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The Journal of Immunology, 2007, 178: 1048-1058.
Copyright © 2007 by The American Association of Immunologists, Inc.

Distinct NKT Cell Subsets Are Induced by Different Chlamydia Species Leading to Differential Adaptive Immunity and Host Resistance to the Infections1

Antony George Joyee, Hongyu Qiu, Shuhe Wang, Yijun Fan, Laura Bilenki and Xi Yang2

Laboratory for Infection and Immunity, Departments of Medical Microbiology and Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We investigated the role of NKT cells in immunity to Chlamydia pneumoniae and Chlamydia muridarum infections using a combination of knockout mice and specific cellular activation approaches. The NKT-deficient mice showed exacerbated susceptibility to C. pneumoniae infection, but more resistance to C. muridarum infection. Activation of NKT reduced C. pneumoniae in vivo growth, but enhanced C. muridarum infection. Cellular analysis of invariant NKT cells revealed distinct cytokine patterns following C. pneumoniae and C. muridarum infections, i.e., predominant IFN-{gamma} in the former, while predominant IL-4 in the latter. The cytokine patterns of CD4+ and CD8+ T cells matched those of NKT cells. Our data provide in vivo evidence for a functionally diverse role of NKT cells in immune response to two intracellular bacterial pathogens. These results suggest that distinct NKT subsets are induced by even biologically closely related pathogens, thus leading to differential adaptive immune response and infection outcomes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Natural killer T cells are a unique subset of T lymphocytes that express markers of NK cells and a semi-invariant TCR. These cells have been identified as a novel lymphocyte population that acts in innate immune responses. Unlike conventional T lymphocytes, NKT recognize glyco- and phospholipids, rather than peptide Ags, presented by the nonclassical MHC class I molecule CD1 (1). The striking feature of NKT cells is that these cells can rapidly secrete large amounts of IFN-{gamma} and/or IL-4 upon activation, by interaction with the appropriate CD1d1/Ag complex (2). These two cytokines are important for the regulation of type 1 and type 2 T cell responses, respectively (3, 4), thus, NKT cells are believed to be able to modulate immune responses following various infections or other disease conditions (5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16). It has been well-established that virtually all mouse (V{alpha}14) and human (V{alpha}24) invariant NKT (iNKT)3 cells can recognize {alpha}-galactosylceramide ({alpha}-GalCer), a glycolipid originally extracted from marine sponges (17, 18). CD1-dependent NKT cells can be classified as two types based on their reactivity to {alpha}-GalCer. The "classical" or iNKT cells which express the semi-invariant {alpha}beta TCR with the use of V{alpha}14 and J{alpha}18 can be activated by {alpha}-GalCer in the context of CD1, while the nonclassical NKT cells with diverse TCR are nonreactive to {alpha}-GalCer (19). In humans, differential cytokine production by distinct NKT cell subsets has been demonstrated (20, 21). NKT-1-like cells show predominant IFN-{gamma} production while NKT-2-like cells produce higher IL-4. Such NKT subsets may influence the nature of adaptive immune responses during different infections.

Chlamydiae are obligate intracellular bacteria, which include two species, Chlamydia pneumoniae and Chlamydia trachomatis, commonly causing human infections. C. pneumoniae causes a wide spectrum of acute and chronic respiratory diseases like bronchitis, obstructive pulmonary disease, sinusitis, etc., while C. trachomatis causes ocular, respiratory, and sexually transmitted diseases. The prevalence of chlamydial infections is very high worldwide. In particular, Abs specific for C. pneumoniae can be detected in the serum of up to 70% of healthy human beings implying that most individuals have had contact with these organisms (22). More recently, C. pneumoniae has been implicated in the pathogenesis of diverse diseases such as atherosclerosis, Alzheimer’s disease, and multiple sclerosis (23, 24, 25). The pathogenesis of these conditions is considered to be immunopathologically mediated. Given the role of C. pneumoniae in many human diseases, an effective vaccine would be of significant public health benefit. However, vaccine development against this important human pathogen has been hindered by the limited understanding of immune mechanisms that lead to protective or adverse immune responses.

The experimental animal models, especially mouse models, to study C. pneumoniae and C. trachomatis infections have been instrumental in studying the components of immune system that contribute to, or is essential for, the control of these intracellular pathogens. Studies in both humans and mice have shown that the activation of type 1 immune responses including cell-mediated immunity and IFN-{gamma} production are essential for protection against chlamydial infection, while type 2 response may be associated with immunopathology (26, 27, 28, 29, 30, 31, 32, 33, 34). However, the precise immune mechanisms involved in resistance or pathogenesis to chlamydial infection has not been fully elucidated. In particular, the role of cellular and soluble components in immune responses during the innate phase of infection remains largely unknown.

We recently reported that NKT cells promote C. trachomatis infection in a murine lung infection model (35). Using a mouse biovar of C. trachomatis, mouse pneumonitis (MoPn) strain (more recently named as Chlamydia muridarum), we found that NKT-deficient CD1-knockout (KO) mice are much more resistant to infection and that the activation of NKT cells by {alpha}-GalCer exacerbated host susceptibility to infection. In the present study, we further investigated the role of NKT cells in C. pneumoniae infection using a combination of approaches. First, we used two types of gene knockout mice that are deficient in NKT cells (NKT-KO and CD1-KO mice) to identify the role of NKT in immunity to C. pneumoniae infection. Next, we analyzed the effect of pharmacological activation of iNKT cells by {alpha}-GalCer, the NKT ligand in the outcome of infection, and finally we performed a direct cellular analysis on iNKT cells by using CD1d tetramers to elucidate the nature of NKT responses following infections with these two different chlamydial species. The results showed that NKT cells, in contrast to what was seen during C. muridarum infection, play a crucial role in mediating protective immune responses against C. pneumoniae. Further analyses showed that NKT cells activated by C. pneumoniae exhibit very distinct cytokine patterns from those activated by C. muridarum infection. These findings suggest that NKT cells may play diverse roles in immune responses to even very closely related pathogens, which may differentially influence adaptive immune responses and infection outcomes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Breeding pairs of J{alpha}18 knock out (NKT-KO) mice with B6 background were provided by Dr. M. Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama City, Japan). These mice were generated by specific deletion of the J{alpha}18 (formerly J{alpha}281) gene segment using homologous recombination and aggregation chimera techniques (36). Breeding pairs of CD1d KO mice were purchased from The Jackson Laboratory. The mice were bred and maintained at the pathogen-free animal care facility (University of Manitoba, Winnipeg, Manitoba, Canada). Control C57BL/6 and BALB/c mice were purchased from Charles River Laboratories or bred at the University of Manitoba animal care facility. Eight- to 10-wk-old mice were used in the study. All experiments were done in compliance with the institutional guidelines issued by the Canadian Council of Animal Care.

