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Infect Immun. 2009 October; 77(10): 4232–4242.
Published online 2009 July 20. doi:  10.1128/IAI.00305-09
PMCID: PMC2747942

Nontypeable Haemophilus influenzae Clearance by Alveolar Macrophages Is Impaired by Exposure to Cigarette Smoke [down-pointing small open triangle]

Abstract

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-α) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-α secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-α secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

The human respiratory tract is one of the largest body surfaces in contact with the environment and, therefore, is a main entry portal for microorganisms. In healthy humans, the lungs are sterile due to the combined actions of a repertoire of defense mechanisms. The components of lung innate immunity include mechanical barriers such as the mucociliary barrier, humoral elements present in the fluid in contact with the lung epithelium such as surfactants, complement, antimicrobial peptides, lysozyme, and lactoferrin, and resident innate immunity cells such as alveolar macrophages and dendritic cells (32, 37). Alveolar macrophages are professional phagocytes and antigen-presenting cells which patrol the lungs as sentinels and are endowed with, among other things, a collection of pattern recognition receptors used to recognize microorganisms containing pathogen-associated molecular patterns. As professional phagocytes, alveolar macrophages recognize, ingest, and process foreign material using a phagolysosomal pathway and thus play an essential role in the clearance of infections (18).

Cigarette smoke is the main risk factor for the development of lung cancer, chronic obstructive pulmonary disease (COPD), and respiratory infections (26). In this context, the so-called “British hypothesis” states that recurrent bronchial infections were the reason, at least partially, that some smokers developed progressive airway obstruction and others did not (12, 13). Exposure to cigarette smoke markedly alters lung immunity by disruption of the mucociliary function, mucus hypersecretion, and disturbance of the mucosal integrity (31). Cigarette smoke also causes oxidative stress which triggers local lung inflammation by activation of epithelial cells, alveolar macrophages, neutrophils, and T lymphocytes (2). These cells secrete inflammatory cytokines, proteases, and reactive oxygen species, causing necrosis, tissue damage, and further amplification of the inflammatory response with enhanced recruitment of neutrophils into the lung. Tissue damage promotes the release of inflammatory mediators and inhibits lung tissue repair functions, further increasing the tissue damage in the lungs of smokers (35, 38, 39). It is generally accepted, although it has not been formally proven, that these alterations could allow access of microorganisms to the otherwise sterile lungs, thereby leading to microbial colonization (28-30). Supporting this hypothesis, mice exposed to cigarette smoke were impaired in the ability to clear a Pseudomonas aeruginosa infection (10). However, there is currently limited information concerning the effect of cigarette smoke at the molecular and cellular levels on the interaction between pathogens and alveolar macrophages.

Glucocorticoids are drugs that are widely used to control many inflammatory and immune diseases, including respiratory diseases. Moreover, adjunctive glucocorticoid therapy is currently being used against a variety of bacterial infections, including otitis media, and COPD (7, 21). However, despite their importance in suppressing inflammatory responses, little is known about the effects of glucocorticoids in host defense against pathogens.

Nontypeable Haemophilus influenzae (NTHI) is a frequent gram-negative asymptomatic colonizer of the upper respiratory tract in healthy humans, but it is also an opportunistic bacterial pathogen. NTHI causes invasive diseases such as meningitis and acute respiratory infections such as otitis media with effusion, sinusitis, pneumonia, and bronchitis (24). Moreover, NTHI is the pathogen isolated most frequently from lower respiratory tract secretions from patients suffering from chronic respiratory diseases such as COPD and chronic bronchitis (30). Lipooligosaccharide (LOS) is the main glycolipid on the NTHI cell surface and comprises a membrane-anchoring lipid A molecule linked to oligosaccharide chains that extend from the bacterial cell surface (27). Phosphocholine (PCho) is a substituent frequently present in NTHI LOS chain extensions (36). This modification has been shown to be a virulence factor that is involved in NTHI adhesion and invasion of the respiratory epithelium and hence promotes pathogen persistence on the mucosal surface of the respiratory tract (33, 34).

The importance of NTHI as a respiratory pathogen has been extensively demonstrated, and alveolar macrophages play an essential role in the clearance of bacterial infections. However, little is known about the interaction between NTHI and alveolar macrophages and about the influence of PCho on this interaction. It is tempting to speculate that NTHI might be able to escape alveolar macrophage-mediated killing and that PCho could play an important role in this process. In addition, given the association between cigarette smoke and respiratory infections caused by NTHI, we hypothesized that cigarette smoke could modify the characteristics of the interaction between NTHI and alveolar macrophages. In the present study, we investigated the features of the interaction between NTHI and alveolar macrophages. Furthermore, we analyzed the impact of cigarette smoke on the ability of alveolar macrophages to engulf and process this respiratory pathogen and whether glucocorticoids affect this interaction.

MATERIALS AND METHODS

Bacterial strains, culture conditions, and reagents.

NTHI strain 398 (formerly strain 157925) is a clinical isolate from a COPD patient (Hospital Son Dureta, Spain) (25); NTHI strain 375 is an otitis media isolate (5). NTHI strains were grown on chocolate agar plates (Biomeriux) or on plates containing brain heart infusion (BHI) agar supplemented with hemin (10 μg/ml) and β-nicotinamide adenine. Bacteria were grown at 37°C with 5% CO2.

An NTHI mutant strain lacking PCho was generated as follows. A 2-kb fragment containing the lic1BC genes was amplified by PCR with Taq polymerase (Promega) using NTHI strain 375 genomic DNA as the template and primers licBC-F1 (5′-GAAACCTATTGCAATCAGACAAATAAA) and licBC-R1 (5′-AACGAACCTTTGATATTCATCACGGTG) and cloned into pGEM-T Easy (Promega) to generate pGEM-T/lic1BC. The lic1BC-containing fragment was disrupted by inverse PCR with Vent polymerase (New England Biolabs) using primers licBC-F2 (5′-TAGCAGCAGGATTAGGCAGCCGATTTA) and licBC-R2 (5′-TAATAAAGTGTAACGATCACTCCTGCA), and an internal 30-bp fragment was replaced by a blunt-ended SmaI erythromycin resistance cassette from pBSLerm (33), resulting in plasmid pGEM-T/lic1BC-ermC. This plasmid was digested with NotI to obtain a 3.1-kb lic1BC-ermC-lic1BC disruption cassette that was used to transform NTHI strain 375 using MIV medium (14). Recombinants were screened by plating bacteria on supplemented BHI agar plates containing erythromycin 10 μg/ml and performing colony PCR. Loss of PCho reactivity was checked by colony blotting with monoclonal antibody TEPC-15 (Sigma).

