| Objective:IL-23/Th17 axis is an important regulator in various inflammatory diseases. However, the role of IL-23 in allergic airway inflammation is not well understood. This study aims to observe whether IL-23-IL-23R signaling is involved in the development of allergic airway inflammation, furthermore, to investigate the possible mechanisms.Methods:(1) mRNA expression of IL-23p19, IL-12p35, IL-12/IL-23p40 and IL-23R was determined by real-time PCR in whole lung tissue from OVA-challenged B6 female mice. Non-challenged B6 female mice were used as control. (2) IL-23 KO and WT female mice were subjected to OVA-sensitizing-induced asthma. Inflammatory infiltrates in lung were assessed by HE staining. Total cells of bronchoalveolar lavage fluid (BALF) from the asthmatic mice were counted. Cellular profiles in BALF upon OVA challenge were assessed by cytospin with May-Gruenwald Giemsa staining. EPO expression in lung was determined by real-time PCR. OVA-specific IgE expression in sera was measured by ELISA. Expression of type-2 cytokines in lung draining lymph node cells and splenocytes after ex vivo OVA restimulation was assessed by ELISA. OVA-specific Th17 cytokine expression was determined by ELISA. (3) Generation two lines of IL-23R Tg mice with IL-23R overexpression in T cells using the human CD2 mini locus promoter. IL-23R mRNA expression was tested by real-time PCR. One of the two lines was extensively analyzed. Naive T cells were FACS-sorted from IL-23R Tg or B6 mice and activated under Thl7 and Thl conditions with or without recombinant mouse IL-23. Four days later, IFN-y and IL-17-producing cells were analyzed by intracellular staining. (4) IL-23R Tg and B6 female mice were subjected to OVA-sensitizing-induced asthma. Inflammatory infiltrates in lung were assessed by HE staining. Total cells of bronchoalveolar lavage fluid (BALF) from the asthmatic mice were counted. Cellular profiles in BALF upon OVA challenge were assessed by cytospin with May-Gruenwald Giemsa staining. EPO expression in lung was determined by real-time PCR. OVA-specific IgE expression in sera was measured by ELISA. Expression of type-2 cytokines in lung draining lymph node cells and splenocytes after ex vivo OVA restimulation was assessed by ELISA. OVA-specific Th17 cytokine expression was determined by ELISA. (5) Naive T cells were FACS-sorted and stimulated with anti-CD3 and irradiated splenic APC from IL-23 KO or WT mice in the presence of IL-2, IL-4, and anti-IFN-y. Five days later, IL-4 and IL-17-producing cells were analyzed by intracellular staining. Cytokine production was measured by ELISA. T-bet, GATA-3, and Foxp3 mRNA expression was analyzed by RT-PCR. (6) Naive T cells were FACS-sorted from IL-23R Tg or B6 mice and stimulated with anti-CD3 and anti-CD28 in the presence of IL-2, IL-4, and anti-IFN-y with or without recombinant mouse IL-23. Five days later, IL-4 and IL-17-producing cells were analyzed by intracellular staining. Cytokine production was measured by ELISA. T-bet, GATA-3, and Foxp3 mRNA expression was analyzed by RT-PCR.Results:(1) IL-23p19, IL-12p35, IL-12/IL-23p40 and IL-23R mRNA were highly induced in the lungs from OVA-challenged mice compared with those from non-immunized mice (P＜0.05). (2) In OVA-sensitizing-induced asthma model, antigen-induced inflammatory cell infiltration was greatly inhibited in the lungs from IL-23 KO mice compared with that from WT mice. Total cell number was significantly decreased in IL-23 KO mice (P＜0.05). Eosinophils, macrophages and neutrophils were significantly decreased in IL-23 KO mice (P<0.05), and lymphcytes were slightly decreased (P>0.05). IL-23 deficiency led to dramatically decreased expression of EPO in IL-23 KO mice (P＜0.05). OVA-specific IgE expression is significantly lower than that in WT mice (P＜0.05). Upon ex vivo OVA restimulation, the expression of IL-4, IL-5, and IL-13 in lung draining lymph node cells and splenocytes from OVA-challenged IL-23 KO mice was significantly lower in comparison with WT cells (P＜0.05). OVA specific Th17 responses were observed at similar low levels in both IL-23 KO and WT mice. (3) IL-23R expression in thymus was significantly higher in IL-23 R Tg mice than in WT mice, one of the two lines was extensively analyzed. Under the Thl7 condition, addition of recombinant mouse IL-23 significantly increased the frequency IL-17-producing cells in IL-23R Tg T cells. Under the Thl condition, addition of recombinant mouse IL-23 greatly inhibited the generation of IFN-y-producing cells in IL-23R Tg T cells. (4) In OVA-sensitizing-induced asthma model, antigen-induced inflammatory cell infiltration was greatly increased in the lungs from IL-23R Tg mice compared with that from WT mice. Total cell number was significantly increased in IL-23R Tg mice (P＜0.05). Eosinophils, macrophages and neutrophils were significantly increased in IL-23R Tg mice (P＜0.05), and lymphcytes were slightly increased (P>0.05). IL-23R overexpression led to dramatically increased expression of EPO in IL-23R Tg mice (P＜0.05). OVA-specific IgE expression is significantly higher than that in WT mice (P<0.05). Upon ex vivo OVA restimulation, the expression of IL-4, IL-5, and IL-13 in lung draining lymph node cells and splenocytes from OVA-challenged IL-23R Tg mice was significantly higher in comparison with WT cells (P＜0.05). OVA specific Th17 responses were observed at similar low levels in both IL-23R Tg and WT mice. (5) CD4+T cells activated with IL-23-deficient APCs exhibited reduced IL-4-producing cell numbers, and visually no IL-17-producing cells were observed. IL-23-deficient APCs led to significantly reduced amounts of IL-4 and IL-5 expression by effector T cells (P＜0.05). GATA-3 mRNA expression was significantly decreased in T cells treated with IL-23 KO APCs compared with those treated with WT APCs (P＜0.05). (6) Addition of recombinant mouse IL-23 increased IL-4-producing cell numbers and protein expression of IL-4 and IL-13 in IL-23R Tg cells (P＜0.05). In WT T cells, only IL-13 was significantly enhanced by IL-23 treatment (P＜0.05). In response to IL-23 stimulation, IL-23R Tg but not WT T cells highly expressed GATA-3 mRNA (P＜0.05).Conclusion:IL-23-IL-23R signaling is involved in the development of allergic airway inflammation. IL-23 signaling may regulate allergic asthma through modulation of Th2 cell differenciation.
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