Organism

C. pneumoniae (AR-39; provided by Dr. K. Wolf, University of Manitoba) was propagated in HL cells in Eagle’s MEM containing 10% FBS and 2 mM L-glutamine. Infected cells were harvested with sterile glass beads and ultrasonically disrupted. After centrifugation at 500 x g for 10 min to remove cell debris, bacteria were concentrated by high-speed centrifugation at 30,000 x g for 25 min, resuspended in sucrose-phosphate-glutamic acid (SPG) buffer, and stored frozen in small aliquots (frozen at –80°C) until used. The infectivity, as measured by inclusion-forming units (IFUs) of the bacterial preparation, was determined in HL cell culture. The same seed stock of C. pneumoniae was used throughout the study. For immunological in vitro assays and Ab ELISAs, highly purified elementary body (EB) preparations were obtained by adopting a Renografin gradient separation method as described previously (37). In brief, the partially purified EB suspension was passed through a 20–50% (v/v) Renografin (Squibb) gradient. The EB band from the interphase was collected, washed twice, and resuspended in SPG buffer. For cytokine induction in culture, a sonicated killed preparation of C. pneumoniae was prepared as previously described (38). Briefly, renografin-purified C. pneumoniae EBs were sonicated for 10 min on ice (Sonic Vibra Cell VC 130; Sonics & Materials). The nonviability of chlamydiae was confirmed by inoculation onto HL cells. This sonicated-killed C. pneumoniae preparation was used for in vitro antigenic restimulation assays for cytokine analysis.

Mice infection and quantitation of in vivo infectivity

Mice were mildly sedated with isoflurane and inoculated intranasally with 5 x 106 IFUs of C. pneumoniae in 40 µl of PBS. At predetermined days after inoculation, the mice were euthanized and the lungs were aseptically isolated and mechanically homogenized using a cell grinder in SPG buffer. The tissue homogenates were centrifuged down at 1900 x g for 30 min at 4°C to remove coarse tissues and debris and the supernatants were stored frozen at –80°C until tested. For C. pneumoniae quantitation, 100 µl of serially diluted organ tissue supernatants were inoculated onto HL cells grown to confluence in 96-well flat-bottom microtiter plates. The plates were centrifuged at 1100 x g followed by incubation at 37°C for another 30 min. The cell layers were then washed and 200 µl of MEM containing cycloheximide (2 µg/ml), gentamicin (10 µg/ml), and vancomycin (25 µg/ml) were added to each well. The plates were incubated for 72 h at 37°C in 5% CO2. After the incubation, the culture medium was removed, cell monolayers were fixed with methanol, and stained with Chlamydia-specific murine mAb and HRP-conjugated goat anti-mouse IgG secondary Abs and developed with substrate (4-chloro-1-naphthol; Sigma-Aldrich). The number of inclusions was counted under a microscope at x100 magnification. Five fields through the midline of each well were counted. C. pneumoniae titers in the organs were calculated based on dilution titers of the original inoculum.

Histological analysis

Lungs were aseptically removed and fixed in 10%-buffered formalin and embedded in paraffin for histological assessment. The tissue sections were stained with H&E and the histological changes and cellular infiltration were determined by light microscopy. For the detection of C. pneumoniae inclusions and analysis of cell types using immunofluorescence staining, one lung was snap-frozen in liquid nitrogen. For the latter analysis, lung sections cut (at a thickness of 10 µm) from the frozen tissue were treated with 2% goat normal serum for blocking and further stained for epithelial cells, macrophages, and chlamydial inclusions. For staining chlamydial inclusions, sections were incubated with mouse anti-Chlamydia Ab (Chemicon International) and then with FITC-conjugated goat anti-mouse IgG secondary Ab (Sigma-Aldrich). Macrophages were stained with PE labeled anti-mouse F4/80 Ab (eBioscience).To stain epithelial cells, tissue sections were further treated with mouse antipan-cytokeratin Ab (Calbiochem-Novabiochem). The sections were washed, incubated with biotinylated goat anti-mouse Ab (DakoCytomation), and then finally developed with AMCA (Vector Laboratories). The stained tissue sections were observed under a fluorescent microscope and analyzed.

In vitro restimulation assays and cytokine measurements

Mice were killed at various days postinfection (p.i.) and the spleens and draining (mediastinum) lymph nodes were aseptically removed. To analyze for cytokine production, single-cell suspensions were prepared from spleen and lymph nodes and cultured at a concentration of 7.5 and 5.0 x 106 cells/ml, respectively, alone or with sonicated killed C. pneumoniae (104 IFU/ml). Duplicate cultures were established from the spleen and lymph node cells of individual mice in each group. The cells were incubated at 37°C in complete culture medium: RPMI 1640 containing 10% heat-inactivated FBS, 25 µg/ml gentamicin, 2 mM L-glutamine, and 5 x 10–5 2-ME (Kodak) for 72 h in a 5% CO2 atmosphere, after which the supernatants were collected, frozen, and later analyzed for cytokines by ELISA using purified (capture) and biotinylated (detection) Abs as previously described (30, 31). Abs purchased from BD Pharmingen were used for ELISA measuring IFN-{gamma}, IL-12, IL-4, and IL-5.