Cell culture and bacterial infection.

MH-S (ATTC CRL-2019) murine alveolar macrophages were grown on RPMI 1640 tissue culture medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 10 mM HEPES at 37°C in a humidified atmosphere containing 5% CO2. U937 (ATTC CRL-2367) human monocytes were cultured exactly as described previously (8). For bacterial infection, cells were seeded in 24-well tissue culture plates 15 h before the experiment at a density of 7 × 105 cells per well. Stationary-phase bacteria were recovered with 1 ml phosphate-buffered saline (PBS) from a chocolate agar plate incubated at 37°C in the presence of 5% CO2 for 16 h. The optical density at 600 nm of the bacterial suspension was adjusted to 1 (approximately 109 CFU/ml). Cells were infected with 50 μl of this suspension at a multiplicity of infection (MOI) of 100:1.

For adhesion experiments, cells were infected for 1 h, washed five times with PBS, and lysed with 300 μl of PBS-0.025% saponin for 10 min at room temperature, and then serial dilutions were plated on supplemented BHI agar plates. The level of adhesion was determined as follows: percent adhesion = (output/input) × 100. For phagocytosis assays, cells were infected for 1 h and washed three times with PBS. Cells were then incubated for an additional hour with RPMI 1640 medium containing 10% FCS, 10 mM HEPES, and 300 μg/ml gentamicin in order to eliminate extracellular bacteria. Cells were then washed three times with PBS and lysed as described above. Phagocytosis data were expressed in CFU/well. For time course experiments, the culture medium was replaced 1 h after gentamicin treatment by RPMI 1640 medium containing 10% FCS, 10 mM HEPES, and 16 μg/ml gentamicin and, when required, processed as described above. In all cases, the data are the averages of at least three independent experiments. When indicated below, cells were preincubated for 1 h with methyl-β-cyclodextrin (MβCD) (1 mM) and LY294002 hydrochloride (75 μM), for 30 min with cytochalasin D (5 μg/ml), or for 2 h with dexamethasone (1 μM) before they were infected as described above. Exposure to these drugs had no effect on cell and bacterial viability under the conditions tested. All drugs were purchased from Sigma.

Purification of human alveolar macrophages.

Participants (12 people who never smoked, 16 smokers with normal lung function, and 14 COPD patients, all male subjects) were recruited consecutively from the endoscopy unit of Hospital Universitario Son Dureta, Spain (a tertiary referral hospital) from patients requiring bronchoscopy for clinical evaluation of a solitary pulmonary nodule or hemoptysis. Participants gave written consent after they were fully informed of the nature, characteristics, risks, and potential benefits of the study, which had been approved previously by the ethics committee of Hospital Son Dureta. Seven patients were being treated with inhaled steroids, but none was receiving oral steroid therapy. We excluded individuals with other chronic lung diseases (asthma, bronchiectasis, and interstitial lung diseases), atopy, or cardiac, hepatic, or renal failure. Table Table11 shows the clinical and functional data for the subjects included in the study. The cumulative smoking exposure was not significantly different between the smokers with COPD and the smokers without COPD. Lung function was normal in people who never smoked and in smokers without COPD, whereas, by definition, patients with COPD had airflow obstruction (which was moderate to severe) (Table (Table1).1). The volume of bronchoalveolar lavage fluid (BALF) recovered was significantly lower for COPD patients than for people who never smoked, but the total cell count was higher for patients with COPD and smokers with normal lung function than for people who never smoked (Table (Table1).1). The differential cell counts were not different for the different groups (Table (Table1).1). All participants underwent bronchoalveolar lavage so that we could obtain alveolar macrophages, as described elsewhere (22). Briefly, BALF samples were processed under sterile conditions within 1 h after collection and were maintained at 4°C until analysis. Cells were recovered from BALF by centrifugation (400 × g for 10 min at 4°C) and suspended at a concentration of 106 cells/ml in RPMI 1640 medium containing 10% FCS, 2 mM l-glutamine, 25 U/ml penicillin, and 25 mg/ml streptomycin. After 2 h of incubation, nonadherent cells were removed, and the remaining alveolar macrophages were consistently (98 to 100%) positive for expression of esterase, a marker of alveolar macrophages (19). Cells were recovered using a rubber policeman, the concentration was adjusted to 105 cells/ml, and the cells were incubated in 24-well plates (2 × 105 cells per well for NTHI phagocytosis assays; 6.8 × 105 cells per well for latex bead phagocytosis assays). Cell viability, as determined by trypan blue exclusion, was always greater than 90%.

TABLE 1.
Main clinical, functional, and BALF dataa

Immunofluorescence and transmission electron microscopy.

Cells were seeded on 12-mm circular coverslips in 24-well tissue culture plates. Infection was carried out as described above. Where indicated, cells were washed with PBS three times and fixed with 3.7% paraformaldehyde (PFA) in PBS (pH 7.4). For early endosome antigen 1 (EEA1) staining, cells were fixed with 2.5% PFA for 10 min at room temperature, followed by 1% PFA with 80% methanol at −20°C for 5 min.

