J Med Microbiol 57 (2008), 1340-1348; DOI: 10.1099/jmm.0.47747-0
© 2008 Society for General Microbiology
ISSN 0022-2615
Paradoxically high resistance of natural killer T (NKT) cell-deficient mice to Legionella pneumophila: another aspect of NKT cells for modulation of host responses
Kayoko Hayakawa1,2,
Kazuhiro Tateda1,
Etsu T. Fuse1,
Tetsuya Matsumoto3,
Yoshikiyo Akasaka4,
Toshiharu Ishii4,
Toshinori Nakayama5,
Masaru Taniguchi6,
Mitsuo Kaku2,
Theodore J. Standiford7 and
Keizo Yamaguchi1
1 Department of Microbiology and Infectious Diseases, Toho University, School of Medicine, Tokyo 143-8540, Japan
2 Department of Infection Control and Laboratory Diagnostics, Internal Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan
3 Department of Microbiology, Tokyo Medical University, Tokyo, Japan
4 Department of Pathology, Toho University School of Medicine, Tokyo, Japan
5 Department of Immunology, Graduate School of Medicine, Chiba University, Chiba, Japan
6 Laboratory of Immune Regulation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan
7 Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109-0360, USA
Correspondence
Kazuhiro Tateda
kazu{at}med.toho-u.ac.jp
Received November 4, 2007
Accepted August 1, 2008
In the present study, we examined the roles of natural killer T (NKT) cells in host defence against Legionella pneumophila in a mouse model. The survival rate of NKT cell-deficient J
281 knock-out (KO) mice was significantly higher than that of wild-type mice. There was no bacterial overgrowth in the lungs, but J281 KO mice showed enhanced pulmonary clearance at a later stage of infection, compared with their wild-type counterparts. The severity of lung injury in L. pneumophila-infected J281 KO mice was less, as indicated by lung permeability measurements, such as lung weight and bronchoalveolar lavage fluid albumin concentration. Recruitment of inflammatory cells in the lungs was approximately twofold greater in J281 KO mice on day 3. Interestingly, higher values of interleukin (IL)-1β and IL-18, and increased caspase-1 activity were noted in the lungs of J281 KO mice from an early time point (6 h). Exogenous -galactosylceramide, a ligand of NKT cells, induced IL-12 and gamma interferon at 6 h, but suppressed IL-1β at later time points in wild-type, whereas no effects were evident in J281 KO mice, as expected. Systemic administration of heat-killed L. pneumophila, but not Escherichia coli LPS, reproduced exaggerated production of IL-1β in the lungs of J281 KO mice. These results demonstrate that NKT cells play a role in host defence against L. pneumophila, which is characterized by enhanced lung injury and decreased accumulation of inflammatory cells in the lungs. The regulation of IL-1β, IL-18 and caspase-1 may be associated with the modulating effect of host responses by NKT cells.
Abbreviations: BAL, bronchoalveolar lavage; -GalCer, -galactosylceramide; KO, knock-out; IFN-
, gamma interferon; IL, interleukin; NK, natural killer; NKT, natural killer T.
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INTRODUCTION
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Legionella pneumophila is a Gram-negative bacillus that causes acute pneumonia, known as Legionnaires' disease. The pneumonia is often serious and life-threatening, and can be nosocomial or community-acquired (Reingold, 1988; Marston et al., 1997). Legionnaires' disease accounts for 2–8 % of community-acquired pneumonia cases (Bartlett and Mundy, 1995; Fang et al., 1990), and it is much more frequent among patients admitted to intensive care units (Rello et al., 1993). Unfortunately, high mortality rates ranging from 10 to 50 % have been observed, especially in immunocompromised individuals (el-Ebiary et al., 1997; Pedro-Botet et al., 1998; Tkatch et al., 1998).
