| Background and Objective:Allergic rhinitits is very popular in developing countries and European countries and China as well. In China, the rate was 10%. In clinic, there are not proper therapeutic targets and preventive methods. TH2 related pathology was the cardinal feature of allergic rhinitis induced by environmental factors in the local epithelium and lot of cytokines and cell subsets contribute to the initiation and development of the disease. Gene microarry reveals that Clara cell 10-KDa Protein (CC10) was the most downregulated in the local mucosa of allergic rhinitis patients. CC10, a member of the secretoglobin family, also referred to as uteroglobin, is a steroid-inducible, multifunctional, secreted protein with anti-inflammatory and immunomodulatory effects. It is constitutively expressed by the epithelial lining of all organs that communicate with the external environment, including bronchi and nose. CC10 can inhibit the activity of phospholipase A2, suppress the expression and function of several cytokines, diminish inflammatory cell chemotaxis, downregulate Th2 cell differentiation, and block prostaglandin D2 receptor-mediated nuclear factor-κB activation. We and others previously also found that compared with responses seen in wild-type mice, CC10-deficient mice develop an intensive pulmonary inflammatory response after sensitization and challenge with Ag and CC10 protein could directly inhibits the differentiation of TH2 cell.TH17 cells are a new subset of T cells recongnized in recent years. TH17 cells are first discovered in experimental autoimmune encephalomyelitis (EAE), and arthritis (CIA). Traditionally, these two kinds of autoimmune disease are considered to be mediated by the TH1 cells. However, studies have found that:removal or neutralization TH1 related cytokines IFN-y or IL-12 could not prevent or mitigate the disease process. However, depeletion of IL-23 could slow the disease process. Subsequent studies confirmed that the lack of IL-23 reduced the in vivo IL-17+T percentage,but did not affect the number of Thl. Thus the correlation of IL-23 and IL-17+ cells in autoimmune disease are further confirmed and further studies found that lack of IL-17+T cells supressed the disease of EAE. Thus, it was th17 cells rather than classical TH1 cells in this environment induced autoimmune disease. In addition to the cytokines IL-17 (IL-17A), TH17 cells also secret IL-17F, and IL-21, IL-22, IL-6, TNF-a and other cytokines. Different T cell subsets have relatively specific markers in that they have different transcription factors. TH1 cells have T-bet, TH2 cells have GATA-3 and Tregs have FoxP3. It was confirmed that the specific transcription factor for Th17 cell was ROR-yt. In the early discovery TH17 cells have been tightly associated with autoimmune diseases including rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus (SLE) and TH17 have also been confirmed in the occurrence and development of tumors. Recent studies found that IL-17 levels are increased in patients with asthma and allergic rhinitis. Further investigation confirmed TH17 cells promote TH2 cell induced recruitment of eosinophils in a mouse model of asthma.In view of the role of CC10 protein in the differentiation of T helper cells and the possible role of TH17 cells in allergic airway diseases, we established a mouse model of allergic rhinitis for the study of the effect of CC10 on TH17 cells and provide a theoretical basis for developing CC10 protein drugs in clinic.Specific research objectives are as follows:1. To establish allergic rhinitis models①To observe the relationship between local CC10 protein and TH17 response in nasal mucosa.②To investigate the local Th17 response in local mucosa in CC10 knock out mouse models.③To study the effect of CC10 administration on Th17 response in vivo. ④To reveal the cell and molecular mechanisms.Methods:1. Subcutaneous injection of ovalbumin in sensitization and challenge with ovalbumin were exloited to establish wild-type and CC10 knockout mouse models of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa.2. CC10 was given intraperitoneally in the sensitized phase In the CC10 knockout mouse model of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa and inguinal lymph nodes.3. CC10 was given nasally in the challenge phase in wild type mouse model of allergic rhinitis. The development of the disease process was discovered by HE staining or PAS staining. The expression of CC10 protein was detected by immunohistochemistry. Real-time PCR and ELISA methods were used to detect TH1, TH2, and TH17 response. flow cytometry method was used to detect the TH17 and TH2 response in nasal mucosa and inguinal lymph nodes.4. wild-type mouse spleen dendritic cells were isolated and treated with CC10 protein and OVA in vitro, ELISA method was used to measure the level of IL-6, TGF-βin the supernatant and Real-time PCR method was used to measure the expression of IL-23 in dendritic cells.5. After in vitro treatment of dendritic cells with CC10 protein and ovalbumin, they were cocultured with T cells sensitized in vivo with OVA, Intracellular cytokine staining was used to detect Th17 response.6. Double Fluorescent staining was used to detect dendritic cells derived IL-6, TGF-βand IL-23 expression in the spleen in the allergic rhnitis models of CC10 gene knockout mice and wild-type mice. 7. Immunohistochemistry method was used to detect epithelial expression of CCL20 and Real-time PCR method was used to measure the expression of IL-23 in the nasal mucosa in CC10 gene knockout mice and wild-type mice model of allergic rhinitis. BEAS-2B cells in vitro were stimulated with TH1, TH2, and proinflammatory cytokines with the subsequent treatment with CC10.Results:1. In allergic rhinitis wild-type mice, the epithelial CC10 positive cells increased with the challenge times, in a gradual downward trend, before the challenge, the epithelial CC10-positive cell