NTHI was stained with rabbit anti-NTHI antibody obtained by repeated immunization of rabbits (Charles Rivers) with a mixture of acetone-killed NTHI strain 398, NTHI strain 375, and NTHI strain 2019 bacteria (34) diluted 1:1,000. The actin cytoskeleton was stained with rhodamine-phalloidin (Invitrogen) diluted 1:200, early endosomes were stained with goat anti-EEA1 (N-19) antibody (Santa Cruz Biotechnology) diluted 1:50, late endosomes were stained with rat anti-Lamp-1 (1D4B) or rat anti-Lamp-2 (ABL-93) antibody (DSHB) diluted 1:150, and DNA was stained with Hoechst stain (Invitrogen) diluted 1:2,500. Donkey anti-rabbit, donkey anti-rat, and donkey anti-goat conjugated secondary antibodies were purchased from Jackson Immunological and diluted 1:200. Acidic compartments were loaded with LysoTracker Red DND-99 (0.5 μM) 45 min before fixation.

Staining was carried out in PBS containing 10% horse serum and 0.1% saponin. Coverslips were washed twice in PBS containing 0.1% saponin and once in PBS and incubated for 30 min with primary antibodies. The coverslips were then washed twice in PBS containing 0.1% saponin and once in PBS and incubated for 30 min with secondary antibodies. Finally, the coverslips were washed twice in PBS containing 0.1% saponin, once in PBS, and once in H2O, mounted with Aqua Poly/Mount (Polysciences), and analyzed with a Leica CTR6000 fluorescence microscope. Images were taken with a Leica DFC350FX camera.

For transmission electron microscopy, cells were seeded in 24-well tissue culture plates. Cells were infected as described above, fixed with glutaraldehyde, and processed for transmission electron microscopy as described previously (15).

Detection of Akt phosphorylation by Western blotting.

Cells were seeded 15 h before the experiment into six-well tissue culture plates at a concentration of 2.8 × 106 cells per well. Cells were infected as described above, washed three times with cold PBS, scraped, and lysed on ice with 100 μl of a lysis buffer, which was 1× sodium dodecyl sulfate sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% [wt/vol] sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol, 0.01% [wt/vol] bromophenol blue). Samples were sonicated, boiled at 100°C for 10 min, and cooled on ice before polyacrylamide gel electrophoresis and Western blotting were performed. Akt phosphorylation was detected with primary rabbit anti-phosphoSer473 Akt antibody (Cell Signaling Technology) diluted 1:1,000 and secondary goat anti-rabbit antibody conjugated to horseradish peroxidase (Thermo) diluted 1:50,000. Tubulin, used as a loading control, was detected with primary mouse anti-tubulin antibody (Sigma) and secondary goat anti-mouse antibody (Pierce) conjugated to horseradish peroxidase diluted 1:1,000. Images were recorded with a GeneGnome HR imaging system (Syngene) and exported to a personal computer for densitometry analysis using ImageJ software (http://rsb.info.nih.gov/ij/download.html). Bands in each lane were selected and analyzed using the Histogram analysis tool, and the mean intensity was recorded. The results were expressed as relative levels of protein (mean intensity of protein/mean intensity of tubulin × 100).

TNF-α determination.

Cells were seeded in 24-well tissue culture plates and infected as described above. When necessary, cells were treated with dexamethasone and/or exposed to 10% cigarette smoke extract (CSE). Supernatants were recovered every 2 h from the beginning of the infection. The amounts of tumor necrosis factor alpha (TNF-α) present in supernatants of culture cells were determined by performing an enzyme-linked immunosorbent assay (Bender MedSystems) with a sensitivity of <2 pg/ml.

Production of CSE and cell exposure.

CSE was prepared from commercial cigarettes (0.8 mg of nicotine, 10 mg of tar, 10 mg of CO; Phillip Morris, Spain) as described previously (25). Briefly, one cigarette was combusted using a syringe-modified apparatus which draws the smoke into a sterile glass containing 5 ml of tissue culture medium. Sixty milliliters of smoke was drawn for 10 s following a 30-s break. This process was repeated five times per cigarette. The CSE was filtered through a 0.22-μm filter. The resulting solution was designated the 100% CSE solution and used within 30 min. Cells were exposed to 10% CSE as follows. Cells were washed once with PBS and incubated for 3 h before bacterial infection with 1 ml RPMI 1640 medium supplemented with 10% FCS and 10 mM HEPES containing 100 μl (10% CSE) or 50 μl (5% CSE) of the 100% CSE solution prepared previously, and exposure was maintained during bacterial contact. When indicated, cells were exposed to CSE before infection and removed when bacteria were added; alternatively, cells were exposed to CSE only during gentamicin treatment. When necessary, dexamethasone (1 μM) pretreatment and exposure to 10% CSE were combined. Exposure to 10% CSE was used for MH-S experiments. For U937 experiments, 5% CSE was used because it did not alter the viability of phorbol myristate acetate (PMA)-treated U937 cells, whereas 10% CSE did alter the viability (there was a 40% decrease in the number of viable cells).

Latex bead phagocytosis.

Cells were seeded in 24-well tissue culture plates as described above. Cells were incubated for 1 h with 1-μm-diameter latex beads conjugated to green fluorescent protein (GFP) (Sigma) by adding 10 or 20 beads per cell, depending on the experiment. Wells were then washed three times with PBS and incubated with RPMI 1640 medium for an additional hour. Wells were fixed with 3.7% PFA and processed for immunofluorescence analysis as described above. When required, cells were exposed for 3 h to 5 or 10% CSE before incubation with the latex beads.

Statistical methods.

Differences between experimental groups were analyzed by using a nonparametric two-sided Mann-Whitney U test or, where indicated below, by using two-way analysis of variance followed by a Bonferroni multiple-comparison test (GraphPad Software Inc.). A P value less than 0.05 was considered significant.

RESULTS

Adhesion of NTHI to alveolar macrophages and phagocytosis of NTHI by alveolar macrophages.