Legionella usually infects humans via inhalation of contaminated aerosols from waterborne environmental sources. In lung tissues, the bacteria multiply in several types of host cells, but especially in macrophages (Horwitz & Silverstein, 1980; Nash et al., 1984; Mody et al., 1993). Protective roles of cytokines, such as interleukin (IL)-12, IL-18, tumour necrosis factor alpha, and gamma interferon (IFN-) have been reported both in animal models of pneumonia and in human infections (Skerrett & Martin, 1994; Gebran et al., 1994; Brieland et al., 1998, 2000; Salins et al., 2001; Tateda et al., 1998). Recent studies have focused on the mechanisms of bacterial flagellin recognition by the intracellular sensor system, the nucleotide-binding oligomerization domain (NOD), which results in IL-1β and IL-18 production through activation of the caspase-1 cascade (Franchi et al., 2006; Miao et al., 2006; Molofsky et al., 2006). Infected macrophages respond in several ways, such as apoptosis, autophagy and pyroptosis, depending on the balance between bacterial virulence and host cellular responses (Fink & Cookson, 2005). Pyroptosis is a newly defined state characterized by non-apoptotic, pro-inflammatory programmed cell death. An important hallmark of pyroptosis in L. pneumophila-infected macrophages is the caspase-1-dependent production of IL-1β and IL-18 (Molofsky et al., 2006). Pyroptosis may resist Legionella infections by limiting bacterial replication in host cells and enhancing recruitment of inflammatory cells to the site of infection (Fink & Cookson, 2005; Swanson & Molofsky, 2005). The clinical significance of pyroptosis in L. pneumophila infections remains unclear.
Natural killer T (NKT) cells constitute a T-cell subpopulation that expresses natural killer (NK) markers, and they have been reported to play an important role in a variety of autoimmune, allergic, antitumour and antimicrobial immune responses (Godfrey & Kronenberg, 2004; Taniguchi & Nakayama, 2000; Taniguchi et al., 2003; van der Vliet et al., 2004). NKT cells are characterized by an invariant T-cell receptor V chain, which consists of a V14 J281 gene segment in mice (Koseki et al., 1991; Exley et al., 1997). Although a natural ligand for these cells remains to be identified, a synthetic glycolipid, -galactosylceramide (-GalCer), presented in the context of CD1d induces a prompt production of Th1 and/or Th2 cytokines. Previous reports have demonstrated that NKT cells function as important innate immune effector cells against a variety of pathogens, such as Leishmania major, Pseudomonas aeruginosa, Streptococcus pneumoniae and Cryptococcus neoformans (Ishikawa et al., 2000; Kawakami et al., 2001, 2003; Nieuwenhuis et al., 2002). However, antibody-mediated blockage or gene-disruption of NKT cells has been associated with increased resistance to certain pathogens, such as Toxoplasma gondii, Listeria monocytogenes and Chlamydia trachomatis, which suggest a more complicated role for NKT cells in host immunological systems (Szalay et al., 1999; Bilenki et al., 2005; Ronet et al., 2005). These data clearly suggest that NKT cell-mediated host responses may be pathogen-dependent, with these cells participating in broad and diverse types of cellular responses.
In the present study, we investigated the roles of NKT cells in L. pneumophila challenge, using J281 gene-disrupted mice [J281 knock-out (KO) mice]. Lethal sensitivity, the bacterial burden and bronchoalveolar lavage (BAL) cell counts in the lungs, in addition to kinetics of cytokines (IL-1β, IL-10, IL-12, IL-18 and IFN-) and caspase-1 activity were compared between J281 KO and control (wild-type) mice.
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METHODS
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Animals.
Specific pathogen-free C57BL/6 mice (6- to 8-week-old) were purchased from Charles River Laboratories (Yokohama, Japan). J281 KO mice generated at Chiba University (Chiba, Japan) were back-crossed eight times with C57BL/6 mice (Cui et al., 1997). All animals were housed in a pathogen-free environment in the Toho University School of Medicine. The mice were provided sterile food and water.
Bacteria.
