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The Journal of Immunology, 2008, 180, 5558 -5568
Copyright © 2008 by The American Association of Immunologists, Inc.
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*Staphylococcal Infections

NK Cells Play a Critical Protective Role in Host Defense against Acute Extracellular Staphylococcus aureus Bacterial Infection in the Lung1

Cherrie-Lee Small*, Sarah McCormick*, Navkiran Gill*, Kapilan Kugathasan*, Michael Santosuosso*, Nickett Donaldson{dagger}, David E. Heinrichs{ddagger}, Ali Ashkar* and Zhou Xing2,*

* Department of Pathology and Molecular Medicine and Division of Infectious Diseases, Center for Gene Therapeutics, and {dagger} Department of Biology, McMaster University, Hamilton, and {ddagger} Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or {gamma}{delta} T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Staphylococcus aureus is a Gram-positive, extracellular bacterium that asymptomatically colonizes the human nasal tract, rectum, mouth, genitals, and skin. When there is a breach in these barriers, S. aureus may cause a variety of diseases including pneumonia, sepsis, and septic arthritis (1, 2). Indeed, Staphylococcal pneumonia accounts for 20–30% of nosocomial infections and remains one of the leading causes of death during influenza epidemics (2, 3). Furthermore, recent reports have indicated a high prevalence of community-acquired infections caused by multidrug-resistant strains of S. aureus in otherwise healthy individuals (3).

The cellular and molecular mechanisms of host defense against pulmonary S. aureus infection still largely remain to be understood. In general, it is believed that as is the case for other extracellular bacterial pathogens, anti-S. aureus host defense depends heavily on innate immunity. In this regard, a limited number of studies using murine models of pulmonary S. aureus infection have investigated microbial pathogenesis and host responses seen in pulmonary staphylococcal pneumonia (4, 5, 6). Staphylococcal infection in the lung of immunocompetent mice usually evokes a strong innate immune response dominated by infiltration of neutrophils and macrophages, mimicking the course of infection in humans (4, 7, 8). Within 24 h of pulmonary staphylococcal infection, alveolar macrophages and other inflammatory cells are recruited to the mucosal site and may efficiently control the infection via various mechanisms including intracellular killing of macrophages and antimicrobial peptides contained in granules of granulocytes (4, 5, 7, 8), suggesting a role for these innate cells in anti-staphylococcus host defense.

In addition to neutrophils and macrophages, however, NK cells have also been speculated to play a role in host defense against acute infections. Although conventionally NK cells are well known for their critical protective role in antiviral innate immunity (9), mounting evidence suggests that NK cells may also assist in the elimination of extracellular bacterial pathogens. Presumably, one way by which NK cells may be associated with antibacterial innate immune responses is via their surface expression of receptors for IL-12, IL-15, and IL-18, the cytokines that are produced primarily by activated phagocytic cells including macrophages. This implies that macrophage and neutrophil activation may render the activation of NK cells that will produce IFN-{gamma} or carry out cytolytic activity (10). Indeed, in vitro studies have demonstrated that upon phagocytosis of extracellular bacterial pathogens such as Escherichia coli and S. aureus, human monocytes could activate NK cells (11, 12). Furthermore, it has been shown that NK cells could be activated directly in vitro by S. aureus or staphylococcal products to produce IFN-{gamma} (12, 13, 14). Regardless of these in vitro findings, the precise role of NK cells in vivo in host defense against extracellular bacterial infection remains to be defined or is controversial. For instance, depletion of NK cells in SCID mice was found to reduce lung pathology and bacteremia following pulmonary pneumococcal infection and, furthermore, depletion of NK cells in wild-type (WT)3 mice also reduced cytokine responses, bacterial burden, and/or mortality in systemic models of bacteria-induced sepsis (15, 16, 17). These results seem to suggest that, contrary to its immune-protective role in host responses to viral infection, NK cell activation instead plays a detrimental role in host defense against extracellular bacterial infection. In contrast, there is also evidence to suggest that NK cells do not play a significant role in host defense against bacterial infection per se but do play a role in the development of arthritis following S. aureus infection (18). Thus, while the issue regarding the role of NK cells in host defense against pulmonary S. aureus infection remains to be investigated, these confounding findings suggest that NK cells may play differential functional role, depending on the nature of the extracellular bacterial pathogens and the route of infection.

IL-15, first described in 1994, has been shown to act on various cells of the immune system and to exert a spectrum of biological effects, sharing some properties of IL-2 (19, 20). However, unlike IL-2 which is produced principally by T cells, IL-15 mRNA is constitutively expressed by several cell types but its protein is produced primarily by monocytes and macrophages (21, 22). A number of studies have used IL-15R{alpha} or IL-15 gene knockout (KO) mice to study the pleiotropic effects of IL-15 on several innate immune cell types. For example, IL-15 is critically required for the development, survival, and activation of the NK type of innate cell, while it may also have an effect on other innate immune cells (21, 23, 24, 25, 26). Although IL-15 has been shown to play an important role in adaptive immunity against intracellular bacterial pathogens including Listeria monocytogenes and Mycobacterium bovis bacillus Calmette-Guérin (BCG) (27, 28, 29), its role in innate immune responses to extracellular bacterial infection, particularly pulmonary extracellular bacterial or S. aureus infection, still remains to be determined.

Our current study set out to investigate the role of NK cells and IL-15 and their relationship in host defense against acute pulmonary S. aureus infection by using WT, IL-15 KO, and NK cell-depleted murine models. Overall, our study has identified a critical role for NK cells in host defense against acute staphylococcal infection in the lung. Furthermore, we have found that IL-15 represents an important cytokine involved in this process via its indispensable effect on NK cell activities.


