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Infect Immun. 2010 June; 78(6): 2667–2676.
Published online 2010 March 29. doi:  10.1128/IAI.01441-09
PMCID: PMC2876567

α-Galactosylceramide Promotes Killing of Listeria monocytogenes within the Macrophage Phagosome through Invariant NKT-Cell Activation[down-pointing small open triangle]

Abstract

α-Galactosylceramide (α-GalCer) has been exploited for the treatment of microbial infections. Although amelioration of infection by α-GalCer involves invariant natural killer T (iNKT)-cell activation, it remains to be determined whether macrophages (M[var phi]) participate in the control of microbial pathogens. In the present study, we examined the participation of M[var phi] in immune intervention in infection by α-GalCer using a murine model of listeriosis. Phagocytic and bactericidal activities of peritoneal M[var phi] from C57BL/6 mice, but not iNKT cell-deficient mice, were enhanced after intraperitoneal injection of α-GalCer despite the absence of iNKT cells in the peritoneal cavity. High levels of gamma interferon (IFN-γ) and nitric oxide (NO) were detected in the peritoneal cavities of mice treated with α-GalCer and in culture supernatants of peritoneal M[var phi] from mice treated with α-GalCer, respectively. Although enhanced bactericidal activity of peritoneal M[var phi] by α-GalCer was abrogated by endogenous IFN-γ neutralization, this was only marginally affected by NO inhibition. Similar results were obtained by using a listeriolysin O-deficient strain of Listeria monocytogenes. Moreover, respiratory burst in M[var phi] was increased after α-GalCer treatment. Our results suggest that amelioration of listeriosis by α-GalCer is, in part, caused by enhanced killing of L. monocytogenes within phagosomes of M[var phi] activated by IFN-γ from iNKT cells residing in an organ(s) other than the peritoneal cavity.

Listeria monocytogenes, a Gram-positive facultative intracellular bacterium, is the causative agent of listeriosis, with an overall mortality rate of 30% (76). A major virulence factor of L. monocytogenes is listeriolysin O (LLO), a 58-kDa protein encoded by the hly gene (26, 42, 65). LLO promotes intracellular survival of L. monocytogenes in professional phagocytes such as macrophages (M[var phi]) by promoting listerial escape from the phagosome into the cytosol (10, 22, 26, 42, 62, 65). Cells of the innate immune system play a pivotal role as a first line of defense against L. monocytogenes infection and among these, mononuclear phagocytes are critical (56, 61). Activation of M[var phi] by gamma interferon (IFN-γ) is mandatory for elimination of L. monocytogenes (31, 35). Nitric oxide (NO) synthesized by inducible NO synthase, which is localized in the cytosol of professional phagocytes, participates in killing of L. monocytogenes (48, 52, 69, 71). Similarly, reactive oxygen intermediates (ROI) play a role in killing of L. monocytogenes within the phagosome (52, 53, 59).

Natural killer T (NKT) cells represent a unique T-lymphocyte population expressing NKR-P1B/C (NK1.1; CD161), which is a type 2 membrane glycoprotein of the C-type lectin superfamily (6). In the mouse, the majority of NKT cells express an invariant T-cell receptor (TCR) α chain encoded by Vα14/Jα18 gene segments and a TCRVβ highly biased toward Vβ8.2, Vβ7, and Vβ2 (invariant NKT [iNKT] cells) (6). In contrast to conventional T cells, which recognize antigenic peptides presented by polymorphic major histocompatibility complex class I or class II molecules, iNKT cells recognize glycolipid antigens, including α-galactosylceramide (α-GalCer), a synthetic glycolipid originally isolated from a marine sponge, presented by the nonpolymorphic antigen presentation molecule CD1d (6, 40). iNKT cells are highly versatile and promptly produce both type 1 and type 2 cytokines, such as IFN-γ and interleukin-4 (IL-4), respectively, upon activation through their TCRs (1, 15-17, 79). IL-15 is an essential growth factor of both iNKT cells and NK cells and, hence, both cell populations are absent in IL-15-deficient (IL-15−/−) mice (58). The numbers of iNKT cells are also markedly reduced in SJL mice because of a large deletion in their TCRVβ genetic region (5, 78).

In vivo administration of α-GalCer causes prompt release of various cytokines by iNKT cells, which are involved in the control of various diseases, e.g., tumor rejection and prevention of autoimmune diseases (33, 41, 67, 70). Although α-GalCer has been reported to enhance host resistance to some microbial pathogens (27-29, 37, 39, 44, 55, 64), its potential role in protection against intracellular bacterial infections remains enigmatic.