Clinical isolates of L. pneumophila Suzuki, a serogroup 1 strain originally isolated at Toho University Hospital (Tateda et al., 1998), were grown for 3–4 days at 37 °C on buffered charcoal–yeast extract (BCYE) agar supplemented with L-cysteine and ferric nitrates. A single colony was transferred to 3 ml buffered yeast extract broth and incubated overnight at 37 °C with constant shaking. The bacterial suspension was transferred to fresh buffered yeast extract broth as serial fivefold dilutions and incubated overnight at 37 °C with constant shaking. After confirmation of bacterial motility, the concentration of bacteria in the broth was determined at OD600. Post-exponential-phase bacteria were used as challenge organisms (Byrne & Swanson, 1998). Heat-killed L. pneumophila were prepared by incubating the bacterial suspension in normal saline at 95 °C for 5 min. After the heat treatment, the sterility of the bacterial suspension was confirmed by plating it on BCYE agar. The numbers of viable bacteria in the challenge suspension and the pre-heat treatment suspension were determined by plating and incubating organisms on BCYE agar for 4 days.
Challenge of L. pneumophila in mice.
Mice were anaesthetized intramuscularly with ketamine (7 mg) and xylazine (15 mg kg–1). The trachea was exposed and 30 µl bacterial suspension or saline was injected directly into the trachea using a sterile 26-gauge needle. The skin incision was closed with a surgical staple.
Intravenous challenge using Escherichia coli LPS or heat-killed L. pneumophila.
E. coli 055 : B5 LPS (50 µg) (Difco) suspended in 200 µl normal saline or 4x108 c.f.u. heat-killed L. pneumophila suspended in 200 µl saline were intravenously injected into mice.
Lung harvesting for analysis.
Mice were sacrificed by CO2 asphyxia at designated time points. Before removal of the lung, the pulmonary vasculature was perfused with 1 ml saline via the right ventricle. After removal, whole lungs were homogenized in 1 ml saline using a tissue homogenizer (Omni International) under a vented hood. Ten microlitre aliquots of each homogenate were inoculated onto BCYE agar after serial 1 : 10 dilutions in saline. The remainder of each homogenate was incubated on ice for 30 min and then centrifuged at 3000 r.p.m. for 10 min. Supernatant was collected, passed through a 0.45 µm filter (Kanto Chemical), and stored at –40 °C until use.
Collection of BAL.
Mice were sacrificed at designated times after inoculation with bacteria. The trachea was exposed and intubated using a 1.7 mm (outer diameter) polyethylene catheter. BAL was performed by instilling 2 ml RPMI 1640 (Gibco-BRL), and BAL fluid was pooled for each animal. Leukocyte numbers were determined using a haemocytometer. Collected cells were attached to glass slides by cytospins, stained with May–Giemsa, and differential cell counts were performed. The remaining BAL fluid was stored at –40 °C until further analysis. In some experiments, albumin concentrations in BAL fluid were determined by a mouse albumin ELISA quantification kit (AKRAL-121; Shibayagi).
ELISA of IL-1β, IFN-, IL-10, IL-12 and IL-18.
Levels of IL-1β, IFN-, IL-10, IL-12 and IL-18 cytokines in lung homogenates were measured using ELISA kits (IL-1β, IFN-, IL-10 and IL-12; R & D Systems; IL-18; Medical and Biological Laboratories). The assays were performed according to the manufacturers' instructions, and the level in each sample was determined in duplicate.
Determination of caspase activity.
Levels of caspase activity were determined in lung homogenates. Caspase-1 and caspase-3 activities were determined by colorimetric assay (R & D Systems), in which a caspase-specific peptide that conjugated to the colour reporter molecule p-nitroanilide was used. The data are expressed as a fold increase, compared to those of control mice (n=5).
Treatment with -GalCer.
-GalCer (Kirin Brewery) was prepared as described previously (Gonzalez-Aseguinolaza et al., 2000). The stock -GalCer solution (220 µg ml–1 in 0.5 % polysorbate 20 in normal saline) was diluted to 10 µg ml–1 with PBS. PBS was used as a control vehicle solution. Both the treatment and control solutions (200 µl) were injected intraperitoneally on the day of infection.
