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. 2015 Mar 6;212(6):948–958. doi: 10.1093/infdis/jiv146

Lung Mucosa Lining Fluid Modification of Mycobacterium tuberculosis to Reprogram Human Neutrophil Killing Mechanisms

Jesús Arcos 1, Lauren E Diangelo 1, Julia M Scordo 1, Smitha J Sasindran 1, Juan I Moliva 1, Joanne Turner 1,2, Jordi B Torrelles 1,2
PMCID: PMC4548464  PMID: 25748325

Abstract

We have shown that human alveolar lining fluid (ALF) contains homeostatic hydrolases capable of altering the Mycobacterium tuberculosis cell wall and subsequently its interaction with human macrophages. Neutrophils are also an integral part of the host immune response to M. tuberculosis infection. Here we show that the human lung mucosa influences M. tuberculosis interaction with neutrophils, enhancing the intracellular killing of ALF-exposed M. tuberculosis and up-regulating the expression of tumor necrosis factor and interleukin 8. In contrast, ALF-exposed M. tuberculosis does not induce neutrophil apoptosis or necrosis, degranulation, or release of extracellular traps, and it decreases the oxidative response. These results suggest an important role for the human alveolar mucosa: increasing the innate capacity of the neutrophil to recognize and kill M. tuberculosis by favoring the use of intracellular mechanisms, while at the same time limiting neutrophil extracellular inflammatory responses to minimize their associated tissue damage.

Keywords: tuberculosis, alveolar lining fluid, neutrophil, innate immunity, lung surfactant

Tuberculosis is caused by the airborne pathogen Mycobacterium tuberculosis, with 8.7 million new tuberculosis cases annually worldwide [1]. Although many studies focus on delineating interaction of M. tuberculosis with host cells, there have been limited studies investigating how the alveolar mucosa, or alveolar lining fluid (ALF), participates in this interaction. The M. tuberculosis bacilli are exposed to ALF in the alveolar space when an individual is first infected with M. tuberculosis, on release from infected host cells, when M. tuberculosis is extracellular within lung cavities, and potentially during transmission in active tuberculosis. We have demonstrated that human ALF contains an array of hydrolases that are capable of altering the M. tuberculosis cell envelope with 2 outcomes: modifications to the M. tuberculosis cell envelope surface and release of M. tuberculosis-derived cell envelope fragments [2]. The M. tuberculosis cell envelope modifications were evident after exposure to human ALF for as little as 15 minutes, which has previously been shown to improve the control of M. tuberculosis infection by human macrophages [2].

Neutrophils are a key first line of defense against microbes [3]. They possess extracellular and intracellular killing mechanisms to control microbial infections [4]. Neutrophil extracellular killing mechanisms include (1) secretion of azurophilic/primary, specific/secondary, and secretory/tertiary granules containing hydrolytic enzymes and antimicrobial peptides [5]; (2) production of reactive oxygen intermediates [6]; and (3) release of neutrophil extracellular traps (NETs), composed of DNA fibers with embedded granules capable of extracellular binding and killing of microbes [7]. Second to phagocytosis, the major neutrophil intracellular killing mechanism is phagosome-lysosome fusion [4].

In humans, neutrophils represent the most abundant cell population harboring M. tuberculosis in bronchoalveolar lavage and sputum samples from patients with active tuberculosis [8, 9], yet the impact of the lung environment and particularly the role of ALF on neutrophil innate responses during microbial infection remain unknown. In the current study, we determined how alterations of the M. tuberculosis cell surface from exposure to human ALF affect the interaction of M. tuberculosis with human neutrophils. In contrast to human macrophages, neutrophils had enhanced recognition of ALF-exposed M. tuberculosis. Despite this, ALF-exposed M. tuberculosis did not trigger degranulation, release of NETs, apoptosis, or the oxidative response of infected neutrophils. Instead, we observed enhance intracellular clearance of ALF-exposed M. tuberculosis, primarily due to increased phagosome-lysosome fusion. ALF-exposed M. tuberculosis also triggered a significant increase in the production of the chemoattractant interleukin 8 (IL-8). Interestingly, ALF-exposed M. tuberculosis–infected neutrophils did not trigger the release of IL-12 by resting macrophages. Thus, our results demonstrate that human ALF-derived modifications of the M. tuberculosis cell envelope contribute to M. tuberculosis intracellular clearance by neutrophils but are not accompanied by some of the characteristic tissue damaging acute inflammatory responses associated with neutrophils.

MATERIALS AND METHODS

Ethics Statement

This study was carried out in strict accordance with US Code of Federal Regulations and Good Clinical Practice guidelines, as approved by the National Institutes of Health and the Institutional Review Board at The Ohio State University.

Chemical Reagents and Antibodies

All reagents were from Sigma-Aldrich except the following phycoerythrin (PE)–mouse anti–human CD63 antibody, PE–mouse immunoglobulin (Ig) G1 isotype control, and PE–annexin V Apoptosis Detection Kit (BD-Biosciences); allophycocyanin–mouse anti–human CD35 and peridinin chlorophyll protein–cyanine 5.5-mouse anti–human CD66b antibodies and their respective isotype controls (Biolegend); 5 (and 6)-carboxy-2′,7′-dichlorofluorescein diacetate and Lyso-Tracker red DND-99 (Invitrogen); human recombinant tumor necrosis factor (TNF) and human TNF, IL-6, IL-12p40, IL-1β, and IL-10 enzyme-linked immunosorbent assay kits (R&D Systems); and rabbit neutrophil elastase antibody and donkey Alexa Fluor 647 antibody to rabbit (Abcam).

