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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 inhibits OVA-inducible Th2 cytokine production in a dose-dependent and peptide-specific manner.Splenocytes, obtained from mice seven days following i.p. immunization with 20 µg OVA + 2 mg alum, were treated with various concentrations of R9-QQP or without peptide and re-stimulated in vitro with 50 µg/mL OVA as indicated. After incubation for 4 days, cell-free tissue culture supernatant samples were assayed for IL-4 (A), IL-5 (C) and IL-13 (E) by ELISA as described in Materials and Methods. Results are those from single titrations for each cytokine and are displayed as the average (± S.D.) of duplicate determinations. For assessing peptide specificity, similar cultures were established as above and splenocytes were treated with 20 µM of R9-QQP, R9-PQM, R9-QQA or no peptide as indicated, and culture supernatants assayed for IL-4 (B), IL-5 (D) and IL-13 (F). Results are displayed as the average (± S.E.M.) of 5-6 replicate experiments utilizing different mice. For each cytokine tested, statistical evaluations were determined by a paired student's t test comparing peptide-treated cells to vehicle-treated/OVA-restimulated cells. *, p < 0.05; **, p < 0.01; #, p>0.05.
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pone-0063645-g001: R9-QQP inhibits OVA-inducible Th2 cytokine production in a dose-dependent and peptide-specific manner.Splenocytes, obtained from mice seven days following i.p. immunization with 20 µg OVA + 2 mg alum, were treated with various concentrations of R9-QQP or without peptide and re-stimulated in vitro with 50 µg/mL OVA as indicated. After incubation for 4 days, cell-free tissue culture supernatant samples were assayed for IL-4 (A), IL-5 (C) and IL-13 (E) by ELISA as described in Materials and Methods. Results are those from single titrations for each cytokine and are displayed as the average (± S.D.) of duplicate determinations. For assessing peptide specificity, similar cultures were established as above and splenocytes were treated with 20 µM of R9-QQP, R9-PQM, R9-QQA or no peptide as indicated, and culture supernatants assayed for IL-4 (B), IL-5 (D) and IL-13 (F). Results are displayed as the average (± S.E.M.) of 5-6 replicate experiments utilizing different mice. For each cytokine tested, statistical evaluations were determined by a paired student's t test comparing peptide-treated cells to vehicle-treated/OVA-restimulated cells. *, p < 0.05; **, p < 0.01; #, p>0.05.

Mentions: Female BALB/c (experiments presented in Figure 1) or C57BL/6 (all other experiments) mice between the ages of 6-12 weeks were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed in microisolator cages until used for experiments.


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 inhibits OVA-inducible Th2 cytokine production in a dose-dependent and peptide-specific manner.Splenocytes, obtained from mice seven days following i.p. immunization with 20 µg OVA + 2 mg alum, were treated with various concentrations of R9-QQP or without peptide and re-stimulated in vitro with 50 µg/mL OVA as indicated. After incubation for 4 days, cell-free tissue culture supernatant samples were assayed for IL-4 (A), IL-5 (C) and IL-13 (E) by ELISA as described in Materials and Methods. Results are those from single titrations for each cytokine and are displayed as the average (± S.D.) of duplicate determinations. For assessing peptide specificity, similar cultures were established as above and splenocytes were treated with 20 µM of R9-QQP, R9-PQM, R9-QQA or no peptide as indicated, and culture supernatants assayed for IL-4 (B), IL-5 (D) and IL-13 (F). Results are displayed as the average (± S.E.M.) of 5-6 replicate experiments utilizing different mice. For each cytokine tested, statistical evaluations were determined by a paired student's t test comparing peptide-treated cells to vehicle-treated/OVA-restimulated cells. *, p < 0.05; **, p < 0.01; #, p>0.05.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3646824&req=5

pone-0063645-g001: R9-QQP inhibits OVA-inducible Th2 cytokine production in a dose-dependent and peptide-specific manner.Splenocytes, obtained from mice seven days following i.p. immunization with 20 µg OVA + 2 mg alum, were treated with various concentrations of R9-QQP or without peptide and re-stimulated in vitro with 50 µg/mL OVA as indicated. After incubation for 4 days, cell-free tissue culture supernatant samples were assayed for IL-4 (A), IL-5 (C) and IL-13 (E) by ELISA as described in Materials and Methods. Results are those from single titrations for each cytokine and are displayed as the average (± S.D.) of duplicate determinations. For assessing peptide specificity, similar cultures were established as above and splenocytes were treated with 20 µM of R9-QQP, R9-PQM, R9-QQA or no peptide as indicated, and culture supernatants assayed for IL-4 (B), IL-5 (D) and IL-13 (F). Results are displayed as the average (± S.E.M.) of 5-6 replicate experiments utilizing different mice. For each cytokine tested, statistical evaluations were determined by a paired student's t test comparing peptide-treated cells to vehicle-treated/OVA-restimulated cells. *, p < 0.05; **, p < 0.01; #, p>0.05.
Mentions: Female BALB/c (experiments presented in Figure 1) or C57BL/6 (all other experiments) mice between the ages of 6-12 weeks were purchased from Jackson Laboratories (Bar Harbor, ME) and were housed in microisolator cages until used for experiments.

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