Statistical analyses.
Statistical significance was determined using the Mann–Whitney test. Survival curves were constructed by the Kaplan–Meier method and were analysed by log rank test. P<0.05 was considered as a significant difference.
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RESULTS AND DISCUSSION
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High resistance of J281 KO mice to L. pneumophila
C57BL/6 control and J281 KO mice were intratracheally challenged with L. pneumophila Suzuki strain (3x106 c.f.u. per mouse), and survival was observed once a day for 14 days after the exposure (Fig. 1
). Wild-type mice started to succumb from day 4, and all 10 mice died by day 8 post-challenge (0/10, 0% survival). In contrast, significantly reduced mortality was observed in J281 KO mice, as the survival rate in these mice was 90 % (9/10) at the end of observation period (P<0.05). Similar results were obtained in three independent experiments. These observations suggested a greater resistance to L. pneumophila challenge in J281 KO mice, compared with the wild-type controls.
Bacterial burden in the lungs of mice after L. pneumophila challenge
After inoculation of the bacteria (1x107 c.f.u. per mouse), mice were sacrificed at the indicated time points, and the number of viable bacteria in the lungs was determined. There were no differences in bacterial numbers in the lungs of wild-type and J281 KO mice for up to 48 h (Fig. 2a). By 72 h, however, there was a modest but significant reduction in bacterial count in the lungs of J281 KO mice, compared with the infected wild-type mice (P<0.05). L. pneumophila c.f.u. in the lungs of J281 KO mice continued to decrease thereafter, whereas lung bacterial c.f.u. in wild-type mice could not be determined at time points after 72 h due to the premature deaths of the mice. No L. pneumophila c.f.u. was isolated in blood or spleen samples of either strain of mice at any time points examined (data not shown).
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Fig. 2. Bacterial burden and evaluation of lung injury in the lungs of L. pneumophila-challenged mice. (a) After intratracheal inoculation of L. pneumophila, mice were sacrificed at the indicated time points and the number of bacteria in the lungs were compared between wild-type (black bars) and J281 KO (white bars) mice (n=5). At 96 h, the bacterial number of wild-type was not determined because mice started to die. (b, c) Total lung weight divided by body weight (b) and albumin concentration in BAL fluid (c) were compared 3 days after bacterial challenge (5x106 c.f.u. per mouse) (n=5). * P<0.05 compared with wild-type mice.
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Evaluation of lung injury in Legionella-challenged mice
To determine the mechanism of enhanced resistance in J281 KO mice, changes in markers of lung injury were evaluated on day 3 post-infection. The markers included lung weight (Fig. 2b
) and BAL fluid albumin levels (Fig. 2c) in wild-type and J281 KO mice. L. pneumophila challenge was associated with an increase in the total lung weight divided by the body weight in wild-type and J281 KO mice; however, lung weights were significantly less in the latter (P<0.05). In addition, albumin concentration in BAL fluid correlated well with the changes in the lung weight, and increases in albumin levels were significantly less prominent in J281 KO mice compared with those observed in wild-type mice (P<0.05). Collectively, these data demonstrate that the lung injury caused by Legionella was less severe in J281 KO mice, which may be associated with the survival benefit observed in this mouse strain.
Leukocyte accumulation in BAL fluid of Legionella-challenged mice
To further understand the relative resistance of J281 KO mice to Legionella, leukocyte accumulation in the air space was examined in mice on days 1 and 3 post-infection (Fig. 3). L. pneumophila challenge resulted in a nearly 30-fold increase of total cell number in BAL fluids of wild-type mice on day 3. Importantly, there was a twofold greater number of inflammatory cells observed in J281 KO mice than that seen in wild-type mice on day 3. Significantly higher values were observed in all cell types (neutrophils, macrophages and lymphocytes) in J281 KO mice, compared with those in wild-type mice with L. pneumophila. These data suggest that more leukocytes accumulated in the lungs of J281 KO mice in response to L. pneumophila.