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

Eight- to 12-wk-old female IL-15 KO mice raised on C57BL/6 background were purchased from Taconic Farms or bred in the barrier facilities at McMaster University, Hamilton, Ontario, Canada. Age- and sex-matched WT C57BL/6 mice purchased from Harlan Laboratories were used as WT controls. IL-15 KO and WT mice were housed in specific pathogen-free facilities. All experiments were conducted in accordance with the animal ethics research board of McMaster University.

Microorganisms

A clinical isolate of the S. aureus B33349 strain was inoculated in tryptic soy broth (TSB) (Difco Laboratories) and incubated at 225 rpm for 16 h at 37°C. The bacteria were collected and resuspended in PBS. The bacterial concentration was determined as colony-forming units by plating 10-fold serial dilutions on tryptic soy agar (TSA) that was cultured for 24 h at 37°C. A virulent clinical isolate of S. aureus strain Newman expressing GFP (Staph-GFP) was used for the phagocytosis assay. Chloramphenicol (10 µg ml–1) was included in tryptic soy broth and TSA medium for the selection of Staph-GFP, and bacterial preparation was conducted as stated above (30).

Pulmonary staphylococcal infection

An experimental model of pulmonary S. aureus infection was established by using an intratracheal (i.t.) instillation procedure as previously described (31). Briefly, mice were anesthetized by inhalation of isoflurane and a small incision was made at the midline of the neck to expose the trachea. A dose between 1 x 107 and 5 x 107 CFU of S. aureus dispersed in 40 µl of PBS was injected i.t. into the lungs of mice.

Body weight and bacterial growth postinfection

The extent of sickness was determined by comparing the body weight changes at various time points postinfection with the original body weight before infection. Mice were sacrificed at 6, 20, 48, and 72 h and 7 days postinfection and the levels of bacterial burden were determined in the whole lung and spleen by plating 10-fold serial dilutions of tissue homogenates on TSA plates. After 24 h of incubation at 37°C, colonies were counted, calculated, and presented as log10 CFU per organ. Peripheral blood was collected by retroorbital bleeding and plated at 1/2 and 1/10 dilutions on TSA plates. Twenty-four hours postincubation, colonies were counted and determined as log10 CFU/ml.

Bronchoalveolar lavage (BAL) and differential cell counts

Lungs were lavaged twice with 250 and 200 µl of PBS through a polyethylene tube cannulated into the trachea as previously described (32). BAL specimens were then centrifuged and supernatants were collected and stored at –20°C. Cell pellets were resuspended in 800 µl of complete RPMI (cRPMI) medium (RPMI 1640 supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% L-glutamine). Cell counts were performed with a hemocytometer, and differential counts were determined using cytocentrifuged specimens prepared from 1 x 105 cells in 100 µl with a modified Wright-Giemsa stain (Polysciences). Three hundred to 500 cells were counted on each cytospin, which was prepared in triplicate. These cells were then classified according to standard morphologic criteria.

Cell isolation from airway lumen, lung, and spleen

Immune cells were isolated from airway lumen, lung, or spleen as previously described (32, 33). Briefly, naive noninfected or infected WT and IL-15 KO mice were sacrificed at 20 h postinfection. Spleens and lungs were removed aseptically, and airway luminal cells were removed by lavaging the lungs. Subsequent to lavage, lungs were perfused through the left ventricle with HBSS (Invitrogen Life Technologies) to remove peripheral blood cells. To obtain mononuclear cells from lung tissue, the lungs were cut into 1-mm pieces and treated with 150U/ml collagenase type I (Sigma-Aldrich), resuspended in HBSS at 200 rpm for 1 h at 37°C, and lung portions were then crushed through 40-µm basket filters and the remaining erythrocytes were lysed with ACK lysis buffer (0.15M NH4Cl, 1.0M KHCO3, 0.1 mM Na2 EDTA (pH 7.4)) and washed with PBS. Airway luminal cells were centrifuged and resuspended in cRPMI. Spleens were also processed for splenocyte isolation. All isolated cells were enumerated on a hemocytometer diluted in 0.5% trypan blue and resuspended to a given concentration in cRPMI.

Flow cytometric analysis

FACS was conducted for analysis of cell surface marker expression as previously described (33, 34, 35). Briefly, 2 x 106 splenocytes or lung cells per well and 0.5 x 106 airway luminal cells per well were plated in 96-well U-bottom plates. Cells were then washed and blocked with CD16/CD32 in 0.5% BSA/PBS for 15 min on ice and then surface stained for 30 min with the following specific Abs: PE-anti-NK1.1, (clone PK136), PE Cy7-anti-CD8a (clone Ly-2), PE-anti-{gamma}{delta} TCR (clone GL3), FITC-anti-CD11c (clone N418), allophycocyanin-Alexa Fluor 750-anti-GR1 (clone RB6-8C5), and allophycyanin-anti-CD11b (clone M1/70), all purchased from eBioscience; PE Cy5-anti-CD3 (clone 145-2C11) and PE Cy7-anti-CD11b (clone M1/70) were purchased from BD Biosciences. Stained cells were analyzed using an LSRII flow cytometer (BD Biosciences), where 250,000 events per sample were collected. The data were then analyzed with FlowJo software version 6.3.4 (Tree Star).

Cytokine analysis

Cell culture and BAL supernatants were measured using murine IL-15 ELISA as previously described (34). Levels of TNF-{alpha} were also determined using Quantikine murine kits from R&D Systems according to the manufacturer’s protocol.

Nitric oxide determination

The nitrite concentration in BAL supernatants was assayed in a 96-well microplate by adding 100 µl (1/5 dilution) of supernatants to 100 µl of Griess reagent (Sigma-Aldrich) as previously detailed (35). The absorbance (A550) was measured 10 min later, and the concentration was determined by referring to a standard curve from 1 to 20 µM sodium nitrite.

