The Interaction Between NKT Cells And NK Cells In Host Defense Chlamydial Infection

Members of Chlamydia family are obligate intracellular gram-negtive bacterial pathogen, which include well-known human pathogens C.trachomatis (Ct) and C.pneumoniae (Cpn). Chlamydiae infects the epithelia of the ocular, respiratory and reproductive tract mucosa, causing a wide range of human diseases. C.trachomatis is a most common bacterial cause of sexually transmitted diseases and has long-term consequences, like chronic pelvic inflammation, infertility and ectopic pregnancy. The species of C.trachomatis also includes trachoma serovar which is the major cause of the preventable infectious blindness. C.pneumoniae is responsible for a series of acute and chronic human diseases, including upper respiratory infection, pneumonia and chronic obstructive pulmonary disease (COPD). C.pneumoniae is also associated with an enhanced risk of developing the atherosclerotic cardiovascular diseases and some neurological disorders.The epidemiologic researches emphasize that Chlamydia infection is still a serious public-health problem worldwide. Antibiotic treatment is effective for Chlamydia infection. But as the infection is frequently asymptomatic and infected people may not often seek for diagnose and treatment, this control strategy is limited in some extend. Thus, the safe and effective vaccine would have a more important impact on the high prevalence of chlamydia infection and the prevention of long-term sequelae. So far, no vaccine is available to prevent human chlamydial dieases due to the lack of clear understanding of immune mechanisms to chlamydial infections.Recent progress in studies on host defense mechanisms has revealed the critical importance of the linkage of innate and adaptive immune responses in controlling/clearance of various infections. Both NK cell and NKT cell are very important components of innate immune system and have been implicated in a wide range of conditions. Although they belong to different cell lineages, NK and NKT cells display striking similarities, such as the expression of the same set of NKR-P1 and Ly49 receptors and the capacity to develop their effector function without prior sensitization. They are critical for initially combating infection and contribute to development of the adaptive immune responses by interacting with dendritic cells and by producing immunoregulatory cytokines. Previous studies have shown NK and NKT cell responses in various types of chlamydial infections. The role of NKT cells in control of chlamydial infection is controversial between C.muridarum (Cm) lung infection and C.pneumoniae (Cpn) lung infection. NK cells play a protective role in murine genital tract Cm infection, but it appears that NK cells are not needed for innate resistance to Cpn in RAG-/- mice. Even these previous studies have shown the involvement of NK cells in host defense against chlamydial, the exact mechanism(s) by which NK cells play their role in host defence against these infections remains largely unclear. In addition, the relationship between these two important innate immune cells, NK and NKT cells, in chlamydial infections has not been reported either.Despite their small population size, NKT cells can swiftly secret large amounts of cytokines upon activation and play a potential role in the modulation of immune response. At present study, using antibody blocking approach and gene-deficient mice, we investigated 1) the modulating effect of NKT cells on the function of NK cells and its relationship with NK subsets during C.muridarum infection 2) whether the modulating effect of NKT cells on NK cells is different in C.pneumoniae lung infection model and the role of NK cell in host defense against Cpn lung infection(一)The modulating effect of NKT cells on IFN-y production and cytotoxic function of NK cells and its relationship with NK subsetsNatural killer T (NKT) cells are a newly identified T cell population which plays a role as effector cell and also has potential immunomodulatory functions. NKT cells have been implicated in the modulation of the function of DCs, macrophages, B cells, conventional T cells as well as NK cells. Several studies have shown modulating effects of NKT cells activated by a-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on NK cells'IFN-y production and cytolytic function in a chlamydial infection model, using NKT cell-deficient mice and antibody blocking (anti-CD1d mAb) approaches.1. the response of NK and NKT cells to C.m in vitro. At different time points, we evaluated the activation of NKT cells and NK