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Fig. 3. Total BAL cell counts for L. pneumophila-challenged mice. Wild-type (black bars) and J281 KO (white bars) mice were intratracheally inoculated with L. pneumophila. BAL analysis was performed on mice sacrificed on days 1 and 3 (n=5). * P<0.05 compared with wild-type mice.
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Cytokine production in the lungs of Legionella-challenged wild-type and J281 KO mice and effects of -GalCer
To further confirm the regulatory role of NKT cells in host defence against L. pneumophila, cytokine production (IL-1β, IL-18, IFN-, IL-12, IL-10), and bacterial number in the lungs of wild-type and J281 KO mice, were determined 6, 12, 24 and 48 h post-infection (Fig. 4). Significantly higher levels of IL-1β and IL-18 were observed in J281 KO mice at the early stages, and these levels were maintained for 24 and 48 h, respectively (Fig. 4a, b). In contrast, the production kinetics of IFN-, IL-12 and IL-10 were not different between wild-type and J281 KO mice, suggesting that there were no changes in Th1/Th2 balance in the absence of NKT cells (Fig. 4c, d, e).
Next, we focused on the influence of NKT cell activation on L. pneumophila challenge, especially pertaining to the production of the various cytokines and susceptibility to death. Mice were intraperitoneally pretreated with -GalCer (2 µg per mouse), which specifically stimulate NKT activity in context of CD1d, prior to the challenge with L. pneumophila. Pretreatment of wild-type mice was associated with significant increases of IFN-, IL-12 and IL-10 in the early stages, whereas reductions of IL-1β and IL-18 were apparent in the later stages. As expected, J281 KO mice failed to respond to -GalCer treatment, which did not induce any significant cytokine changes. No changes of viable bacterial count in the lungs of wild-type and J281 KO mice were observed during these periods, in spite of -GalCer treatment (Fig. 4f). No enhanced susceptibility to death was apparent following -GalCer activation of NKT cells (data not shown). These data suggested that absence of NKT cells or NKT cell-derived factors may be associated with an increase in resistance to L. pneumophila and a skewed production of cytokines.
Caspase-1 and caspase-3 activity in the lungs of wild-type and J281 KO mice
Since significant increases of IL-1β and IL-18 were observed in the early stages of infection in the lungs of J281 KO mice, we next examined the expression of caspase-1, an enzyme required for maturation of IL-1β and IL-18, and caspase-3, a key enzyme for induction of apoptosis (Fig. 5). Importantly, significantly higher levels of caspase-1 activity were demonstrated at the early time points (6 and 12 h) in the lungs of J281 KO mice, whereas no substantial alteration in caspase-3 activity was observed during the observation period.
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Fig. 5. Capase-1 and caspase-3 activity in the lungs of mice. Wild-type (black bars) and J281 KO mice (white bars) were intratracheally infected with L. pneumonia (1x107 c.f.u. per mouse). Caspase-1 (a) and caspase-3 (b) activities in total lung homogenates were determined at the indicated time points after bacterial challenge (n=5). Data are expressed as fold increase compared with those of control mice (n=5). *P<0.05 compared with wild-type mice.
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IL-1β production in the lungs of wild-type and J281 KO mice in response to heat-killed L. pneumophila
To further understand the interaction between L. pneumophila and NKT cells, we examined whether heat-killed L. pneumophila could trigger IL-1β production in the lungs of J281 KO mice. In preliminary experiments, the intratracheal administration of heat-killed organisms into the lungs induced only a minimal amount of IL-1β in both mouse strains. Therefore, an intravenous route was used to administer heat-killed bacteria. Wild-type and J281 KO mice were injected intravenously with heat-killed L. pneumophila (4x108 c.f.u. per mouse) (Fig. 6a) or E. coli LPS (50 µg per mouse) as a control (Fig. 6b). The levels of IL-1β in the lung homogenates were evaluated 2 and 4 h after the injections. As shown in Fig. 6(a), the concentration of IL-1β was significantly higher in the lungs of J281 KO mice than that of wild-type mice when heat-killed L. pneumophila was used as a stimulant. In contrast, a trend toward higher levels of IL-1β was observed in wild-type mice in response to E. coli LPS. These data suggest that heat-stable L. pneumophila antigen may be responsible, at least partly, for the increased production of IL-1β in J281 KO mice.