Determination of C. pneumoniae-specific Ab levels

Ab titers for C. pneumoniae-specific IgG1 and IgG2a responses were measured using an alkaline phosphatase-based ELISA as previously described (31, 32). Briefly, microtiter plates were coated overnight with killed C. pneumoniae EBs. After blocking and washing, serially diluted sera were incubated for 2 h at 37°C. Biotinylated goat anti-mouse Ab was added after washing. Following overnight incubation at 4°C, alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories; Bio/Can Scientific) was added for 45 min at room temperature. The enzyme substrate p-nitrophenyl phosphate (Sigma-Aldrich) (in 0.5 mM MgCl2, 10% diethanolamine (pH 9.8)) was added and the reaction was allowed to proceed for 60 min. The plates were read by using an ELISA reader at 405 nm. Results are expressed as ELISA titers using the endpoint (cutoff at OD 0.5) of the titration curves compared with a constant internal standard run in each assay.

Flow cytometric analysis of cell surface markers

Mouse-specific Abs were purchased from BD Biosciences. Anti-CD3-PE-Cy7, anti-CD3-PE, anti-CD4-PE, anti-CD8-PE-Cy7, anti-NK1.1-PE, CD-19-FITC, and corresponding isotype controls were purchased from BD Biosciences. Cells were stained for surface markers in a staining buffer (Dulbecco’s PBS (D-PBS); Sigma-Aldrich) without Ca2+ and Mg2+ containing 2% FCS and 0.09% NaN3) on ice for 30 min in the dark. Staining for NKT cells was done by using PE-mCDd/PBS57 ligand tetramer (provided by the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility, Atlanta, GA). After incubation with PE-Ab tetramer and PE-Cy7-anti-CD3{epsilon} Abs on ice for 30 min in the dark, the cells were fixed using 2% paraformaldehyde for 1 h, washed twice, resuspended in D-PBS with 2% FCS and 1 mM EDTA. NKT cells were identified as tetramer-positive and CD3{epsilon}-positive cell population. Sample data was collected using a FACSCalibur flow cytometer (BD Biosciences) and the data were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining

Single-cell suspensions of the lung were prepared by collagenase and DNase digestion of the lung tissue. Briefly, the lung tissues were minced into small pieces and incubated in RPMI 1640 containing 2 mg/ml collagenase type XI (Sigma-Aldrich) and 100 µg/ml DNase I (Sigma-Aldrich) for 60 min at 37°C. The digested tissue suspension was passed through a 70-µM cell strainer (BD Biosciences). Splenocytes were obtained by pressing the spleen through 70-µM strainer. For all cells, erythrocytes were lysed with ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) followed by two washes in RPMI 1640 with 5% FCS and resuspended in complete RPMI 1640 medium.

For intracellular cytokine staining, lung mononuclear cells and splenocytes were stimulated with PMA (50 ng/ml) and ionomycin (1 µg/ml) and incubated for 6 h in complete RPMI 1640 medium at 37°C. For the last 4 h incubation, monensin (eBioscience) was added to accumulate cytokines intracellularly. Cell staining was performed in a volume of 100 µl in 96-well plates. In brief, cultured cells (2 x 106) were washed twice and incubated with FcR block Abs (anti-16/32; eBioscience) for 15 min at 4°C to block nonspecific staining. Cell surface staining was performed as described above. The cells were then fixed and permeabilized with Cytofix/Cytoperm (BD Pharmingen) as per manufacturer’s instructions and stained intracellularly with anti-IFN-{gamma}-allophycocyanin (XMG 1.2) and anti-IL-4-FITC (BVD6-24G2) mAbs (eBioscience) or with corresponding isotope control Abs in permeabilization buffer (BD Pharmingen). Finally, the cells were washed, resuspended in D-PBS containing 2% FCS and 1 mM EDTA, and analyzed by flow cytometry.

{alpha}-GalCer administration and assessment of immune responses p.i.

{alpha}-GalCer was provided by Kirin Brewery. Mice received a single i.v. injection of 4 µg of {alpha}-GalCer diluted in a 0.2-ml volume of PBS. Control mice were injected with an identical volume of vehicle solution alone (0.025% polysorbate 20 in PBS). Mice were infected with C. pneumoniae 2 h after {alpha}-GalCer or polysorbate injection.

Statistical analysis

IFU and serum Ab titers were transformed to logarithmic values. The differences in IFU detection, cytokine production, and Ab levels between groups were analyzed by the unpaired, two-tailed Student t test to determine significance with Prism software (PRISM 4; GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Increased susceptibility to C. pneumoniae infection in NKT-KO mice

To assess whether NKT cell deficiency renders mice susceptible to C. pneumoniae infection, groups of NKT-KO and control C57BL/6 mice were intranasally infected with C. pneumoniae. The results showed that, compared with wild-type (WT) mice, the NKT-KO mice suffered much greater weight loss. The WT mice lost ~18% of body weight at day 3 p.i. and started to regain the lost weight from day 4 p.i. and recovered the original body weight by 2 wk following the infection. In contrast, the KO mice showed a continuous decline of body weight and lost >30% of their body weight at day 9 following infection (Fig. 1A). We next quantified the in vivo growth of C. pneumoniae in the lungs. The NKT-KO mice showed significantly higher bacterial loads in the lung than WT mice at day 9 p.i. (Fig. 1B). At a later time point (day 15 p.i.), the WT mice showed a dramatic reduction in the pulmonary bacterial load, which dropped >99%, while the KO mice still had significant bacterial burden in lungs which was close to 1000-fold higher than that in the WT mice (Fig. 1C). These data demonstrate that NKT-KO mice experience more severe disease and in vivo pathogen growth, thus more susceptible to C. pneumoniae infection.


