Limits...
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.

Show MeSH

Related in: MedlinePlus

Mice treated with R9-QQP display reduced airway hyperresponsiveness (AHR).(A) Regimen of OVA immunization and challenge, and peptide treatment with description of treatment for each group of mice as detailed in Materials and Methods. Abbreviations used to represent control groups of mice used for this and subsequent figures are defined as follows: NC = negative control, PC = positive control. (B) Twenty-four hours after the final challenge with OVA, airway resistance was measured for each mouse using the Scireq FlexiVent system in response to increasing concentrations of aerosolized methacholine (MCh). Results represent the group average (± SEM of independent experiments) airway resistance values representing 6 non-sensitized (……) and 17 OVA-sensitized (____) mice in response to various concentrations of MCh. (C) Airway resistance was measured as in panel B using groups of mice that had been rendered asthmatic and treated with R9-QQP or the control peptides R9-QQA or R9-PQM (data were pooled for the two control peptides) or with vehicle as defined in panel A. Bar graphs display the average (± SEM of independent experiments) airway resistance in response to 12 mg/mL MCh. ‘N’ represents the number of independent experiments and ‘n’ represents the total number of animals for each group. *, p<0.05; #, p>0.05.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3646824&req=5

pone-0063645-g002: Mice treated with R9-QQP display reduced airway hyperresponsiveness (AHR).(A) Regimen of OVA immunization and challenge, and peptide treatment with description of treatment for each group of mice as detailed in Materials and Methods. Abbreviations used to represent control groups of mice used for this and subsequent figures are defined as follows: NC = negative control, PC = positive control. (B) Twenty-four hours after the final challenge with OVA, airway resistance was measured for each mouse using the Scireq FlexiVent system in response to increasing concentrations of aerosolized methacholine (MCh). Results represent the group average (± SEM of independent experiments) airway resistance values representing 6 non-sensitized (……) and 17 OVA-sensitized (____) mice in response to various concentrations of MCh. (C) Airway resistance was measured as in panel B using groups of mice that had been rendered asthmatic and treated with R9-QQP or the control peptides R9-QQA or R9-PQM (data were pooled for the two control peptides) or with vehicle as defined in panel A. Bar graphs display the average (± SEM of independent experiments) airway resistance in response to 12 mg/mL MCh. ‘N’ represents the number of independent experiments and ‘n’ represents the total number of animals for each group. *, p<0.05; #, p>0.05.

Mentions: Sensitization to and challenge with the model allergen ovalbumin (OVA, grade V; Sigma; St. Louis, MO; Cat. # A-5503) was performed by intraperitoneal (i.p.) injection of C57BL/6 mice with 20 µg OVA/2 mg alum (Pierce/Thermo Scientific, Inc; Rockford, IL; Cat. # 77161). In order to induce asthma, mice sensitized to OVA were subsequently challenged by intranasal (i.n.) instillation of 10 µg OVA/20 µL PBS into anesthetized mice on days 7, 8, and 9 post sensitization. For peptide treatment, mice sensitized and challenged with OVA as above were treated with R9-QQP or control peptides (R9-PQM or R9-QQA) by i.p. injection of 25 mg peptide per kg of mouse weight (∼0.5 mg/mouse) 24 hours and again 30 minutes before OVA sensitization. This was followed by additional i.p. injections of peptides 24 hours before each intranasal challenge with OVA on days 7, 8, and 9 and by i.n. delivery (200 µg in 20 µl PBS) 2 hours before intranasal OVA challenge. According to the above protocol total peptide delivery was 2.5 mg i.p. and 0.6 mg i.n. per mouse. On day ten of the protocol mice and collected tissues were used for assessment of various asthma parameters. The regimen of OVA sensitization and challenge, and treatment with peptide is outlined in Figure 2A.


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 airway hyperresponsiveness (AHR).(A) Regimen of OVA immunization and challenge, and peptide treatment with description of treatment for each group of mice as detailed in Materials and Methods. Abbreviations used to represent control groups of mice used for this and subsequent figures are defined as follows: NC = negative control, PC = positive control. (B) Twenty-four hours after the final challenge with OVA, airway resistance was measured for each mouse using the Scireq FlexiVent system in response to increasing concentrations of aerosolized methacholine (MCh). Results represent the group average (± SEM of independent experiments) airway resistance values representing 6 non-sensitized (……) and 17 OVA-sensitized (____) mice in response to various concentrations of MCh. (C) Airway resistance was measured as in panel B using groups of mice that had been rendered asthmatic and treated with R9-QQP or the control peptides R9-QQA or R9-PQM (data were pooled for the two control peptides) or with vehicle as defined in panel A. Bar graphs display the average (± SEM of independent experiments) airway resistance in response to 12 mg/mL MCh. ‘N’ represents the number of independent experiments and ‘n’ represents the total number of animals for each group. *, p<0.05; #, p>0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0063645-g002: Mice treated with R9-QQP display reduced airway hyperresponsiveness (AHR).(A) Regimen of OVA immunization and challenge, and peptide treatment with description of treatment for each group of mice as detailed in Materials and Methods. Abbreviations used to represent control groups of mice used for this and subsequent figures are defined as follows: NC = negative control, PC = positive control. (B) Twenty-four hours after the final challenge with OVA, airway resistance was measured for each mouse using the Scireq FlexiVent system in response to increasing concentrations of aerosolized methacholine (MCh). Results represent the group average (± SEM of independent experiments) airway resistance values representing 6 non-sensitized (……) and 17 OVA-sensitized (____) mice in response to various concentrations of MCh. (C) Airway resistance was measured as in panel B using groups of mice that had been rendered asthmatic and treated with R9-QQP or the control peptides R9-QQA or R9-PQM (data were pooled for the two control peptides) or with vehicle as defined in panel A. Bar graphs display the average (± SEM of independent experiments) airway resistance in response to 12 mg/mL MCh. ‘N’ represents the number of independent experiments and ‘n’ represents the total number of animals for each group. *, p<0.05; #, p>0.05.
Mentions: Sensitization to and challenge with the model allergen ovalbumin (OVA, grade V; Sigma; St. Louis, MO; Cat. # A-5503) was performed by intraperitoneal (i.p.) injection of C57BL/6 mice with 20 µg OVA/2 mg alum (Pierce/Thermo Scientific, Inc; Rockford, IL; Cat. # 77161). In order to induce asthma, mice sensitized to OVA were subsequently challenged by intranasal (i.n.) instillation of 10 µg OVA/20 µL PBS into anesthetized mice on days 7, 8, and 9 post sensitization. For peptide treatment, mice sensitized and challenged with OVA as above were treated with R9-QQP or control peptides (R9-PQM or R9-QQA) by i.p. injection of 25 mg peptide per kg of mouse weight (∼0.5 mg/mouse) 24 hours and again 30 minutes before OVA sensitization. This was followed by additional i.p. injections of peptides 24 hours before each intranasal challenge with OVA on days 7, 8, and 9 and by i.n. delivery (200 µg in 20 µl PBS) 2 hours before intranasal OVA challenge. According to the above protocol total peptide delivery was 2.5 mg i.p. and 0.6 mg i.n. per mouse. On day ten of the protocol mice and collected tissues were used for assessment of various asthma parameters. The regimen of OVA sensitization and challenge, and treatment with peptide is outlined in Figure 2A.

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