Effects Of Pulmonary Dendritic Cells Expressing TGF-?1 On Allergic Asthma In Mice And Its Mechanism

Objective:Pulmonary dendritic cells?DCs?are vital innate immune cells in the lung and play a key role in initiating adaptive immune responses preventing from foreign antigen,infections,pathogenic bacteria,and tissue damage.As professional antigen-presenting cells,DCs play important roles in the induction and progression of allergic asthma when encountering antigens.Transforming growth factor?1?TGF-?1?also can act as proinflammatory or antii-nflammatory cytokine in allergic asthma.And DC is a source of TGF-?1,so we hyothesized that lung DCs regulate the development of allergic asthma through TGF-?1.In this study,mice were immunized with Ovalbumin?OVA?plus aluminum hydroxide?ALUM?adjuvant and subsequently challenged with OVA aerosol to induce allergic asthma.The effect of TGF-?1 expression in lung DC on the allergic asthma in mice and its mechanism was studied by using cre-loxp technique to construct the knockout mice with DC-selective deletion of TGF-?1.Methods:1.7-8 weeks old female C57BL/6 mice were sensitized intraperitoneally with OVA plus ALUM on day-28,day-14,and On day 0,1%OVA atomization was performed once a day for twenty minutes and continuously atomized for three days to induce dynamic allergic asthma in mice.The mice were sacrificed at the same time after the first atomization?day0,i.e.,saline;day1;day2;day3;day5;day7;day9?.The dynamic changes of inflammatory cells in lung tissue and alveolar lavage fluid?BAL?were measured by flow cytometry.2.The mice were sacrificed according to the method 1,and the mice were sacrificed at the same time after the first time atomization?day0,day1;day2;day3;day5;day7;day9?.The dynamic changes of lung DC in mice were detected by flow cytometry.3.CD11c-Cre^+/-mice were crossed with TGF-?1^fl/fl mice to establish the DC-selective deletion of TGF-?1 mice called CD11c^cre×TGF-?1^fl/fll/fl mice?Cre+?and TGF-?1^fl/fl mice?Cre-?,and then mice were identified by PCR.4.Gene knockout mice?Cre+?and their littermally control mice?Cre-?were constructed according to method 3 and the early?day2?allergic asthma was induced according to method 1.Mice sensitized with OVA/ALUM and induced with saline were used as model control groups.After two days of first challenge?day2 allergic asthma?,flow cytometry was used to detect the effect of lung DC-deficient TGF-?1 on infiltration of inflammatory cells in the lung and BAL of the early?day2?allergic asthma.5.Similarly,after three days of initial stimulation?day3 allergic asthma?,flow cytometry was used to detect inflammatory cell changes in the lung and BAL of allergic asthma mice with DC-deficient TGF-?1.The levels of IL-4,IL-5 and IL13 of BAL were detected by enzyme-linked immunosorbent assay?ELISA?.The pathological changes of HE and mucus secretion of PAS were observed.6.The counts and proliferation leves of Treg and its subsets in BAL and DLN in the late?day3?allergic asthma were detected by Flow Cytometry.7.Lung tissue of late?day3?allergic asthma was isolated and total RNA was extracted.Then we used the Q-PCR to detect the expression of asthma-related chemotaxis?CCL11,CCL17 and CCL22?in lung tissue.Results:1.OVA/ALUM allergic asthma is an eosinophils-based allergic reaction.In the atomization period,with the increase of the number of atomization,the eosinophils gradually increased in the lung,and in day3 eosinophils rose sharply and reached the peak,then it gradually decreased;and in BAL,we did not detect the infiltration of eosinophils in day0 and day1 but we detected a small amount eosinophils infiltrated in day2 and in day3it rose sharply and reached its peak.2.In mice allergic asthma,the number of total DCs and their subgroups CD103⁺CD11b^-DC and CD103^-CD11b⁺DCs in the lungs increased sharply in day3 and peaked at day5 compared with day0 group,which indicates that lung DC and eosinophils and other inflammatory cells were positively correlated.3.In order to study the effect of TGF-?1 expression in lung DC on allergic asthma,we constructed DC knockout mice with DC-selective deletion of TGF-?1,and we successfully screened knockout mice and control mice by PCR.4.In mice with early?day2?allergic asthma,the expression of TGF-?1 in lung DC did not affect infiltration of eosinophil-based inflammatory cells in the lung and BAL.5.In mice with late?day3?allergic asthma,eosinophils-based inflammatory cells in BAL and lung increased sharply in Cre+mice.The IL-5 of BAL supernatant increased sharply.HE and PAS staining showed that pulmonary inflammatory cells increased infiltration and mucus secretion increased.Results indicated that lung DC-deficient TGF-?1 promoted eosinophil-based type II inflammation.6.The further detection of treg ratio and proliferation indicated that DC-deficient TGF-?1 resulted in the increased ratio of Treg and tTreg in BAL and the proliferation of Treg and foxp3^-effector T cells in the late?day3?allergic asthma mice.While in the DLN,the cell population and its proliferation did not change significantly.Indicate that lung DC-deficient TGF-?1 promoting the eosinophil-based type II inflammation in the late?day3?allergic asthma is not due to the decline in Treg.7.Q-PCR showed that the expression of CCL11,CCL17 and CCL22 in the lungs of mice on day3 was increased in lung DC in Cre+mice.It suggested that lung DC-deficient TGF-?1 promoted the eosinophil-based type II inflammation in the late?day3?allergic asthma,and it was related to the enhancement of expression of chemokines related to pulmonary asthma.Conclusion:Lung DC-deficient TGF-?1 promotes the eosinophil-based type II inflammatory response predominantly in the late stage of allergic asthma?day3?rather than the early?day2?allergic asthma.Its pathogenesis is not achieved by the decline of Treg,and may be associated with the increased expression of asthma-related chemokines CCL11,CCL17,CCL22.