In vivo NK cell depletion

WT mice were injected i.p. with 200 µg of an monoclonal anti-NK1.1 Ab (PK136) in 500 µl of PBS daily for two days using a previously described protocol (36). We found that this protocol effectively depleted NK1.1 cells, and such depletion was sustained for at least 5 days. On the 4th day after initial treatment, mice were infected i.t. with S. aureus. The control mice were also set up with S. aureus infection. Twenty hours postinfection, the lung, spleen, and peripheral blood were collected and subjected to a CFU assay.

In vitro coculture of macrophages and NK cells

Single cell suspensions from lungs and spleens of noninfected WT mice were isolated as stated above. Lung macrophages/dendritic cells were enriched from lung mononuclear cell preparations by purification of CD11b+ leukocytes using MACS single-column purification (Miltenyi Biotech) according to the manufacturer’s protocol. NK cells were purified (85–90% purity) from whole splenocytes using a PAN-NK (CD49b+) positive selection kit from StemCell Technologies according to the manufacturer’s protocol. Purified NK cells (1 x 105) were cultured in the presence or absence of macrophages (3 x 105) with or without 4 µg/ml staphylococcal peptidoglycan (PGN) (Sigma-Aldrich) and 200 U/ml rIL-2 for 24 h at 37°C in 5% CO2 as we described previously (37).

Intracellular cytokine staining

Intracellular cytokine staining was conducted as previously described (31, 33, 35). GolgiPlug (5 µg/ml brefeldin A) (BD Biosciences) was added to all PGN-stimulated cultures either 6 h before the end of the 24-h incubation or together with PGN for a 6-h incubation. Cells were subsequently washed and blocked for 15 min with CD16/CD32 in 0.5% BSA/PBS and then surface stained with the appropriate Abs. Cells were permeabilized according to the manufacturer’s protocol (BD Biosciences) and subsequently stained with PE Cy7-anti-TNF-{alpha} (clone TN3-19) or PE-anti-IL-15 for 30 min. Stained cells were washed and analyzed using an LSR II flow cytometer. The data were subsequently analyzed with FlowJo software (Tree Star).

Phagocytosis of S. aureus by macrophages determined by fluorescent microscopy and flow cytometry

Lungs from noninfected WT and IL-15 KO mice were lavaged as described above to obtain alveolar macrophages. Macrophages at 1 x 105 cells/well were allowed to adhere to glass coverslips in 6-well plates for 3 h at 37°C in 5% CO2. The cells were then washed twice with PBS to remove nonadherent cells and resuspended in 2 ml of RPMI 1640 medium supplemented with 10% FBS and 1% glutamine. Recombinant Staph-GFP was prepared as described above and resuspended to 1 x 107 cells/ml. Opsonization was performed by adding 10% naive B6 and IL-15 KO serum to bacteria for 30 min at 37°C under slow rotation. Bacteria were then washed twice with PBS to remove excess serum and added to macrophage culture (macrophages to bacteria at 1:100) for 2 h at 37°C. After incubation, internalization was stopped by placing cells on ice. Cells were washed twice with ice-cold PBS and fixed with 100% methanol for 15 min in the dark at 4°C. The cells were then washed again twice with PBS and stained in the dark with diluted Hoechst (1/200) for 10 min at room temperature. The cells were again washed with PBS and once with distilled water before the coverslips containing adherent cells were mounted onto microscope slides using Aqua Polymount (Polysciences). Slides were imaged on a Zeiss Axiovert 200 inverted fluorescent microscope using Carl Zeiss AxioVision autofocus software version 2.0 at x40 and x63 magnification. The extent of phagocytosis (phagocytic index) was also expressed as the percentage of macrophages that had engulfed Staph-GFP. All treatments were performed in triplicate. In separate experiments, alveolar macrophages at 0.2 x 105 cells/well were incubated in the presence or absence of NK cells purified from spleens of naive WT mice in a ratio of one NK to three macrophages for 24 h at 37°C in 5% CO2. Cells were then washed with ice-cold PBS and further incubated with opsonized Staph-GFP for 4 h at 37°C. Reaction was stopped by placing cells on ice and penicillin-streptomycin was added for 30 min to remove any membrane-bound bacteria. Cells were washed and fixed at room temperature. Intracellular cell fluorescence (50,000 events) was measured using flow cytometry. Macrophages were identified as CD11bhighCD11clow and NK cells as NK1.1+CD3–. The percentage of FITC-positive macrophages was used as a measure for the phagocytic activity of these cells against unstained cells.

Statistical analysis

Quantitative data were expressed as the mean ± SEM of triplicate samples or at least n = 3 and were representative of at least two separate experiments that yielded similar results. One-way ANOVA, Tukey’s test, or Fisher’s least significant difference test using SigmaStat (SPSS Science) were used to determine the significant differences of cytokine titers, CFU in organs, and total cell numbers between control and experimental or infected groups. Significant differences in percentage of body weight loss between WT and IL-15 KO mice were determined by repeated measures of one-way ANOVA and Tukey’s test using SigmaStat. Any p values of <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Enhanced NK but not NKT or other lymphocytic subset responses in the lung to S. aureus infection

