Sampling of air pollution PM.

The PM_2.5 and PM₁₀ samples used in this study were collected in April 2012 on the rooftop (about 15 m above the ground) of the National Environmental Research and Training Center (CENICA) site in Iztapalapa, Mexico City. Iztapalapa is the most populated of 16 municipalities in Mexico City and characterized by dense traffic, unregulated small industrial emissions, and elevated levels of air contamination (20). PM were sampled using continuously operating high-volume PM₁₀ and PM_2.5 samplers (GMW model 1200, volumetric flow control high-volume PM10 sampler; Sierra Andersen, Smyrna, GA, USA) with an airflow rate of 1.13 m³/min ± 10%. PM_2.5 and PM₁₀ samples were collected on 20.3- by 25.4-cm cellulose nitrate membranes (Sartorius, Goettingen, Germany) with a nominal pore size of 3 μm. Nitrocellulose membranes were removed from the samplers on alternate days, at least twice a week, and PM mechanically recovered from the membranes and pooled by month and particle size (PM_2.5 and PM₁₀). Microbiological analysis of the PM samples in the clinical microbiology laboratory at the Robert Wood Johnson University Hospital showed the presence of a variety of bacterial and fungal species, such as Bacillus, Zygomycetes, and Aspergillus species. To permit cell exposure of cultures and eliminate viable bacteria and fungi, PM were sterilized by autoclaving (121°C and 100 kPa for 20 min using a Steris SV-1262). No differences were found in the type and variety of the bacterial and fungal species contained in the PM_2.5 and PM₁₀ samples. Preliminary analyses in conjunction with Gedi Mainelis' laboratory at Rutgers University showed that the presence of the bacterial species in the PM samples derived from the sampling of ambient air and was not due to artifactual contamination of the PM in the sampling and filter removal process.

Preparation of PM suspensions.

PM_2.5 and PM₁₀ were weighed on a semimicrobalance (CPA225D; Sartorius, Bohemia, NY, USA) and placed into prewashed and baked (4 h at 200°C) glass flasks. Stock suspensions of PM_2.5 and PM₁₀ (1 mg/ml) were prepared by 5 min of sonication (model 351OR-DTH; Branson, Danbury, CT, USA) in F-12K medium enriched with 2% FBS and further diluted in complete culture medium to final concentrations of 0.1, 1, 10, and 50 μg/ml.

Preparation of M. tuberculosis for in vitro infections.

Suspensions of M. tuberculosis (avirulent strain H37Ra) were prepared in Middlebrook 7H9 broth medium supplemented with albumin-dextrose-catalase (ADC) (BD Bioscience, Franklin Lakes, NJ, USA) and 0.2% glycerol. After 21 days of incubation at 37°C on an orbital shaker, M. tuberculosis stock suspensions were harvested, aliquoted, and kept frozen at −86°C until use. The M. tuberculosis concentrations used in each of the infection experiments were confirmed from each thawed M. tuberculosis aliquot by CFU assay of serial dilutions on 7H10 solid agar plates after 21-day incubations at 37°C. Single-cell M. tuberculosis suspensions for A549 infection experiments were prepared as follows. Frozen M. tuberculosis stock suspension aliquots were thawed and centrifuged for 5 min at 6,000 × g, and the resulting M. tuberculosis pellets declumped by vortexing (5 min) with five sterile 3-mm glass beads in 1 ml of RMPI 1640 medium enriched with 10% pooled human AB serum (Gemini Bio-Products, West Sacramento, CA, USA) (19). The remaining M. tuberculosis clumps were removed with an additional centrifugation step at 350 × g for 5 min. The M. tuberculosis suspension volumes required to obtain the desired multiplicities of infection (MOI; i.e., the ratio of M. tuberculosis cells per A549 cell [0.1:1, 1:1, and 10:1]) were calculated based on the CFU numbers known to be present in the M. tuberculosis suspension supernatants. The actual CFU numbers used for in vitro infections were confirmed in each experiment.

A549 cell exposure to PM, infection with M. tuberculosis, and CFU assays.