Figure 1
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  		FIGURE 1. More severe body weight loss and higher organism growth in the lungs after C. pneumoniae infection in NKT-KO mice than in the WT C57BL/6 mice. Mice were intranasally infected with C. pneumoniae (5 x 106 IFUs) and were monitored daily for body weight changes (A). Each point represents the mean ± SD of four mice. The original body weights of the mice were similar between the groups. Mice were sacrificed on day 9 (B) or day 15 (C) p.i. and the lungs were collected and the bacterial loads were quantitatively analyzed as described in Materials and Methods. Results are shown as mean ± SD. One of three independent experiments with similar results is shown. *, p < 0.05, ***, p < 0.001, WT vs KO mice.

 
More severe pathological changes in NKT-KO mice

The histological analysis of lung tissues showed very severe pathological changes and tissue damage in the NKT-KO mice, which correlated with higher bacterial loads in the lungs. On day 9 p.i., the alveolar architecture in KO mice was severely disrupted and showed much dense and diffused cellular infiltration characterized by high proportion of neutrophils and other polymorphonuclear cells (Fig. 2A). Most alveoli were filled with inflammatory exudates. In contrast, the control C57BL/6 mice showed much less lung pathology and the tissue inflammation and cellular infiltration was more localized. The cellular infiltrate mainly comprised macrophages and lymphocytes (Fig. 2B). On day 15 p.i., both groups showed signs of recovery in the lung, but the NKT-KO mice to a much less degree. Notably, some multinucleated giant cells were observed in the lung tissue of WT mice, but not in NKT-KO mice at this stage (Fig. 2C). Consistent with the determination of chlamydial IFUs in lung homogenates, NKT-KO mice showed more inclusions in the lung on both day 9 (Fig. 2D) and day 15 (Fig. 2E) p.i. Importantly, it appeared that more inclusions were visible in macrophages (Fig. 2, D and E, green color) in NKT-KO mice than those in WT mice, which suggests the failure of the macrophages in effectively controlling chlamydial growth within the cells.


Figure 2
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  		FIGURE 2. More serious pathological response in NKT-KO than in WT mice. The lungs from mice intranasally infected with C. pneumoniae (5 x 106 IFUs) at day 9 or 15 p.i. were analyzed for histological changes by H&E staining (A–C) and immunofluorescence staining (D and E) as described in Materials and Methods. A, Day 9 p.i.; B, day 15 p.i.; C, day 9 p.i.; D, day 9 p.i.; and E, day 15 p.i. The H&E-stained slides were analyzed in low (x200, A and B) and high power (x400, C) magnification under light microscopy. White arrow indicates multinucleated giant cells; black arrows indicate epithelioid cells; green arrows indicate macrophage death due to necrosis in the KO mice. The immunofluorescence staining slides were analyzed under fluorescence microscopy (x400). Green color, Chlamydial inclusions; red color, macrophages; blue color, epithelial cells.

 
NKT cells show an IFN-{gamma}-polarized cytokine response following C. pneumoniae infection

To elucidate the mechanism by which NKT cells contributed to host defense during C. pneumoniae infection, we performed a direct cellular level analysis on NKT kinetics and cytokine patterns using ligand-loaded CD1d tetramer. NKT cells showed an expansion of ~5-fold after C. pneumoniae infection. The number and percentage of NKT in the lungs peaked at day 3 p.i., after which they decreased and reached basal levels about days 9 and 10 p.i. (Fig. 3A). Intracellular cytokine staining analysis of lung (Fig. 3B) and spleen (Fig. 3C) NKT cells at 3 days p.i. from the WT mice for IFN-{gamma} and IL-4 showed significantly increased proportion of IFN-{gamma}-producing cells compared with that in the uninfected mice. This difference was more obvious in NKT cells from the lung than those from the spleen (Fig. 3, B and C). Thus, these results suggest that C. pneumoniae infection can skew the cytokine response of NKT cells to IFN-{gamma} polarization.


Figure 3
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  		FIGURE 3. NKT show an IFN-{gamma}-polarizing cytokine pattern following C. pneumoniae infection. A, CD1-d tetramer staining. Expansion of NKT cells after C. pneumoniae infection in vivo. C57BL/6 mice were infected with C. pneumoniae intranasally as described in the legend to Fig. 1 and killed at various days p.i. Lung lymphocytes were prepared as described in Materials and Methods and stained with PE-Cy7-labeled anti-CD3e Abs and PE-labeled mCD1d/PBS57 ligand tetramer. The NKT cells were identified as CD3+ tetramer (+) cells by flow cytometry. B and C, Intracellular cytokine staining. Mice were killed at 3 days after intranasal infection with C. pneumoniae (Cpn). The lung (B) and spleen (C) cells were analyzed for NKT cytokine responses by four-color flow cytometry. Intracellular cytokine staining was performed using Cytofix/Cytoperm kit from BD Biosciences with allophycocyanin-conjugated anti-IFN-{gamma} Abs and FITC-conjugated anti-IL-4 Abs or corresponding isotope controls. The analysis was performed on NKT cells gated as CD3+ tetramer (+) cells. D and E, Graphs represent IFN-{gamma} production NKT cells from lungs (D) and spleen (E) from uninfected and C. pneumoniae-infected mice. Data show the mean ± SD and represent three independent experiments with three mice in each group. **, p < 0.01, and ***, p < 0.001, Student’s t test.

