3-Methyl-4-Nitrophenol Promotes TIM-4 Expression In Dendritic Cells To Promote Allergic Rhinitis

Objective:To build a mouse model of allergic rhinitis using OVA and MNP,to investigate the function of MNP in the induction of allergic rhinitis,and to provide the theoretical basis for the treatment and prevention of allergic rhinitis.Methods:1.Establishing the model of allergic rhinitis in mice by using OVA and MNP,the OVA-induced AR animal model was actively sensitized with three subcutaneous(sc)injections of 100μg OVA and aluminum hydroxide in a total volume of 0.1 m L on days0,7,and 14.Seven days after the completion of the sensitization,these mice were intranasally challenged(OVA 200μg)for 7 consecutive days.Mice in the control group and MNP group underwent the same procedure but received normal saline solution and MNP instead of OVA.For the severe group,MNP was injected subcutaneously into OVA-sensitized animals once on days 0,7,and 14,and intranasally challenged(OVA200μg)for seven days.2.Recording and scoring of nasal symptoms in mice.Using ELISA to detect the expression levels of specific Ig E and Ig G1 in mouse serum and the Th2 type cytokine IL-4,IL-5 and IL-13 in nasal lavage fluid and alveolar lavage fluid.Using nasal mucosa sections to observe and count eosinophils in the nasal mucosa;To observe the inflammatory cells such as mast cells in lung tissue.3.The quantity and maturation of dendritic cells in mouse lungs were measured using flow cytometry.The amounts of TIM-4 and CCR7 expressed by dendritic cells in mouse lung tissues were measured using Western blot and RT-qPCR.4.In vitro experiments were performed to identify the signal transduction pathway of up-regulated expression of TIM-4 in dendritic cells,and the role of AKT and STAT6pathway.To detect Phospho-Akt,Phospho-STAT6 and TIM-4 after use of the small-molecule inhibitor LY294002(a classic inhibitor of PI3K/AKT)in MNP-stimulated DC2.4 cells.Results:1.The OVA group had a higher behavioral score than the Control group(P<0.001),whereas the OVA+MNP group had a significantly higher behavioral score than the OVA group(P<0.05).OVA-specific Ig E(P<0.05)and Ig G1(P<0.001)serum levels were substantially higher in the OVA group compared to the control group,and they were also significantly higher in the OVA+MNP group compared to the OVA group.The levels of Th2-type cytokines IL-5(P<0.01)and IL-13(P<0.01)in mice nasal lavage fluid were higher in the OVA group than in the control group;in the OVA+MNP group,the levels of IL-5 and IL-13 were higher than in the OVA group,but no statistical significance was identified.According to the nasal mucosa HE data,mice in the OVA group showed higher eosinophil infiltration than mice in the Control group(P<0.001);mice in the OVA+MNP group had more eosinophil infiltration than mice in the OVA group(P<0.05).2.Th2-type cytokines IL-4(P<0.05)and IL-13(P<0.01)were higher amounts in the OVA group’s alveolar lavage fluid than in the Control group.Although being greater,the levels of Th2-type cytokines IL-4 and IL-13 in the alveolar lavage fluid of the OVA+MNP group were not statistically significant when compared to the OVA group.In comparison to the control group,the nasal mucosa HE results showed that the OVA group had structural damage to the alveoli,thickened vessel walls,inflammatory cell infiltration,increased eosinophils,and PAS staining showing increased mucus secretion, whereas the OVA+MNP group had a more serious airway inflammatory response with a lot more inflammatory cell infiltrates and significantly increased mucus secretion.3.When compared to the control group,the OVA group had a greater percentage of CD11c~+dendritic cells(P<0.05),while the OVA+MNP group had a considerably larger percentage of CD11c~+dendritic cells than the OVA group(P<0.05).The OVA group had a larger proportion of CD11c~+CD86~+dendritic cells than the control group(P<0.05),and the OVA+MNP group had significantly more CD11c~+CD86~+dendritic cells than the OVA group(P<0.01).4.The results indicated that TIM-4(P<0.01)and CCR7 levels were higher in the OVA group’s lung tissue compared to the Control group,whereas CCR7 levels were higher but not statistically significant.The levels of TIM-4(P<0.05)and CCR7(P<0.05)were significantly increased in the OVA+MNP group compared to the OVA group.5.To detect the expression of TIM-4 m RNA in DC2.4 cells,Western blot and RT-qPCR were used.MNP-stimulated cells had significantly higher levels of TIM-4expression than untreated cells(P<0.05).The results of the Western blot for Phospho-Akt and Phospho-STAT6 levels showed that Phospho-AKT and Phospho-STAT6 peaked at 30min and 6 h,respectively.The Western blot revealed that the expression of Phospho-AKT and Phospho-STAT6 was similar in cells without treatment and with the addition of inhibitor and MNP,whereas the expression of Phospho-AKT and Phospho-STAT6 was slightly decreased in cells with the addition of inhibitor alone,while the expression of Phospho-AKT and Phospho-STAT6 was highest in cells with the addition of MNP alone.The results indicated that TIM-4 expression was similar in cells without treatment,with the addition of inhibitor and MNP,and slightly decreased in cells with the addition of inhibitor alone,with the addition of MNP alone resulting in the greatest TIM-4expression.Conclusion:MNP induces allergic rhinitis in mice and promotes the aggregation and maturation of dendritic cells.MNP promotes the expression of TIM-4 on the surface of dendritic cells by activating the PI3K/AKT/STAT6 pathway,and dendritic cells migrate to the T-cell area,where TIM-4 produced on their surface binds to TIM-1 expressed on na?ve CD4~+T cells,promoting their development into Th2 cells and aggravating allergic rhinitis.