A549 cells were exposed to either PM alone or to PM prior to infection with M. tuberculosis. For exposure to PM alone, after two washing steps with 1× phosphate-buffered saline (PBS), semiconfluent A549 cells in 12-well plates (Corning) were exposed to PM_2.5 or PM₁₀ at final concentrations of 0 (unexposed controls), 0.1, 1, 10, and 50 μg/ml in 1 ml of 2% FBS-enriched F-12K medium and incubated (37°C) for 18 h. For exposure to PM prior to infection with M. tuberculosis, semiconfluent A549 cells were exposed to PM_2.5 or PM₁₀ (0, 0.1, 1, 10, and 50 μg/ml) (37°C for 18 h), washed twice with 1× PBS, and then, in F-12K medium enriched with 2% FBS, infected with M. tuberculosis at an MOI of 10 (37°C for an additional 18 h). In detail, 2 h after the addition of M. tuberculosis suspension to the A549 cells, cells were washed twice with warm PBS to remove extracellular M. tuberculosis. The A549 monolayers were then incubated for an additional 16 h (37°C and 5% CO₂). Assessments of M. tuberculosis growth and, thus, the mycobactericidal capacity of PM-exposed A549 cells were done as described previously (21, 22). Briefly, A549 cells were washed twice with 1× PBS and then lysed with 0.1% SDS (10 min at room temperature) to release any remaining viable intracellular M. tuberculosis cells. The SDS action was neutralized by the addition of Middlebrook 7H9 broth enriched with 20% bovine serum albumin (BSA) to each well. Four serial cell lysate dilutions (1:10) were then prepared and plated in triplicate (10 μl each) onto 7H10 agar plates. The plates were incubated at 37°C for CFU assessments at 21 days using a stereomicroscope at a magnification of ×40 (Fisher Scientific).

Viability assay.

The viability of A549 cells following PM exposure and M. tuberculosis infection was assessed by an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] proliferation assay using the CellTiter 96 aqueous one solution cell kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. Briefly, A549 cells were plated in triplicate 96-well plate wells per condition, allowed to grow to semiconfluence, washed twice with 1× PBS, and exposed to PM_2.5 or PM₁₀ at concentrations of 0, 0.1, 1, 10, 50, and 100 μg/ml in 100 μl of complete culture medium. The viability of the A549 cells was determined at 24, 48, and 72 h following exposure to PM alone or at 36 h following the initial 18-h exposures to PM and subsequent 18-h infection with M. tuberculosis. After 20 μl of CellTiter 96 aqueous one solution reagent (Promega, WI, USA) was added to each well, the plates were incubated (37°C and 5% CO₂) for 1 h. The concentrations of formazan (a compound resulting from the bioreduction of tetrazolium, indicating metabolically active cells) were then measured at 490 nm with a Multiskan FC (Fisher Scientific) plate reader. The results are presented as the mean percentages from three independent experiments performed on three different days.

Preparation of mRNA and cDNA.

Total RNA was extracted from PM-exposed and M. tuberculosis-infected A549 cells, as well as unexposed or uninfected A549 cells, using an RNeasy minikit (Qiagen, Germantown, MD), and DNA was removed from the RNA by RNase-free DNase treatment (Qiagen). The concentration and purity of the RNA were determined by using a Nanodrop spectrophotometer (Nanodrop Technologies, Inc.) prior to transcription of 400 ng of total RNA into cDNA using MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA), according to the manufacturer's protocol.

qRT-PCR.

The fold changes in IL-8, MCP-1, HBD-2, and HBD-3 mRNA expression following exposure to PM_2.5 or PM₁₀, M. tuberculosis infection, or PM exposure prior to M. tuberculosis infection were quantified by quantitative real-time PCR (qRT-PCR) using SYBR green dye (Applied Biosystems, Foster City, CA). Oligonucleotide primers for the β-actin, IL-8, MCP-1, HBD-2, and HBD-3 genes (Table 1) were designed using Primer Express (Applied Biosystems) and purchased from Integrated DNA Technologies (Coralville, IA, USA). The PCR mixtures (final volume of 10 μl) contained 2 μl of template and 8 μl of the reaction mixture (SYBR green, water, and primer). Quantitative fluorogenic amplification of cDNA was performed using the ABI ViiA 7 real-time PCR system (Applied Biosystems). A two-step cycling program consisting of 1 cycle at 95°C for 10 min and 40 cycles at 95°C for 15 s, with a final step of 60°C for 1 min, was used. Primer quality was ensured by controlling primer dissociation curves. mRNA fold changes were calculated using the cycle threshold (C_T) relative quantitation method (ΔΔC_T).