We have recently described that α-GalCer ameliorates murine listeriosis, which is, in part, caused by accelerated infiltration of inflammatory cells into the liver (18), although iNKT cells themselves exacerbate disease (19). Because M[var phi] play a central role in the elimination of L. monocytogenes, we considered the possibility that M[var phi] participate in enhanced resistance to L. monocytogenes infection caused by α-GalCer treatment. In the present study, we examined the influence of α-GalCer on listericidal activities of M[var phi] using a virulent and an avirulent strain of L. monocytogenes.

MATERIALS AND METHODS

Mice.

C57BL/6, IL-15−/−, SJL, and recombination-activating gene 1−/− (RAG-1−/−) mice were purchased from Japan SLC (Hamamatsu, Japan), the Jackson Laboratory (Bar Harbor, ME), Charles River Laboratories Japan (Yokohama, Japan), and the Jackson Laboratory, respectively, and maintained under specific-pathogen-free conditions at our animal facilities. Weight-matched female mice were used at 8 to 12 weeks of age in accordance with institutional guidelines of Gunma University, Max Planck Institute for Infection Biology and Nara Medical University. Unless otherwise stated, C57BL/6 mice were used.

Bacteria and infection.

The parental wild-type strain of L. monocytogenes used in the present study was EGD. The Δhly mutant strain was constructed from the wild-type strain of L. monocytogenes by homologous recombination as described previously (30). L. monocytogenes EGD and Δhly strains recovered from infected liver were grown in tryptic soy broth (Difco Laboratories, Detroit, MI) at 37°C for 18 h, and aliquots were frozen at -80°C for later use. The final concentration of viable bacteria was enumerated by plate counts on tryptic soy agar (Difco Laboratories). Mice were infected intraperitoneally (i.p.) with 2 × 104 L. monocytogenes bacteria. Unless otherwise stated, strain EGD was used.

Antibodies.

Monoclonal antibodies (MAbs) against Fcγ receptor (FcγR) and IFN-γ (2.4G2 and XMG1.2, respectively) were purified from hybridoma culture supernatants by ammonium sulfate precipitation and affinity chromatography on protein A- or G-Sepharose (Amersham Biosciences, Freiburg, Germany). Fluorescein isothiocyanate (FITC)-conjugated anti-CD3epsilon MAb (145-2C11) was purchased from BD PharMingen (Hamburg, Germany). Alexa 594-conjugated anti-rabbit IgG antibody (Ab) and rabbit anti-Listeria sp. Ab were obtained from ViroStat (Portland, ME) and Molecular Probes (Eugene, OR), respectively.

α-GalCer-loaded CD1d tetramer.

α-GalCer-loaded CD1d (α-GalCer/CD1d) tetramers were prepared by using the baculovirus expression system, using a CD1d construct with a BirA biotinylation site, followed by a His6 tag, as described previously (19, 50).

α-GalCer treatment.

α-GalCer was kindly provided by Kirin Pharma Co. Ltd. (Tokyo, Japan). Polysorbate 20 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Mice were treated i.p. with α-GalCer dissolved in 1% polysorbate 20 (1 μg/200 μl) or with 1% polysorbate 20 (200 μl) as a vehicle. In another experiment, cells were incubated in the presence of α-GalCer dissolved in 1% polysorbate 20 (100 ng/ml) or polysorbate 20.

Preparation of cells and peritoneal fluids.

Mice were anesthetized with ether, and peritoneal cells (PC) were harvested from the peritoneal cavity of mice after i.p. injection with RPMI 1640 (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) containing 10% fetal calf serum (FCS; Bio West, France) (5 ml). After a washing step, cells were incubated in six-well tissue culture plates (Corning, Inc., Canton, NY) at 37°C for 2 h, and nonadherent and adherent cells (regarded as M[var phi]) were collected separately. To obtain thioglycolate (TG)-induced M[var phi], mice were injected i.p. with 3 ml of TG (Eiken Chemical Co., Ltd., Tokyo, Japan) 4 days before cell preparation. To obtain peritoneal washout, mice were injected i.p. with 3 ml of RPMI 1640 containing 10% FCS, peritoneal fluids containing PC were harvested from the peritoneal cavity, and supernatants were collected after centrifugation. Unless otherwise stated, TG-uninduced M[var phi] were used. Hepatic leukocytes (HL) were prepared as described previously (15).

Determination of bactericidal activity of M[var phi].

M[var phi] were incubated in 24-well tissue culture plates (Corning) at 106/well for 2 h and infected with L. monocytogenes at 107 bacteria/well (multiplicity of infection [MOI] = 10). After 60 min of incubation, the cells were washed three times with prewarmed RPMI 1640 containing 10% FCS and gentamicin (Sigma-Aldrich, Tokyo, Japan) (5 μg/ml) to remove noningested bacteria. A portion of cells was treated with 0.5% saponin (Sigma-Aldrich) and plated on tryptic soy agar plates after sonication, and CFU were determined after 48 h of culture at 37°C (P60). For determination of bactericidal activity, cells were further incubated in RPMI 1640 containing 10% FCS and gentamicin (5 μg/ml) for 30 min, treated with 0.5% saponin (Sigma-Aldrich), and plated on tryptic soy agar plates after sonication. CFU counts were determined after 48 h of culture at 37°C (B30). Bactericidal activity was calculated as follows: 100 - [(B30/P60) × 100] (%). The data are expressed as a killing index calculated as follows: experimental group {100 - [(B30/P60) × 100]}/control group {100 - [(B30/P60) × 100]}.