 
NKT cells influence the adaptive immune responses and mediate the development of type 1 T cell responses

The cytokine pattern of NKT cells in the innate phase during C. pneumoniae infection may contribute to the activation and differentiation of the major T cell lineages, CD8+ and CD4+ T cells, thus influencing the development of type 1 or type 2 immune responses. To further evaluate whether NKT cells influence T cells (CD8+ and CD4+) in the context of cytokine response to C. pneumoniae infection, we analyzed the cellular cytokine patterns of CD4+ and CD8+ T cells in the WT and NKT-KO mice in the adaptive immune phase of infection. At day 10 p.i., mice were killed and the cytokine profile of CD4+ and CD8+ T cells was analyzed. Intracellular cytokine staining for IFN-{gamma} and IL-4 showed a distinct pattern in IFN-{gamma} production by CD8+ T cells between the WT and KO mice. The WT mice showed a significantly higher proportion of IFN-{gamma}-producing CD8+ T cells compared with the NKT-KO mice (Fig. 4A). In addition, CD4+ T cells of WT mice showed a Th1 cytokine pattern with significantly higher IFN-{gamma} (Fig. 4B) and lower IL-4 levels, while CD4+ T cells from NKT-KO mice showed a Th2-like pattern with more IL-4 (Fig. 4D), but less IFN-{gamma} (Fig. 4B). Of note, CD8+ T cells were the major producers of IFN-{gamma} compared with CD4+ T cells, which supports previously published work (39, 40, 41) showing the importance of the CD8+ T cell response as a major protective mechanism against this pathogen. Overall, these results suggested that NKT cells could modulate both CD8+ and CD4+ T cell responses toward the establishment of the protective type 1 response during C. pneumoniae infection.


Figure 4
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  		FIGURE 4. NKT cells influence the development of IFN-{gamma}-producing CD8+ and CD4+ T cells. Mice were intranasally infected with C. pneumoniae (5 x 106 IFUs) with or without {alpha}-GalCer pretreatment. At day 10 p.i., splenocytes were analyzed for Th1 (IFN-{gamma}) and Th2 (IL-4) cytokines by intracellular cytokine staining as described in Materials and Methods. Cytokine production pattern of CD8+ and CD4+ T cells in NKT-KO and WT mice in the adaptive immune phase following C. pneumoniae infection with or without {alpha}-GalCer treatment. A and B, IFN-{gamma}-producing CD8+ and CD4+ T cells in NKT-KO and WT mice following infection. C, Graph represents IFN-{gamma} production pattern by CD8+ and CD4+ T cells from NKT-KO and WT mice following C. pneumoniae infection. D and E, Effect of {alpha}-GalCer treatment on IFN-{gamma} production by CD8+ and CD4+ T cells in the NKT-KO and WT mice infected with C. pneumoniae. F, Graph represents IFN-{gamma} production pattern by CD8+ and CD4+ T cells following C. pneumoniae infection with prior {alpha}-GalCer treatment. G and H, IL-4 production by CD8+ and CD4+ T cells in NKT-KO and WT mice infected with C. pneumoniae. I, Graph represents IL-4 production pattern by CD8+ and CD4+ T cells following C. pneumoniae infection. J and K, Effect of {alpha}-GalCer treatment on IL-4 production by CD8+ and CD4+ T cells in the NKT-KO and WT mice following C. pneumoniae infection. L, Graph representing IL-4 production pattern by CD8+ and CD4+ T cells following C. pneumoniae infection with prior {alpha}-GalCer treatment. Data show the mean ± SD. At least three independent experiments with three mice in each group were performed and one representative experiment is shown. *, p < 0.05, **, p < 0.01, and ***, p < 0.001, Student’s t test. WT vs KO mice.

 
To further confirm the effect of NKT on Chlamydia-driven cytokine production patterns in the population level, we examined the cytokine production by spleen and draining lymph node cells in the infected mice. As illustrated in Fig. 5A, WT mice displayed a type 1 cytokine production pattern, characterized by significantly higher levels of IFN-{gamma} and IL-12 compared with the NKT-KO mice. In addition, they also showed significantly lower levels of type 2 cytokines, IL-4 and IL-5. Conversely, the NKT-KO mice exhibited a type 2 cytokine pattern with significantly higher levels of IL-4 and IL-5, but lower levels of both IFN-{gamma} and IL-12. In combination with the data from flow cytometry analysis, these results confirm that NKT cells can modulate cytokine responses in a population level during C. pneumoniae infection. We further measured the levels of C. pneumoniae-specific IgG1 and IgG2a Abs in serum samples from NKT-KO and C57BL/6 control mice before and after infection. After infection (day 15), the WT mice showed significantly higher levels of serum IgG2a Abs than NKT-KO mice. In addition, WT mice also showed lower levels of IgG1 Abs when compared with the KO mice (Fig. 5B). These results suggest that NKT cells play an important role in regulating the production of Th1-associated Ab isotope responses in this model of infection. In aggregate, the results indicate that NKT cells contribute to the development and/or promotion of the protective immune responses to C. pneumoniae infection.


Figure 5
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  		FIGURE 5. Altered cytokine and Ab responses in NKT-KO mice. A, Mice (four mice per group) were infected intranasally with C. pneumoniae as described in the legend to Fig. 1 and were sacrificed 9 days p.i. Spleen and draining lymph node cells were cultured with sonicated-killed C. pneumoniae as described in Materials and Methods. Th1 (IFN-{gamma}) and proinflammatory (IL-12) cytokines and Th2 (IL-4, IL-5) cytokines in 72-h culture supernatants were determined by ELISA. Data are presented as the mean ± SD of each group. One representative experiment of three independent experiments is shown. B, Serum samples were collected at day 16 p.i. and tested for C. pneumoniae-specific IgG1 and IgG2a. Ab titers were transformed to log 10 values. Pooled data for three experiments (12 mice in each group) are presented. Data show the mean ± SD. *, p < 0.05, **, p < 0.01, and ***, p < 0.001. NKT-KO vs WT mice.

