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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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Mice treated with R9-QQP display reduced lung inflammation.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (Hematoxylin and Eosin) cross sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box in upper right corner of each micrograph displays additional digital magnification of corresponding region outlined in original image. Peribronchial inflammatory cells are identified by dark purple staining of nuclei and are indicated by red arrowheads. (B) Normalized averages (± SEM) of inflammation scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 45 bronchioles and blood vessels were stained and analyzed as in (A). 100% inflammation score correlated with a raw score of 2.3, assessed as described in Materials and Methods. (C) Representative micrographs (400×magnification) of cells obtained from the BAL fluids of mice treated as described in Figure 3 and stained with Wright-Giemsa dye. Inset boxes in upper right corner of each micrograph shows additional digital magnification of corresponding region outlined in original images. Arrowheads indicate eosinophils as identified by the presence of red cytoplasmic granules. (D) Average (± SEM) of total cell numbers obtained from the indicated (n) number of mice under each experimental condition. *, p < 0.05; **, p < 0.0001; #, p>0.05.
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pone-0063645-g004: Mice treated with R9-QQP display reduced lung inflammation.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (Hematoxylin and Eosin) cross sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box in upper right corner of each micrograph displays additional digital magnification of corresponding region outlined in original image. Peribronchial inflammatory cells are identified by dark purple staining of nuclei and are indicated by red arrowheads. (B) Normalized averages (± SEM) of inflammation scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 45 bronchioles and blood vessels were stained and analyzed as in (A). 100% inflammation score correlated with a raw score of 2.3, assessed as described in Materials and Methods. (C) Representative micrographs (400×magnification) of cells obtained from the BAL fluids of mice treated as described in Figure 3 and stained with Wright-Giemsa dye. Inset boxes in upper right corner of each micrograph shows additional digital magnification of corresponding region outlined in original images. Arrowheads indicate eosinophils as identified by the presence of red cytoplasmic granules. (D) Average (± SEM) of total cell numbers obtained from the indicated (n) number of mice under each experimental condition. *, p < 0.05; **, p < 0.0001; #, p>0.05.

Mentions: Goblet cell hyperactivity and mucus accumulation are triggered by the infiltration and accumulation of inflammatory cells that release soluble lipid mediators and sustained production of pathogenic Th2 cytokines [17]. Treatment of OVA-allergic mice with R9-QQP resulted in 71% reduction in the inflammation score as assessed in lung tissue sections that were stained with hematoxylin and eosin (Figure 4A and B). This observation was consistent with the reduction in total cell numbers seen in BAL fluids (Figure 4C and D). Furthermore, microscopic evaluation of inflammatory cells present in BAL fluids indicated that a significant percentage of infiltrating leukocytes were eosinophils (Figure 4C, PC panel).


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)

Mice treated with R9-QQP display reduced lung inflammation.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (Hematoxylin and Eosin) cross sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box in upper right corner of each micrograph displays additional digital magnification of corresponding region outlined in original image. Peribronchial inflammatory cells are identified by dark purple staining of nuclei and are indicated by red arrowheads. (B) Normalized averages (± SEM) of inflammation scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 45 bronchioles and blood vessels were stained and analyzed as in (A). 100% inflammation score correlated with a raw score of 2.3, assessed as described in Materials and Methods. (C) Representative micrographs (400×magnification) of cells obtained from the BAL fluids of mice treated as described in Figure 3 and stained with Wright-Giemsa dye. Inset boxes in upper right corner of each micrograph shows additional digital magnification of corresponding region outlined in original images. Arrowheads indicate eosinophils as identified by the presence of red cytoplasmic granules. (D) Average (± SEM) of total cell numbers obtained from the indicated (n) number of mice under each experimental condition. *, p < 0.05; **, p < 0.0001; #, p>0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063645-g004: Mice treated with R9-QQP display reduced lung inflammation.(A) Representative micrographs (200×magnification) of bronchioles prepared by staining (Hematoxylin and Eosin) cross sections of lung tissue of mice that had been treated as indicated in Figure 2A and detailed in Materials and Methods. Inset box in upper right corner of each micrograph displays additional digital magnification of corresponding region outlined in original image. Peribronchial inflammatory cells are identified by dark purple staining of nuclei and are indicated by red arrowheads. (B) Normalized averages (± SEM) of inflammation scores for the indicated number (n) of mice calculated as described in Materials and Methods; for each mouse, approximately 45 bronchioles and blood vessels were stained and analyzed as in (A). 100% inflammation score correlated with a raw score of 2.3, assessed as described in Materials and Methods. (C) Representative micrographs (400×magnification) of cells obtained from the BAL fluids of mice treated as described in Figure 3 and stained with Wright-Giemsa dye. Inset boxes in upper right corner of each micrograph shows additional digital magnification of corresponding region outlined in original images. Arrowheads indicate eosinophils as identified by the presence of red cytoplasmic granules. (D) Average (± SEM) of total cell numbers obtained from the indicated (n) number of mice under each experimental condition. *, p < 0.05; **, p < 0.0001; #, p>0.05.
Mentions: Goblet cell hyperactivity and mucus accumulation are triggered by the infiltration and accumulation of inflammatory cells that release soluble lipid mediators and sustained production of pathogenic Th2 cytokines [17]. Treatment of OVA-allergic mice with R9-QQP resulted in 71% reduction in the inflammation score as assessed in lung tissue sections that were stained with hematoxylin and eosin (Figure 4A and B). This observation was consistent with the reduction in total cell numbers seen in BAL fluids (Figure 4C and D). Furthermore, microscopic evaluation of inflammatory cells present in BAL fluids indicated that a significant percentage of infiltrating leukocytes were eosinophils (Figure 4C, PC panel).

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