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. 2009 Oct;77(10):4232-42.
doi: 10.1128/IAI.00305-09. Epub 2009 Jul 20.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Pau Martí-Lliteras  1 , Verónica Regueiro, Pau Morey, Derek W Hood, Carles Saus, Jaume Sauleda, Alvar G N Agustí, José Antonio Bengoechea, Junkal Garmendia
Affiliations

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke

Pau Martí-Lliteras et al. Infect Immun. 2009 Oct.

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-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha 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-alpha 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.

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Figures

FIG. 1.
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, and viable counting on supplemented BHI agar plates. The level of adhesion was determined as follows: percent adhesion = (output/input) × 100. (B) NTHI strains 398, 375, and 375 Δlic1BC were used to infect MH-S alveolar macrophages. Bacterial adhesion was quantified as described above. (C) Immunofluorescence microscopy showing NTHI adhesion to MH-S alveolar macrophages (arrows). NTHI was stained with rabbit anti-NTHI antibody (α-NTHi) and donkey anti-rabbit antibody conjugated to Cy2 (green). The actin cytoskeleton was stained with phalloidin-RRX (Phall-RRX) (red). (D) Transmission electron microscopy showing NTHI strain 398 adhered to the MH-S alveolar macrophage surface. (E) Phagocytosis of NTHI by MH-S alveolar macrophages. NTHI strain 398 was used to infect MH-S mouse alveolar macrophages (MOI, 100:1; 1 h). Wells were washed and incubated with medium containing gentamicin for 1 h. Bacterial uptake was quantified as described above. When required, cells were pretreated for 1 h with MβCD (1 mM) or LY294002 (75 μM) and for 30 min with cytochalasin D (Cyt D) (5 μg/ml). (F) Detection of Akt phosphorylation by Western blotting with rabbit anti-phosphoSer473 Akt antibody and goat anti-rabbit antibody conjugated to horseradish peroxidase (upper panel). Cell extracts were prepared from cells that were not infected (−) or were infected for 10, 15, 20, or 30 min with NTHI strain 398 in the absence (left lanes) or in the presence (right lanes) of LY294002, a PI3K inhibitor. Tubulin was used as a loading control (bottom panel).
FIG. 2.
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 μg/ml gentamicin for up to 8 h. Intracellular bacteria were quantified at 2-h intervals as described in the text. (B) Phagolysosomal maturation during NTHI infection of MH-S alveolar macrophages. For colocalization of NTHI and EEA1, NTHI was stained with rabbit anti-NTHI antibody and donkey anti-rabbit antibody conjugated to Cy2 (green) and EEA1 was stained with goat anti-EEA1 antibody and donkey anti-goat antibody conjugated to rhodamine (red). For colocalization of NTHI and the late endosomal markers Lamp-1 and Lamp-2, NTHI and host cell nuclei were stained with Hoechst stain (blue) and Lamp-1 and Lamp-2 were stained with rat anti-Lamp-1 or rat anti-Lamp-2 antibody and donkey anti-rat antibody conjugated to rhodamine (red). To determine the location of intracellular NTHI in acidic subcellular compartments, NTHI and host cell nuclei were stained with Hoechst stain (blue), and acidic subcellular compartments were loaded with LysoTracker Red DND-99 (red). To determine incorporation of NTHI material in host endocytic compartments, NTHI was stained with rabbit anti-NTHI antibody and donkey anti-rabbit antibody conjugated to Cy2 (green), Lamp-1 was stained with rat anti-Lamp-1 antibody and donkey anti-rat antibody conjugated to rhodamine (red), and DNA was stained with Hoechst stain (blue). Colocalization of NTHI and Lamp-1 occurred in empty compartments, where bacterial DNA could not be visualized.
FIG. 3.
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 the bacterial adhesion to nonexposed cells was defined as 100%. (B) Phagocytosis of NTHI by MH-S alveolar macrophages exposed to 10% CSE (3 h before infection) and/or 1 μM dexamethasone (Dex) (2 h before infection). NTHI strain 398 was used to infect macrophages for 1 h. Wells were washed and incubated with medium containing gentamicin for 1 h. Bacterial uptake was quantified by lysis and serial dilution. (C) Phagocytosis of NTHI by MH-S alveolar macrophages preexposed to 10% CSE. Cells were exposed to 10% CSE for 3 h and washed, and fresh medium was added. NTHI strain 398 was used to infect macrophages. Wells were washed and incubated with medium containing gentamicin for 1 h. Bacterial uptake was quantified as described above. (D) Phagocytosis of GFP-latex beads by MH-S alveolar macrophages exposed to 10% CSE 3 h before infection. One-micrometer-diameter GFP-latex beads were