Despite the medical importance of pulmonary S. aureus infection, the majority of recent experimental studies have thus far focused on systemic or peripheral S. aureus infection related to sepsis, septic arthritis, and skin infections (38, 39). To determine the innate immune responses in acute S. aureus pneumonia, we developed a murine model of pulmonary staphylococcal infection elicited by using a nonlethal dose of a clinical strain of S. aureus. We observed an early neutrophilic infiltration in the airway lumen that peaked at 20 h and became undetectable by 72 h postinfection (Fig. 1A). Increased neutrophilic responses coincided with declining numbers of macrophages at 20 and 48 h postinfection (Fig. 1B). In comparison, a low level of lymphocytic response was not evident until 72 h (Fig. 1C). To begin investigating the role of NK cells in innate immune responses to pulmonary staphylococcal infection, we examined NK cell responses both in the bronchoalveolar space and the lung interstitium of WT mice. We found that while the airway lumen of uninfected mice had few NK cells (NK1.1+CD3–), staphylococcal infection triggered a vigorous NK cell influx into the bronchoalveolar space (Fig. 2A) (p < 0.05). Furthermore, although the naive lung interstitium contained a significant number of NK cells, the number of these cells was markedly elevated upon staphylococcal infection (Fig. 2B) (p < 0.01). As there is evidence that NKT cells and {gamma}{delta} T cells may also play a role in host defense (40, 41, 42, 43) and some of these T cell subsets and conventional CD8+ T cells or their functions may be impaired in IL-15 KO mice (22), we also examined these lymphocytic subsets in the lungs of WT mice. Differently from the sharply increased NK cells in the bronchoalveolar space of infected mice, the number of NKT (NK1.1+CD3+), {gamma}{delta} ({gamma}{delta}+CD3+), and CD8+ (CD3+CD8+) T cells kept to a minimum in the bronchoalveolar space before and after infection (Fig. 2A). In a similar way, differently from the markedly increased NK cells in the lung interstitium in response to infection, the number of NKT or {gamma}{delta} T cells remained small or undetectable while the number of CD8 T cells was unaltered before and after infection (Fig. 2B), in basic agreement with the findings from a number of pulmonary infection models (42, 44, 45, 46, 47). These findings thus strongly implicate NK cells in host innate immune responses to pulmonary staphylococcal infection.


Figure 1
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  		FIGURE 1. Inflammatory cellular responses in the lung of WT mice following pulmonary S. aureus infection. C57BL/6 mice were sacrificed at various time points postinfection and their lungs were lavaged. Total inflammatory cells in BAL fluids were determined and the differential cell numbers of neutrophils (A), alveolar macrophages (MØ) (B), and lymphocytes (C) were enumerated on cytospins. Data are expressed as the mean ± SEM of 8–10 mice/time from four independent experiments.

 

Figure 2
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  		FIGURE 2. Comparison of NK cell and lymphocyte subset responses in the airway lumen and lung tissue following pulmonary S. aureus infection. C57BL/6 mice were sacrificed at 20 h postinfection and lungs were lavaged. The BAL and lung interstitial cells were subject to immunostaining and FACS. Absolute numbers of {gamma}{delta} T cells (CD3+{gamma}{delta}T+), NK cells (NK1.1+CD3–), NKT cells (NK1.1+CD3+), and CD8+ T cells (CD3+CD8+) were determined in BAL (A) and lung (B) of noninfected naive and infected WT mice. Data are expressed as the mean ± SEM of two independent experiments. *, p < 0.05; **, p < 0.01; compared with noninfected controls.

 
Increased production of IL-15 in the lung during S. aureus infection

Having demonstrated innate cellular responses to pulmonary staphylococcal infection, we examined innate cytokine responses in the lung. We first examined the level of TNF-{alpha}, which is a well-known proinflammatory cytokine with multiple functions in innate immunity including the induction of adhesion molecule expression, chemokines, and leukocyte recruitment (48). The level of TNF-{alpha} in the lung increased at 6 h and peaked at 20 h (Fig. 3A), which coincided with the peak influx of polymorphonuclear (PMN) and NK cells into the airway lumen (Figs. 1A and 2). Because we observed a marked influx or accumulation of NK cells to the airway lumen and lung (Fig. 2) and it is well known that IL-15 is a critical cytokine for NK cell chemotaxis and activation (22, 23, 49), we examined whether IL-15 was induced in the lung following staphylococcal infection. Indeed, markedly increased levels of IL-15 in the bronchoalveolar space were observed at both 20 and 48 h postinfection, which slightly lagged behind TNF-{alpha} responses (Fig. 3B). In correlation with this finding, we also investigated whether BAL-derived macrophages/monocytes expressed membrane-bound IL-15 by FACS. Twenty hours postinfection, macrophages in the BAL were stained for the surface expression of CD11b, GR1, and CD11C and subsequently permeabilized and stained for IL-15. After excluding GR1high neutrophils from analysis, CD11bhighCD11clow macrophages were found to strongly express membrane-associated IL-15 without stimulation (Fig. 3C). Stimulation by PGN further up-regulated such IL-15 expression in these cells from infected lungs, whereas the cells from noninfected lungs expressed no detectable IL-15 (p < 0.01) (Fig. 3D). This enhanced production of IL-15 both in the lung and on macrophages correlated with the marked accumulation of NK cells within the airway lumen and lung tissue seen at 20 h postinfection (Fig. 2), suggesting that IL-15 plays a role in host defense against staphylococcal infection via its promoting effect on NK cell chemotaxis and activation.


Figure 3
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  		FIGURE 3. Cytokine production in the lung or macrophages of WT mice during S. aureus infection. A and B, Levels of TNF-{alpha} (A) and IL-15 (B) were measured by ELISA in BAL fluids collected at various times postinfection. Data are expressed as the mean ± SEM of 5–10 mice per time from four independent experiments. *, p < 0.05; §, p < 0.005; compared with 0 h controls. C, In separate experiments, mice were sacrificed at 20 h postinfection, the lungs were lavaged, and BAL cells were collected. Without any stimulation the CD11bhighCD11clowGR1low BAL macrophages were immunostained for IL-15 and analyzed by FACS. Representative histogram of IL-15-positive cells is presented. D, The BAL macrophages collected from the BAL of noninfected and infected mice were stimulated with PGN and stained for IL-15 and analyzed by FACS. Data are expressed as absolute numbers per BAL sample of IL-15-positive macrophages (the mean ± SEM of two independent experiments; *, p < 0.05; compared with noninfected controls).

