a paradigm for dissecting signal transduction pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Microbial Sciences, San Diego State University, San Diego, California, United States of America.

ABSTRACT
The inducible T cell kinase (ITK) regulates type 2 (Th2) cytokines that provide defense against certain parasitic and bacterial infections and are involved in the pathogenesis of lung inflammation such as allergic asthma. Activation of ITK requires the interaction of its SH3 domain with the poly-proline region of its signaling partner, the SH2 domain containing leukocyte phosphoprotein of 76 kilodaltons (SLP-76). The specific disruption of the ITK-SH3/SLP-76 poly-proline interaction in vitro by a cell-permeable competitive inhibitor peptide (R9-QQP) interferes with the activation of ITK and the transduction of its cellular functions in T lymphocytes. In the present investigation, we assessed the effects of R9-QQP treatment on the induction of an in vivo immune response as represented by lung inflammation in a murine model of allergic asthma. We found that mice treated with R9-QQP and sensitized and challenged with the surrogate allergen ovalbumin (OVA) display significant inhibition of lung inflammation in a peptide-specific manner. Thus, parameters of the allergic response, such as airway hyper-responsiveness, suppression of inflammatory cell infiltration, reduction of bronchial mucus accumulation, and production of relevant cytokines from draining lymph nodes were significantly suppressed. These findings represent the first demonstration of the biological significance of the interaction between ITK and SLP-76 in the induction of an immune response in a whole animal model and specifically underscore the significance of the ITK-SH3 domain interaction with the poly-proline region of SLP-76 in the development of an inflammatory response. Furthermore, the experimental approach of intracellular peptide-mediated inhibition might be applicable to the study of other important intracellular interactions thus providing a paradigm for dissecting signal transduction pathways.

Show MeSH
Related in: MedlinePlus