The Proliferation,Activation And Function Of The γδT Cells Or Its Subsets In The Lung Of Mice Infected With Chlamydial Muridarum

Objective:γδT cells play a pivotal role in host innate response, not only as the bridge between innate and adaptive immunity, but also acts in anti-infection, anti-tumor, immune regulation and immune tolerance. The mice were inoculated intranasally with Chlamydia muridarum to induce the Chlamydia pneumonia in this study. The proliferation, activation and function of γδT cells in lung were firstly investigated during Cm airway infection. Then the γδT cell subsets and the variations in the lung of mice following Cm infection were investigated. The proliferation, activation and cytokines production by the two of γδT cell subsets, Vγ1+T or Vγ4+T cells were finally discussed to define the role of the γδT cells and its subsets in chlamydia pneumonia of mice. These studies will provide theoretical basis for the immunological mechanism of intracellular bacterial infection. Methods:WT mice were inoculated intranasally with 1×103 inclusion-forming units(IFU) of Chlamydia muridarum to induce the murine model of Chlamydia pneumonia.1. Mice were sacrificed at different time points post infection. The lung mononuclear cells were isolated and stained with anti-CD3, anti-TCRγδ and anti-CD69. The percentage of γδT cells and CD69 expression were detected by flow cytometry. IFNγ and IL-17 production by γδT cells in the lung of mice following Cm infection were detected by using intracellular cytokine staining to define the role of γδT cells in Chlamydia infection.2. WT mice and TCRδ-/- mice were intranasally infected with Cm. The survival qualities and weight changes postinfection are monitored every day. Chlamydia growth in the lung was detected by ELISA. Histological analysis was processed by staining lung sections with H&E to evaluate the inflammation levels.3. The lung tissue of mice was frozen by liquid nitrogen, and mRNA expression of γδT cell subset were detected by using RT-PCR technology to define γδT cell subsets distribution in lung and the changes post Cm airway infection.4. Mice were sacrificed at different time points post Cm infection and lung mononuclear cells were prepared and stained with anti-CD3, anti-TCRγδ, anti-TCRVγ1 and anti-TCRVγ4. The percentage and absolute number of Vγ1+T or Vγ4+T cell subsets and CD69 expression were detected by using flow cytometry to define the proliferation and activation of two γδT cell subsets in Chlamydia infection. IFNγ and IL-17 production by Vγ1+T or Vγ4+T cells in the lung of mice following Cm infection were detected by using intracellular cytokine staining to study the fuction of γδT cell subsets in Chlamydia infection. Results:Chlamydial pneumonia was induced in mice by intranasal inoculation of Cm. The percentage and absolute number of CD3+TCRγδ+T cells gradually increased following Cm infection and reached the highest level on the day7(p<0.01). Cm infection induced lung γδT cells activate, the expression of CD69 significantly increased on the day3 and day7(p<0.01,p<0.05). γδT cells produced IFNγ and IL-17 at early stage of infection and participated in the host against Chlamydial lung infection. In contrast with WT mice, TCRδ-/- mice showed more severe disease which indicates the γδT cells play a protective role during Cm infection. According to the different TCRγ chain,mice γδT cells are divided into Vγ1+T, Vγ2+T, Vγ4+T, Vγ5+T, Vγ6+T and Vγ7+T cell subsets, and there is no definite conclusion on the tissue distribution of these cells. We firstly study the distribution of γδT cell subsets in lung of normal mice. RT-PCR results showed there are 5 kinds of γδT cell subsets including Vγ1+T、Vγ2+T、Vγ4+T、Vγ5+T and Vγ6+T cells in lung of mice and the contents are compared to Vγ2 > Vγ4 > Vγ1 > Vγ6 > Vγ5. Vγ4+T and Vγ1+T cell subsets significantly increased after Cm lung infection, Vγ4+T cells reached the highest level on the 3rd day and then declined(p<0.001) and Vγ1+T cells reached the highest level on the 7th day(p<0.01). Vγ6+T also increased after infection and proliferate significantly in 7th days. Vγ2+T and Vγ5+T cells have no significant change in the lung after Cm infection. Vγ1+T and Vγ4+T cells in lung tissue were further detected by flow cytometry, it showed that the percentage and absolute number of Vγ1+T cells increased gradually and reached the highest level on day7(p<0.01), and the percentage and absolute number of Vγ4+T cells increased rapidly and reached the highest level on day3(p<0.001), then declined. The expression of CD69 showed the activation percentage of Vγ1+T cells and Vγ4+T cells reached the peak on the 7th and 3rd day respectively(p<0.01). Cm infection mainly induced Vγ4+T cells proliferation in early stage, and then Vγ1+T cells become the main γδT cell subsets gradually. Cytokine detection showed Vγ1+T cells could secrete IFNγ but do not product IL-17, and Cm infection induce IFNγ secretion reach the peak on 7th day(p<0.01). Vγ4+T cells could secrete IFNγ and IL-17, and the cytokine levels increased rapidly after Cm infection and reached the peak on 3rd day(p<0.001), then declined to the basic level in middle stage of Cm infection. The levels of cytokine secretion of Vγ1+T cells, Vγ4+T cells and Vγ1-Vγ4-γδT cells were compared and showed Vγ4+T cells are the main subset of γδT cells in secreting IFNγ and IL-17 at early stage of the immune response induced by Chlamydia lung infection in mice. Conclusions:Chlamydia airway infection induced γδT cells to proliferate, activate and to product IFNγ and IL-17 in lung at early period of the infection, which play an immune protective role against Cm infecion. Lung γδT cells are composed of Vγ1+T、Vγ2+T、Vγ4+T、Vγ5+T and Vγ6+T subsets. Vγ1+T and Vγ4+T cells are the main γδT cell subsets of proliferation following Cm lung infection. Vγ1+T cells could secrete IFNγ but have no ability to secrete IL-17 and Cm infection induced the increased secretion of IFNγ. Vγ4+T cells could secrete IFNγ and IL-17 simultaneously during Cm airway infection. Vγ4+T cells became the major IL-17 and IFNγ-producing γδT cell subsets at the early period of Cm airway infection.