The Regulation Of γδT Cells Producing Large Amounts Of IL-17 On Treg Cells During Chlamydia Lung Infection

Cbjective:γδT cells bridge innate and adaptive immunity as a major cells of host innate immune response. Some studies showed that the proliferation and function of Treg cells were partly inhibited by IL-17⁺γδT cells. In this study, the influence and mechanism of IL-17-producing γδT cells on Treg cells proliferation was investigated on Chlamydia pneumonitis induced by respiratory infection with Cm by using TCRδ-/- mice. The research results will provide theoretical basis for the cross regulation of γδT cells with Treg cells and add new content for the interaction between innate and adaptive immunity.Nfethods:WT and TCRδ^-/- mice were inoculated intranasally with 1×10³ inclusion-forming units （IFU） of Chlamydia muridarum （Cm） to induce the Chlamydia pneumonitis. Bodyweight change of infected mice was monitored daily. Mice were sacrificed at different days post infection. Bronchoalceolar lavage（BAL） and lung mononuclear cells were prepared and stained with FITC-anti-CD3 and APC-anti-TCRy5. The percentage of CD3⁺TCRγδT cells was detected by flow cytometry. IL-17 production by CD3⁺γδT cells and Vyl and Vy4 subsets were determined by intracellular cytokine analysis. The level of IL-17 in BAL of WT and TCRδ-/- mice were detected by ELISA. In order to explore the infulence of γδT cells on Treg cells proliferation during Cm infection, CD4⁺CD25⁺T/CD25⁺Foxp3⁺cells in the lung and spleen were detected by flow cytometry. The mRNA expression of Foxp3N IL-2N TGF-β and IL-10 were detected by RT-PCR.Results:Chlamydial pneumonitis was induced in mice by intranasal inoculation of 1×10³ IFU of Cm. The percentage of CD3⁺TCRy5⁺T cells was increased on the first day （*P<0.05）after Cm infection and reached the highest level on day 7（***P<0.001）, and then declined until to the basic line on the day 10 post infection. Intracellular cytokine staining results showed that γδT cells secreted a large amounts of IL-17 on the first day after Cm infection （**P<0.01）, reached the peak on 3 day（***P<0.001）, and the level of IL-17 secreted by γδT cells significantly reduced on the fifth day（*P<0.05）. The secretion of IL-17 which produced by two important γδT cell subsets Vyl and Vy4 in the lung was further investigated following Cm infection. The results showed that Vyl basically secreted no IL-17, and Vy4 secreted a large amount of IL-17, especially reached to the peak on the third day and sharp decrease to the basic level on the tenth day. Moreover, the results also showed that γδT cells are the primary source of IL-17 infection in early stage of infection. The level of IL-17 in BAL was declined after γδT cells deletion （*P<0.05）. Cm infection can induce the proliferation of Treg cells in spleen and lung. Interestingly, the percentage of Treg cells on spleen tissues of TCRδ-/-mice was increased compared to WT mice on day （**p<0.01）,but it was decreased on day 7（**p<0.01）. The mRNA expression of Treg cells related transcription factor Foxp3 in lung were consistent with the changes of spleen Treg cells proliferation. IL-2 mRNA expression of lung was very low on day 3 and no significant difference between WT and TCRδ-/- mice, but it was higher in TCRδ-/-mice on day 7 compared to WT mice（**P<0.01）. IL-10 mRNA expression of lung also increased on day 3（* P<0.05） and decreased on day 7（*P<0.05） in TCRδ-/- mice compared with WT mice. There is no significant difference on TGF-β mRNA expression in the lung between WT and TCRδ-/- mice.Conelusions:Cm respiratory infection can induce γδT cells proliferation and secretion of large amounts of IL-17 in the lung, meanwhile Vy4 γδT cell is the main γδT cells subsets of IL-17 secretion. Cm infection can induce the proliferation of Treg cells in sites of inflammation and peripheral immune organs. The regulation of γδT cells to Treg cells was present during the Cm respiratory infection. However, the regulation mechanisms were distinct in different stages of infection. IL-17⁺γδT cells maybe suppress proliferation and function of Treg cells at early stage during Cm infection, then they may be to promote Treg cells response by IL-2/IL-2R pathway at the medium-term of infection. In addition, γδT cells mainly mediated immunoregulation of Treg cells by affecting the expression of functional cytokine IL-10 during Cm infection.