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. 2013:3:1754.
doi: 10.1038/srep01754.

IL-6 activated integrated BATF/IRF4 functions in lymphocytes are T-bet-independent and reversed by subcutaneous immunotherapy

Sonja Koch  1 , Stephanie Mousset, Anna Graser, Sarah Reppert, Caroline Übel, Cornelia Reinhardt, Theodor Zimmermann, Ralf Rieker, Hans A Lehr, Susetta Finotto
Affiliations

IL-6 activated integrated BATF/IRF4 functions in lymphocytes are T-bet-independent and reversed by subcutaneous immunotherapy

Sonja Koch et al. Sci Rep. 2013.

Abstract

IL-6 plays a central role in supporting pathological T(H2) and T(H17) cell development and inhibiting the protective T regulatory cells in allergic asthma. T(H17) cells have been demonstrated to regulate allergic asthma in general and T-bet-deficiency-induced asthma in particular. Here we found an inverse correlation between T-bet and Il-6 mRNA expression in asthmatic children. Moreover, experimental subcutaneous immunotherapy (SIT) in T-bet((-/-)) mice inhibited IL-6, IL-21R and lung T(H17) cells in a setting of asthma. Finally, local delivery of an anti-IL-6R antibody in T-bet((-/-)) mice resulted in the resolution of this allergic trait. Noteworthy, BATF, crucial for the immunoglobulin-class-switch and T(H2),T(H17) development, was found down-regulated in the lungs of T-bet((-/-)) mice after SIT and after treatment with anti-IL-6R antibody, indicating a critical role of IL-6 in controlling BATF/IRF4 integrated functions in T(H2), T(H17) cells and B cells also in a T-bet independent fashion in allergic asthma.