ELISA.

IL-8, MCP-1, and HBD-2 concentrations were assessed by enzyme-linked immunosorbent assay (ELISA) in 96-well plates (Peprotech, Rock Hill, NJ) according to the manufacturer's instructions. Briefly, capture antibodies were diluted with 1× PBS to concentrations of 0.5 μg/ml (HBD-2 and IL-8) or 0.25 μg/ml (MCP-1) and added (100 μl/well) to 96-well plates for overnight incubation. The plates were then washed 4 times with washing buffer (300 μl/well) and incubated with blocking buffer (300 μl/well) for 1 h at room temperature. Following an additional washing step, standards or samples were added (100 μl/well) and the plates incubated for 2 h (IL-8 and MCP-1) or 2.5 h (HBD-2) and washed, detection antibodies were added and the plates incubated for 2 h (0.5 μg/ml, 100 μl/well) and washed again, and avidin peroxidase (100 μl of a 1:2,000 dilution/well) was added and the plates incubated for 30 min at room temperature. After a final washing step, the plates were incubated with ABTS [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] substrate (100 μl/well, 20 min at room temperature) and read in quadruplicate using a Fisher Scientific Multiskan FC microplate reader (405 nm, with a correction wavelength of 650 nm).

SA-β-Gal staining assay.

Cell senescence was assessed using the senescence-associated β-galactosidase (SA-β-Gal) staining kit (Cell Signaling Technology, MA, USA) according to the manufacturer's instructions. Briefly, semiconfluent A549 cells in 6-well plates in 1.5 ml of F-12K medium enriched with 2% FBS were incubated overnight and subsequently exposed to 0, 0.1, 1, 10, and 50 μg/ml of PM_2.5 or PM₁₀ for 18 h. The supernatants were then removed, the A549 cells washed twice with 1× PBS, and 1 ml of fixative solution (20% formaldehyde, 2% glutaraldehyde in PBS) added to each well (15-min incubation at room temperature) prior to two washing steps with 1× PBS. One milliliter of β-galactosidase staining solution (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside in N-N-dimethylformamide) was then added, and the cells incubated overnight at 37°C in a CO₂-free incubator with 1 ml of β-galactosidase staining solution. To assess the proportions of senescent cells, 200 cells per condition were counted under a microscope (×200 magnification) (Axiovert 40C; Carl Zeiss) and the percentage of cells stained in blue (senescent cells) assessed.

TEM of A549 cells.

To assess the cellular uptake and localization of PM and M. tuberculosis cells, A549 cells were prepared for transmission electron microscopy (TEM) as follows. A549 cells (10⁶/well in 6-well plates) were incubated overnight (in complete medium at 37°C and 5% CO₂), washed, and subsequently exposed to either PM_2.5 or PM₁₀ (10 μg/ml) for 18 h as the only treatment or were then infected with M. tuberculosis at an MOI of 10 for an additional 18 h. The A549 cells were then removed by trypsinization and fixed in 2.5% glutaraldehyde–4% paraformaldehyde in 0.1 M cacodylate (90 min at room temperature). After washing with PBS once, A549 cells were postfixed in buffered 1% osmium tetroxide, dehydrated in a graded series of acetone, and embedded in Epon resin. Fixed cells were cut into 90-nm thin sections using a Leica EM UC6 ultramicrotome. Sectioned grids were then stained with a saturated solution of uranyl acetate and lead citrate. Images were captured with an AMT XR111 digital camera (Advanced Microscopy Techniques, Woburn, MA) on a Philips CM12 transmission electron microscope.

Cytotoxic effects of PM_2.5 and PM₁₀.

