The Role Of α-galactosylceramide Stimulated NKT Cells In Genital Tract Infection With Chlamydia Muridarum

Chlamydiae, an obligate intracellular bacterial pathogen, causes various human diseases. Chlamydia trachomatis genital tract infection is the most prevalent bacterial cause of sexually transmitted diseases in the world, with an estimated100million new cases each year in women. The genital tract chlamydial infection can result in severe complications, including pelvic inflammatory disease, ectopic pregnancy, and infertility. Moreover, chlamydial genital infection increases the risk of acquiring human immunodeficiency virus-related AIDS and human pappilloma virus-induced cervical carcinoma. Therefore, genital tract chlamydial infection is an important public health concern with a heavy financial burden. Previous studies have demonstrated that the Thl immune response, especially IFN-y production, is critical for protection against chlamydia infection. However, there is no clear and detail understanding of immune mechanisms to chlamydial infections.Natural killer T (NKT) cells are a unique lymphocyte subpopulation characterized by co-expression of surface markers with conventional T cells and NK cells. The most characteristic function of NKT cells is their ability to be rapidly activated and to produce cytokines in response to T-cell receptor engagement. Stimulation of NKT cells also results in the activation of other cells, including dendritic cells, NK cells, macrophages, B cells and conventional T cells. Because of their potent immuno-modulatory properties, NKT cells are considered to influence a wide range of diverse disease conditions such as autoimmune diseases, allergy and tumor rejection, as well as infections.NKT cells recognize lipid antigens that are presented by the nonclassical MHC I molecule CD1d. α-galactosylceramide (a-GalCer), a synthetic glycolipid ligand, which specifically binds to CD Id can stimulate classical NKT (invariant NKT) cells with semi-invariant TCR. In contrast, the nonclassical NKT cells with diverse TCR and CD1d-independent NKT-like cells do not respond to a-Galcer.Recent evidence suggests that NKT cells play an important immune regulatory role in the response to various microbial pathogens. Recent studies showed that NKT cells play a crucial protective role in the host defense against chlamydial infections. For example, NKT-knockout mice experience more severe disease and in vivo pathogen growth in lung C. pneumoniae infection. Moreover, the activation of NKT cells generated protection against C. trachomatis L2-induced arthritis. However, in a mouse model of C. muridarum lung infection, NKT cells was found to enhance the Th2immune response and promote chlamydial infection in vivo. These different observations reflect the functional complexity of NKT cells and the necessity for analyzing the role of NKT in different infection models including different infection routes.The genital tract is the natural route of sexually transmitted C. trachomatis infection, and genital tract mucosa has unique immunological features different from the mucosal immune system of lung or other organs. In the current study, we investigated the role of NKT cells in genital tract infection with C. muridarum for studying chlamydial genital infections in mouse models. Since α-GalCer is a specific ligand of NKT cells and has been shown in numerous studies for its stimulating effect on NKT cells, we used α-GalCer to enhance the function of NKT cells and investigated the mechanisms by which NKT cells modulate the immune responses against genital tract chlamydial infection.Ⅰ. The role of α-GalCer stimulated NKT cells in genital tract infection with C. muridarum1. α-GalCer enhance the activation of NKT cells during C. muridarum genital infection We first investigated whether α-GalCer could influence the activation of NKT cells. BALB/c mice were infected intravaginally with1×105IFUs of C. muridarum2h after α-Galcer or vehicle intravenous injection. The a-GalCer-treated mice showed a significant increase in percentages of NKT (CD1d tetramer+TCRβ+) cells compared with the vehicle control (5.6%vs1.55%,p<0.001).In addition, the expression of CD69, an early activation marker, was significantly upregulated in α-GalCer-treated mice than that of control (p<0.05). These data suggest that α-GalCer effectively promoted NKT cells activation during C. muridarum genital infection.2. α-Galcer-treated mice show enhanced clearance of C. muridarum genital infection and reduced inflammatory pathologic changesWe evaluated the role of NKT cells in host defense against the infection. The body weight loss of the α-GalCer-treated mice was less than that of control. The IFU level was significantly different between α-GalCer-treated group and vehicle-treated control group(p<0.05). Mice pretreated with α-GalCer showed clear reduction in chlamydial shedding at as early as day3postinfection. The difference expanded with time until day9post-infection when the α-GalCer-treated mice showed approximately10-folds lower chlamydial burden than the ones without α-GalCer-treatment.We further compared the histopathological changes in the genital tract between the two groups. Gross examination showed