 
Markedly increased susceptibility of IL-15-deficient hosts to pulmonary S. aureus infection

To further investigate the role of NK cells in host defense against pulmonary staphylococcal infection, we used IL-15 KO mice. IL-15 KO mice have demonstrated deficiency in NK cells (22, 23, 50) and have been used to study NK functions during intracellular infection (27) but have not been used to study NK functions in acute extracellular bacterial infection. We found that upon pulmonary S. aureus infection, IL-15 KO mice became severely ill as indicated by their ruffled fur and significant and sustained body weight losses within 72 h in contrast to healthy looking WT mice with only mild and temporary body weight changes (p < 0.005) (Fig. 4). Associated with the severe sickness of IL-15 KO mice was a much higher and more sustained bacterial burden in their lungs at all time points examined (Fig. 5A). There was also a greater extent of bacteremia in these mice at 48 h (Fig. 5B). Thus, by 6 days postinfection when WT mice had completely cleared S. aureus infection from the lungs and spleens, IL-15 KO mice were still unable to clear infection from these organs (Fig. 5, A and C). Collectively, these results indicate that lack of IL-15 and NK cells in IL-15KO mice leads to increased susceptibility to pulmonary S. aureus infection.


Figure 4
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  		FIGURE 4. Comparison of the extent of sickness of WT and IL-15 KO mice following pulmonary S. aureus infection. Body weight was monitored at various times postinfection. Results were reported as the mean percentage of body weight loss ± SEM of 5 mice/group. §, p < 0.005; compared with WT control at the same time point.

 

Figure 5
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  		FIGURE 5. Bacterial burden in WT and IL-15 KO mice following pulmonary staphylococcal infection. Mice were sacrificed at various time postinfection and the lung (A), peripheral blood (B), and spleen (C) were taken and subjected to a bacterial CFU assay. Data are expressed as the mean ± SEM of 8–10 WT mice or four IL-15 KO mice per time and are representative of two independent experiments. *, p < 0.05; §, p < 0.005; as compared with WT controls.

 
Because IL-15 KO mice were more susceptible to S. aureus infection, we next investigated whether the absence of IL-15 in these mice had an impact on acute inflammatory responses to S. aureus infection. We examined both the cellular and cytokine responses in the lung at 20 h after S. aureus exposure. Compared with WT mice, IL-15 KO mice had more PMN and macrophages in the lung than WT controls (p < 0.05) (Table I). Of note, regardless of increased PMN and macrophage responses in the lung, the levels of TNF-{alpha} were significantly lower in the lungs of IL-15 KO mice compared with those in WT mice (p < 0.05) (Fig. 6A). However, consistent with the unimpaired neutrophilic and macrophage infiltration in the lung of IL-15 KO mice, the content of NO in the lung of these mice was not significantly different from that in WT counterparts (Fig. 6B). Taken together, these results suggest that because IL-15-deficient hosts had increased numbers of neutrophils and macrophages in the lung, the increased susceptibility of these mice to pulmonary staphylococcal infection is unlikely to be accounted for by the impairment of responses of these innate immune cells but rather is likely due to the lack of NK cells in these mice.


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  		Table I. Neutrophil and macrophage responses in the lung of WT and IL-15 KO micea

 

Figure 6
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  		FIGURE 6. TNF-{alpha} and NO production in the lungs of WT and IL-15 KO mice during pulmonary S. aureus infection. Levels of TNF-{alpha} (pg/ml) (A) and nitrite (µM) (B) in BAL fluids collected at 20 h postinfection were measured by ELISA and a chemical reaction assay, respectively. Data are expressed as the mean ± SEM of 5–10 WT mice or four IL-15 KO mice per group. *, p < 0.05; as compared with WT controls.

 
Unimpaired macrophage function in IL-15 KO mice

As there is evidence to suggest that IL-15 may also have biologic effects on other innate cells such as macrophages (24), we examined the function of lung macrophages from IL-15 KO mice. Lung tissue CD11b+ macrophages from naive IL-15 KO mice were able to produce enhanced levels of TNF-{alpha} to a similar extent as their WT counterparts upon stimulation with staphylococcal PGN in the presence or absence of IL-12 (Fig. 7A). Similar results were also obtained with alveolar macrophages (data not shown). To further examine the functionality of macrophages, we assessed the level of phagocytosis of live S. aureus by macrophages from naive IL-15 KO mice and compared it with the level from their WT counterparts by using a strain of S. aureus expressing GFP. Comparable levels of internalization of S. aureus were observed in both IL-15 KO and WT macrophages (Fig. 7, B and C). Moreover, consistent with these findings, similar levels of "bactericidality" were observed (data not shown). Together, these data suggest that the function of lung macrophages in IL-15 KO mice is not impaired in response to S. aureus infection, thus lending further support to our observation that the increased susceptibility of IL-15 KO mice to pulmonary staphylococcal infection is due to the lack of NK cells.


Figure 7
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  		FIGURE 7. Comparison of functions of lung macrophages (MØ) from WT and IL-15 KO mice. A, TNF-{alpha} production by naive CD11b+ cells isolated from the lungs of WT and IL-15 KO mice upon stimulation with staphylococcal PGN with or without IL-12. After 48 h of incubation the supernatants were collected and TNF-{alpha} was assayed by ELISA. Data are expressed as the mean ± SEM of triplicate samples. B, The ability of alveolar macrophages to phagocytose S. aureus. Alveolar macrophages (Hoechst blue) from WT and IL-15 KO mice were incubated with Staph-GFP (FITC-green) for 2 h at 37°C and fixed with 100% methanol. Representative fluorescent microscopic photographs of WT and IL-15 KO macrophages that engulfed Staph-GFP are shown at x63 original magnification. C, Index of phagocytosis was determined under a fluorescent microscope by enumerating the number of macrophages that had engulfed bacteria and is expressed as a percentage. Data are expressed as the mean ± SEM of triplicate determinations of 100 macrophages per group.