The ability of NTHI to adhere to the surface of alveolar macrophages was assessed by infecting MH-S mouse alveolar macrophages with NTHI clinical isolate 398 (25). Infections were carried out using bacteria grown to stationary phase and an MOI of 100:1 for 1, 2, or 3 h. Bacteria were enumerated by plating after macrophage lysis, and the highest levels of bacterial adhesion were observed after 3 h of infection (Fig. (Fig.1A).1A). Immunofluorescence microscopy showed that bacteria adhered efficiently to the host cell surface, and approximately 85% of the macrophages were infected after 1 h (Fig. (Fig.1C1C and data not shown). Bacterial adhesion to the macrophage surface was also observed by transmission electron microscopy (Fig. (Fig.1D).1D). A significant decrease in cell viability was observed after 3 h of infection; for this reason, the adhesion conditions used for all of the subsequent experiments were an MOI of 100:1 for 1 h.

FIG. 1.
Adhesion of NTHI to alveolar macrophages and phagocytosis of NTHI by alveolar macrophages. (A) NTHI strain 398 was used to infect MH-S mouse alveolar macrophages at an MOI of 100:1 for 1, 2, or 3 h. Bacterial adhesion was quantified by lysis, serial dilution, ...

Given that it has previously been shown that PCho is involved in NTHI adhesion and invasion of the respiratory epithelium (33), we asked if this LOS modification could also mediate NTHI adhesion to alveolar macrophages. Colony blot analysis using a mouse anti-PCho monoclonal antibody (TEPC-15) showed that NTHI strains 398 and 375 displayed high and medium levels of PCho expression on their surfaces, respectively (data not shown). As shown in Fig. Fig.1B,1B, NTHI strains 398 and 375 adhered to alveolar macrophages at comparable levels. Moreover, NTHI strain 375 Δlic1BC, a strain 375 isogenic mutant lacking PCho, adhered to alveolar macrophages as efficiently as wild-type strain 375 (Fig. (Fig.1B).1B). Together, our data suggested that PCho does not play a significant role in the adhesion of NTHI to alveolar macrophages.

We next assessed the molecular mechanisms used by alveolar macrophages to engulf NTHI. For this purpose, MH-S cells were infected with NTHI strain 398 for 1 h in the presence or absence of drugs which specifically inhibit host cell functions. Cytochalasin D significantly reduced the engulfment of NTHI compared to control infections, indicating that NTHI phagocytosis involved F-actin assembly (Fig. (Fig.1E).1E). MβCD, which depletes cholesterol from host cell membranes, was used to analyze the involvement of cell plasma membrane lipid rafts in NTHI phagocytosis. As shown in Fig. Fig.1E,1E, cholesterol depletion impaired NTHI engulfment by MH-S alveolar macrophages. Finally, we studied the contribution of the phosphatidylinositol 3-kinase (PI3K) signaling pathway to NTHI phagocytosis. Pretreatment of MH-S cells with LY294002, a specific inhibitor of PI3K activity, resulted in blockage of NTHI uptake (Fig. (Fig.1E).1E). Akt is a downstream effector of PI3K which is phosphorylated upon activation of the PI3K signaling cascade. Western blot analysis revealed that NTHI infection induces the phosphorylation of Akt (Fig. (Fig.1F).1F). As expected, LY294002 inhibited NTHI-dependent phosphorylation of Akt (Fig. (Fig.1F).1F). The three drugs, cytochalasin D, MβCD, and LY294002, also reduced the phagocytosis of NTHI strain 375 by MH-S cells (data not shown). In summary, these results showed that NTHI phagocytosis by alveolar macrophages is an event that depends on the host actin cytoskeleton and on the presence of cholesterol on the host cell membrane. Moreover, the PI3K-Akt host signaling pathway is activated during NTHI infection and is required for bacterial phagocytosis.

Alveolar macrophages clear NTHI infection by phagolysosomal fusion.

Time course experiments were carried out to determine the fate of intracellular NTHI. MH-S cells were infected with NTHI strain 398, and intracellular bacteria were enumerated by plating at 2-h intervals. As shown in Fig. Fig.2A,2A, the bacterial load was reduced over the time, and bacteria were almost totally cleared at 8 h after gentamicin treatment. Similar results were obtained for NTHI strain 375 (data not shown).

FIG. 2.
(A) Dynamics of NTHI clearance by MH-S alveolar macrophages. Cells were infected with NTHI strain 398 for 1 h (MOI, 100:1). Wells were washed and incubated for 1 h with medium containing 300 μg/ml gentamicin and then with medium containing 16 ...

To investigate the maturation of an NTHI-containing phagosome, we analyzed colocalization of intracellular bacteria with early and late endosomal markers using immunofluorescence microscopy. We found that intracellular NTHI strain 398 was located in subcellular compartments with early endosome features as early as 10 min postinfection, based on colocalization with EEA1. Bacteria were seen in EEA1-positive compartments up to 50 min postinfection. Labeling for the late endosome markers Lamp-1 and Lamp-2 around the bacteria was positive at 60 min postinfection and was observed until the pathogen was cleared. Bacteria were located in acidic vacuolar compartments during the entire course of their intracellular life until they were cleared by the macrophage lysosomal machinery, as shown by colocalization with the acidotropic probe LysoTracker Red DND-99, a weak base conjugated to a red fluorophore that freely permeates cell membranes and remains trapped in acidified organelles. Similar data were obtained for NTHI strain 375 (Fig. (Fig.2B2B and data not shown). We observed in these experiments that material that had a bacterial origin appeared to be incorporated into host cell subcellular compartment membranes, as shown by colocalization of Lamp-1 and NTHI labels in empty endocytic compartments (Fig. (Fig.2B,2B, bottom panel). This observation could have been a consequence of bacterial processing and material recycling by macrophage phagolysosomes. Collectively, these results showed that alveolar macrophages clear NTHI infection by intracellular processing via their phagolysosomal pathway.

Exposure to CSE impairs the capacity of alveolar macrophages to clear NTHI infection.

