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Role of Tyk-2 in Th9 and Th17 cells in allergic asthma.

Übel C, Graser A, Koch S, Rieker RJ, Lehr HA, Müller M, Finotto S - Sci Rep (2014)

Bottom Line: In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13.We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production.Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Cellular and Molecular Immunology of the Lung, Institute of Molecular Pneumology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany [2].

ABSTRACT
In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.

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Tyk-2 inhibited T cell proliferation in asthma.(a). and (b). CFSE labelled spleen CD4+ T cells from naïve and asthmatic Tyk-2(−/−) mice were analyzed for their proliferation (n = 4–5; M = Mitosis: M1: p = 0,0000, M2: p = 0,0000, M3: p = 0,0001, M4: p = 0,0005, M5: p = 0,0459). (c–e). CD4+T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-6 ((c): n = 2–3; p = 0.0014, p = 0,0053, p = 0,0052), IL-21 ((d): n = 2–3; p = 0.0015), TGF-β ((e): n = 3)). (f). TGF-β levels in the supernatants of total lung cells (n = 3). (g). CD4+ T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-4 (n = 2–4; p = 0,019 p = 0,005 p = 0,001 p = 0,0046). (h). Total lung cells from wild type and Tyk-2−/− mice were incubated with antibodies against CD4 and pSTAT3 (n = 4–5; p = 0.002) and analyzed by flow cytometry. (i). Total lung RNA was analyzed after cDNA synthesis by qPCR for the expression of Stat5 mRNA (n = 3). (j). Western blot analysis was performed on total lung protein extracts from OVA-treated mice. The membrane was probed with antibodies against SOCS3 and β-Actin as normalization control (n = 5; p = 0.00003).
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f2: Tyk-2 inhibited T cell proliferation in asthma.(a). and (b). CFSE labelled spleen CD4+ T cells from naïve and asthmatic Tyk-2(−/−) mice were analyzed for their proliferation (n = 4–5; M = Mitosis: M1: p = 0,0000, M2: p = 0,0000, M3: p = 0,0001, M4: p = 0,0005, M5: p = 0,0459). (c–e). CD4+T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-6 ((c): n = 2–3; p = 0.0014, p = 0,0053, p = 0,0052), IL-21 ((d): n = 2–3; p = 0.0015), TGF-β ((e): n = 3)). (f). TGF-β levels in the supernatants of total lung cells (n = 3). (g). CD4+ T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-4 (n = 2–4; p = 0,019 p = 0,005 p = 0,001 p = 0,0046). (h). Total lung cells from wild type and Tyk-2−/− mice were incubated with antibodies against CD4 and pSTAT3 (n = 4–5; p = 0.002) and analyzed by flow cytometry. (i). Total lung RNA was analyzed after cDNA synthesis by qPCR for the expression of Stat5 mRNA (n = 3). (j). Western blot analysis was performed on total lung protein extracts from OVA-treated mice. The membrane was probed with antibodies against SOCS3 and β-Actin as normalization control (n = 5; p = 0.00003).

Mentions: To investigate the role of Tyk-2 in T cells in a setting of allergic asthma, we sorted out lung CD4+ T cells from asthmatic mice and analyzed their proliferation after CFSE staining in a four days cell culture. As shown (Fig. 2 a and b), CD4+ T cells isolated from the lung of Tyk-2(−/−) mice proliferated more than those from the lung of wild type littermates, indicating increased effector T cells in the absence of Tyk-2. We then analyzed IL-6, a T regulatory cell inhibiting cytokine25, and found it elevated in the supernatants of cultured lung CD4+ T cells in the absence of Tyk-2 in experimental allergic asthma (Fig. 2c). Similarly, IL-21, a cytokine known to counteract T regulatory cells26, was found increased in the supernatants of lung CD4+ T cells of Tyk-2(−/−) mice (Fig. 2d), while TGF-β, a T regulatory inducing cytokine was found unchanged before and after allergen challenge (Fig. 2e,f). The Th9 subset differentiates in vitro by the addition of TGF-beta and IL-427. We next analyzed IL-4 which, consistent with a role of Tyk-2 in Th2 cells, was found significantly up-regulated in the supernatants of lung CD4+ cells isolated from the Tyk-2(−/−) mice as compared to those isolated from wild type littermates (Fig. 2g). Taken together, these data support a central role of Tyk-2 in inhibiting Th9 differentiation and in general its T cell anti-proliferative function in allergic asthma.


Role of Tyk-2 in Th9 and Th17 cells in allergic asthma.

