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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 a lung DLN Th2 cytokine response.Cytokine levels of (A) IL-13, (B) IL-5, and (C) IL-4 in cultures of OVA (50 µg/ml) or medium restimulated lung-draining lymph node cells obtained from mice treated as described in Figure 2A and indicated in the inset legend. One×105 (panels A and B) or 106 (panel C) cells per well of round-bottom 96-well microtiter trays were incubated for 4 days at 37°C and cell-free culture supernatants were assayed by ELISA as described in Materials and Methods. Results are displayed as the average (± SEM) cytokine amounts for the indicated number (N) of independent experiments each representing DLN cells pooled from 2-4 mice. **, p < 0.01; #, p>0.05; *, p < 0.05.
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pone-0063645-g006: R9-QQP inhibits a lung DLN Th2 cytokine response.Cytokine levels of (A) IL-13, (B) IL-5, and (C) IL-4 in cultures of OVA (50 µg/ml) or medium restimulated lung-draining lymph node cells obtained from mice treated as described in Figure 2A and indicated in the inset legend. One×105 (panels A and B) or 106 (panel C) cells per well of round-bottom 96-well microtiter trays were incubated for 4 days at 37°C and cell-free culture supernatants were assayed by ELISA as described in Materials and Methods. Results are displayed as the average (± SEM) cytokine amounts for the indicated number (N) of independent experiments each representing DLN cells pooled from 2-4 mice. **, p < 0.01; #, p>0.05; *, p < 0.05.

Mentions: T lymphocytes that are recruited to the lungs in response to OVA stimulation and challenge (Figure 5D and E) secrete Th2 cytokines such as IL-4, IL-5, and IL-13 (Figure 6). Among other events, sustained levels of these cytokines are known to induce eosinophilia, goblet cell hyperactivity and mucus production [16], [17]. Since ITK is known to regulate the transcriptional activation and secretion of Th2 cytokines [2], [3], we tested the effects of R9-QQP treatment on the ability of antigen-specific T cells from OVA stimulated and challenged mice to produce IL-4, IL-5 and IL-13. Due to the limited numbers of T cells recruited to the inflamed lung tissue, we isolated T cells from the lung draining lymph nodes (DLN). Isolated lymphocytes from the peribronchial and mediastinal DLN of OVA-sensitized and control mice that had been treated with R9-QQP, no peptide, or R9-QQA control peptide were re-stimulated in vitro with OVA, but without additional peptide treatment. The results in Figure 6 demonstrate that all three Th2 cytokines were significantly reduced in cultures prepared from the DLN of mice that were treated with R9-QQP but not those from the DLN of mice that were treated with control peptide or no treatment. Reduction was 85% in the case of IL-13 (Figure 6A), 71% for IL-5 (Figure 6B) and 73% for IL-4 (Figure 6C).


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 a lung DLN Th2 cytokine response.Cytokine levels of (A) IL-13, (B) IL-5, and (C) IL-4 in cultures of OVA (50 µg/ml) or medium restimulated lung-draining lymph node cells obtained from mice treated as described in Figure 2A and indicated in the inset legend. One×105 (panels A and B) or 106 (panel C) cells per well of round-bottom 96-well microtiter trays were incubated for 4 days at 37°C and cell-free culture supernatants were assayed by ELISA as described in Materials and Methods. Results are displayed as the average (± SEM) cytokine amounts for the indicated number (N) of independent experiments each representing DLN cells pooled from 2-4 mice. **, p < 0.01; #, p>0.05; *, p < 0.05.
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Related In: Results  -  Collection

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

pone-0063645-g006: R9-QQP inhibits a lung DLN Th2 cytokine response.Cytokine levels of (A) IL-13, (B) IL-5, and (C) IL-4 in cultures of OVA (50 µg/ml) or medium restimulated lung-draining lymph node cells obtained from mice treated as described in Figure 2A and indicated in the inset legend. One×105 (panels A and B) or 106 (panel C) cells per well of round-bottom 96-well microtiter trays were incubated for 4 days at 37°C and cell-free culture supernatants were assayed by ELISA as described in Materials and Methods. Results are displayed as the average (± SEM) cytokine amounts for the indicated number (N) of independent experiments each representing DLN cells pooled from 2-4 mice. **, p < 0.01; #, p>0.05; *, p < 0.05.
Mentions: T lymphocytes that are recruited to the lungs in response to OVA stimulation and challenge (Figure 5D and E) secrete Th2 cytokines such as IL-4, IL-5, and IL-13 (Figure 6). Among other events, sustained levels of these cytokines are known to induce eosinophilia, goblet cell hyperactivity and mucus production [16], [17]. Since ITK is known to regulate the transcriptional activation and secretion of Th2 cytokines [2], [3], we tested the effects of R9-QQP treatment on the ability of antigen-specific T cells from OVA stimulated and challenged mice to produce IL-4, IL-5 and IL-13. Due to the limited numbers of T cells recruited to the inflamed lung tissue, we isolated T cells from the lung draining lymph nodes (DLN). Isolated lymphocytes from the peribronchial and mediastinal DLN of OVA-sensitized and control mice that had been treated with R9-QQP, no peptide, or R9-QQA control peptide were re-stimulated in vitro with OVA, but without additional peptide treatment. The results in Figure 6 demonstrate that all three Th2 cytokines were significantly reduced in cultures prepared from the DLN of mice that were treated with R9-QQP but not those from the DLN of mice that were treated with control peptide or no treatment. Reduction was 85% in the case of IL-13 (Figure 6A), 71% for IL-5 (Figure 6B) and 73% for IL-4 (Figure 6C).

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