A549 cells were exposed to PM_2.5 or PM₁₀ at a concentration of 0 (no PM, negative control), 0.1, 1, 10, 50, or 100 μg/ml for 24, 48, and 72 h (Fig. 1A), and their viabilities were assessed by MTS assay. Exposures of A549 cells to PM_2.5 or PM₁₀ for 24 h did not result in any significant decreases in mitochondrial activity, a measure of cellular viability. However, A549 cell viability decreased significantly upon exposure to PM_2.5 at 10, 50, or 100 μg/ml following 48- and 72-h exposures. Interestingly, the viability of A549 cells exposed to PM₁₀ was significantly reduced only after 72 h at 10, 50, or 100 μg/ml (Fig. 1A).

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FIG 1

Cytotoxic effects of PM_2.5 and PM₁₀ in A549 cells. (A) A549 cells were exposed to 0, 0.1, 1, 10, 50, or 100 μg/ml of PM_2.5 or PM₁₀ for 24, 48, and 72 h, and viabilities were determined by MTS assay. (B) Similarly, the effects on A549 cell viability of 18 h of incubation with PM_2.5 or PM₁₀ prior to a subsequent M. tuberculosis infection for 18 h were measured by MTS assay. Data represent mean results ± SDs from three independent experiments. Statistically significant differences between results for unexposed and PM-exposed cells are shown with single (P < 0.05) or double (P < 0.01) asterisks.

To assess cytotoxicity in the context of PM exposure and M. tuberculosis infection, A549 cells were exposed to PM_2.5 or PM₁₀ at 0, 0.1, 1, 10, or 50 μg/ml for 18 h, followed by M. tuberculosis infection (MOI of 10) for an additional 18 h. A549 cells incubated in complete culture medium for 36 h served as a control. The viabilities of PM- and M. tuberculosis-exposed A549 cells were compared with that of control A549 cells. No statistically significant differences were observed between cells infected with M. tuberculosis alone (MOI of 10; 0 μg/ml PM) and control cells. A549 cells exposed to PM_2.5 or PM₁₀ and subsequently infected with M. tuberculosis showed significant decreases in viability at 10 and 50 μg/ml for PM_2.5 and at 10 μg/ml for PM₁₀ compared to the viability of M. tuberculosis-infected cells with no PM (P < 0.05). PM_2.5 induced greater toxicity than PM₁₀ in cells infected with M. tuberculosis (Fig. 1B).

Effects of PM_2.5 and PM₁₀ exposure on antimicrobial peptide and chemokine expression.

To assess the effects of PM exposures on antimicrobial peptide and chemokine production, A549 cells were exposed to PM_2.5 or PM₁₀ (0.1, 1, 10, or 50 μg/ml) for 18 h or left unexposed (0 μg/ml). HBD-2, HBD-3, IL-8, and MCP-1 mRNA and protein expression levels were assessed by qRT-PCR and ELISA, respectively. The HBD-2, HBD-3, IL-8, and MCP-1 mRNA expression profiles of PM-exposed A549 cells were compared to that of unexposed control A549 cells (0 μg PM). The HBD-2 mRNA fold changes and protein production in PM_2.5-exposed cells followed bell-shaped (quantal) dose-responses (Fig. 2A and ​andB)B) (18). It is worth noting that the HBD-2 protein expression profile correlated with HBD-2 mRNA fold changes (Fig. 2A and ​andB).B). In contrast to PM_2.5 exposure, PM₁₀ exposure inhibited HBD-2 protein production in a concentration-dependent manner (Fig. 2B). PM₁₀ induced significantly increased HBD-3 mRNA expression at exposure concentrations higher than 1 μg/ml, while exposure to PM_2.5 did not result in significant changes compared to the mRNA expression in unexposed control cells (Fig. 2C). HBD-3 protein expression could not be studied as no ELISA was available.

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FIG 2

Effects of PM exposure on chemokine and antimicrobial peptide production in A549 cells. A549 cells were incubated with 0, 0.1, 1, 10, or 50 μg/ml of PM_2.5 or PM₁₀ for 18 h, and mRNA fold changes and protein production assessed by qRT-PCR and ELISA, respectively. The mRNA fold changes and protein production of HBD-2 (A and B), HBD-3 (C), IL-8 (D and E), and MCP-1 (F and G) are shown. Data represent mean results ± SDs from three independent experiments. Statistically significant differences between results for unexposed and PM-exposed cells are shown with single (P < 0.05) or double (P < 0.01) asterisks.