Flow cytometry.

Cells were incubated with anti-FcγR MAb and then stained with conjugated MAb at 4°C for 15 min. Stained cells were washed with phosphate-buffered saline containing 0.1% bovine serum albumin (Thermo, Hamilton, New Zealand) and 0.1% sodium azide (Wako Pure Chemical Industries), fixed with 1% paraformaldehyde (Merck, Darmstadt, Germany), acquired by FACSCalibur (BD Biosciences, Mountain View, CA), and analyzed with CellQuest software (BD Biosciences). Cells were stained with phycoerythrin (PE)-labeled α-GalCer/CD1d tetramer for 15 min at room temperature after blocking.

Enzyme-linked immunosorbent assay.

IFN-γ levels were determined by Quantikine (R&D Systems, Inc., Minneapolis, MN) according to the manufacturer's instructions. The detection limit was 2 pg/ml.

NO measurement.

NO was measured using the Griess reagent system (Promega, Madison, WI) according to the manufacturer's instructions. The detection limit was 2.5 μM.

Immunohistochemistry.

M[var phi] (105/well) incubated in a Lab-Tek Chamber Slide (Nalge Nunc International, Naperville, IL) were infected with L. monocytogenes (106 bacteria/well; MOI = 10) for 1, 2, or 4 h. After a wash with prewarmed RPMI 1640 containing 10% FCS and gentamicin (5 μg/ml), the cells were stained with Alexa 488-conjugated phalloidin (Molecular Probes) and/or rabbit anti-Listeria Ab at room temperature for 2 h, followed by Alexa 594-conjugated anti-rabbit IgG Ab at room temperature for a further 2 h. Specimens were observed under fluorescence microscope and photographed.

Inhibition experiment.

M[var phi] were incubated in 24-well tissue culture plates (Corning) at 106/well in the presence or absence of NG-monomethyl-l-arginine (500 μM; Calbiochem, Darmstadt, Germany) or anti-IFN-γ neutralizing MAb (50 μg/ml) for 24 h.

Reverse transcription-PCR.

TCRVα14 mRNA expression was analyzed as described previously (20), with slight modifications. Briefly, total RNA extracted with an RNAgents total RNA isolation system (Promega) was primed for reverse transcription with random hexamers. To normalize cDNA content for further comparative analysis, β-actin-specific PCR was performed with serial dilutions of cDNA. The β-actin-specific primers were 5′-TGGAATCCTGTGGCATCCATGAAC-3′ (forward) and 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′ (reverse).

After a denaturation step at 94°C for 3 min, PCR cycles were run at 94°C for 30 s, 58°C for 30 s, and 72°C for 1 min for 30 cycles, followed by an extension step at 72°C for 7 min. β-Actin PCR products were analyzed by 1.2% agarose gel electrophoresis and ethidium bromide staining. For analysis of TCRVα14 mRNA expression, a normalized amount of cDNA yielding equivalent amounts of β-actin-specific PCR products was applied. The cDNA was denatured at 94°C for 4 min, and PCR cycles were run at 94°C for 30 s, 55°C for 30 s, and 72°C for 40 s for 30 cycles. The PCR products were subjected to electrophoresis on a 1.8% agarose gel. The 3′ and 5′ primers for the TCRVα14 PCR were (3′) Cα (5′-GAAGCTTGTCTGGTTGCTCCAG-3′) and (5′) Vα14 (5′-CTAAGCACAGCACGCTGCACA-3′). All PCRs were performed in a PTC-150 MiniCycler (Bio-Rad, Tokyo, Japan).

Luminol-dependent chemiluminescence.

The activity of respiratory burst in peritoneal M[var phi] was evaluated by using luminol-dependent chemiluminescence (CL) as described previously (23, 46). In brief, a peritoneal M[var phi] suspension (1.2 × 106 to 1.5 × 106 cells in 400 μl of Hanks balanced salt solution [HBSS] containing 1% gelatin [GHBSS]) was mixed with 30 μl of luminol solution (10−6 M; Laboscience Co., Tokyo, Japan) in a counting tube, which was kept at 37°C for 5 min with gentle agitation. L. monocytogenes grown in tryptic soy broth at 37°C for 18 h was sedimented (1,200 × g) for 20 min and washed twice in HBSS before being adjusted to a concentration of 6 × 108 CFU/ml. The bacteria were opsonized for 60 min at 37°C with 10% normal pooled mouse serum in GHBSS and washed twice with GHBSS before use for the CL assay. To generate CL, opsonized L. monocytogenes (108 CFU in 150 μl of GHBSS) was added to the cell mixture. Light release was measured by using a Lumiphotometer TD-4000 (Laboscience) over a 15-min period, and the peak response (in relative light units [RLU]) was noted.