It is a well-established fact that smoking favors chronic colonization of the lower airways by opportunistic pathogens such as NTHI (30). Having established that alveolar macrophages clear ingested NTHI, we explored whether cigarette smoke could affect the capacity of alveolar macrophages to eliminate NTHI infections. First, we tested the effect of CSE on the adhesion of NTHI strain 398 to MH-S cells. The adhesion of NTHI to cells exposed to 10% CSE was 25% lower than the adhesion of NTHI to nonexposed cells (Fig. (Fig.3A).3A). Interestingly, CSE-treated MH-S cells phagocytosed significantly fewer NTHI bacteria than nonexposed cells (Fig. (Fig.3B).3B). A similar observation was made when MH-S cells were preexposed to CSE and a subsequent NTHI infection was performed in the absence of CSE (Fig. (Fig.3C).3C). Control experiments demonstrated that exposure of NTHI to 10% CSE for a period of time identical to the duration of the assay did not affect the viability of the bacteria (data not shown). Similarly, infection of non-CSE-exposed MH-S cells with NTHI preexposed to 10% CSE did not reveal a significant effect on the phagocytic ability of macrophages (data not shown). Moreover, 10% CSE did not affect the viability of MH-S cells because the percentages of viable cells were similar for CSE-exposed cells and nonexposed cells, as determined by measuring the release of lactate dehydrogenase into culture supernatants (data not shown). Importantly, this phagocytic defect was not observed when 1-μm-diameter GFP-latex beads were used instead of NTHI (Fig. (Fig.3D),3D), ruling out the possibility that CSE could trigger a general phagocytic defect. We then investigated the molecular mechanism behind the phagocytic defect caused by CSE. We had previously shown that activation of PI3K-P-Akt signaling is required for NTHI phagocytosis by MH-S cells (Fig. (Fig.1F).1F). Therefore, we examined whether CSE could affect the activation of this signaling cascade after bacterial infection by using phosphorylation of Akt as the readout. Western blot analysis showed that the levels of NTHI-induced phosphorylated Akt were lower in CSE-treated cells than in nontreated cells (Fig. (Fig.3E).3E). It is noteworthy that bacteria ingested by MH-S cells preexposed to CSE were cleared by intracellular processing via the macrophage phagolysosomal pathway in a manner similar to the manner observed for nontreated cells (see Fig. S1 in the supplemental material).

FIG. 3.
(A) Adhesion of NTHI to MH-S alveolar macrophages exposed to 10% CSE. NTHI strain 398 was used to infect MH-S mouse alveolar macrophages. Bacterial adhesion was quantified as described in the text. The results are expressed as percentages, and ...

We next assessed the impact of CSE on the fate of intracellular NTHI by exposing infected cells to 10% CSE during gentamicin treatment. The bacterial clearance after 4 h of gentamicin treatment was three times higher for cells exposed to CSE than for nontreated cells, suggesting that maturation of NTHI-containing phagolysosomes occurs faster upon exposure to CSE (Fig. (Fig.3F3F).

Glucocorticoids do not restore the capacity of CSE-treated alveolar macrophages to clear NTHI infection.

Glucocorticoids are used as standard therapy for patients suffering from smoking-related diseases due to their efficient control of inflammatory disorders caused by smoking (1, 3). However, our knowledge about the effect of glucocorticoids on the host-pathogen interaction is limited. Treatment of alveolar macrophages with dexamethasone, a synthetic glucocorticoid hormone, did not affect NTHI phagocytosis (Fig. (Fig.3B),3B), and addition of dexamethasone during gentamicin treatment did not have a significant impact on the ability of macrophages to clear an NTHI infection (Fig. (Fig.3F).3F). We next asked whether dexamethasone could restore NTHI phagocytosis to CSE-treated MH-S cells. As shown in Fig. Fig.3B,3B, dexamethasone treatment did not restore the phagocytic ability of CSE-treated cells. Moreover, addition of dexamethasone to CSE-exposed cells during gentamicin treatment did not have an effect on the macrophage-NTHI interaction (Fig. (Fig.3F).3F). Together, these results showed that glucocorticoids do not counteract the deleterious effect of CSE on the ability of alveolar macrophages to engulf NTHI.

Alveolar macrophage inflammatory and phagocytic responses during NTHI infection are uncoupled events.

Upon activation, alveolar macrophages secrete inflammatory cytokines such as TNF-α and interleukin-1, promoting early local inflammation in response to bacterial infections (18, 32). We sought to determine whether CSE could also impair the secretion of cytokines by alveolar macrophages upon challenge with NTHI. To this end, we measured the levels of TNF-α secreted by alveolar macrophages at different time points after infection with NTHI strain 398 in the presence or absence of 10% CSE. NTHI triggered TNF-α secretion by MH-S cells, which reached a plateau at 4 h postinfection (Fig. (Fig.4).4). NTHI also induced the secretion of TNF-α by MH-S cells treated with 10% CSE, although the plateau levels reached were significantly lower than those for infected cells not exposed to CSE. Ten percent CSE did not induce secretion of TNF-α by alveolar macrophages (data not shown). As expected, dexamethasone significantly reduced the levels of TNF-α secreted by NTHI-infected cells to the levels of noninfected cells, whether the cells were exposed to 10% CSE or not (Fig. (Fig.4).4). Given that CSE exposure reduces the ability of macrophages to engulf NTHI, it can be concluded that inflammation and phagocytosis are uncoupled events during infection of alveolar macrophages by NTHI.

FIG. 4.
Quantification by an enzyme-linked immunosorbent assay of TNF-α secreted into the supernatant by MH-S alveolar macrophages after infection with NTHI strain 398 (•), by NTHI-infected MH-S cells exposed to 10% CSE ([filled lozenge]), by ...

Exposure to CSE impairs the ability of PMA-treated U937 cells to phagocytose NTHI.

We next asked if the observations made with mouse alveolar macrophages could be extended to human cells. To this end, we used PMA-treated U937 cells as a model of human alveolar macrophages (8). Macrophage-like U937 cells phagocytosed NTHI strain 398 efficiently. However, exposure to 5% CSE dramatically reduced the ability of the cells to ingest NTHI (Fig. (Fig.5A).5A). U937 cells treated with 5% CSE engulfed numbers of latex beads similar to the numbers of beads engulfed by nonexposed U937 cells (Fig. (Fig.5B),5B), suggesting that CSE does not cause a general phagocytic defect. Similar to the results for MH-S cells, treatment with dexamethasone did not restore the ability of CSE-exposed macrophage-like U937 cells to phagocytose NTHI (Fig. (Fig.5A5A).