 
NKT activation by {alpha}-GalCer enhances the protective immune responses following infection

The above results generated from studies using NKT-KO mice demonstrated an important role of NKT in host resistance to C. pneumoniae infection. To further confirm this conclusion, we used an alternative approach, which tested NKT function in the condition of enhanced NKT activation. WT mice were treated with {alpha}-GalCer, which specifically activates NKT, and the effect of treatment on the outcome of infection was examined. As shown in Fig. 6A, {alpha}-GalCer pretreatment significantly reduced the number of bacteria in the lungs of WT mice to ~10-fold when compared with mice given vehicle treatment. Moreover, the reduction in the bacterial load in the former was correlated with enhanced type 1 cytokine response. As illustrated in Fig. 6B, {alpha}-GalCer treatment strongly enhanced the production of IFN-{gamma} and IL-12 in the WT mice in comparison with the vehicle-treated controls. Moreover, significant reduction in the levels of type 2 cytokines, IL-4 and IL-5, was also seen in these mice. Furthermore, as shown in Fig. 6, C and D, intracellular cytokine analysis showed that prior treatment with {alpha}-GalCer followed by C. pneumoniae infection enhanced the IFN-{gamma} production by NKT after infection, while {alpha}-GalCer treatment alone typically enhanced both IFN-{gamma} and IL-4 production by NKT cells (in comparison with that in naive mice and C. pneumoniae infected, but {alpha}-GalCer-untreated mice as shown in Fig. 3, B and C). The IFN-{gamma} polarization effect on NKT by C. pneumoniae infection was observed in NKT from the lung (Fig. 6C) and spleen (Fig. 6D). In addition, CD4+ and CD8+ T cell cytokine pattern analysis showed that prior {alpha}-GalCer treatment enhanced IFN-{gamma} production by both types of cells in WT, but not in NKT-KO, mice (Fig. 4, E and F). Moreover, histological findings showed that {alpha}-GalCer treatment substantially reduced pulmonary pathological changes in the infected mice (data not shown). In contrast to these observations in the WT mice, the protective effect of {alpha}-GalCer treatment was not detected in the NKT-KO mice, confirming the specificity of {alpha}-GalCer on NKT activation. Taken together, these results further confirm the direct involvement of activated NKT cells in eliciting strong protective type 1 responses against C. pneumoniae infection.


Figure 6
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  		FIGURE 6. Effect of in vivo stimulation of NKT cells by {alpha}-GalCer on host response to C. pneumoniae. A and B, Naive C57BL/6 and NKT-KO mice (four mice per group) were injected i.v. with 4 µg of {alpha}-GalCer in PBS or with polysorbate vehicle control. The mice were intranasally infected with C. pneumoniae (Cpn; 5 x 106 IFUs), 2 h after {alpha}-GalCer or vehicle injection. Mice were sacrificed on day 9 p.i. for quantification of C. pneumoniae growth in the lungs (A) and for the analysis of C. pneumoniae-specific cytokine production by spleen cells (B) using the methods described in Materials and Methods. C and D, WT C57BL/6 mice were treated with {alpha}-GalCer (4 µg) followed by intranasal infection with C. pneumoniae (Cpn; 5 x 106 IFUs) or mock treatment. The lung (C) and spleen (D) cells were analyzed for NKT cytokine patterns using four-color flow cytometry as described in the legend to Fig. 3. One of three independent experiments with similar results is shown. *, p < 0.05. **, p < 0.01.

 
Similar outcome of infection in CD1-KO mice as in NKT-KO mice

To further confirm the role of NKT in C. pneumoniae infection, we next examined CD1-KO mice, another type of NKT-deficient mice. The rationale for using CD1-KO mice is 2-fold. First, the CD1-KO mice had a different genetic background (BALB/c) from the NKT-KO mice, thus the role of NKT can be examined in a different mouse strain. This is an important point because in certain murine models of infection, the protective or pathological outcome of infection varies in mouse strains with different genetic background, such as C57BL/6 vs BALB/c mice (42). Therefore, it was also our aim to see whether the difference in genetic background had an influence on the protective role of NKT in C. pneumoniae infection. The other and more important rationale is that our previous finding on the role of NKT in C. muridarum infection was based on a study using CD1-KO mice (35). To conclude that NKT play distinct roles in C. pneumoniae and C. muridarum infection, the potential variation due to the differences in the types of NKT-deficient mice should be excluded. In the present study, our results showed that, similar to our finding in NKT-KO mice, CD1-KO mice also showed increased susceptibility to C. pneumoniae infection. As shown in Fig. 7, CD1-KO KO mice showed greater body weight loss compared with the control BALB/c mice (Fig. 7A), which was associated with significantly higher pathogen loads in the lungs (Fig. 7B) and decreased type 1 cytokine responses (Fig. 7C). Further, {alpha}-GalCer treatment had no beneficial effect on the outcome of infection in CD1-KO mice, but showed significantly enhanced protective immunity against C. pneumoniae in WT BALB/c mice (data not shown). These results confirm that NKT/CD1d indeed play a protective role in the course of C. pneumoniae infection, which is not affected by the genetic backgrounds of mice, at least in the tested strains. In addition, the results indicate that the distinct role of NKT in C. pneumoniae and C. muridarum infections observed in our studies was not due to the difference in the types of NKT-deficient mice used.


Figure 7
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  		FIGURE 7. Similar outcome in CD1 KO mice as that of NKT-KO mice after C. pneumoniae infection. CD1-KO and BALB/c control mice were intranasally infected with C. pneumoniae (5 x 106 IFUs) and were monitored for body weight changes (A). At day 9 p.i. infection, chlamydial loads in the lungs (B) and organism-driven cytokine production by spleen cells (C and D) were analyzed using the methods described in Materials and Methods. *, p < 0.05, BALB/c vs CD1-KO mice.

 
NKT cytokine response in C. muridarum infection is different from that in C. pneumoniae infection

As mentioned earlier, our previous report on the role of NKT in C. muridarum infection was based on the findings with CD1 KO mice on BALB/c background (35). Moreover, in the previous study, although a Th2 promoting role of NKT was observed in C. muridarum infection (35), the cytokine profile of NKT cells following infection was not examined. Therefore, we next performed experiments with NKT-KO mice to exclude the possibility of mouse strain difference reflecting in different outcomes to these two pathogens. NKT-KO mice and the WT C57BL/6 control mice were infected with C. muridarum and were analyzed for body weight loss, in vivo organism growth in the lungs and cytokine pattern. Our results showed that the WT, but not NKT-KO, mice experienced more body weight loss (Fig. 8A), higher lung pathogen loads (Fig. 8B) and increased production of IL-4, whereas the IFN-{gamma} levels were similar between the groups (Fig. 8, C and D). These results are consistent with our previously reported findings in CD1 KO mice (35).