Statistical analysis.

The statistical significance was determined by using a Mann-Whitney U test, and a P value of <0.05 was regarded as significant.

RESULTS

α-GalCer treatment ameliorates murine listeriosis.

C57BL/6 mice were left untreated or were treated with α-GalCer or vehicle and then infected with L. monocytogenes. CFU in peritoneal cavities, livers, and spleens were determined at different time points thereafter. Considerable numbers of viable bacteria were detected in peritoneal cavities of vehicle-treated and untreated mice 1 h postinfection (p.i.), and these were reduced by 1 order of magnitude after α-GalCer treatment (Fig. (Fig.1).1). Viable bacteria in peritoneal cavities became undetectable (<50 CFU) at 4 h p.i. regardless of α-GalCer treatment (data not shown). High numbers of viable bacteria were detected in the livers and spleens of vehicle-treated and untreated mice on day 4 p.i., and these were reduced up to 100-fold by α-GalCer treatment (Fig. (Fig.1).1). Thus, α-GalCer treatment ameliorated listeriosis.

FIG. 1.
Influence of in vivo administration of α-GalCer on listerial growth in the peritoneal cavities, livers, and spleens of C57BL/6 mice. Mice were left untreated or were treated with α-GalCer or vehicle on day −1 and infected with ...

In vivo administration of α-GalCer enhances bactericidal and phagocytic activities of M[var phi] in an iNKT-cell-dependent manner.

M[var phi] play a central role in elimination of L. monocytogenes (56, 61). We therefore examined the influence of α-GalCer on the bactericidal activity of M[var phi]. Peritoneal M[var phi] from mice left untreated or treated with α-GalCer or vehicle were infected with L. monocytogenes, and their bactericidal activities were compared. Since it has been shown that gentamicin has the potential to kill L. monocytogenes in M[var phi] (13), we first examined whether L. monocytogenes in M[var phi] was killed by gentamicin. To address this issue, we compared numbers of viable bacteria in M[var phi] cultured in the presence or absence of gentamicin. In contrast to previous findings (13), the numbers of viable bacteria in M[var phi] were comparable between the two groups (data not shown). Consistent with this, many actin tails were found in TG-induced M[var phi] 4 h after in vitro culture in the presence of gentamicin (see Fig. Fig.3D).3D). Thus, the viability of L. monocytogenes in M[var phi] was apparently not affected by gentamicin in our experimental system. Because viable L. monocytogenes bacteria in resident M[var phi] from C57BL/6 mice were maximal at 60 min after phagocytosis and viable bacteria became virtually undetectable a further 60 min after incubation regardless of mouse strains, we determined phagocytic and bactericidal activities of M[var phi] at 60 and 30 min after infection, respectively. The data are expressed as a killing index and not as numbers of killed bacteria to standardize bactericidal activity, because it is possible that numbers of killed bacteria are higher in M[var phi] that phagocytose many bacteria than in those that phagocytose few bacteria. The bactericidal activity (for 30 min after 60 min of phagocytosis) of M[var phi] from α-GalCer-treated C57BL/6 mice was significantly higher than that from vehicle-treated and untreated C57BL/6 mice (Fig. (Fig.2A,2A, upper panel). Accordingly, M[var phi] showed morphological changes after α-GalCer treatment (see Fig. Fig.3A).3A). In contrast to C57BL/6 mice, bactericidal activities of M[var phi] from IL-15−/− and SJL mice were virtually unchanged after α-GalCer treatment (Fig. (Fig.2A,2A, upper panel). Bactericidal activities of M[var phi] from untreated C57BL/6, IL-15−/−, and SJL mice were comparable, suggesting that M[var phi] from these mouse strains have similar listericidal potentials (data not shown). Hence, α-GalCer enhanced bactericidal activity of M[var phi] in an iNKT-cell-dependent fashion.

FIG. 2.
Influence of in vivo administration of α-GalCer on listericidal and phagocytic activities of peritoneal M[var phi]. C57BL/6, IL-15−/−, and SJL mice were left untreated or were treated with α-GalCer or vehicle, and peritoneal ...
FIG. 3.
Influence of in vivo administration of α-GalCer on the phagocytic activity of resident and TG-induced peritoneal M[var phi]. (A) C57BL/6 mice were left untreated or were treated with α-GalCer or vehicle, and peritoneal M[var phi] were ...