IL-8 (Fig. 2D and ​andE)E) mRNA fold changes and protein production were positively correlated with the concentrations of both PM_2.5 and PM₁₀ (P < 0.05). However, for MCP-1 (Fig. 2F), this correlation was only observed in mRNA fold changes and not in protein expression. These findings are consistent with other reports (25,–28) showing proinflammatory immune responses in epithelial cells exposed to PM obtained from diverse sources.

Effects of PM_2.5 and PM₁₀ exposure on M. tuberculosis-induced antimicrobial peptide and chemokine expression.

To model the effects of air pollution exposure prior to M. tuberculosis infection on HBD-2, HBD-3, IL-8, and MCP-1 production, A549 cells were exposed to PM_2.5 and PM₁₀ for 18 h and then infected with M. tuberculosis for an additional 18 h. The mRNA and protein expression levels were assessed by qRT-PCR and by ELISA of culture supernatants, respectively. The levels of HBD-2 and HBD-3 expression in response to M. tuberculosis infection were decreased in PM-exposed A549 cells compared to the levels in cells only infected with M. tuberculosis (Fig. 3A to ​toC).C). Inhibition of HBD-2 and HBD-3 expression was observed in both PM_2.5- and PM₁₀-exposed A549 cells at concentrations as low as 0.1 μg/ml (P < 0.05) (Fig. 3A to ​toCC).

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FIG 3

Effects of PM exposure on M. tuberculosis-induced chemokine and antimicrobial peptide production in A549 cells. The mRNA fold changes and protein production of A549 cells incubated with PM_2.5 and PM₁₀ at 0, 0.1, 1, 10, or 50 μg/ml for 18 h followed by 18 h of M. tuberculosis infection (MOI 10) were determined by qRT-PCR and ELISA, respectively. Graphs represent mRNA fold changes and protein production of HBD-2 (A and B), HBD-3 (C), IL-8 (D and E), and MCP-1 (F and G). Data represent mean results ± SDs from three independent experiments. Statistically significant differences between results for unexposed and PM-exposed cells are shown with single (P < 0.05) or double (P < 0.01) asterisks.

The IL-8 mRNA expression patterns were different in PM_2.5- and PM₁₀-exposed M. tuberculosis-infected A549 cells. While PM_2.5 induced IL-8 mRNA expression, PM₁₀ inhibited IL-8 mRNA expression in M. tuberculosis-infected A549 cells. Interestingly, this opposing effect seen in PM_2.5- and PM₁₀-exposed cells was not observed when culture supernatants were assessed for the corresponding protein expression. Both PM_2.5 andPM₁₀ exposures induced significantly higher levels of IL-8 protein (P < 0.01) than M. tuberculosis infection alone. The discrepancies between IL-8 protein and mRNA expression levels may be due to the short half-life of IL-8 mRNA (29) (Fig. 3D and ​andE).E). The observed reduction of IL-8 mRNA in PM₁₀-exposed cells (Fig. 3D) may be due to the induction of cellular senescence discussed below (see Fig. 6). In response to M. tuberculosis infection, both the mRNA and protein level corresponding to MCP-1 increased significantly (P < 0.05) in A549 cells exposed to PM_2.5 at concentrations of 10 and 50 μg/ml. On the other hand, a significant increase in MCP-1 mRNA expression was observed in A549 cells exposed to PM₁₀ at 50 μg/ml (Fig. 3F and ​andGG).

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FIG 6

PM induces senescence in A549 cells in a dose-dependent manner. Percentages of senescent cells were determined by SA-β-Gal staining. The proportions of SA-β-Gal-positive cells increased in a dose-dependent manner when A549 cell were exposed to 0, 0.1, 1, 10, or 50 μg/ml of PM_2.5 or PM₁₀ for 18 h. The proportions of senescent cells were higher in PM₁₀- than in PM_2.5-exposed cells. Mean results ± SD from three independent experiments are shown. Statistically significant differences between results for unexposed and PM-exposed cells are shown with single (P < 0.05) or double (P < 0.01) asterisks.