Figure 8
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  		FIGURE 8. Promoting role of NKT cells in C. muridarum infection. A, Less body weight loss following C. muridarum infection in NKT-KO mice. B–D, In vivo organism growth and cytokine pattern after C. muridarum infection. NKT-KO and C57BL/6 mice were intranasally infected with C. muridarum (MoPn; 1000 IFUs) sacrificed on day 12 p.i. and analyzed for in vivo chlamydial growth in the lungs (B) and cytokine production by spleen cells (C and D). One representative experiment of three independent experiments with similar results is shown. *, p < 0.05, NKT-KO vs C57BL/6 mice. E, IL-4-dominant cytokine response of NKT cells after C. muridarum infection. C57BL/6 mice were infected with C. muridarum with or without prior {alpha}-GalCer treatment (4 µg). The mice were killed at day 3 p.i. and lung lymphocytes were analyzed by intracellular cytokine staining for NKT cytokine pattern as described in the legend to Fig. 3. The figures show analysis on gated CD3+ tetramer (+) cells (E). One of the three independent experiments with similar results is shown.

 
To further examine whether the cytokine profile of NKT activated by C. trachomatis (MoPn) is different from those activated by C. pneumoniae, we directly examined the NKT cytokine response following MoPn infection using CD1d tetramers. As shown in Fig. 8E, intracellular cytokine staining of lung NKT cells from MoPn-infected WT C57BL/6 mice showed a predominant IL-4 cytokine response. Further, prior treatment with {alpha}-GalCer increased the proportion of IL-4-producing NKT cells in C. muridarum-infected mice (Fig. 8). This cytokine pattern is in sharp contrast to the cytokine profile of NKT (IFN-{gamma} dominant) following C. pneumoniae infection in the absence (Fig. 3) or presence of {alpha}-GalCer stimulation (Fig. 6). Taken together, these findings confirm that the initial cytokine response of NKT is different following C. pneumoniae or C. muridarum infection, which may be the basis for the role played by NKT cells in the development of adaptive immune responses to, and the outcome of, the infection.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The most significant finding in this study is the difference in NKT cytokine patterns and disease outcomes following infection with two different chlamydial species. It has provided in vivo evidence that distinct NKT subsets (NKT-1 like or NKT-2 like) are activated in the same organ (lung) following infection with two closely related intracellular pathogens, which contributes to the development of differential adaptive immune responses to, and different outcomes of, the infections. Our previous study showed that NKT promote susceptibility to C. muridarum lung infection (35). The comparison of cytokine production patterns and the types of immune response between CD1-KO and WT mice and the findings on the effect of {alpha}-GalCer treatment suggested that NKT enhance type 2 responses to C. muridarum infection (35). In sharp contrast, the present data revealed a crucial protective role of NKT in C. pneumoniae infection. Our analysis on both lung and spleen NKT showed that these cells produced higher IFN-{gamma} than IL-4 following C. pneumoniae infection, which was associated with type 1 cytokine production by both CD8 and CD4 T cells. To find an answer to why the role of NKT is different in C. pneumoniae and C. muridarum infection models, we also dissected the cellular cytokine response of NKT in C. muridarum infection using intracellular cytokine staining analysis. Interestingly, the data showed that NKT display a distinct pattern of cytokine response depending on the pathogen. Thus, our data provide in vivo evidence that depending on the nature of the infectious stimuli, distinct NKT subsets can be generated, thus subsequently influencing the development of cellular responses to either type 1 or type 2 directions and the outcome of infections. Moreover, our data show that, similar to that observed in human studies (20, 21), functionally distinct NKT cell subsets in the mouse also have differential cytokine production profiles. This finding is particularly significant because it came from a study using two biologically related pathogens.

In light of these findings, our results pose significant implications on the functional properties of NKT in host response to infections. From the chlamydial infection point of view, these results suggest that the lung infection models of C. muridarum may not reflect the innate and/or adaptive immune responses to C. pneumoniae infection. Even though both pathogens share many biological features, the cellular response and immunological course after infection are different apart from the differences in growth characteristics and host specificity of these two pathogens. Using the method of T cell intracellular cytokine staining, we confirmed the reported findings on the difference of adaptive immune responses between C. muridarum and C. pneumoniae infections. For example, in C. muridarum, both CD4+ and CD8+ T cells have been reported to be protective, with the role of CD4+ T cells being more important (43, 44, 45). In contrast, CD8+ T cells have a prominent role in protective responses that has been demonstrated both in the clearance of primary infection (28) and against reinfection with C. pneumoniae (29), while CD4+ T cells, in the absence of CD8+ T cells, may even enhance the bacterial growth in the lungs (28). Indeed, our present results showed that CD8+ T cells are the major cell type producing a substantial amount of IFN-{gamma} during the adaptive immune phase of C. pneumoniae infection. Taken together, our present and previous data showing a differential NKT response suggest that adequate attention should be paid to potential differences in immunity to various chlamydial infections in designing strategies against Chlamydia.

The exact mechanism of how different NKT responses are generated to these two Chlamydia infections remains elusive. At least three possibilities, which are not necessarily exclusive, may exist: 1) C. pneumoniae and C. muridarum may preferentially induce differentiation of NKT0-like cells into NKT1-or NKT2-like cells, although this may be against the current dogma in mouse NKT cell biology, which holds that NKT cells cannot be polarized; 2) the same set of NKT is activated, but they produce skewed cytokine profiles because of the different nature of the two chlamydial species (DNA homology of these species is under 10%); and 3) distinct subsets of NKT cells producing different sets of cytokines are recruited to the lung. From a broader view, these data suggest that NKT activation effect or the activation itself is pathogen or even strain specific. This may be helpful in reconciling some inconsistent findings in previous studies (15, 46). For example, one previous study showed that NKT play a protective role in HSV infection (15). However, more recently, another study reported that NKT cells are not critical for host protection against this pathogen, because the absence of NKT in the NKT-KO did not affect the outcome of infection when compared with the WT mice (46). Although both of these studies have analyzed the role of NKT in HSV-1 infection, the strains of HSV-1 used were different. Thus, it is likely that NKT cells are activated by certain epitopes specific for pathogens or even strains of the same pathogen species. This antigenic variability may account for differential functional roles of NKT cells in infectious diseases. To address this issue more precisely, characterization of glycolipid Ags from various microbial pathogens and identification of antigenic effects on NKT may throw more light on immunological activity exerted by NKT cells. It is probable that some microbial glycolipid Ags preferentially activate NKT cells to produce more IFN-{gamma}, while some activate IL-4-producing NKT cells. Thus, the different functional NKT subsets, such as NKT-1-like or NKT-2-like cells, may be activated depending on the type or extent of activation stimuli or antigenic epitopes, which in turn may reflect in the variability of NKT-mediated immunological outcomes.