We next examined the influence of α-GalCer on phagocytic activity of M[var phi] by counting viable bacteria (colony count method) after 60 min of phagocytosis. Considerable numbers, but low efficacy of phagocytosis (ca. 0.1%), of viable bacteria were detected in M[var phi] from untreated and vehicle-treated C57BL/6 mice, and these numbers were significantly lower in M[var phi] from α-GalCer-treated C57BL/6 mice (Fig. (Fig.2B).2B). To verify whether phagocytic activity of M[var phi] from C57BL/6 mice was indeed reduced by α-GalCer treatment, peritoneal M[var phi] from C57BL/6 mice that were left untreated or were treated with α-GalCer or vehicle were infected with L. monocytogenes, and the numbers of L. monocytogenes bacteria phagocytosed by M[var phi] were determined immunohistochemically using anti-Listeria Ab. In contrast to the colony count method, the numbers of L. monocytogenes bacteria ingested by M[var phi] from α-GalCer-treated C57BL/6 mice were markedly higher than those from vehicle-treated and untreated C57BL/6 mice (Fig. (Fig.3A3A and Table Table1).1). No alterations were found in the numbers of L. monocytogenes bacteria ingested by M[var phi] from IL-15−/− and SJL mice after α-GalCer treatment (Fig. (Fig.2B).2B). Hence, α-GalCer enhanced the phagocytic activity of M[var phi] in an iNKT-cell-dependent manner.

TABLE 1.
Numbers of L. monocytogenes bacteria ingested by peritoneal M[var phi] from C57BL/6 mice and numbers of M[var phi] infected with L. monocytogenesa

iNKT cells in organs other than peritoneal cavity participate in enhanced bactericidal activity of peritoneal M[var phi] by α-GalCer treatment.

We wondered whether enhancement of bactericidal activity of peritoneal M[var phi] by α-GalCer involved iNKT cells present in the peritoneal cavity. Total PC from C57BL/6 mice were stimulated in vitro with α-GalCer and infected with L. monocytogenes, and bactericidal activity of M[var phi] was determined. In contrast to in vivo treatment, bactericidal activity of M[var phi] was unchanged by in vitro stimulation with α-GalCer (data not shown). We therefore rule out the possibility that enhancement of bactericidal activity of peritoneal M[var phi] by α-GalCer involved iNKT cells in the peritoneal cavity. We wondered whether the lack of enhanced bactericidal activity of peritoneal M[var phi] by α-GalCer was caused by the absence of iNKT cells in the peritoneal cavity. We therefore attempted to detect iNKT cells in PC using α-GalCer/CD1d tetramers. HL were used as a positive control, because iNKT cells are most abundant in the liver compared to other organs (16). α-GalCer/CD1d tetramer-reactive T cells were undetectable in peritoneal cavities not only of IL-15−/− and SJL mice (data not shown) but also of C57BL/6 mice regardless of α-GalCer treatment (Fig. (Fig.4A),4A), although high numbers of α-GalCer/CD1d tetramer-reactive T cells were detected in the livers of untreated C57BL/6 mice, and these became undetectable after α-GalCer treatment (Fig. (Fig.4A)4A) (18, 19). To verify whether iNKT cells were indeed absent in the peritoneal cavity, Vα14 mRNA expression in PC was analyzed. As positive and negative controls, Vα14 mRNA expression of HL from C57BL/6 mice and RAG-1−/− mice lacking T cells, including iNKT cells, was also analyzed. TCRVα14 mRNA was detected in HL from C57BL/6 mice but was undetectable in PC from C57BL/6 mice regardless of α-GalCer treatment, as well as in HL from RAG-1−/− mice (Fig. (Fig.4B).4B). These results indicate that iNKT cells responsible for enhanced bactericidal activity of M[var phi] after α-GalCer treatment reside in organs other than the peritoneal cavity.

FIG. 4.
Proportions of iNKT cells in the peritoneal cavity and liver, and TCRVα14 mRNA expression in nonadherent PC and HL before and after in vivo administration of α-GalCer. Mice were left untreated or treated with α-GalCer. (A) Nonadherent ...

IFN-γ from iNKT cells participates in enhanced bactericidal activity of M[var phi] by α-GalCer treatment.

We wondered whether cytokines secreted by iNKT cells participate in enhanced bactericidal activity of M[var phi] by α-GalCer. Resident peritoneal M[var phi] from C57BL/6 mice were incubated in the presence of peritoneal fluids from C57BL/6 mice left untreated or treated with α-GalCer or vehicle, and the bactericidal activities were determined. Bactericidal activities of M[var phi] were unchanged by peritoneal fluids from vehicle-treated or untreated mice, whereas those of M[var phi] were significantly enhanced by peritoneal fluids from α-GalCer-treated mice (Fig. (Fig.5A).5A). These results suggest that cytokines enhance bactericidal activity of M[var phi] in the peritoneal cavities of α-GalCer-treated mice.