Effects of PM_2.5 and PM₁₀ on M. tuberculosis growth control in A549 cells.

HBD-2 and HBD-3, which are produced by A549 cells in response to M. tuberculosis infection, contribute to A549-mediated M. tuberculosis growth control. To examine whether the observed suppression of HBD-2 and HBD-3 production in PM-exposed A549 cells (Fig. 3A to ​toC)C) alters the intracellular growth control of M. tuberculosis, A549 cells were exposed to PM_2.5 or PM₁₀ (0.1, 1, 10, or 50 μg/ml) or left unexposed, followed by M. tuberculosis infection (MOI of 0.1, 1, and 10), and CFU assays were performed. To ensure that the growth control assay results were not affected by extracellular M. tuberculosis remaining on A549 cells prior to cell lysis and plating of the cell lysates on agar plates, multiple washing steps were performed and the wash fluids assessed for CFU content. No M. tuberculosis (CFU) was found in the wash fluids of the last three of the total of six washing steps done for each sample prior to A549 cell lysis (data not shown). The CFU counts assessed in our experiments (Fig. 4A and ​andB)B) thus represent M. tuberculosis growth/survival in the intracellular environment of A549 cells and are not falsely increased by extracellular bacteria left over from the experimental infection process. The M. tuberculosis CFU numbers were significantly higher in PM_2.5-exposed than in unexposed (0 μg/ml) M. tuberculosis-infected (MOI of 10) cells (Fig. 4A) at all PM concentrations (P < 0.05), indicating suppression of intracellular M. tuberculosis growth control upon PM_2.5 exposure. Furthermore, in cells exposed to 10 or 50 μg/ml PM_2.5 prior to M. tuberculosis infection, impairment of growth control was observed even at MOI as low as 1 and 0.1. In PM₁₀-exposed A549 cells (Fig. 4B), a PM dose-dependent decrease in M. tuberculosis growth control was observed in cells infected with an MOI of 0.1 but not in cells infected with higher doses of M. tuberculosis (MOI of 1 and 10). Although at present we cannot explain the latter observation, it could be related to reduced M. tuberculosis uptake by the cells and/or induction of cellular senescence (discussed below). Interestingly, M. tuberculosis CFU numbers were lower in A549 cells exposed to 10 or 50 μg/ml PM₁₀ (Fig. 4B) than in A 549 cells exposed to the same concentrations of PM_2.5 (Fig. 4A). Taken together, these data suggest that the differential effects of PM on M. tuberculosis growth control in A549 cells were probably related to differences in the physicochemical characteristics of the two PM fractions. As expected, the observed reduction of A549-mediated M. tuberculosis growth control appeared to be correlated with the downregulation of HBD-2 and HBD-3 expression upon exposure to PM (Fig. 3A to ​toC),C), confirming the importance of the antimicrobial peptide function for M. tuberculosis growth control by A549 cells.

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FIG 4

PM exposure decreases intracellular mycobacterial growth control. A549 cells were incubated with 0, 0.1, 1, 10, or 50 μg/ml of PM_2.5 (A) or PM₁₀ (B) for 18 h, followed by infection with M. tuberculosis at an MOI of 0.1, 1, or 10 for an additional 18 h. Recombinant human interleukin-1β (rhIL-1β) was used as a positive control for HBD-2 induction. CFU assays were performed to determine the effect of PM on the mycobacterial growth control of A549 cells. CFU numbers are shown as mean results ± SDs from three independent experiments. Statistically significant differences between results for unexposed and PM-exposed cells are shown with single (P < 0.05) or double (P < 0.01) asterisks.

Effects of PM_2.5 and PM₁₀ on A549 cell morphology.

