The Glycolipid Exoantigen Derived From Chlamydia Activates Invariant Natural Killer T Cells And The Function Of Il-22Against Chlamydia Infection

Part One:The Glycolipid Exoantigen (GLXA) Derived from Chlamydia muridarum Activates Invariant Natural Killer T CellsObjectiveChlamydiae are obligate intracellular bacterial pathogens. The genus Chlamydia includes two species, Chlamydia trachomatis and Chlamydia pneumoniae, which cause various human diseases. C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases. A murine biovar of C.trachomatis (known as mouse pneumonitis [MoPn] agent and now classified as a new species, Chlamydia muridarum) has been used to study the mechanisms of C. trachomatis pathogenesis and immunity in a mouse pulmonary and genital infection models. Further, C. pneumoniae causes respiratory diseases like bronchitis, sinusitis and pneumonia. To date, there is no effective vaccine available against human chlamydial diseases. Considering the public health significance of chlamydial diseases, it is highly desirable to have an effective and safe chlamydial vaccine. However, one of the main constraints in the way of vaccine development is poor understanding of the role of chlamydial components in host-chlamydial interactions. NKT (Natural killer T) cells represent an innate subset of T lymphocytes and recognize glycolipid antigens presented by CD1d, which is a nonclassical MHC class I molecule expressed on antigen presenting cells (APC). We have previously reported that iNKT are activated in vivo and play an important role in immune responses to chlamydial infections. GLXA is a glycolipid component of the chlamydial membrane and intracellular inclusion bodies, which has been implicated in chlamydial-host cell interaction. Taking account of these facts, we hypothesized that GLXA might specifically activate iNKT. To test the hypothesis, we investigated the role of GLXA derived from Chlamydia muridarum in iNKT activation using in vitro as well as in vivo settings.We want to further identify the immune mechnism of chlamydial infection and develop vaccine against chlamydial by targeting GLXA.MethodsWe prepared GLXA antigen from the supernatant of C. muridarum infected cell. Supernatants harvested from uninfected cell monolayers were prepared in an identical manner and used as cell mock control. SDS-PAGE and Western blot analysis were done to identify GLXA. We investigated the role of GLXA in iNKT activation using in vitro as well as in vivo settings.1、in vitro1) CD1d fusion protein iNKT hybridoma culture:96-well plates were incubated with mouse CDld fusion protein, and GLXA and iNKT hybridoma cells were added to each well and co-culture. The culture supernatant was measured for IL-2by ELISA.2) LMC-BMDC co-culture:DC were generated in vitro from bone marrow cells. BMDC were cultured in the presence of GLXA for24h. The treated BMDC were co-cultured with LMC for48h. The levels of IFN-y and IL-4in the culture supernatants were measured by ELISA. We use the iNKT knock out mice and CD1d antibody blocking system to do further identification in this co-culture system.2、in vivo1) Intravenous injection:C57BL/6(WT) and iNKT KO mice were intravenously treated with GLXA. The serum levels of IL-4and IFN-y were measured at2h and12h respectively after the treatment. The measurement of the cytokine levels was done by ELISA.2) Intraperitoneal injection:Mice were injected with GLXA intraperitoneally. After72h, spleens and livers were harvested from the animals, and processed into single-cell suspensions. The cells were then stained with the appropriate Abs for flow cytometric analysis. Results1、Preparation of GLXA antigen:Silver staining and Western blot of the antigen sample demonstrated a banding pattern similar to GLXA as described previously, cell mock preparation failed to show the bands although.2、in vitro1) CD1d fusion protein iNKT hybridoma culture:On stimulation with GLXA-treated CD1d, in contrast to the cell mock or PBS, iNKT produced IL-2in a dose-dependent manner, suggesting that GLXA can specifically stimulate iNKT hybridoma.2) LMC-BMDC co-culture:We found that GLXA stimulated LMC from WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock. To confirm that the cytokines produced in the co-culture were secreted by iNKT, we isolated the LMC from iNKT KO and co-cultured it with BMDC. The data showed that the LMC from iNKT KO mice produced significantly lower quantities of IFN-y and IL-4compared with those from WT mice, indicating that the cytokines were mainly produced by iNKT. We further examined whether the cytokine production by the iNKT was CD1d-dependent by blocking CD1d using anti-CD1d Abs in the BMDC-LMC co-culture system. On addition of the Abs, the GLXA-treated co-culture showed a dramatic decrease in IFN-y and IL-4production compared with the controls, demonstrating the dependence of iNKT activation by GLXA on CD1d molecules3、in vivo1) Intravenous injection:GLXA stimulated WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock, whereas there was no significant difference in the cytokine production between GLXA and cell mock in iNKT KO mice. These data suggested that the cytokines produced in the serum were largely secreted by iNKT, and that iNKT were activated by GLXA in this