FIG. 5.
IFN-γ levels in peritoneal fluids and NO concentrations in M[var phi] culture supernatants after α-GalCer stimulation, and the influence of IFN-γ neutralization or NO inhibition on bactericidal activity of M[var phi]. (A) C57BL/6 ...

Since IFN-γ plays a central role in M[var phi] activation and this cytokine is essential in defense against L. monocytogenes (31, 35), we measured IFN-γ concentrations in peritoneal fluids. IFN-γ was undetectable in peritoneal fluids drawn from untreated C57BL/6 mice, but high levels of IFN-γ were detected in fluids from α-GalCer-treated C57BL/6 mice (Fig. (Fig.5B).5B). Although IFN-γ was detectable in peritoneal fluids from vehicle-treated mice, it was less abundant than that for α-GalCer-treated mice.

To determine whether IFN-γ is involved in enhanced bactericidal activity of M[var phi] by α-GalCer, IFN-γ was neutralized. Bactericidal activity of peritoneal M[var phi] from α-GalCer-treated C57BL/6 mice was significantly, although incompletely, reduced in the presence of anti-IFN-γ neutralizing MAb (Fig. (Fig.5D).5D). These results suggest that IFN-γ participates, at least in part, in enhanced bactericidal activity of M[var phi] after α-GalCer treatment.

NO does not play a major role in enhanced bactericidal activity of M[var phi] by α-GalCer treatment.

NO is produced by M[var phi] after L. monocytogenes infection and plays a crucial role in intracellular killing of L. monocytogenes (48, 52, 69, 71). Moreover, NO production is induced by IFN-γ (4, 7, 38, 48). We thus examined NO concentrations in peritoneal fluids. NO was undetectable in peritoneal fluids not only from vehicle-treated and untreated C57BL/6 mice but also from α-GalCer-treated C57BL/6 mice (data not shown). However, considerable levels of NO were detected in culture supernatants of peritoneal M[var phi] from α-GalCer-treated, but not untreated, C57BL/6 mice (Fig. (Fig.5C).5C). Some NO was detected in culture supernatants of peritoneal M[var phi] from vehicle-treated mice, but at lower levels than after α-GalCer-treatment.

To examine the role of NO in bactericidal activity of M[var phi] by α-GalCer, an NO inhibitor was used. Bactericidal activity of peritoneal M[var phi] from α-GalCer-treated C57BL/6 mice was not prevented by NO inhibitor, although the effect of NO inhibitor was slightly enhanced by anti-IFN-γ neutralizing MAb (Fig. (Fig.5D).5D). These results argue against a major role of NO in enhanced bactericidal activity of M[var phi] by α-GalCer treatment.

Respiratory burst within the phagosome is promoted by α-GalCer.

ROI are produced by M[var phi] in response to L. monocytogenes infection, and they play a central role in listerial killing within phagosomes (52, 53, 59). To determine the role of ROI experimentally, the respiratory bursts in M[var phi] from α-GalCer- and vehicle-treated C57BL/6 mice were compared. The maximal CL responses were significantly higher in peritoneal M[var phi] from α-GalCer-treated mice than in those from vehicle-treated mice (Table (Table2).2). The CL emission during phagocytosis by peritoneal M[var phi] from α-GalCer-treated mice reached a maximum within 160 s (see Table Table2)2) and then gradually decayed (data not shown). In contrast, the emission by peritoneal M[var phi] from vehicle-treated mice was slower (see Table Table2)2) and rapidly decayed after reaching its maximum (data not shown). Thus, α-GalCer promoted a profound respiratory burst in M[var phi] after ingestion of L. monocytogenes.

TABLE 2.
CL response to L. monocytogenes by peritoneal M[var phi] from C57BL/6 mice treated with α-GalCer or vehiclea

α-GalCer promotes killing of L. monocytogenes within the phagosomes of peritoneal M[var phi].