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Fig. 6. Levels of IL-1β production in the lungs of mice in response to heat-killed L. pneumophila. Wild-type (black bars) and J281 KO mice (white bars) were injected intravenously with heat-killed L. pneumophila (4x108 c.f.u. per mouse) (a) or E. coli LPS (50 µg per mouse) (b). The levels of IL-1β in the lung homogenates were evaluated 2 and 4 h after the injection (n=5). *P<0.05 compared with wild-type mice.
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In the present study, our findings clearly indicate that NKT cell-deficient J281 KO mice are more resistant than control C57BL/6 mice to L. pneumophila. In particular, a striking reduction in mortality was observed in J281 KO mice, as compared to control mice. These data are in line with the findings of others demonstrating that NKT cells can be either beneficial or detrimental to host anti-bacterial defences, depending upon the inciting pathogen used. Our data further suggest the possibility that NKT cells may contribute to the production of the pyroptosis-related cytokines IL-1β and IL-18, by modulating the caspase-1 cascade.
Previously, we reported that acute lung injury may be a major cause of death in mice with L. pneumophila pneumonia (Tateda et al., 2003). As expected, a significant elevation of acute lung injury markers, including lung weight and albumin levels in BAL fluids, was observed in the lungs of mice with L. pneumophila. Importantly, J281 KO mice demonstrated lower lung weights and BAL albumin levels, indicative of reduced lung injury. The relatively modest differences in L. pneumophila c.f.u. between the strains at the 72 h time point is unlikely to account for the more impressive differences in survival and magnitude of lung injury observed in this study.
The mechanisms accounting for the greater resistance of J281 KO mice to L. pneumophila have not been completely defined. We observed enhanced leukocyte accumulation and a more modest but significant reduction of bacterial burden in the lungs of J281 KO mice on day 3. Although significantly higher levels of IL-1β and IL-18, but not IL-12 and IFN-, in the lungs of J281 KO mice were noted as early as 6 h post-challenge, the differences were modest and transient. These data suggested involvement of other factors, such as cell accumulating signals, mediators and responses. IL-1β and IL-18 are reported to have several biological roles in inflammatory and immunological events, which may be associated with exaggerated inflammatory cell accumulation in the lungs of J281 KO mice. The present data also demonstrated enhanced activation of caspase-1 in J281 KO mice at between 6 and 12 h, which may suggest a contribution of NKT cells to the pyroptosis cascade. Mechanisms by which NKT cells modulate the pyroptosis cascade are an ongoing area of investigation.
Accumulating data demonstrated that NKT cells are a critical component for induction of innate immunity against a variety of pathogens. A characteristic feature of NKT cells is the prompt production of Th1 (IL-12, IFN-) and/or Th2 (IL-4, IL-10) cytokines in response to invading pathogen, although how the Th1/Th2 cytokine balance is determined at the cellular level is incompletely understood. Moreover, the complexity of NKT cell immunology is illustrated by the fact that blocking of NKT cells by specific antibody or NKT gene-disruption in mice made them more resistant to some organisms. For example, in systemic Listeria monocytogenes infection, antibody-mediated inhibition of NKT cells reduced TGF-β2 production, while concomitantly increasing Th1 cytokines (IL-12, IFN-), which was associated with amelioration of survival (Szalay et al., 1999). Bilenki and collaborators have reported that NKT cell activation promotes Chlamydia trachomatis infection in mice, in which exaggerated IL-4, IL-5 and IL-10 production and skewed Th2-type host responses were observed (Bilenki et al., 2005). In the present study, a shifting of Th1/Th2 balance was not demonstrated in J281 KO mice, and in our hands IL-4 was not detected in the lungs of either group of mice during the observation period. In contrast, exogenous -GalCer treatment induced a prompt response of IL-10, IL-12 and IFN- in control mice, but not in J281 KO mice, although these alterations were not associated with changes in the number of bacteria in the lungs. These data indicate that NKT cells have a promoting role in L. pneumophila challenge, as they do in Chlamydia and Listeria. However, the previously proposed biased production of Th1/Th2 cytokines may not explain the resistant mechanism(s) in J281 KO mice.