To assess whether the exposure to PM and infection with M. tuberculosis induce morphological or ultrastructural changes and alter bacterial uptake in the A549 cells, TEM was performed (Fig. 5). We observed uptake of PM_2.5 and PM₁₀ in exposed A549 cells. PM_2.5 and PM₁₀ varied by size, density, and shape (Fig. 5F and ​andI,I, dashed arrows). PM_2.5 presents in an irregular structure, while PM₁₀ maintains a well-defined spherical structure that is attributable to PM from industrial sources (30). Interestingly, while M. tuberculosis uptake appeared to lead to endocytic vesicle formation (with a double membrane surrounding the bacteria), no vesicle formation was noted surrounding PM_2.5 or PM₁₀ (Fig. 5E, ​,F,F, ​,H,H, and ​andI),I), probably indicating differences in the mechanisms underlying cellular uptake of M. tuberculosis and PM. The M. tuberculosis uptake appeared to be greater in cells exposed to PM_2.5 than in cells exposed to PM₁₀ (data not shown), a finding that correlated with our CFU data (Fig. 4), indicating that cellular M. tuberculosis uptake may have been related to the size of the PM. In addition, A549 cells revealed major morphological abnormalities upon PM exposure, particularly following PM₁₀ exposure. The abnormalities in cell morphology appeared to be microvillus shedding and elongation and swelling of mitochondria (Fig. 5J and ​andK),K), which have been described to be indicative of cellular senescence (31,–33). These abnormalities were not observed in PM-unexposed control A549 cells (Fig. 5L) or A549 cells only infected with M. tuberculosis (Fig. 5A), suggesting that the ultrastructural changes observed here were due to the PM exposure.

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FIG 5

Transmission electron microscopy (TEM) of PM and M. tuberculosis uptake in A549 cells. (A) Overview (×3,800) of uptake of multiple M. tuberculosis cells (white arrow) by an A549 cell. (B and C) Magnifications (×10,000 [B] and ×17,000 [C]) of boxed area from panel A showing endocytic vacuoles containing M. tuberculosis in an infected A549 cell. (D) Overview (×3,800) of a PM_2.5-exposed A549 cell (18-h exposure to 10 μg/ml PM followed by M. tuberculosis infection at an MOI of 10 for an additional 18 h). (E) Magnification (×10,000) of boxed area from panel D showing an endocytic vacuole containing M. tuberculosis cells (white arrow) and PM_2.5 (black dashed arrow). (F) Magnification (×17,000) of boxed area from panel E showing PM_2.5 within the cell. (G) Overview (×3,800) of a PM₁₀-exposed (10 μg/ml for 18 h), M. tuberculosis-infected (MOI of 10) A549 cell showing cytoplasmic PM₁₀ (black dashed arrow) and M. tuberculosis cells (white arrow) in endocytic vacuoles. (H) Magnification (×10,000) of boxed area from panel G showing PM₁₀ (black dashed arrow) and multiple M. tuberculosis cells (white arrow). (I) Magnification (×17,000) of boxed area from panel H showing PM₁₀ (black dashed arrow) and multiple M. tuberculosis cells (white arrow). Multiple M. tuberculosis cells are visible in endocytic vacuoles surrounded by double membranes, while PM₁₀ (black dashed arrow) appears to be directly inserted in the cytoplasm, with endocytic vacuole formation completely missing. (J) Magnification (×10,000) of a PM₁₀-exposed A549 cell showing ultrastructural changes that appear to represent microvillus shedding (circled). (K) Magnification (×22,000) of an A549 cell exposed to PM₁₀ (black dotted arrow) showing mitochondrial elongation and swelling (white asterisks). (L) Magnification (×3,800) of a PM-unexposed and M. tuberculosis-uninfected A549 cell.

This work was supported by NIEHS grant R01ES020382-02 (S. Schwander) and Rutgers Center for Environmental Exposures and Disease NIEHS grant number P30ES005022.

We thank Gediminas Mainelis, Department of Environmental Sciences at Rutgers School of Environmental and Biological Sciences, for contributing to the analysis of the PM material.

C. E. Rivas-Santiago, S. Sarkar, Á. Osornio-Vargas, R. Quintana-Belmares, T. J. Kirn, and S. Schwander participated in the conception and design of the study. C. E. Rivas-Santiago, S. Sarkar, Á. Osornio-Vargas, Q. Meng, P. Ohman Strickland, J. C. Chow, J. G. Watson, M. Torres, and S. Schwander participated in analysis and interpretation. Drafting of the manuscript for important intellectual content was by C. E. Rivas-Santiago, S. Sarkar, Á. Osornio-Vargas, Q. Meng, M. Torres, and S. Schwander.