To evade phagosomal destruction, L. monocytogenes escapes from the phagosome into the cytosol by means of LLO (10, 22, 26, 42, 62, 65). However, in the cytosol, NO is capable of killing L. monocytogenes (48, 52, 69, 71). We therefore determined whether α-GalCer promotes the killing of L. monocytogenes after escape into the cytosol of M[var phi]. Subsequent to listerial escape from the phagosome, actin tails are formed in the cytosol of M[var phi] (12, 30, 32, 63, 74). We used immunohistochemistry, specifically fluorescein-labeled phalloidin, for detection of actin tails in peritoneal M[var phi]. In contrast to previous findings (12, 30, 32, 63, 74), the actin tail was undetectable in M[var phi] even 4 h p.i. regardless of α-GalCer treatment (Fig. (Fig.3B,3B, upper panel). This not only indicates that L. monocytogenes does not escape from the phagosome into the cytosol but also suggests that the killing of L. monocytogenes occurs in the phagosome, prior to entry into the cytosol. To decide whether α-GalCer promotes killing of L. monocytogenes within the phagosome, the Δhly L. monocytogenes strain, which has lost the capacity to escape from the phagosome into the cytosol due to the absence of LLO, was used. Bactericidal and phagocytic activities of M[var phi] from C57BL/6 mice, but not from IL-15−/− and SJL mice, were enhanced by α-GalCer treatment (Fig. (Fig.2,2, lower panel, and Fig. Fig.3B,3B, lower panel). We wondered whether our failure to obtain evidence for phagosomal escape of L. monocytogenes was related to the source of M[var phi] used in our experiments. We thus used TG-induced peritoneal M[var phi]. In contrast to resident M[var phi], the actin tail was detected in TG-induced M[var phi] infected with strain EGD but not with the Δhly strain (Fig. 3C and D). The proportion of M[var phi] with the actin tail was rare, although detectable, 1 h after infection. However, the numbers of M[var phi] with actin tails were increased thereafter, and actin tails became detectable in most of infected M[var phi] 4 h after infection (see Fig. Fig.3D).3D). Our results suggest that α-GalCer promotes the killing of L. monocytogenes within the phagosomes of M[var phi].

DISCUSSION

We show here that M[var phi] activation by iNKT cells is involved in the amelioration of listeriosis after α-GalCer intervention. Listericidal activity of peritoneal M[var phi] was enhanced by α-GalCer, at least in part, via IFN-γ secreted by iNKT cells. We conclude that effects of α-GalCer in listeriosis are influenced by enhanced bactericidal M[var phi] activities and mediated by IFN-γ from iNKT cells.

In our experiments, IL-15−/− mice were used as iNKT cell-deficient mice. Since IL-15−/− mice are devoid not only of iNKT cells but also of NK cells, we cannot formally exclude the possible contribution of NK cells to amelioration of listeriosis and to enhanced bactericidal activity of M[var phi] by α-GalCer. However, amelioration of listeriosis and enhanced bactericidal activity of M[var phi] by α-GalCer were not found in SJL mice, with a genetic background different from that of C57BL/6 mice, and amelioration of listeriosis by α-GalCer was not found in Jα18−/− mice, which harbor NK cells but not iNKT cells (19). Thus, we assume that iNKT cells are essential for amelioration of listeriosis and M[var phi] activation by α-GalCer.

Numbers of viable L. monocytogenes bacteria in M[var phi] from α-GalCer-treated C57BL/6 mice were significantly lower than those from vehicle-treated and untreated mice 60 min after infection (as determined by the colony count method). Nevertheless, by immunohistochemical analysis, considerable numbers of L. monocytogenes bacteria were detected in M[var phi] from α-GalCer-treated C57BL/6 mice, and these were markedly higher than those from vehicle-treated and untreated mice. The discrepancy is probably caused by the difference in killing activity of each M[var phi], i.e., L. monocytogenes organisms are killed by M[var phi] from α-GalCer-treated mice immediately after infection. Indeed, the numbers of viable L. monocytogenes bacteria in M[var phi] from α-GalCer-treated C57BL/6 mice were markedly higher than those from vehicle-treated and untreated mice 20 min after infection (M. Emoto et al., unpublished observations). These results indicate that α-GalCer has a great potential to ingest and kill L. monocytogenes by M[var phi] through iNKT cell activation. However, because it is possible that pinocytosis is accelerated by α-GalCer, we cannot formally exclude the possibility that this effect is influenced by gentamicin, although comparable numbers of viable L. monocytogenes bacteria were detected in M[var phi] cultured in the presence or absence of gentamicin.

Abundant numbers of iNKT cells have been detected in the peritoneal cavities of C57BL/6 mice by flow cytometry (36). In contrast, we did not detect iNKT cells in the peritoneal cavity even at mRNA levels. The discrepancy is probably caused by nonspecific staining in the study of Ito et al. (36), because cells are diagonally stained and a distinct population of NKT cells is invisible. Since (i) iNKT cells secrete IFN-γ in response to α-GalCer (6, 40), (ii) high levels of IFN-γ were detected in peritoneal fluids of α-GalCer-treated C57BL/6, but not IL-15−/− and SJL, mice, and (iii) iNKT cells were undetectable in the peritoneal cavity even at mRNA levels, we assume that IFN-γ was derived from iNKT cells residing in an organ(s) other than the peritoneal cavity. Indeed, high numbers of IFN-γ-secreting cells were detected among liver iNKT cells 1 h after α-GalCer treatment (18). Consistent with this notion, IFN-γ was detected in the peripheral blood of C57BL/6, but not IL-15−/− and SJL, mice after in vivo administration of α-GalCer (21, 43, 54; Emoto et al., unpublished). It is possible that iNKT cells infiltrate the peritoneal cavity after α-GalCer treatment. Because iNKT cells were undetectable in the peritoneal cavity after α-GalCer treatment not only at the cellular level but also at the mRNA level (this study) and because most L. monocytogenes bacteria are trapped in the liver and spleen immediately after i.p. infection (see Fig. Fig.1),1), we assume that IFN-γ detected in the peritoneal cavity is derived from other organs, such as the liver and spleen.