FIG. 5.
(A) Phagocytosis of NTHI by U937 macrophages exposed to 5% CSE. NTHI strain 398 was used to infect macrophages. Wells were washed and incubated with medium containing gentamicin for 1 h. Bacterial uptake was quantified by lysis and serial dilution ...

Alveolar macrophages from smokers and COPD patients have an impaired ability to engulf NTHI.

In order to assess if the deleterious effect of CSE on the phagocytic ability of alveolar macrophages occurs in vivo, we tested the abilities of alveolar macrophages isolated from the lungs of COPD patients, smokers, and people who never smoked to ingest NTHI. Figure Figure6A6A shows that alveolar macrophages obtained from smokers and COPD patients engulfed NTHI strain 398 at comparable levels, and in both cases the levels were significantly lower than the levels observed for alveolar macrophages from people who never smoked. In contrast, alveolar macrophages obtained from people who never smoked, smokers, and COPD patients engulfed similar numbers of latex beads (Fig. (Fig.6B),6B), suggesting that smoke does not cause a general phagocytic defect. We then asked whether the activation of PI3K-P-Akt signaling after bacterial infection is affected in alveolar macrophages from smokers and COPD patients. Western blot analysis showed that the levels of NTHI-induced phosphorylated Akt were lower in alveolar macrophages obtained from smokers and COPD patients than in alveolar macrophages obtained from people who never smoked (Fig. (Fig.6C6C).

FIG. 6.
Phagocytosis of NTHI by human alveolar macrophages. Human alveolar macrophages obtained by bronchoalveolar lavage from people who never smoked (control; n = 8), smokers (n = 12), and COPD patients (n = 10) were seeded on tissue ...

Together, these data support our hypothesis that continuous exposure to cigarette smoke impairs the phagocytic ability of professional phagocytes in the lung.

DISCUSSION

In this study we examined the features and dynamics of infection of alveolar macrophages by NTHI, a common respiratory pathogen. We demonstrated that bacterial uptake requires host cell actin polymerization, integrity of plasma membrane lipid rafts, and activation of the PI3K signaling cascade. Our results revealed that alveolar macrophages clear NTHI infection by maturation of the NTHI-containing compartment via the phagolysosomal route by subsequent acquisition of early and late endosomal markers in an acidified subcellular environment. Parallel to phagolysosomal maturation, alveolar macrophages secrete TNF-α upon NTHI infection. These conclusions were reached by using murine alveolar macrophages (MH-S cell line).

Two previous studies independently assessed the interaction between NTHI and professional phagocytes. One study analyzed whether J774 mouse macrophage-like cells cleared a collection of otitis media NTHI clinical isolates and found that NTHI could be recovered from macrophages 24 to 72 h after phagocytosis (9). The second study showed that NTHI was ingested by neutrophils, which activated a respiratory burst and interleukin-8 secretion; however, rather than kill the bacteria, the neutrophils themselves were killed by necrosis (20). To the best of our knowledge, this is the first study where alveolar macrophages were used to address NTHI interactions. Our observations indicate that in the absence of external stresses, alveolar macrophages clear an NTHI infection, which could contribute to the maintenance of lung sterility in healthy individuals. We also found that PCho, a LOS substituent shown to play a relevant role in different aspects of NTHI pathogenicity, such as respiratory epithelium colonization and resistance to killing by antimicrobial peptides (17, 33), does not seem to play a major role in the interaction between NTHI and alveolar macrophages under the experimental conditions used in this study. Thus, we could not detect significant differences in the adhesion of NTHI strains expressing different levels of PCho or no PCho at the bacterial surface.

Another important finding of this study is that exposure of alveolar macrophages to CSE impaired the engulfment of NTHI. However, similar to the results for macrophages not exposed to CSE, bacteria ingested by CSE-pretreated cells were eliminated by phagolysosomal fusion. It has been demonstrated in a number of studies that CSE alters lung immunity, and its effects include disruption of the mucociliary function, mucus hypersecretion, and disturbance of the mucosal integrity (35, 38, 39). Although the hypothesis has not been formally proven, it is generally accepted that these changes should lead to an increased risk of infection, which is supported by epidemiological studies. A recent study showed that mice exposed to cigarette smoke displayed delayed bacterial clearance, increased inflammation, and increased morbidity (10). Thus, Drannik and coworkers suggested that the impaired bacterial clearance by animals exposed to smoke was not due to defects in the phagocytic ability of alveolar macrophages, because alveolar macrophages from animals exposed to smoke engulfed latex beads as efficiently as cells from mice not exposed to smoke (10). In our work, we made similar observations, but importantly, we demonstrated that CSE-treated alveolar macrophages indeed have a reduced ability to engulf bacteria compared to nontreated cells. This highlights the fact that care should be taken when conclusions about host-pathogen interactions are drawn using data obtained with inert particles.

Mechanistically, we observed that exposure to CSE reduced Akt phosphorylation levels after NTHI infection and, subsequently, PI3K signaling. Considering that blockage of PI3K activation by a specific inhibitor reduced NTHI phagocytosis, our data suggest that PI3K blockage by CSE could explain the decrease in bacterial phagocytosis. However, we cannot exclude the possibility that CSE may affect additional host signaling pathways which could modulate bacterial phagocytosis together with PI3K. Intriguingly, exposure to CSE increased bacterial killing, once bacteria had been taken up. At present, we can only speculate on a molecular explanation for this observation because the effect of cigarette smoke on the host-pathogen interaction has been poorly characterized. Given that CSE contains oxygen free radicals and has an oxidant effect (23), exposure to this agent could also amplify the alveolar macrophage oxidative burst, contributing to faster bacterial clearance. Future studies will address these hypotheses.