 
Marked increased susceptibility of NK cell-depleted WT mice to pulmonary S. aureus infection

Given that IL-15 KO mice lack both IL-15 and NK cells, we further addressed the requirement of NK cells for anti-staphylococcus host defense by using an NK cell depletion approach. By this approach, NK cells would be depleted by using anti-NK1.1 Abs in WT hosts where IL-15 responses are kept intact. We found that upon pulmonary staphylococcal infection of the WT mice depleted of NK cells, the level of staphylococcal infection markedly increased not only in the lung (p < 0.05, compared with NK-competent WT control) but also in the spleen (p < 0.05) (Fig. 8, A and C), although no differences were observed in the bacterial load within the blood (Fig. 8B). To verify that the depletion of NK cells in these mice had no major effect on IL-15 production, we measured the level of IL-15 in the BAL collected from the lung of these mice at 20 h postinfection. We found that NK-depleted WT mice did not have impaired IL-15 responses in the lung and, in fact, the level of IL-15 in the lungs of NK-depleted mice was slightly higher, albeit not statistically significant, than that in NK-competent WT mice (Fig. 8D). Taken together, these findings lend strong support to the finding from the IL-15 KO mouse model that NK cells play a critical role in innate immune protection against pulmonary staphylococcal infection. Furthermore, these findings also indicate that IL-15 is required for host defense primarily via its effect on NK cells, but not other innate immune cells.


Figure 8
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  		FIGURE 8. Bacterial burden and IL-15 production in WT control and WT mice depleted of NK cells following pulmonary infection with S. aureus. A–C, Twenty hours postinfection the lungs (A), peripheral blood (B), and spleens (C) were taken and subjected to a bacterial colony unit (CFU) assay. Data are expressed as the mean ± SEM of 10 WT control mice and four NK-depleted WT mice. *, p < 0.05; compared with WT controls. D, IL-15 production in the lung in pulmonary S. aureus infection. Levels of IL-15 in BAL were measured by ELISA. Data are expressed as the mean ± SEM of 4–6 mice per group.

 
Enhanced macrophage phagocytosis of S. aureus by NK cells

It is believed that NK cells may perform differential effector activities in the elimination of pulmonary pathogens (12, 51). To better understand the potential mechanism by which NK cells contribute to host defense in pulmonary staphylococcal infection, we determined whether NK cells could enhance the ability of alveolar macrophages to phagocytose live S. aureus organisms. We observed that while, as expected, NK cells had a poor ability to uptake S. aureus, alveolar macrophages actively phagocytosed S. aureus (Fig. 9, A and B). However, the presence of NK cells markedly enhanced further the ability of alveolar macrophages to phagocytose S. aureus (Fig. 9C). Altogether, these findings suggest that the cognate interaction between NK cells and lung macrophages facilitates the better control of bacterial infection by phagocytic innate cells.


Figure 9
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  		FIGURE 9. Enhanced macrophage phagocytosis of S. aureus in the presence of NK cells. Alveolar macrophages from naive C57BL/6 mice were incubated for 24 h in the presence or absence of NK cells purified from the spleens of these mice. Staph-GFP was added to macrophages and/or NK cells and phagocytosis was allowed for 4 h at 37°C. Penicillin-streptomycin was added to samples for 30 min to remove any membrane-bound bacteria. Cells were then immunostained and analyzed by FACS. Macrophages were identified as CD11bhighCD11clow cells and NK cells as NK1.1+CD3– cells. Fifty thousand events were measured to assess the percentage of NK cells (A) or macrophages (B and C) that phagocytosed Staph-GFP. The gray shading represents unstained cells as control and the solid line represents the cells positive for Staph-GFP fluorescence. Data are representative of duplicate determinations.

 
Production of proinflammatory cytokines by NK cells upon stimulation by staphylococcal products

To further understand the effector activities of NK cells in the course of pulmonary staphylococcal infection, we examined the ability of NK cells to produce proinflammatory cytokine TNF-{alpha} upon exposure to staphylococcal products in vitro and in vivo. This issue became particularly relevant to this study because we observed that S. aureus-infected, NK-deficient IL-15 KO mice had significantly reduced production of TNF-{alpha} in the lung (Fig. 6A). By using in vitro approaches, we observed that upon staphylococcal PGN stimulation, NK cells isolated from naive WT mice were activated to produce TNF-{alpha} (Fig. 10). As it has been previously shown that the cell to cell contact of human NK cells with monocytes facilitates NK activation (12), we assessed NK cell activation in a NK and lung macrophage coculture system and found that, indeed, many more NK cells cocultured with lung macrophages released TNF-{alpha} upon PGN stimulation (Fig. 10). Having observed NK cell activation by exposure to staphylococcal products in vitro, we next examined whether this would also be the case in vivo during pulmonary staphylococcal infection. Thus, at 20 h postinfection we analyzed NK cells collected from the airway lumen of WT mice and found that the majority of NK cells were TNF-{alpha} positive (Fig. 11). Collectively, these results suggest that direct exposure to S. aureus could lead to the activation of NK cells and that such activation can be further enhanced by the presence of lung macrophages. Furthermore, these findings indicate that the decreased production of TNF-{alpha} observed in the lungs of infected IL-15 KO mice (Fig. 6A) resulted from the lack of NK cells.