NO participates in the killing of L. monocytogenes in M[var phi] (48, 52, 69, 71). Consistent with this, considerable NO levels were detected in culture supernatants of peritoneal M[var phi] from α-GalCer-treated mice. However, the enhanced listericidal activity of peritoneal M[var phi] by α-GalCer was only marginally affected by NO inhibitor. At first sight, these findings seem to argue against a major role of NO in killing of L. monocytogenes. However, as shown here, the escape of L. monocytogenes from the phagosome into the cytosol was not detectable in peritoneal M[var phi]. Since (i) NO is produced in the cytosol (45, 60) and (ii) killing of L. monocytogenes in TG-induced peritoneal M[var phi] that permit escape of L. monocytogenes from the phagosome into the cytosol was inhibited by NO inhibitor (Emoto et al., unpublished), it is possible that NO plays only a minor role in prompt phagosomal killing as observed in our model.

α-GalCer promoted a profound respiratory burst in M[var phi] after ingestion of opsonized L. monocytogenes. ROI are produced by M[var phi] in response to L. monocytogenes infection, and they play a central role in listerial killing within phagosomes (52, 53, 59). We therefore assume that enhanced listerial killing in M[var phi] by α-GalCer is similarly caused by rapid and abundant production of ROI.

Further, in our hands, actin tails were undetectable in resident peritoneal M[var phi] even 4 h after L. monocytogenes infection in vitro. This finding could be taken as evidence against listerial escape from the phagosome into the cytosol (10, 22, 26, 42, 62, 65). However, actin tails were detected in TG-induced M[var phi] already 1 h p.i. in vitro. We thus consider the possibility that L. monocytogenes escapes from the phagosome in inflammatory, but not resident, peritoneal M[var phi]. Resident and inflammatory M[var phi] differ in maturation, differentiation, and activation status (14, 34, 57, 68, 72, 73, 75). Bone marrow-derived monocytes are immature compared to resident M[var phi] (8, 11, 47, 51, 77), and M[var phi] colony-stimulating factor provides an essential signal for M[var phi] maturation (8, 11, 47, 51, 77). After the i.p. administration of TG or proteose peptone, monocytes from bone marrow infiltrate the peritoneal cavity (2, 9). We assume that L. monocytogenes escapes from the phagosome into the cytosol in freshly immigrant monocytes, but not in resident peritoneal M[var phi]. Consistent with this notion, escape of L. monocytogenes from the phagosome has been described to occur in bone marrow-derived M[var phi] (32, 63). LLO is essential for the escape of L. monocytogenes from the phagosome into the cytosol (10, 22, 26, 42, 62, 65), and its activity is highly dependent on low pH (3, 24, 25, 66) and on the abundance of ROI (49, 59). Bacterial numbers ingested by M[var phi] were comparable for the hly-expressing strain L. monocytogenes EGD and for the L. monocytogenes Δhly strain (see Fig. 3B and C), indicating that phagocytosis occurred independently of LLO. The numbers of engulfed L. monocytogenes bacteria were higher in TG-induced M[var phi] than in resident peritoneal M[var phi] (see Fig. 3B and C). We assume that inflammatory M[var phi] have a higher phagocytic potential than resident peritoneal M[var phi]. Since L. monocytogenes escaped from the phagosome into the cytosol in TG-induced M[var phi] but not resident peritoneal M[var phi], we assume that killing capacities are influenced by the degree of escape from the phagosome into the cytosol. Because bacterial escape from the phagosome into the cytosol is determined by whether the actin tail can be detected or not, it would be better to verify this with a confocal microscope to obtain direct evidence.

In conclusion, we describe here profound bactericidal activities of M[var phi] after α-GalCer treatment. M[var phi] activation was mediated by IFN-γ from iNKT cells. Our findings therefore define α-GalCer as an anti-intracellular bacterial agent.

Acknowledgments

This study was supported by a Grant-in-Aid for Scientific Research (no. 17590383) from the Japan Society for the Promotion of Science; The Waksman Foundation of Japan, Inc.; the Japan Research Foundation of Clinical Pharmacology; and the German Science Foundation (SFB421).

We are grateful to M. Kronenberg and Kirin Pharma Co., Ltd., for providing mouse CD1d/β2-microglobulin-expressing baculovirus and α-GalCer, respectively. We thank Y. Shimozawa for technical assistance and M. Stäber for MAb purification. We also thank M. L. Grossman for critical reading of the manuscript.

Notes

Editor: J. B. Bliska

Footnotes

[down-pointing small open triangle]Published ahead of print on 29 March 2010.

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