Although care should be taken to extrapolate findings obtained using in vitro approaches to an in vivo scenario, it is worth pointing out that in this study we obtained similar results regarding the effect of exposure to cigarette smoke on bacterial phagocytosis by using mouse alveolar macrophages and human macrophage-like cell lines and alveolar macrophages obtained from humans (ex vivo samples). It is worthwhile discussing the clinical implications of the findings obtained in this study. The fact that CSE impairs the phagocytosis of NTHI by alveolar macrophages could help the pathogen escape macrophage-mediated killing and therefore persist in the lung. In fact, we postulate that this could be one of the explanations for the association between cigarette smoking and the risk of respiratory infections. Supporting this hypothesis, we show here that alveolar macrophages from smokers, with or without airway obstruction, had an impaired ability to ingest NTHI compared to cells from people who never smoked. Interestingly, it has been reported that the negative effect of cigarette smoke on phagocytosis is restricted to the lung compartment, since the phagocytosis of NTHI by blood macrophages from patients with airway obstruction was indistinguishable from that by blood macrophages from people who never smoked (4). In this study, we explored this finding in depth, expanding the analysis to the molecular mechanisms involved in the interaction between NTHI and alveolar macrophages and to the modulation of these mechanisms by exposure to external agents.

Finally, we explored the effect of glucocorticoids, which are widely used in the treatment of smoking-related diseases, on the interaction between NTHI and alveolar macrophages. Dexamethasone did not restore CSE-induced phagocytic impairment, although, as expected, it reduced the amount of TNF-α secreted by alveolar macrophages upon NTHI infection. It is important to note that this effect could amplify airway colonization by bacterial pathogens. Evidence indicates that establishment of pneumonia is greatly facilitated by suppression of proinflammatory cytokine expression (6). Supporting this, it has recently become apparent that treatment of COPD patients with inhaled glucocorticoids is associated with an increased risk of hospitalization due to pneumonia, followed by death (11).

In conclusion, this study revealed novel effects of cigarette smoking on alveolar macrophage physiological functions which could contribute to lung bacterial colonization by opportunistic pathogens, such as NTHI.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank M. Apicella for kindly providing NTHI strain 2019 and plasmid pBSLerm and A. Jahn for kindly helping with collection of the BALF samples.

J.G. is a recipient of a Contrato de Investigador del Instituto de Salud Carlos III from Fondo de Investigaciones Sanitarias. This work was funded by grants from Fondo de Investigaciones Sanitarias (grants CP0500027 and PI061251) and from Fundación Mutua Madrileña to J.G. Centro de Investigación Biomédica en Red de Enfermedades Respiratorias is an initiative of Instituto de Salud Carlos III, Spain.

Notes

Editor: J. N. Weiser

Footnotes

[down-pointing small open triangle]Published ahead of print on 20 July 2009.

Supplemental material for this article may be found at http://iai.asm.org/.