cells by measuring surface CD69 expression and intracellular IFN-γproduction. During the incubation, NKT and NK cells from EB-stimulated bulk spleen cells up-regulated CD69 expression and produced IFN-γ. These results indicated that live EB can induce both NKT cells and NK cells activation and IFN-γsecretion in vitro.2. the influence of C.m-induced NKT activation on NK cell.To analyze whether NKT can influence EB-induced NK activation, we stimulated bulk spleen cells from Jα18-/-mice and WT mice with live EB and tested NK activation marker CD69 expression and IFN-γproduction using FACS and ICCS. A reduced CD69 expression and IFN-γproduction were monitored on NK cells from Jα18-/- mice after EB stimulation, indicating that the absence of NKT cells partially inhibited EB-induced NK cell activation. Furthermore, the addition of blocking CD1d mAb mildly inhibited CD69 expression and IFN-γsecretionby NK cells in bulk spleen cells. Together, the experiments confirmed the significant modulating effect of NKT cells on NK cell activation and cytokine production.3. The response of NK and NKT cells during C.m lung infection.After showing activation of both NKT and NK cells by Cm infection and the influence of NKT cells on NK cells in the in vitro system, we further tested NKT and NK cell responses in vivo. We observed that both NKT cells and NK cells expanded following Cm lung infection. NKT cells significantly up-regulated early activation maker CD69 expression after infection. In addition, we found increased CD69 expression on NKT cells and NK cells following in vivo infection. The results indicate that Cm lung infection can induce the activation and expansion of both NKT cells and NK cells, confirming the finding of in vitro experiments.4. The function change on NK cells during C.m infection.Having shown the activation and expansion of NK cells in mice with Cm lung infection, we further examined the function of NK cells on IFNy production and cytotoxicity following the infection. lung NK cells from Cm-infected mice showed quick enhancement of IFNy production and degranulation. The results suggest that NK cells not only produce IFNy but also display cytotoxicity during Cm lung infection.5. the regulatory effect of NKT on NK function following C.m infection.To further test the influence of NKT cells on NK cells in vivo following chlamydial infection, we first examined the expansion of NK cells in the spleen and lung following Cm infection in the WT and Jα18-/-mice. NK cells in the Jα18-/- mice was significantly reduced in both percentages and absolute numbers in the spleen and lung following Cm lung infection.We then compared WT and Jα18-/-mice in IFNy production and degranulation by NK cells following Cm lung infection to examine the impact of NKT cells on NK cell function. significantly lower percentages of splenic and pulmonary NK cells in Jα18-/- mice produced IFNy than WT mice. In contrast, the percentages of degranulating splenic and pulmonary NK cells in Jα18-/- mice were significantly higher than those in WT mice. Taken together, the results suggest NKT cells have a positive effect on NK cell expansion but have differential effect on NK cells'IFNy production (promoting) and cytotoxicity (inhibitory) following Cm lung infection.6. Whether the differential effect of NKT on NK cells are associate with NK subsetsTo examine whether the differential effect was caused by potential differential modulating effect of NKT cells on NK cell subsets which might have different patterns for the two aspects of NK cell function, we first measured the percentage of different NK subsets in Jα18-/- and WT mice based on the surface expression of CD27 and CD11b following Cm lung infection. We found that the proportion of NK subsets was altered in the spleen of Jα18-/- mice following Cm infection, suggestting the NKT cell do have some impact on the maturation of NK cells.We further examined IFNγproduction and surface CD 107a expression on different NK subsets by flow cytometry. We found that CD27highCD11bhigh subset was the one with highest IFNy production and CD 107a expression among the subsets. Furthermore, we found that the differential modulating effect of NKT cells on NK cells'IFNy production and cytotoxicity was observed in immature and mature NK subsets although it was more dramatic in the relatively mature CD11bhighCD27highNK cell subset. These results suggested that NKT cell deficiency leads to changes in NK cell subset expansion but the differential effect on IFNy production and cytotoxicity was not restricted to particular NK cell subsets.