IL-17 is required in the initiation of allergic asthma
To investigate the role of IL-17 in allergic asthma we first used receptor IL-17R gene–deficient (IL-17R KO) mice in an OVA-induced model of asthma. Reduced lung and airway recruitment of granulocytes, predominantly eosinophils, were found in IL-17R KO mice compared with WT mice (). Furthermore, eosinophil peroxidase (EPO) activity in lung tissues () and OVA-specific serum IgE concentrations were reduced () in the absence of IL-17R signaling. The reduced asthma like sequellae of antigen challenge in IL-17R KO compared with WT mice correlates with a reduced regional Th2 cytokine production. Regional lymph nodes from antigen-sensitized and intranasally challenged mice were restimulated ex vivo (in cell culture), resulting in reduced IL-5 production in cells from IL-17R KO compared with WT mice (). The composition of CD3+, CD11b+, and CD11c+ lymph node cells was similar in IL-17R KO and WT mice (), indicating that reduced antigen-induced cell activation rather than recruitment of immune cells to the lymph nodes of IL-17R KO mice causes attenuated Th2 responses. Therefore, signaling through IL-17R is critically required to develop allergic asthma.
Figure 1. Role of IL-17 in development of allergic inflammation, as assessed in receptor IL-17R KO mice. OVA/alum-sensitized C57BL/6 mice (WT) and IL-17R KO (IL-17R−/−) mice were challenged three times intranasally with OVA. 48 h after the third (more ...)
IL-17 is induced in the lung upon local allergen challenge
Pulmonary IL-17 was induced in allergen-sensitized compared with nonsensitized mice and further increased upon intranasal challenge (). IL-17 production correlated with pulmonary IL-23 induction (). In cell cultures derived from allergen-sensitized and challenged mice, IL-23 was able to induce IL-17 in mediastinal lymph node () but not in splenocyte cultures (), suggesting a local pulmonary role of IL-17. Therefore, we show in a model of allergic asthma that IL-17 is locally augmented by allergen challenge and induced by IL-23.
Figure 2. IL-17 is induced in the lung upon allergen challenge. Mice were sensitized and challenged with OVA, and IL-17 and IL-23 were determined by ELISA in the lung homogenate (A and B). In naive, nonsensitized mice, no IL-17 was detected (A). In C and D, cells (more ...)
Exogenous IL-17 reduces methacholine response upon antigen challenge
OVA-sensitized C57BL/6 mice challenged intranasally with OVA, but not NaCl or IL-17 alone, developed a robust response to aerosolized methacholine given in enhanced respiratory pause (Penh) values () (38
). Penh values measured provide an estimate for airway obstruction and may indicate airways hyperreactivity and inflammation. Recombinant murine IL-17 (0.1 μg) given together with the OVA challenge inhibited methacholine response by 58 ± 18% (P < 0.03; ), and this effect was similar at high IL-17 dose (10 μg). Moreover, the response of mice sensitized with OVA plus adjuvant aluminium hydroxide and challenged intranasally with OVA was also reduced by IL-17 (unpublished data).
Figure 3. Exogenous IL-17 inhibits methacholine response upon allergen challenge. OVA-sensitized C57BL/6 mice were challenged intranasally with either saline, OVA alone, IL-17 alone, OVA with IL-17, or OVA with IL-17 plus neutralizing IL-17 antibodies. 24 h after (more ...)
IL-17–induced inhibition of methacholine response was fully reversed by neutralizing IL-17 antibody treatment (), confirming the specificity of exogenous IL-17 effect. IL-17 antibody administration augmented the methacholine response above controls (P < 0.01) (, bar 5 vs. 3), suggesting that endogenous IL-17 was also neutralized and contributed to methacholine response.
IL-17 did not inhibit systemic OVA-specific serum IgE concentrations, which reflect intact T cell–B cell interactions (). Therefore, the data suggest that IL-17 given with antigen challenge has an inhibitory effect on methacholine response.
IL-17 decreases lung eosinophil recruitment
We then investigated the regulatory role of IL-17 in antigen-induced lung inflammation. 24 h after methacholine response analysis, inflammatory cell recruitment into the lung tissue and the alveolar space was analyzed (). The total bronchoalveolar lavage (BAL) cell numbers increased up to 24-fold in OVA-challenged compared to saline-treated control mice (15 × 104 to 354 × 104 cells/BAL). The higher cell number was mainly caused by eosinophil recruitment, increasing more than 700-fold (P < 0.01; ).