REFERENCES

1. Barnes, P. J. 2003. Therapy of chronic obstructive pulmonary disease. Pharmacol. Ther. 97:87-94. [PubMed]
2. Barnes, P. J. 2008. Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol. 8:183-192. [PubMed]
3. Barnes, P. J., K. Ito, and I. M. Adcock. 2004. Corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase. Lancet 363:731-733. [PubMed]
4. Berenson, C. S., M. A. Garlipp, L. J. Grove, J. Maloney, and S. Sethi. 2006. Impaired phagocytosis of nontypeable Haemophilus influenzae by human alveolar macrophages in chronic obstructive pulmonary disease. J. Infect. Dis. 194:1375-1384. [PubMed]
5. Bouchet, V., D. W. Hood, J. Li, J. R. Brisson, G. A. Randle, A. Martin, Z. Li, R. Goldstein, E. K. Schweda, S. I. Pelton, J. C. Richards, and E. R. Moxon. 2003. Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media. Proc. Natl. Acad. Sci. USA 100:8898-8903. [PubMed]
6. Burleson, G. R., and F. G. Burleson. 2008. Testing human biologicals in animal host resistance models. J. Immunotoxicol. 5:23-31. [PubMed]
7. Butler, C. C., and J. H. Der Voort. 2001. Steroids for otitis media with effusion: a systematic review. Arch. Pediatr. Adolesc. Med. 155:641-647. [PubMed]
8. Cosio, B. G., L. Tsaprouni, K. Ito, E. Jazrawi, I. M. Adcock, and P. J. Barnes. 2004. Theophylline restores histone deacetylase activity and steroid responses in COPD macrophages. J. Exp. Med. 200:689-695. [PMC free article] [PubMed]
9. Craig, J. E., A. Cliffe, K. Garnett, and N. J. High. 2001. Survival of nontypeable Haemophilus influenzae in macrophages. FEMS Microbiol. Lett. 203:55-61. [PubMed]
10. Drannik, A. G., M. A. Pouladi, C. S. Robbins, S. I. Goncharova, S. Kianpour, and M. R. Stampfli. 2004. Impact of cigarette smoke on clearance and inflammation after Pseudomonas aeruginosa infection. Am. J. Respir. Crit. Care Med. 170:1164-1171. [PubMed]
11. Ernst, P., A. V. González, P. Brassard, and S. Suissa. 2007. Inhaled corticosteroid use in chronic obstructive pulmonary disease and the risk of hospitalization for pneumonia. Am. J. Respir. Crit. Care Med. 176:162-166. [PubMed]
12. Fletcher, C. M. 1959. Chronic bronchitis. Its prevalence, nature, and pathogenesis. Am. Rev. Respir. Dis. 80:483-494. [PubMed]
13. Fletcher, C. M., P. C. Elmes, A. S. Fairbairn, and C. H. Wood. 1959. The significance of respiratory symptoms and the diagnosis of chronic bronchitis in a working population. Br. Med. J. 2:257-266. [PMC free article] [PubMed]
14. Herriott, R. M., E. M. Meyer, and M. Vogt. 1970. Defined nongrowth media for stage II development of competence in Haemophilus influenzae. J. Bacteriol. 101:517-524. [PMC free article] [PubMed]
15. Kruskal, B. A., K. Sastry, A. B. Warner, C. E. Mathieu, and R. A. Ezekowitz. 1992. Phagocytic chimeric receptors require both transmembrane and cytoplasmic domains from the mannose receptor. J. Exp. Med. 176:1673-1680. [PMC free article] [PubMed]
16. Reference deleted.
17. Lysenko, E. S., J. Gould, R. Bals, J. M. Wilson, and J. N. Weiser. 2000. Bacterial phosphorylcholine decreases susceptibility to the antimicrobial peptide LL-37/hCAP18 expressed in the upper respiratory tract. Infect. Immun. 68:1664-1671. [PMC free article] [PubMed]
18. Mizgerd, J. P. 2008. Acute lower respiratory tract infection. N. Engl. J. Med. 358:716-727. [PMC free article] [PubMed]
19. Munger, J. S., G. P. Shi, E. A. Mark, D. T. Chin, C. Gerard, and H. A. Chapman. 1991. A serine esterase released by human alveolar macrophages is closely related to liver microsomal carboxylesterases. J. Biol. Chem. 266:18832-18838. [PubMed]
20. Naylor, E. J., D. Bakstad, M. Biffen, B. Thong, P. Calverley, S. Scott, C. A. Hart, R. J. Moots, and S. W. Edwards. 2007. Haemophilus influenzae induces neutrophil necrosis: a role in chronic obstructive pulmonary disease? Am. J. Respir. Cell Mol. Biol. 37:135-143. [PubMed]
21. Niewoehner, D. E., M. L. Erbland, R. H. Deupree, D. Collins, N. J. Gross, R. W. Light, P. Anderson, and N. A. Morgan. 1999. Effect of systemic glucocorticoids on exacerbations of chronic obstructive pulmonary disease. Department of Veterans Affairs Cooperative Study Group. N. Engl. J. Med. 340:1941-1947. [PubMed]
22. Pons, A. R., A. Noguera, D. Blanquer, J. Sauleda, J. Pons, and A. G. Agustí. 2005. Phenotypic characterisation of alveolar macrophages and peripheral blood monocytes in COPD. Eur. Respir. J. 25:647-652. [PubMed]
23. Rahman, I. 2003. Oxidative stress, chromatin remodeling and gene transcription in inflammation and chronic lung diseases. J. Biochem. Mol. Biol. 36:95-109. [PubMed]
24. Rao, V. K., G. P. Krasan, D. R. Hendrixson, S. Dawid, and J. W. St. Geme III. 1999. Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae. FEMS Microbiol. Rev. 23:99-129. [PubMed]
25. Regueiro, V., M. A. Campos, P. Morey, J. Sauleda, A. G. Agustí, J. Garmendia, and J. A. Bengoechea. 2009. Lipopolysaccharide-binding protein and CD14 are increased in the bronchoalveolar lavage fluid of smokers. Eur. Respir. J. 33:273-281. [PubMed]
26. Ruiz, M., S. Ewig, M. A. Marcos, J. A. Martínez, F. Arancibia, J. Mensa, and A. Torres. 1999. Etiology of community-acquired pneumonia: impact of age, comorbidity, and severity. Am. J. Respir. Crit. Care Med. 160:397-405. [PubMed]
27. Schweda, E. K., J. C. Richards, D. W. Hood, and E. R. Moxon. 2007. Expression and structural diversity of the lipopolysaccharide of Haemophilus influenzae: implication in virulence. Int. J. Med. Microbiol. 297:297-306. [PubMed]
28. Sethi, S. 2000. Bacterial infection and the pathogenesis of COPD. Chest 117:286S-291S. [PubMed]
29. Sethi, S., and T. F. Murphy. 2001. Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. Clin. Microbiol. Rev. 14:336-363. [PMC free article] [PubMed]
30. Sethi, S., and T. F. Murphy. 2008. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N. Engl. J. Med. 359:2355-2365. [PubMed]
31. Sopori, M. 2002. Effects of cigarette smoke on the immune system. Nat. Rev. Immunol. 2:372-377. [PubMed]
32. Standiford, T. J. 1997. Cytokines and pulmonary host defenses. Curr. Opin. Pulm. Med. 3:81-88. [PubMed]
33. Swords, W. E., B. A. Buscher, S. Ver, I., A. Preston, W. A. Nichols, J. N. Weiser, B. W. Gibson, and M. A. Apicella. 2000. Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor. Mol. Microbiol. 37:13-27. [PubMed]
34. Swords, W. E., M. R. Ketterer, J. Shao, C. A. Campbell, J. N. Weiser, and M. A. Apicella. 2001. Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling. Cell. Microbiol. 3:525-536. [PubMed]
35. Wang, H., X. Liu, T. Umino, C. M. Skold, Y. Zhu, T. Kohyama, J. R. Spurzem, D. J. Romberger, and S. I. Rennard. 2001. Cigarette smoke inhibits human bronchial epithelial cell repair processes. Am. J. Respir. Cell Mol. Biol. 25:772-779. [PubMed]
36. Weiser, J. N., N. Pan, K. L. McGowan, D. Musher, A. Martin, and J. Richards. 1998. Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J. Exp. Med. 187:631-640. [PMC free article] [PubMed]
37. Whitsett, J. A. 2002. Intrinsic and innate defenses in the lung: intersection of pathways regulating lung morphogenesis, host defense, and repair. J. Clin. Investig. 109:565-569. [PMC free article] [PubMed]
38. Wickenden, J. A., M. C. Clarke, A. G. Rossi, I. Rahman, S. P. Faux, K. Donaldson, and W. MacNee. 2003. Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis. Am. J. Respir. Cell Mol. Biol. 29:562-570. [PubMed]
39. Witherden, I. R., E. J. Vanden Bon, P. Goldstraw, C. Ratcliffe, U. Pastorino, and T. D. Tetley. 2004. Primary human alveolar type II epithelial cell chemokine release: effects of cigarette smoke and neutrophil elastase. Am. J. Respir. Cell Mol. Biol. 30:500-509. [PubMed]

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