Figure 10
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  		FIGURE 10. TNF-{alpha} production by NK cells upon stimulation with staphylococcal PGN with or without coculture with lung macrophages. Macrophages (MØ) and NK cells were purified from whole lung and spleen of naive WT mice, respectively. NK cells were cultured in the presence or absence of macrophages with or without PGN and rIL-2. After 24-h incubation, cells were immunostained and analyzed by intracellular cytokine staining and FACS. A, Representative dot plots of NK1.1+CD3–TNF-{alpha}+ NK cells cultured with or without PGN stimulation and/or macrophages are shown. B, The frequency of NK1.1+CD3–TNF-{alpha}+ NK cells cultured with or without PGN stimulation and/or macrophages; *, p < 0.05; {ddagger}, p < 0.0005; compared with unstimulated counterparts. C, TNF-{alpha} content in culture supernatants was measured by ELISA; *, p < 0.05; compared with PGN-stimulated NK cells alone. Data in B and C are expressed as the mean ± SEM of three independent experiments.

 

Figure 11
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  		FIGURE 11. TNF-{alpha} expression by NK cells (NK1.1+CD3–) in the lung following pulmonary S. aureus infection. Mice were sacrificed at 20 h postinfection, the lungs were lavaged, and BAL cells were stimulated, immunostained, and analyzed by intracellular cytokine staining and FACS. A and B, Representative dot plots were shown in noninfected (A) and infected (B) WT mice where NK cells (NK1.1+CD3–) were analyzed for TNF-{alpha} production. C, Absolute numbers of NK1.1+CD3–TNF-{alpha}+ cells in the BAL were determined based on the percentages of TNF-{alpha}+ NK cells. Data are expressed as the mean ± SEM of three mice/group. {ddagger}, p < 0.0005; compared with noninfected controls.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Innate host resistance to pulmonary pathogens mainly requires a well and highly coordinated leukocyte and cytokine response at the mucosal surface to allow for efficient elimination of respiratory pathogens. NK cells are instrumental players in innate immunity and are well known for their roles in host defense against intracellular pathogens. However, the precise role of NK cells in host defense against extracellular bacterial infection in vivo has remained to be defined or is controversial. In the current study we sought to investigate the role of NK cells in innate immunity against acute pulmonary S. aureus infection by using several approaches including the use of WT, IL-15 KO, and NK cell-depleted mouse models. We found that NK cells, but not NKT cells or other lymphocytic subsets, were not only greatly increased in the lung but mobilized into the airway lumen in response to pulmonary staphylococcal infection. Such NK cell responses were closely associated with enhanced IL-15 production in the lung and macrophages. IL-15 gene KO mice deficient in NK cells were susceptible to staphylococcal infection despite unimpaired neutrophil and macrophage responses in the lung. In contrast, the WT hosts depleted of NK cells were also susceptible to S. aureus infection while they remained fully capable of IL-15 production. Of note, enhanced levels of infection resulting from NK depletion were observed only in the lung and spleen but not in the blood. Likewise, infected IL-15 KO mice at 20 h also demonstrated a level of bacteremia similar to that of WT mice. Thus, it is likely that NK cells may not be required for limiting pathogen dissemination into the blood from the lung, whereas they are critically required for controlling bacterial infection within the tissue. Apart from the approaches undertaken in our current study, adoptive transfer of NK cells back into IL-15 KO mice may seem to be another useful approach, but the fact that NK cells absolutely require IL-15 for survival in vivo would preclude such consideration (52, 53). Nevertheless, together our findings suggest that NK cells are critically required for protective innate immune responses to pulmonary S. aureus infection, whereas IL-15 represents an important cytokine involved in this process via its indispensable effect on NK cell activities.

In our current study we observed a significant increase in the number of NK cells at the infected site, but the NKT cell population was almost undetectable both in the airway lumen and the lungs of WT mice during pulmonary staphylococcal infection. In this regard, our observation is in agreement with those from the previous studies showing that NKT cells play a negligible role in host immune protection in a number of models of pulmonary infections by mycobacteria and Pseudomonas (44, 45, 46, 47). It has been found that NKT-deficient mice did not show any significant difference in their ability to clear Pseudomonas aeruginosa, an extracellular Gram-negative bacterium, from the lungs compared with WT mice (46). Similarly, there was a minimum of recruitment of {gamma}{delta} and conventional CD8 T cells to the airway lumen during S. aureus infection. Although conventional CD8 T cells are present in the lung interstitium, there was no increase in the number of these T cells, at least in the critical phase of host defense upon infection (up to 48 h). Altogether, our study demonstrates the most crucial role of NK cells in host defense against acute pulmonary staphylococcal infection. However, it is noteworthy that our study may not entirely exclude a role by {gamma}{delta} T cells in our model, as this subset of T cells was suggested to play a role in the regulation of host defense or inflammation in different models of pulmonary extracellular bacterial infection (42, 43).