IL-17 administration at 0.1 μg significantly reduced the total BAL cells by 91% (P < 0.01), the eosinophil counts by 95% (P < 0.01; ), and lymphocyte counts by 84% (P < 0.01; ). These regulatory IL-17 effects were fully reversed by the administration of neutralizing IL-17 antibody (, bar 4). IL-17 antibody administration augmented eosinophil counts above controls (P < 0.01) (, bar 5 vs. 2), suggesting that endogenous IL-17 was also neutralized, which contributed to eosinophil recruitment. Application of isotype-matched control antibody with the OVA challenge had no effect on OVA-induced response (unpublished data).
Microscopically the lung tissue of OVA-challenged mice showed peribronchial cell recruitment together with hyperplasia of bronchial smooth muscle. IL-17 reduced peribronchial eosinophil infiltration and the mucus hypersecretion and hyperplasia of goblet cells () from 4 to 1 using scoring scale from 0 to 5 (). Reduction of eosinophil infiltration correlated with reduced EPO activity in the lung tissue of IL-17–treated animals (). These effects were reversed by neutralizing IL-17 antibodies. Neutralizing IL-17 antibodies given alone to OVA-challenged mice augmented eosinophil infiltration and mucus secretion above OVA-challenged controls (, C and D, bar 5 vs. 2), suggesting that endogenous IL-17 was also neutralized, which contributed to pathology.
Figure 4. IL-17 inhibits cell recruitment to the lungs and mucus hypersecretion. Lung sections of OVA-sensitized C57BL/6 mice killed 2 d after the third challenge with either saline, OVA, OVA with IL-17 antibodies, OVA with IL-17, or OVA with IL-17 plus IL-17 antibodies (more ...)
IL-17 inhibits pulmonary CC-chemokine expression in vivo
CC-chemokines, eotaxin, Rantes, and TARC, contribute to allergic inflammation, as demonstrated in neutralization experiments and gene-deficient mice (39
). IL-17 reportedly inhibits Rantes expression in TNF-stimulated lung fibroblasts and in colonic myofibroblasts (33
). We tested whether IL-17 inhibits the mononuclear cell–chemokine production in allergic lungs.
OVA-sensitized and intranasally challenged mice showed increased TARC, eotaxin, and Rantes production in the lung tissue (). Exogenous IL-17 abolished TARC and eotaxin expression completely and reduced Rantes induction by 50% (). As expected, the inhibition was largely reversed by neutralizing IL-17 antibody treatment (), demonstrating the specificity of the IL-17 effects. Neutralizing IL-17 antibodies given alone to OVA-challenged mice augmented TARC expression above OVA-challenged controls (, A and B, bar 2 vs. 3), suggesting that endogenous IL-17 also affects TARC production.
Figure 5. IL-17 inhibits pulmonary contents of the mononuclear cell attracting chemokines TARC, eotaxin and Rantes. OVA-sensitized C57BL/6 mice were challenged three times intranasally with either saline, OVA alone, OVA plus IL-17–neutralizing antibodies, (more ...)
In the allergen-inflamed BAL, TARC (), but neither eotaxin nor Rantes (unpublished data), were detected. Reduced TARC production correlated with reduced lymphocyte and eosinophil counts. Our findings suggest that IL-17 attenuates antigen-induced asthma by inhibiting the production of CC-chemokines, TARC, eotaxin, and Rantes.
IL-17 decreases CC-chemokine expression in lung explant cell culture and inhibits DC function
IL-17–mediated inhibition of the chemokines TARC and eotaxin observed in vivo was consolidated in cell cultures. Mice were sensitized, intranasally challenged with OVA, and 2 d after the last challenge lung explant cell cultures, mediastinal lymph node cells, and splenocyte cultures thereof were restimulated ex vivo with 50 μg/ml OVA in the presence or absence of IL-17 (). In lung explant cell cultures, IL-17 reduced spontaneous and OVA-induced expression of eotaxin and TARC (). In lung macrophages, specifically eotaxin (), but not TARC (), was expressed, implying that TARC may be expressed by nonadherent leukocytes, likely DCs, a major source of TARC.
Figure 6. IL-17 inhibits TARC and eotaxin production ex vivo in lung explants. Lung explants (A and C) and lung adherent cells (B and D) were prepared from OVA-sensitized and challenged animals. Cells were treated with saline, 50 μg/ml OVA alone, IL-17 (more ...)