number was 100±2/mm, while one day after the first challenge, CC10-positive cells numbered 90±3/mm, and on day 17 and day 19, the CC10 positive cells were 80±6/mm and 50±4/mm. there was a significant statistical difference, compared with that of the saline group with P<0.05 and 0.01 respectively.2. In the wild-type allergic mouse model, local RORyt, IL-17A, IL-17F, IL-22 mRNA levels increased by3 fold (p<0.05),4 fold (p<0.01),5 fold (p<0.05) and 7 fold (p<0.05) respectively compared to the saline control group. IL-17A protein levels in the nasal lavage fluid increased with the challenge times, with a growing trend, On day 14 there was 200±2pg/ml and 200±10pg/ml on day 15, there was no statistical difference compared with the control group. there was statistically significant difference on 17 day (400±7pg/ml) and day19(1600±11pg/ml), compared with the control group with p <0.05 and p＜0.01 respectively. In the nasal lavage fluid, IL-17 was mainly produced by TH17 cells (CD4+IL-17+) which accounted for 10.2%, with statistically significant difference compared the saline group.3. In the CC10 knockout mouse model of allergic rhinitis, nasal lavage fluid cell counts showed that the total number of inflammatory cells were 1.75×106个,the number of eosinophils were 0.9×106, and lymphocytes were 0.7×106个, and there was statistically significant difference compared with the control group. there was statistically significant difference Nasal mucosa local IL-4, IL-5, IL-13 and mRNA levels (p<0.05) relative to wild-type control group and the expression of IFN-ymRNA has no significant difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+), accounted for 27.2%, and there was statistically significant difference compared with the wild-type control group. 4. After day 0 and 7 sensitization, mice were killed and inguinal lymph nodes were isolated. Mononuclear cells measured by flow cytometry method showed the proportion of TH2 cells was 20.3%, TH17 cells 17.9%, and there was statistically significant difference (p<0.05) compared to wild-type mice, while the percentage of TH1 cells had no significant difference between the two groups.5. When CC10 protein was administered in sensitized mice, after OVA challenge, the number of epithelial goblet cells, total inflammatory cells, eosinophils and lymphocytes in the nasal lavage were significantly decreased compared to those of saline control group, there was a significantly statistical difference. local IL-4, IL-5, IL-13 and mRNA levels of nasal mucosa was statistically significant different relative to wild-type control group (p <0.05) and the expression of IFN-γmRNA has no significant difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+)accounted for 20.2%, with the control group (5.3%) and there was statistically significant difference.6. Wild-type mouse spleen T lymphocytes were polarized with corresponding cytokine TGF-β, IL-6 and IL-23, IL-4 neutralizing antibody, IFN-y neutralizing antibody and anti-CD3 anti-CD28 monoclonal antibody to TH17 cells, and then the CC10 protein were treated. the result showed that TGF-β, IL-6 and IL-23, IL-4 neutralizing antibody, IFN-y neutralizing antibody and anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody significantly increase the differentiation of TH17 cells, but the CC10 protein could not inhibit this effect.7. At dayO and 7 after the two sensitization, the mice were killed and the mouse spleens were isolated. mouse spleen tissue sections by double staining showed that dendritic cells derived TGF-β, IL-6 and IL-23 levels in the CC10 knockout mice were significantly higher than thosein wild-type mice.8. Spleen CD11C+dendritic cells sorted by micobeads in wild-type sensitized mice were stimulated with CC10 protein and ovalbumin in vitro for 24 hours, TGF-β, IL-6 levels in the supernatant of the dendritic cells treated by CC10 protein were significantly reduced, and dendritic cell derived IL-23p19mRNA were also significantly lower.9. Dendritic cells treated with CC10 protein in vitro and OVA were cocultured with mouse spleen T cells from the sensitized mice for 48 hours, TH17 cells decreased from 20.1% to 7.6%. 10.When the mice were given CC10 protein in challenge stage, the number of goblet cells in the epithelium and total inflammatory cells, eosinophils and lymphocytes in nasal lavage significantly decreased compared to the number of saline control group. local IL-4, IL-5, IL-13 and mRNA levels in nasal mucosa was statistically significant differenr relative to that of wild-type control group (p<0.05) and the expression of IFN-ymRNA has no significant statistical difference. In the nasal lavage fluid, TH17 cells (CD4+IL-17+), accounted for 12%, with the control group (5.3%) and there was statistically significant difference.11. Nasal lavage cytoslides by immunofluorescence staining showed CD11C positive dendritic cells in CC10 knockout mice expressed significantly higher IL-23 than those in wild-type control group, Moreover, Real-time quantitative PCR method showed that local IL-23p19 and IL-23P40 mRNA level in the local mucosa was significantly higher than that of wild-type control group.12. Immunohistochemistry method showed that local CCL20 protein expression in nasal mucosa in CC10 gene knockout mouse model was significantly higher than that of the control. in vitro CC10 protein significantly downregulated BEAS-2B cell derived CCL20 induced by IL-β,TNF-α, IL-4, IL-13, IFN-γ.Conclusion:1. TH17 cells and the function were enhanced in the local mucosa in allergic rhinitis mouse model.2. CC10 protein inhibits TH17 cell differentiation by downregulating dendritic derived cytokines in the sensitization phase.3. CC10 protein inhibits the multiplication of Th17 cells in the challenge stage by regulating the secretion of IL-23 in dendritic cells.4. CC10 protein acts on the epithelial cells to regulate local TH17 cell chemotaxtics.
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