incubated with macrophages (20 beads/cell) for 1 h. Wells were then washed, incubated with fresh medium for 1 h, fixed with PFA, and stained with phalloidin-RRX (red) and Hoechst stain (blue). For quantification, cells which had ingested latex beads (green) were considered positive and empty cells were considered negative. Nine hundred cells from three coverslips (300 cells/coverslip) from three independent experiments were counted. The results are expressed as percentages of positive cells. (E) Detection of Akt phosphorylation (P-Akt) by Western blotting with rabbit anti-phosphoSer473 Akt antibody and goat anti-rabbit antibody conjugated to horseradish peroxidase (upper panel). Cell extracts were prepared from cells that were not infected (lane −) or from cells that were infected for 10, 15, 20, or 30 min with NTHI strain 398 (left lanes) or after 3 h of exposure to 10% CSE (right lanes). The lower panel is an immunoblot showing tubulin levels under the same conditions. The results are representative of three independent experiments. The relative ratios of P-Akt obtained after densitometry analysis of gels are indicated below the lower panel. (F) Effect of exposure to 10% CSE on NTHI clearance by alveolar macrophages. NTHI strain 398 was used to infect macrophages. Wells were washed and incubated with medium containing gentamicin with or without 10% CSE and/or 1 μM dexamethasone for 4 h. Intracellular bacteria were quantified as described above.
FIG. 4.
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 (⧫), by NTHI-infected MH-S cells treated with 1 μM dexamethasone (□), and by NTHI-infected MH-S cells simultaneously exposed to 10% CSE and dexamethasone (▵). Statistical analysis was carried out by using two-way analysis of variance followed by a Bonferroni multiple-comparison test. A P value of <0.05 was considered statistically significant. Significant differences between the amount of secreted TNF-α after NTHI infection by nontreated cells and the amount of secreted TNF-α after NTHI infection by CSE-treated cells are indicated by asterisks. Significant differences between the amount of secreted TNF-α after NTHI infection by nontreated cells and the amount of secreted TNF-α after NTHI infection by dexamethasone-treated cells are indicated by gray triangles.
FIG. 5.
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 plating. Dex, dexamethasone. (B) Phagocytosis of GFP-latex beads by U937 macrophages exposed to 5% CSE. Control cells or cells exposed to 5% CSE for 3 h were incubated for 1 h with 1-μm-diameter GFP-latex beads (green), using 20 beads per cell. Wells were washed, incubated with medium for 1 h, fixed, and stained with phalloidin-RRX (red) and Hoechst stain (blue). For quantification, cells which had ingested latex beads were considered positive and empty cells were considered negative. The distribution was determined using at least 300 cells. The results are expressed as percentages of positive cells.
FIG. 6.
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 culture plates and used to carry out phagocytosis assays in which the cells were infected with NTHI strain 398 (MOI, 100:1; 1 h). Wells were washed and incubated with fresh medium containing gentamicin for 1 h. Bacterial uptake was quantified by lysis and serial dilution plating (B) Phagocytosis of GFP-latex beads by human alveolar macrophages. Cells obtained by bronchoalveolar lavage from people who never smoked (control; n = 4), smokers (n = 4), and COPD patients (n = 4) were seeded on tissue culture plates and used to carry out phagocytosis assays in which the cells were incubated for 1 h with 1-μm-diameter GFP-latex beads (green), using 20 beads per cell. Wells were washed, incubated with medium for 1 h, fixed, and stained with phalloidin-RRX (red) and Hoechst stain (blue). For quantification, cells which had ingested latex beads were considered positive and empty cells were considered negative. Six hundred cells from two coverslips (300 cells/coverslip) were counted for each patient sample. The results are expressed as percentages of positive cells. (C) Detection of Akt phosphorylation (P-Akt) by Western blotting with rabbit anti-phosphoSer473 Akt antibody and goat anti-rabbit antibody conjugated to horseradish peroxidase (upper panel). Cells were obtained from the same BALF that were used for the experiment whose results are shown in panel B. Cell extracts were prepared from cells that were not infected or from cells that were infected for 15 or 30 min with NTHI strain 398. The lower panel is an immunoblot showing the tubulin levels under the same conditions. The relative ratios of P-Akt obtained after densitometry analysis of gels are indicated in the graph.
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