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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 mucus production by goblet cells surrounding bronchioles.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (PAS) cross-sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset boxes display additional digital magnification of corresponding region outlined in original image and red arrowheads indicate the presence of mucus producing goblet cells as defined by cytoplasm staining fuchsia. (B) Normalized averages (± SEM) of mucus severity scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 30 bronchioles were stained and analyzed as in panel A. 100% mucus severity score correlated with a raw score of 3.3, assessed as described in Materials and Methods. *, p < 0.05; #, p>0.05.
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pone-0063645-g003: R9-QQP specifically inhibits mucus production by goblet cells surrounding bronchioles.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (PAS) cross-sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset boxes display additional digital magnification of corresponding region outlined in original image and red arrowheads indicate the presence of mucus producing goblet cells as defined by cytoplasm staining fuchsia. (B) Normalized averages (± SEM) of mucus severity scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 30 bronchioles were stained and analyzed as in panel A. 100% mucus severity score correlated with a raw score of 3.3, assessed as described in Materials and Methods. *, p < 0.05; #, p>0.05.

Mentions: As mentioned above, the asthmatic response is characterized by mucus accumulation as well as the recruitment of inflammatory infiltrates to the lungs [16]. In the current study, we observed that mucus production was reduced in the bronchi of OVA-allergic mice that were treated with R9-QQP (Figure 3A; compare PC and R9-QQP panels) while mice treated with the control peptide R9-QQA were unaffected (Figure 3A; compare PC and R9-QQA panels). As displayed in Figure 3B, cumulative data from multiple experiments demonstrated that treatment with R9-QQP reduced the mucus severity score by 88% as compared to OVA-allergic mice that were treated with vehicle (PC). In addition, treatment with control peptide R9-QQA did not result in a significant reduction of the mucus severity score for this group of mice.


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 mucus production by goblet cells surrounding bronchioles.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (PAS) cross-sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset boxes display additional digital magnification of corresponding region outlined in original image and red arrowheads indicate the presence of mucus producing goblet cells as defined by cytoplasm staining fuchsia. (B) Normalized averages (± SEM) of mucus severity scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 30 bronchioles were stained and analyzed as in panel A. 100% mucus severity score correlated with a raw score of 3.3, assessed as described in Materials and Methods. *, p < 0.05; #, p>0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646824&req=5

pone-0063645-g003: R9-QQP specifically inhibits mucus production by goblet cells surrounding bronchioles.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (PAS) cross-sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset boxes display additional digital magnification of corresponding region outlined in original image and red arrowheads indicate the presence of mucus producing goblet cells as defined by cytoplasm staining fuchsia. (B) Normalized averages (± SEM) of mucus severity scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 30 bronchioles were stained and analyzed as in panel A. 100% mucus severity score correlated with a raw score of 3.3, assessed as described in Materials and Methods. *, p < 0.05; #, p>0.05.
Mentions: As mentioned above, the asthmatic response is characterized by mucus accumulation as well as the recruitment of inflammatory infiltrates to the lungs [16]. In the current study, we observed that mucus production was reduced in the bronchi of OVA-allergic mice that were treated with R9-QQP (Figure 3A; compare PC and R9-QQP panels) while mice treated with the control peptide R9-QQA were unaffected (Figure 3A; compare PC and R9-QQA panels). As displayed in Figure 3B, cumulative data from multiple experiments demonstrated that treatment with R9-QQP reduced the mucus severity score by 88% as compared to OVA-allergic mice that were treated with vehicle (PC). In addition, treatment with control peptide R9-QQA did not result in a significant reduction of the mucus severity score for this group of mice.

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