Übel C, Graser A, Koch S, Rieker RJ, Lehr HA, Müller M, Finotto S - Sci Rep (2014)

Tyk-2 inhibited T cell proliferation in asthma.(a). and (b). CFSE labelled spleen CD4+ T cells from naïve and asthmatic Tyk-2(−/−) mice were analyzed for their proliferation (n = 4–5; M = Mitosis: M1: p = 0,0000, M2: p = 0,0000, M3: p = 0,0001, M4: p = 0,0005, M5: p = 0,0459). (c–e). CD4+T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-6 ((c): n = 2–3; p = 0.0014, p = 0,0053, p = 0,0052), IL-21 ((d): n = 2–3; p = 0.0015), TGF-β ((e): n = 3)). (f). TGF-β levels in the supernatants of total lung cells (n = 3). (g). CD4+ T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-4 (n = 2–4; p = 0,019 p = 0,005 p = 0,001 p = 0,0046). (h). Total lung cells from wild type and Tyk-2−/− mice were incubated with antibodies against CD4 and pSTAT3 (n = 4–5; p = 0.002) and analyzed by flow cytometry. (i). Total lung RNA was analyzed after cDNA synthesis by qPCR for the expression of Stat5 mRNA (n = 3). (j). Western blot analysis was performed on total lung protein extracts from OVA-treated mice. The membrane was probed with antibodies against SOCS3 and β-Actin as normalization control (n = 5; p = 0.00003).
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f2: Tyk-2 inhibited T cell proliferation in asthma.(a). and (b). CFSE labelled spleen CD4+ T cells from naïve and asthmatic Tyk-2(−/−) mice were analyzed for their proliferation (n = 4–5; M = Mitosis: M1: p = 0,0000, M2: p = 0,0000, M3: p = 0,0001, M4: p = 0,0005, M5: p = 0,0459). (c–e). CD4+T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-6 ((c): n = 2–3; p = 0.0014, p = 0,0053, p = 0,0052), IL-21 ((d): n = 2–3; p = 0.0015), TGF-β ((e): n = 3)). (f). TGF-β levels in the supernatants of total lung cells (n = 3). (g). CD4+ T cells were isolated from the lungs of naïve and OVA-treated wild type and Tyk-2(−/−) mice and cultured for 24 h with α-CD3 and α-CD28 antibodies and supernatants were analyzed by ELISA for IL-4 (n = 2–4; p = 0,019 p = 0,005 p = 0,001 p = 0,0046). (h). Total lung cells from wild type and Tyk-2−/− mice were incubated with antibodies against CD4 and pSTAT3 (n = 4–5; p = 0.002) and analyzed by flow cytometry. (i). Total lung RNA was analyzed after cDNA synthesis by qPCR for the expression of Stat5 mRNA (n = 3). (j). Western blot analysis was performed on total lung protein extracts from OVA-treated mice. The membrane was probed with antibodies against SOCS3 and β-Actin as normalization control (n = 5; p = 0.00003).
Mentions: To investigate the role of Tyk-2 in T cells in a setting of allergic asthma, we sorted out lung CD4+ T cells from asthmatic mice and analyzed their proliferation after CFSE staining in a four days cell culture. As shown (Fig. 2 a and b), CD4+ T cells isolated from the lung of Tyk-2(−/−) mice proliferated more than those from the lung of wild type littermates, indicating increased effector T cells in the absence of Tyk-2. We then analyzed IL-6, a T regulatory cell inhibiting cytokine25, and found it elevated in the supernatants of cultured lung CD4+ T cells in the absence of Tyk-2 in experimental allergic asthma (Fig. 2c). Similarly, IL-21, a cytokine known to counteract T regulatory cells26, was found increased in the supernatants of lung CD4+ T cells of Tyk-2(−/−) mice (Fig. 2d), while TGF-β, a T regulatory inducing cytokine was found unchanged before and after allergen challenge (Fig. 2e,f). The Th9 subset differentiates in vitro by the addition of TGF-beta and IL-427. We next analyzed IL-4 which, consistent with a role of Tyk-2 in Th2 cells, was found significantly up-regulated in the supernatants of lung CD4+ cells isolated from the Tyk-2(−/−) mice as compared to those isolated from wild type littermates (Fig. 2g). Taken together, these data support a central role of Tyk-2 in inhibiting Th9 differentiation and in general its T cell anti-proliferative function in allergic asthma.

Bottom Line: In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13.We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production.Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung.

View Article: PubMed Central - PubMed

Affiliation: 1] Laboratory of Cellular and Molecular Immunology of the Lung, Institute of Molecular Pneumology, University of Erlangen-Nürnberg, 91054 Erlangen, Germany [2].

ABSTRACT
In a murine model of allergic asthma, we found that Tyk-2((-/-)) asthmatic mice have induced peribronchial collagen deposition, mucosal type mast cells in the lung, IRF4 and hyperproliferative lung Th2 CD4(+) effector T cells over-expressing IL-3, IL-4, IL-5, IL-10 and IL-13. We also observed increased Th9 cells expressing IL-9 and IL-10 as well as T helper cells expressing IL-6, IL-10 and IL-21 with a defect in IL-17A and IL-17F production. This T helper phenotype was accompanied by increased SOCS3 in the lung of Tyk-2 deficient asthmatic mice. Finally, in vivo treatment with rIL-17A inhibited local CD4(+)CD25(+)Foxp3(+) T regulatory cells as well as Th2 cytokines without affecting IL-9 in the lung. These results suggest a role of Tyk-2 in different subsets of T helper cells mediated by SOCS3 regulation that is relevant for the treatment of asthma, cancer and autoimmune diseases.

Show MeSH
Related in: MedlinePlus