L. pneumophila-mediated activation of NK cells and their robust IFN- production dependent on MyD88 expression have been reported to represent the crucial mechanism of in vivo control of L. pneumophila infection (Sporri et al., 2006). Another study also indicated that NK cells respond in vivo and in vitro to stimulation by producing IFN- and by increased cytolytic activity (Blanchard et al., 1988). In the present study, however, there was no difference in IFN- production between wild-type and J281 KO mice. Our findings suggest that unlike NK cells, IFN- production may not be the key factor in modulating host responses by NKT cells.
The present data may not correctly reflect human L. pneumophila infection because there was no growth of bacteria in the lungs. The important fact is that murine macrophages exhibit strain-dependent levels of in vitro resistance to Legionella replication, ranging from the highly susceptible A/J strain to the resistant C57BL/6 strain (Diez et al., 2003; Growney & Dietrich, 2000; Wright et al., 2003). Consistent with these data, previously we have reported 10–100-fold replication of L. pneumophila in the lungs of A/J mice, but not C57BL/6 mice (Tateda et al., 2001). In the present study, we have used wild-type and J281 KO mice in a C57BL/6 background, and as expected no growth of the bacteria was observed in the lungs. In spite of these experimental limitations, several investigators have examined pathogenesis of L. pneumophila infection in C57BL/6 background mice (Molofsky et al., 2006; Archer & Roy, 2006). Another important factor to consider with respect to human relevance of this model is the challenge dose of the bacteria. We have used 3x106 c.f.u. per mouse for survival experiments. This number is quite similar to that in the previous reports (Zamboni et al., 2006: Archer & Roy, 2006), where 1x106 c.f.u. per mouse was used for pulmonary L. pneumophila infection in C57BL/6 mice. Importantly, we have obtained substantially the same results in lower challenge dose experiments (3x105 c.f.u. per mouse), no increase of bacteria and higher levels of IL-1β and IL-18 in J281 KO mice (data not shown).
Heat-killed L. pneumophila was prepared as a crude LPS, and its biological activity compared to that of E. coli LPS. The data obtained from these experiments were that heat-killed L. pneumophila, but not E. coli LPS, reproduced exaggerated IL-1β in the lungs of J281 KO mice at early time points after stimulation. L. pneumophila LPS is known to have a specific structure characterized with a longer fatty acid side chain and stronger hydrophobic nature (Knirel et al., 1996; Moll et al., 1997; Girard et al., 2003). We and other investigators have reported that L. pneumophila LPS is recognized through TLR-2, but not TLR-4, in macrophages (Archer & Roy, 2006; Akamine et al., 2005; Braedel-Ruoff et al., 2005; Fuse et al., 2007). Given that macrophages are both a major niche in vivo for L. pneumophila infection and cellular sources of IL-1β/IL-18, the present data suggest an important interplay between NKT cells and macrophages. Intracellular and intercellular regulation of the pyroptosis cascade in macrophages, probably involving TLR- and NOD-mediated signalling, may be crucial for host defence systems against an intracellular pathogen such as L. pneumophila.
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ACKNOWLEDGEMENTS
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We thank Soichiro Kimura and Yoshikazu Ishii for their helpful discussions. The authors express deep appreciation to Michele S. Swanson and Paul H. Edelstein for their critical suggestions. This work was supported by the Naito Foundation and grants from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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INTRODUCTION
METHODS
) were challenged intratracheally with L. pneumophila Suzuki strain (3x106 c.f.u. per mouse), and mortality was observed for 14 days post-infection (n=10). Similar results were obtained in three independent experiments. * P<0.05 compared with the wild-type mice.