Our findings provide evidence on the role of NKT cells in influencing the direction of adaptive immune responses, including CD4 and CD8 T cells, to C. pneumoniae infection in vivo. Thus, these findings demonstrate that NKT cells function not only in the innate immune phase, but also in bridging to the type 1 acquired immune responses, which leads to the host protection against C. pneumoniae infection. In terms of the mechanism by which NKT manipulate adaptive immune responses, because it is known that IFN-{gamma} has a potent activity on Ag processing and presentation by APCs, it is likely that IFN-{gamma}, secreted by NKT, enhances the function of APCs, such as dendritic cells to produce IL-12. Such an activity would result likely in preferential activation/differentiation of C. pneumoniae-specific type-1 T cells and would ultimately increase the level of protection. Because the activation of NKT in vivo may also trigger a prompt series of cellular activation events including the activation of other innate cells such as NK cells allowing a more functional network of immune components, it need not be the case that NKT cells are the sole or even the major source of the IFN-{gamma}, although these cells may be critical for the initial induction of IFN-{gamma} synthesis which is important for directing immune responses to control infection. It should also be noted that although the NKT KO mice indeed showed slower clearance of C. pneumoniae infection and more serious disease (greater body weight loss) than WT mice, they also showed a trend of recovery in later stage (Fig. 1). The result suggests that the role of NKT in host resistance to C. pneumoniae infection may be mainly in the early stage of infection for the rapid induction of protective immunity, but may not be essential for the resolution of the infection. However, because the fast clearance of the pathogen is critical in preventing pathological changes in chlamydial diseases, the important role played by NKT cells in the early stage should not be underestimated.

Although showing that iNKT cells mediate protective immune responses in C. pneumoniae infection, our data also provide evidence that the type of immune response enhanced by the same NKT stimulator, {alpha}-GalCer, may vary depending on the nature of the pathogen. These findings have important clinical and therapeutic implications. A single injection of {alpha}-GalCer before intranasal infection substantially reduced C. pneumoniae loads in the lungs of WT mice and enhanced type 1 immunological responses. Of note, {alpha}-GalCer treatment significantly alleviated the lung pathology and promoted faster normalization of alveolar architecture of lungs in the treated WT mice compared with the vehicle-treated controls (data not shown). However, in both NKT-KO and CD1-KO mice infected with C. pneumoniae, the beneficial effect of {alpha}-GalCer treatment was seen to be abolished, indicating that {alpha}-GalCer displays its adjuvant-like action by specifically activating V{alpha}14 NKT cells in the context of CD1d molecules. This indicated that the administration of {alpha}-GalCer substantially enhances the level of protective immunity to this infection. In contrast, following C. muridarum (MoPn) infection, the administration of {alpha}-GalCer appears to enhance type 2 response characterized by increased IL-4 and IL-5 production (35). These observations raise a potential challenge to the attempt to use {alpha}-GalCer as an adjuvant in immunization, i.e., how to appropriately use {alpha}-GalCer to enhance the preferable protective type immunity against a particular infection.

In conclusion, the results of the present study unveiled the important role of iNKT cells in host protection against C. pneumoniae lung infection. Moreover, these findings provide in vivo evidence that even biologically, closely related pathogens may activate different functional NKT subsets which can further polarize the immune cells in adaptive immune responses, such as CD4+ and CD8+ cells. Further studies revealing the nature of the pathogens/stimuli which involve the development and/or promotion of differential NKT responses will greatly enhance our understanding on the linkage between innate and adaptive immune responses in infectious diseases.


   Acknowledgments
 
We thank Dr. Katerina Wolf for initially providing us the AR-39 strain; Dr. Y. Koezuka (Kirin Pharmaceuticals) for {alpha}-GalCer; and the National Institute of Allergy and Infectious Disease MHC Tetramer Core Facility for Cd1d tetramers for our experiments. We are grateful to Dr. Masaru Taniguchi (RIKEN Research Center for Allergy and Immunology, Yokohama City, Japan) for providing the J{alpha}18 KO mice.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by operating grants from the Canadian Institutes for Health Research (CIHR) and the Manitoba Health Research Council. X.Y. is Canada Research Chair in Infection and Immunity. A.G.J. is a recipient of Postdoctoral Fellowship Award from the International Centre for Infectious Diseases/CIHR National Training Program. L.B. is a recipient of a CIHR Doctoral Studentship and a trainee of the CIHR National Training Program in Allergy/Asthma. Back

2 Address correspondence and reprint requests to Dr. Xi Yang, Laboratory for Infection and Immunity, Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Room 523, BMSB, 730 William Avenue, Winnipeg, Manitoba R3E 0W3, Canada. E-mail address: yangxi{at}cc.umanitoba.ca Back

3 Abbreviations used in this paper: iNKT, invariant NKT; {alpha}-GalCer, {alpha}-galactosylceramide; MoPn, mouse pneumonitis; KO, knockout; IFU, inclusion-forming unit; EB, elementary body; D-PBS, Dulbecco’s PBS; p.i., postinfection; WT, wild type. Back

Received for publication July 13, 2006. Accepted for publication October 30, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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