In our study, we observed increased levels of IL-15 in the airway lumen of WT mice in response to staphylococcal infection that correlated with the influx of NK cells into the airway lumen, thus suggesting a crucial role for IL-15 in the recruitment of NK cells to the lung during S. aureus infection. This observation supports the current understanding that IL-15 is a key cytokine not only in the proliferation and survival but also in the chemotaxis of NK cells (49, 53, 54, 55). Although the regulation of IL-15 expression in our model of pulmonary staphylococcal infection remains to be fully understood, we have shown high levels of soluble IL-15 in infected lungs by using ELISA. This observation with contrasts our previous study reporting the difficulty in detecting the secreted form of IL-15 in cell culture supernatants (34). To examine the cell membrane-bound IL-15 and to address the potential cellular source of IL-15 in the lung, we investigated whether alveolar macrophages/monocytes isolated from infected lungs may produce IL-15 by FACS. It is known that IL-15 is produced in two forms: 1) the secretable form, which can be found in the Golgi, early endosomes, and the endoplasmic reticulum; and 2) the nonsecretable form, which is stored intracellularly in the nucleus and cytoplasm (56). The secretable form of IL-15 could also be readily detected in the cell membrane and up-regulated upon stimulation with IFN-{gamma}, GM-CSF, or LPS (57). In our present study, by permeabilizing the cells before staining for IL-15, we were able to detect both intracellular and membrane-associated IL-15 protein only in macrophages/monocytes isolated from infected lung but not in those from noninfected lungs. This suggests that lung macrophages represent a significant source of IL-15 during staphylococcal infection. Such macrophage-derived IL-15 could be trans-presented in complex with IL-15R{alpha} on the cell surface, thus enabling it to activate NK cells as previously reported in other studies (11, 58, 59, 60). Furthermore, we did observe that IL-15 responses lagged slightly behind TNF-{alpha} responses in the lung, thus suggesting that TNF-{alpha} may also play a role in the up-regulation of macrophage IL-15 production.

The protective role of both IL-15 and NK cells has previously been demonstrated in murine models of intracellular bacterial infections and in models of endotoxic sepsis by systemic E. coli infection (27, 61, 62, 63, 64). However, to date there has been a lack of studies examining the relationship between IL-15 and NK cells in host resistance against pulmonary extracellular bacterial infection. In our current study, we addressed this question by using both IL-15 KO and NK-depletion mouse models. Our study revealed that IL-15 KO mice suffered increased susceptibility to staphylococcal infection and there was a concomitantly decreased TNF-{alpha} response in these mice despite the significant increase in other innate leukocytes such as macrophages recruited to the airway lumen. Because our study further demonstrated an unimpaired function of IL-15–/– macrophages and the ability of NK cells to produce significant amounts of TNF-{alpha} in vitro or ex vivo in response to staphylococcal exposure, it is very likely that reduced TNF-{alpha} production in the lungs of IL-15 KO mice resulted directly from the lack of NK cells. In contrast, because mounting evidence also suggests that IL-15 may also act on other innate immune cells in addition to NK cells (24, 25), we further examined the relationship between IL-15 and NK cells and their role in anti-S. aureus host defense by depleting NK cells from WT mice before pulmonary staphylococcal infection. We found that while NK depletion did not affect IL-15 responses in the infected lungs of these mice, it led to marked increased susceptibility to pulmonary S. aureus infection similar to that observed in IL-15 KO hosts. Therefore, our findings indicate that IL-15 plays a critical role in host defense against pulmonary extracellular bacterial infection primarily via its effect on NK cells and that any potential effects of IL-15 on other innate immune cells cannot compensate for the lack of NK cells. Collectively, our current study ascribes a novel important function to NK cells as well as IL-15 in pulmonary innate immunity against acute bacterial infection and helps clarify the confusion derived from the previous studies that instead suggested a detrimental role by NK cells in host defense against acute extracellular bacterial infections (15, 16, 17). Our study also suggests that knowledge with regard to the role of NK cells in antibacterial host defense cannot be adequately extrapolated or generalized from model systems involving different pathogens, routes of infection, or organ tissues, and rather it needs to be investigated and interpreted in a pathogen- and organ-specific manner. Although the mechanisms by which NK cells contribute to anti-S. aureus host defense in the lung still remain to be completely understood at this point, it is possible that one way is via NK production of proinflammatory cytokines, such as TNF-{alpha}, and NK cytotoxicity (12). Indeed, not only did we observe decreased TNF-{alpha} production but also markedly increased numbers of PMN and macrophages in the lung of infected NK-deficient IL-15 KO mice. We further demonstrated the production of TNF-{alpha} by NK cells and enhanced NK cell TNF-{alpha} production by coincubation with lung macrophages. Furthermore, we provided the evidence that NK cells could directly increase lung macrophage phagocytosis of S. aureus. Therefore, as we have shown that lung macrophages are a significant source of IL-15, it is likely that lung macrophage-derived IL-15 is required for NK cell recruitment and activation, including TNF-{alpha} release. Activated NK cells, in turn via their cytokine production and cell-to-cell contact, modulate macrophage activities including bacterial phagocytosis (65, 66). This contention is further supported by our observation that lack of IL-15 per se has little direct effect on lung macrophage functionality.

In summary, our study has identified a critical role for NK cells in host defense against acute staphylococcal pneumonia. Given that currently there is no vaccine available for S. aureus infection, which is often clinically antibiotic-resistant, our findings suggest that future therapeutic modalities may be designed to involve the use of IL-15 or to enhance the function of NK cells.


   Acknowledgments
 
We are grateful to Dr. Juliet Daniel for allowing us access to a fluorescent microscope and Christopher Shaler for tireless help in generating quality graphics.


   Disclosures
Top
 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 study is supported by funds from the Canadian Institutes for Health Research. Back

2 Address correspondence and reprint requests to Dr. Zhou Xing, Room 4012, Michael G. DeGroote Centre for Learning and Discovery, Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, L8N 3Z5, Canada. E-mail address: xingz{at}mcmaster.ca Back

3 Abbreviations used in this paper: WT, wild type; BAL, bronchoalveolar lavage; cRPMI, complete RPMI medium; i.t., intratracheal; Staph-GFP, S. aureus strain Newman expressing GFP; KO, knockout; PGN, peptidoglycan; PMN, polymorphonuclear; TSA, tryptic soy agar. Back

Received for publication November 7, 2007. Accepted for publication February 13, 2008.


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 Materials and Methods
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