process.2) Intraperitoneal injection:Compared with those from the cell mock-treated, iNKT from GLXA-treated mice showed significantly higher expression of CD69. On flow cytometric analysis of the pattern of intracellular cytokine production by iNKT in vivo, we found that iNKT from the GLXA-treated mice produced large amounts of IFN-y and IL-4compared with the cell mock-treated mice. These data show that GLXA stimulates iNKT in vivo. ConclusionsThe findings from the present study show that GLXA, a glycolipid antigen derived from the chlamydia, can activate iNKT, and this activated function depend on CD1d molecules. GLXA may be responsible for activation of NKT cells as a mager antigen. The effect of GLXA on activation of iNKT cells remains to be elacidated.SignificanceThis finding provides significant new knowledge on the molecular and cellular mechanisms of immune response to chlamydial infection. Our research laid the theoretical foundition using GLXA as a target to design vaccine against chlamydial infection. Part Two:The Role of IL-22against Chlamydial Lung InfectionObjectiveAs a member of IL-10cytokine family, Interleukin (IL)-22is an inflammatory cytokine and it plays an important role in modulation tissue responses during inflammation. Recent studies have identified that IL-22is involved in the host defense mechanism against infections caused by bacteria, fungi, viruses and parasites. It is generally accepted that IL-22plays a protective role in extracellular bacterial infections, such as Citrobactor rodentium, Klebsiella pneumoniae, and segmented filamentous bacterium (SFB). However, the role of IL-22in the host defense against intracellular bacteria infections is not clear. Therefore, further investigation is required to evaluate the function of IL-22in an intracellular bacteria infection model. Chlamydiae are obligate intracellular bacterial pathogens. Chlamydia trachomatis and Chlamydia pneumonia are responsible for various human diseases. C. pneumoniae causes respiratory diseases such as bronchitis, sinusitis and pneumonia, and C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases.The mouse pneumonitis agent strain is designated as Chlamydia muridarum (Cm). Cm has been widely used in mouse models of respiratory and genital tract infections in research laboratories. Studies from the respiratory chlamydial infection model have shown that the Thl-dominant and IFN-y-dependent immunity is a major host protective determinant for controlling chlamydial infection.Our previous studies indicated that Thl7also plays an important role in host defense against chlamydial infection by promoting Thl-type cell responses. Therefore, the development of Th1and Th17cell immune responses is important for host to effectively control the infection and clear the pathogens. IL-22and IL-17are both produced by the Th17lineage, and data to date suggest that IL-17and IL-22cooperate in some infection models. So far, there is no report on the functional role of IL-22during the development for chlamydial lung infection. In this report, we evaluated the role of IL-22using neutralized anti-IL-22mAb in chlamydial lung infection model. Methods1、IL-22neutralized:To assess whether IL-22is important for host defense against Cm infection in the lung, Anti-mouse IL-22mAb was used to neutralize endogenous IL-22after Cm lung infection.1) Flow cytometry:detect the changes of Th, Th17and Treg cells.2) ELISA:detect the levels of IL-6, IL-10, IL-12p40, IL-17and IFN-y in Cell culture supernatants.3) RT-PCR:detect the changes of IL-22, IL-17, IFN-y, T-bet and RORyt at RNA levels.4) HE staining:detect the pathological changes of lung tissue.2、IL-22treated:To confirm the role of IL-22in the development of chlamydial pulmonary infection, we treated the C57BL/6mice with mouse recombinant IL-22or PBS following Cm lung infection.1) Flow cytometry:detect the changes of Th1and Th17cells.2) ELISA:detect the levels of IL-17and IFN-y in Cell culture supernatants.3) HE staining:detect the pathological changes of lung tissue.Results1、IL-22neutralized:IL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. The mice treated with neutralizing anti-IL-22mAb exhibited severe disease, which showed significantly lower body weight, higher organism growth, and much more severe pathological changes in the lung compared to isotype control group. Immunological analysis showed lower Chlamydia-specific Thl and Th17responses with higher CD4+CD25+Foxp3+regulatory T cells responses in IL-22-neutrolized mice. Moreover, IL-10production was decreased in IL-22-deficient mice compared to WT mice.2、IL-22treated:IL-22treatment in Chlamydia infected mice dramatically inhibited the development of pulmonary infection and the growth of bacteria. The exogenous IL-22promoted the production of Th17cells after Cm infection.ConclusionsIL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. IL-22has a protective role during chlamydial lung infection. The protective role of IL-22in host defense against chlamydial infection was complied by promoting the Th17immune response. SignificanceIL-22is an important inflammatory cytokine in host defense against chlamydial infection in the lung. Our study raises the possibility for new treatments to use IL-22to control or inhibit chlamydial lung infection.