that the incidence of hydrosalpinx and dilatation of uterine horn was significantly reduced in the α-GalCer-treated mice. Detailed histopathological comparisons were performed by light microscopy. The a-GalCer-treated mice showed less severe tissue inflammation, in contrast, the vehicle-treated mice showed more severe inflammatory cellular, tissue inflammatory exudates and hyperemia infiltration.Therefore, NKT cells play a protective role in the host defense against C. muridarum genital infection in both pathogen clearance and pathological changes. Ⅱ. The mechanism by which activated a-GalCer-activated NKT cells play a promoting role in host resistance to C. muridarum genital tract infection1. NKT cells become activated during C. muridarum genital infection We investigated whether NKT cells could respond to chlamydial genital infection. At different time points after infection, the mice showed a significant increase in percentages of NKT (CD1d tetramer+TCRβ+) cells compared with the uninfection control. In addition, the expression of CD69was significantly upregulated after infection. These data suggest that C. muridarum genital infection effectively induce NKT cells activation.2. C. muridarum genital infection induces IFN-γ polarization of NKT cellsNext, we investigated the intracellular cytokine profile of NKT cells during genital chlamydial infection. Compared with the unfected control, the infected mice showed a significantly higher expression level of IFN-γ. Therefore, the pattern of cytokine production suggested that C. muridarum infection can skew the cytokine response of NKT cells to IFN-γ polarization.3. NKT activation by α-GalCer enhances IFN-γ polarization of NKT cells following genital chlamydial infectionWe further investigated the influence of α-GalCer on the cytokine pattern of NKT cells during chlamydial infection. Intracellular cytokine analysis showed that prior treatment with α-GalCer followed by C. muridarum infection enhanced the IFN-γ production by NKT after infection (p<0.001). Notably, NKT cells activated by α-GalCer alone (without C. muridarum infection) showed typically high levels of both IFN-γ and IL-4production. Therefore, the pattern of cytokine production by NKT cells was determined by C. muridarum genital tract infection, while α-GalCer treatment enhanced the IFN-γ polarization of NKT cells during genital chlamydial infection.4. α-GalCer-treated mice show enhanced type1immune response during C. muridarum genital tract infectionTo explore whether the improved protective effect in host defense against C. muridarum genital infection in α-GalCer-treated mice was associated with an enhanced Chlamydia-specific Thl immune response, we evaluated cytokine production in genital tract tissues and local lymph node cells of the different groups of mice. First, real-time PCR results suggested α-GalCer-treated mice showed a significantly higher expression level of IFN-y and IL-12mRNA but decreased IL-10expression in genital tissues compared with the control.We next determined the level of cytokine protein by ELIS A. IFN-y production by lymph node cells and in genital secretions from a-GalCer-treated mice was significantly higher than those from vehicle-treated mice post-infection. Similarly, a-GalCer-treated mice showed high levels of IL-12production. In contrast, IL-10production was significantly lower in a-GalCer-treated mice than in vehicle-treated mice.Therefore, NKT cells activated by a-GalCer significantly increase Thl immune response in the genital infection model.5. α-GalCer-treated mice show increased IFN-y production by NK and T cells during chlamydial infectionWe further examined the influence of NKT cell activation on NK and T cells during genital chlamydial infection. Compared with cells from vehicle-treated mice, NK cells from lymph nodes isolated from α-GalCer-treated mice showed significantly enhanced IFN-γ production after infection. This suggested that enhanced NKT cells activation by α-GalCer promoted IFN-γ production of NK cells. Next, we compared intracellular IFN-γ and IL-4production by CD4+T and CD8+T cells in splenocytes from α-GalCer-treated mice and control mice. CD4+T and CD8+T cells from α-GalCer treatment mice produced a greater amount of IFN-γ than did untreated mice after infection. In contrast, the control mice induced more IL-4production by CD4and CD8T cells. Therefore, NKT cell activation by α-GalCer contributed to enhanced type1T cell response during C. muridarum genital tract infection.In conclusion, our results demonstrate that enhanced activation of NKT cells by the administration of a-Galcer led to an enhancement of protection against C. muridarum genital tract infection The beneficial effect of NKT cells is associated with a shift in cytokine profile of NKT cells to IFN-y polarization induced by C. muridarum genital tract infection. The results also suggested that activated NKT cells have an promoting effect on IFN-y production by NK and conventional T cells in this infection. These findings are helpful to enrich our understanding on the mechanism of chlamydial infection, moreover, the investigation provides novel strategy and experiment foundation for developing prophylactic vaccine against chlamydial infection.