(二)DCs are involved in modulation of NKT cells on NK cellsPrevious study has reported NKT cells play the distinct role and induce different disease outcome during infection with two Chlamydial species, C.muridarum and C.pneumoniae. We further investigated whether the modulating effect of NKT cells on NK cells function is different during C.pneumoniae lung infection.1. NK cells respond to Cpn respiratory infection.To test whether NK cells respond to Cpn infection, we collected the lung mononuclear cells from the mice exposed to Cpn at various time points and analyzed the expansion, activation and IFN-y production of NK cells by flow cytometry. Both percentage and absolute number of NK cells in lung and spleen was increased at day 1 and reached to the peak at day 3 after Cpn infection, after which there was a slight decrease and the number of NK cells reached to the base level about day 9.To determine whether NK cells exhibit an activated phenotype in response to Cpn infection, we measured, in a kinetic manner, the expression of two early activation markers, CD69 and CD25, and IFN-y production on NK cells in the lung and spleen of Cpn-infected mice. We found that during the course of Cpn respiratory infection, NK cells from lung and spleen exhibit an activated phenotype and produce a large amount of IFN-y in response to Cpn infection.2. NKT regulate NK activation and IFN-y productionOur previous study reported that NKT cell population in lung was activated and produced IFN-y rapidly after Cpn respiratory infection. Moreover, NKT cells activated by a-GalCer induced CD69 up-regulation on NK cells, and this induction was eliminated in CDId-deficient mice. We analyzed the number, the phenotypic activation and IFN-y production of NK cells in WT and NKT KO (Jα18-/-) mice. We found that:1)there is no significant difference in NK cell number between these two mouse strains before and after Cpn infection.2) NK cell from infected NKT KO mice remained significant lower levels of CD69 and CD25 expression compared to those from WT mice.3) there was a notably reduced number of pulmonary NK cells in NKT KO mice stained positively for IFNy, compared with those in infected WT mice. Thus, the results suggested that NKT cells have a positive effect on NK cell activation and IFNy production after Cpn lung infection.3. The modulating effect of NKT cells on NK subsets.Having found the promoting effect of NKT cells on NK cells activation and IFNy production, we then decided to examine whether the phenotypic and functional changes of NK cells in NKT deficiency mice were associated with different NK subsets during Cpn infection. we measured the percentage of different NK subpopulations from NKT KO mice and WT mice based on the surface expression of CD27 and CD11b before and after Cpn infection. The results showed that NKT cell deficiency leads to changes in NK cell subset expansion but the effect on IFNy production was not restricted to particular NK cell subsets4. The function of DCs during NKT and NK interaction.Dendritic cells are critical to initiate the immune response as sentinels in immune system. DC has been implicated in induction NK cell activation though both cell to cell contact and soluble factors. We assumed that the alteration of NK cells function in NKT deficient mice is due to the functional changes in DC. We found that naive NK cells produced decreased IFNy when they were co-cultured with DC from Cpn-infected NKT deficient mice, compared with those with DC from Cpn-infected WT mice. The data suggested that DCs were involved in compromised NK IFNγproduction in NKT deficient mice.5. The role of NK cell in host defense against Cpn lung infectionPrevious study have reported that NK cells is not needed in innate resistance to Cpn infection in RAG-1-/-mice. To investigate whether NK cells contribute to host resistance to Cpn lung infection in immune intact mice, we used anti-asialo-GM1 treatment to deplete NK cells in vivo. We found that depletion of NK cells exacerbated C.pneumoniae lung infection. NK depleted mice showed more severe body weight loss and bacterial burden in lung compared with WT mice. Immunological anlaysis on CD4 and CD8 T cells showed NK depletion leads to reduction of type 1 T cell response following Cpn infection. These data indicate the protective role of NK cells in host defense against C.pneumoniae infection.Taken together, these findings demonstrate the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting. Our study will also likely enrich our understanding on the interaction between different types of innate and adaptive immune cells in a broader perspective.