Therefore, we analyzed whether IL-17 reduces DC functions. TARC was produced in unstimulated and TNF-activated bone marrow–derived DCs (CD11c+) () but not in bone marrow–derived macrophages (CD11c−) (). IL-17 reduced spontaneous and TNF-induced expression of TARC (). Furthermore, dendritic antigen uptake of fluorescence-labeled OVA was reduced by IL-17 by 46% ().
Figure 7. IL-17 inhibits TARC production and OVA uptake in DCs. DCs (A and C) and macrophages (B) were differentiated in vitro from naive bone marrow–derived cells. Cells were treated with saline, IL-17 alone, 3 ng/ml TNF, or TNF with 30 ng/ml IL-17 for (more ...)
Therefore, the data show that IL-17 reduces TARC and eotaxin expression directly in lung cells, providing a key effector mechanism of how IL-17 inhibits allergy in vivo. The absence of TARC production upon OVA stimulation in regional lymph node and splenocyte cultures () supports the relevance of TARC in lungs rather than lymphoid organs.
Figure 8. IL-17 inhibits IL-5, IL-13, and Rantes production in mediastinal lymph nodes (MLNs). MLN (A–D) and splenocyte (E–H) cultures were prepared from OVA-sensitized and challenged animals. Cells were treated with saline, 50 μg/ml OVA (more ...)
IL-17 reduces Th2 cytokine production
Since IL-17 reduces functions of the APCs, the DCs, we addressed whether IL-17 reduces antigen-induced Th2 cytokine production in vivo and in cell cultures. In vivo, IL-17 reduced allergen-induced IL-5 production in lung tissues by 72 ± 6% (P < 0.05) and in the BAL fluid by 100% from 54.3 ± 29.8 pg/ml to below detection limit (1 pg/ml). BAL IL-4 concentrations were reduced by 98 ± 0.5% in the presence of IL-17, from 13.0 ± 3.3 pg/ml to 0.3 ± 0.5 pg/ml. The Th2 cytokine expression of IL-5 and IL-13 in restimulated regional lymph node cell cultures was also inhibited by IL-17 by >50% () but not in splenocyte cultures (). Similarly, IL-17 reduced OVA-induced Rantes expression by regional lymph node cells () but not splenocyte cultures (). Therefore, the data show that IL-17 markedly inhibits antigen-stimulated lung and regional lymph node cells, providing a key target mechanism of how IL-17 inhibits allergy.
IL-17 was augmented in IL-4Rα KO mice and reduced allergic asthma
Since IL-4 signaling has a major effect on the allergic response (42
) and has been implicated in vitro to act on Th17 development (10
), we addressed the question of whether IL-4Rα KO mice have altered IL-17 expression. Allergen challenge in IL-4Rα KO mice increased IL-17 concentrations from 20 ± 4 pg/ml in WT mice to 32 ± 7 pg/ml (). Pulmonary IL-23 concentrations upon OVA challenge were equal in IL-4Rα KO and WT mice, indicating that IL-4 acts directly on IL-17 production (unpublished data).
Figure 9. IL-4 signaling suppresses pulmonary IL-17 production. OVA/alum-sensitized BALB/c mice (WT) and IL-4Rα KO (IL4R−/−) mice were challenged twice intranasally with either saline or OVA alone. In parallel, the IL-4Rα KO mice (more ...)
IL-4Rα KO mice had reduced chemokine TARC production and allergic inflammation upon OVA challenge. Blocking of endogenous IL-17 by neutralizing antibodies reversed the reduced pulmonary TARC production, eosinophil recruitment to the airways, and EPO activity in the lungs in IL-4Rα KO mice (). This indicates that the inhibition of asthma in IL-4Rα KO mice is partially caused by endogenous IL-17. This marked effect of endogenous IL-17 in IL-4Rα KO confirms the effect found on TARC expression of endogenous IL-17 in WT mice shown in .
In parallel, IL-17 neutralization reduced neutrophil recruitment to the airways, myeloid peroxidase activity in the lungs, and neutrophil chemokine KC production. This demonstrates that augmented neutrophil recruitment in IL-4Rα KO mice is partially caused by endogenous IL-17 (). These data suggest a novel down-regulatory role of endogenous IL-17 in the effector phase of allergic asthma, which may, however, be accompanied by increased neutrophil accumulation in the absence of IL-4 signaling.