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Figures

Figure 1
Figure 1. Increased IL-6 in asthma in the absence of T-bet.
(a) Correlation between mRNA values of healthy pre-school control children (left panel) and asthmatic (right panel) children.(b) Experimental design of a murine model of allergic asthma in wild-type and T-bet(−/−) mice. Mice received 100 μg OVA/Alum intraperitoneally (i.p.) and 2 mg/ml OVA intranasally (i.n.). (c) Increased expression of Il-6 mRNA in murine lung tissue by qPCR in T-bet(−/−) naïve (PBS) and asthmatic mice (OVA). (d) Increased IL-6 in murine lung CD4+ T cells in T-bet(−/−) asthmatic mice after intracellular flow cytometric analysis.
Figure 2
Figure 2. IL-6 induces BATF in the absence of T-bet in lung CD4+ T cells from naïve and asthmatic mice.
(a) Increased levels of IgE in murine blood serum in T-bet(−/−) mice (n = 9–17 mice per group). (b) Increased Batf mRNA expression measured by qPCR in isolated murine lung CD4+ T cells from T-bet(−/−) mice (n = 3–5 mice per group). (c) Increased expression of Rorγt mRNA in murine lung tissue of T-bet(−/−) mice. (d, e) Batf and Rorγt mRNA expression after quantitative real time PCR in naïve lung murine CD4+ T cells cultured with α-CD3 and α-CD28 alone (Med) or with IL-6 (20 ng/ml) (Med+IL-6) (n = 3–5 mice per group). BATF and not RORγt were induced by IL-6 in lung CD4+ T cells isolated from T-bet(−/−) mice. Students t test was used to calculate statistical significances. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Results are expressed as mean ± s.e.m.
Figure 3
Figure 3. Increased IL-21, IL-17A and IL-10 expression in lung CD4+ T cells of T-bet(−/−) asthmatic mice.
(a) Increased Il-21 mRNA expression in lung CD4+ T cells isolated from asthmatic T-bet(−/−) mice. (b) Increased Il-21R mRNA expression in murine lung tissue from asthmatic T-bet(−/−) mice. (c) Increased Expression of Irf4 mRNA in asthmatic murine lung tissue. (d) IL-17A level measured in the supernatants of murine lung CD4+ T cells isolated from T-bet(−/−) asthmatic mice and cultured for 24 h with α-CD3 and α-CD28 (n = 4–6 mice per group). (e) Increased number of lung CD4+IL-10+ T cells in T-bet(−/−) asthmatic mice after intracellular flow cytometric analysis. (f) Increased IL-10 release by lung CD4+ T cells from T-bet(−/−) mice measured by ELISA in the supernatants of these cells after 24 hours of cell culture with α-CD3, α-CD28 antibody challenge. Students t test was used to calculate statistical significances. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Results are expressed as mean ± s.e.m.
Figure 4
Figure 4. IL-2 expression is increased in the lung of T-bet(−/−) asthmatic mice.
(a) Increased IL-2 protein expression in the supernatants of lung cells isolated from T-bet(−/−) asthmatic mice and cultured with α-CD3 and OVA (500 μg/ml) for 24 hours as indicated. (b) Decreased CD4+CD25+Foxp3+ Treg cells in T-bet(−/−) mice sensitized and challenged with OVA as described in Figure 1 and after flow cytometric analysis. Students t test was used to calculate statistical significances. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Results are expressed as mean ± s.e.m.
Figure 5
Figure 5. Decreased mRNA expression of different transcription factors and cytokines involved in TH17 differentiation after immunotherapy (SIT) in T-bet(−/−) mice.
(a) Experimental design of the murine asthma model with immunotherapy.Mice received 100 μg OVA/Alum intraperitoneally (i.p.), 1000 μg OVA subcutaneous (s.c.) and 2 mg/ml OVA intranasally (i.n.). (b–j) mRNA expression of Il-6 (b), Rorγt (c), Batf (d), Irf4 (e), Il-21 (f), Il-21R (g), Il-17A (h), Il-10 (i) and Il-2 (j) in lung tissue of T-bet(−/−) mice before and after SIT (n = 3–6 mice per group). Results in this figure are expressed as mean ± s.e.m. Students t test was used to calculate statistical significances in this figure. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Figure 6
Figure 6. Reduction of IgE, inflammation and TH2 cytokines in α-IL-6R antibody treated asthmatic T-bet(−/−) mice.
(a) Experimental design of a murine asthma model and the α-IL-6R antibody therapy.Mice received 100 μg OVA/Alum intraperitoneally (i.p.) and 2 mg/ml OVA intranasally (i.n.). Some of the mice also received 75 μg of α-IL-6R antibody or IgG. (b) Decreased pathological Score of the inflammation and corresponding histological sections of the lungs stained with H&E in anti-IL-6R antibody treated mice. (c) Decreased levels of IgE in blood serum of T-bet(−/−) mice treated with α-IL-6R antibody in vivo. One of three similar experiments are shown with three to five mice per group. (d,e) Decreased number of CD19+ B cells (d) and CD4+ T cells (e) isolated from the lungs of T-bet(−/−) sensitized and challenged mice (n = 4–21 mice per group) and in vivo treated with anti-IL6R antibody. (f–h) ELISA of IL-4 (f), IL-5 (g) and IL-13 (h) in the supernatants of total lung cell cultures from OVA-challenged and α-IL-6R antibody treated mice. Cells were cultured for 24 h with α-CD3 and α-CD28 (n = 7–8 mice per group). Statistical significances in this figure were evaluated with a Students t test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Data are mean ± s.e.m.
Figure 7
Figure 7. Reduced BATF, c-maf and IL-21 expression after in vitro treatment with α-IL-6R antibody.
(a) In vitro treatment with OVA (500 μg/ml) and additionally anti-IL-6 R antibody or IgG (15 μg/ml). (b,c) Reduced expression of Batf (b), but not Rorγt (c) after treatment with α-IL-6R antibody.(d) c-maf mRNA expression in total lung cells from untreated, OVA-treated and/or α-IL-6R antibody treated wild-type and T-bet(−/−) mice (n = 3–5 mice per goup). (e) Decreased Il-21 mRNA expression in lung CD4+ T cells isolated from wild-type and T-bet(−/−) mice after α-IL-6R antibody treatment. Cells were cultured for 24 h with α-CD3 and α-CD28 (n = 10–16 mice per group). Data are mean ± s.e.m. Students t test was used to calculate statistical significances. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Figure 8
Figure 8. Decreased IL-21R and IL-10 in T-bet deficient mice treated in vivo with anti-IL-6R antibody.
(a) Experimental design of anti-IL-6R antibody treatment in vivo.100 μg OVA/Alum was administered to the mice intraperitoneally (i.p.) and 2 mg/ml OVA intranasally (i.n.). Some of the mice also received 75 μg of α-IL-6R antibody or IgG. (b) Irf4 mRNA expression in lung tissue of T-bet(−/−) mice (n = 8–9 mice per group). (c) IL-17A measured by ELISA in the supernatants of total lung cell cultures of T-bet(−/−) mice sensitized and challenged in vivo with OVA and treated in vivo with α-IL-6R antibody. (d) Decreased expression of Il-21R mRNA in lung tissue from T-bet deficient mice treated with anti-IL-6R antibody (n = 3–6 mice per group). (d) Decreased IL-10 in the supernatants of total lung cell cultures of T-bet(−/−) mice sensitized with OVA treated in vivo with α-IL-6R antibody . Students t test was used to calculate statistical significances. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. Results are expressed as mean ± s.e.m.
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