Growth Conditions of M. tuberculosis

green fluorescent protein–M. tuberculosis (provided by Marcus Horwitz, University of California, Los Angeles) was grown as described elsewhere [2].

Human ALF Isolation

Human ALF was obtained from human bronchoalveolar lavage fluid; as described elsewhere [2] (further details in Supplementary Material).

Exposure of M. tuberculosis to Human ALF and ALF Hydrolases for Infection

Single-cell suspensions of M. tuberculosis (1 × 108) were obtained and exposed to 0.9% sodium chloride (NaCl; control buffer), human ALF in 0.9% NaCl, or a mixture of the most bioactive ALF hydrolases (alkaline and acid phosphatase and a nonspecific esterase, collectively termed Mix) at their physiological concentrations in vivo, as reported elsewhere [2]. On exposure to these conditions for 12 hours at 37°C, 5% carbon dioxide (CO2), bacilli were gently washed, suspended as single bacteria in Hank's buffered salt solution (HBSS), and recounted using a Petroff-Hausser counting chamber before being used for neutrophil infections [2]. As a reference, single-cell suspensions used to make inocula were incubated in medium alone (Supplementary Figure 1). In all reported experiments, exposed M. tuberculosis was produced fresh, and data were reproduced using ALFs from multiple healthy human donors.

Isolation and Preparation of Human Neutrophils

Neutrophils were obtained from healthy human donors, as described elsewhere [10]. Purified neutrophils were suspended in HBSS (with purity confirmed by microscopy), counted in a hemocytometer, and kept on ice for immediate use.

Association Studies of Exposed Bacteria With Human Neutrophils

Association of exposed M. tuberculosis with neutrophil monolayers (denoting combined binding and uptake) was performed in the absence or presence of cytochalasin D (10 µmol/L) [11] at a multiplicity of infection (MOI) of 10:1 in HBSS for 10 and 30 minutes at 37°C [12, 13]; association was determined by counting bacilli associated with ≥500 consecutive neutrophils per coverslip using phase-contrast and fluorescence microscopy, as described elsewhere [2].

Total and Intracellular Killing of Exposed M. tuberculosis

Killing experiments were completed as described elsewhere [13]. Resting or preactivated (with 10 ng/mL human TNF for 30 minutes) neutrophils (4 × 105 cells per well) were infected for various time periods with exposed M. tuberculosis (MOI, 1:1). Total killing (extracellular and intracellular) was determined at 30–360 minutes. Cultures were left unwashed until lysis on ice, with the same volume of cold 1% Triton-X100 in Dulbecco's phosphate-buffered saline (Life Technologies) added for 5 minutes. To determine intracellular killing, neutrophils were infected as described above for 30 minutes, infected monolayers were then washed to remove unbound M. tuberculosis, and extracellular bound M. tuberculosis was killed by adding 50 µg/mL of gentamycin for 30 minutes at 37°C and 5% CO2 [14]. For some experiments, monolayers were preexposed to and infected in the presence of 10 mmol/L ammonium chloride (NH4Cl) [15]. Neutrophils containing intracellular bacteria were then lysed, and lysates were serially diluted 10-fold in Water-Tween-Albumin buffer (1% Tween 80 and 1% bovine serum albumin in sterile, endotoxin-free water), plated, and cultured on 7H11 agar plates at 37°C for 21 days.

Phagosome-Lysosome Fusion of M. tuberculosis–Containing Phagosomes and Autophagosome Formation in Neutrophils

Neutrophils were infected with exposed M. tuberculosis (MOI, 10:1) for 30 minutes at 37°C and 5% CO2, fixed with 2% paraformaldehyde, permeabilized, blocked, and stained with the late endosomal/lysosomal marker CD63 (0.5 μg/mL) or IgG1 isotype control, as described elsewhere [2, 16]. To assess M. tuberculosis–containing phagosome acidification, neutrophils were preincubated with 75 nmol/L Lyso-Tracker before infection. To assess autophagosome formation, infected neutrophils were stained with rabbit anti–human LC3 antibody (2 μg/mL) or IgG1 isotype control, followed by a secondary donkey anti–rabbit Alexa Fluor 647 antibody (1:500). The percentage of exposed M. tuberculosis phagosomes that colocalized with CD63, LC3 or Lyso-Tracker was quantified by counting >50 consecutive phagosomes per coverslip in each test group.

RNA Isolation and Gene Expression at Quantitative Reverse-Transcription Polymerase Chain Reaction

After neutrophils were infected with exposed M. tuberculosis (MOI, 5:1) for 6 hours and lysed in TRIzol reagent (Invitrogen) for 10 minutes, total RNA was extracted. Gene expression for TNF, IL-6, and IL-8 was assessed by qRT-PCR as described elsewhere [17] (details in Supplementary Material).

Neutrophil Respiratory Burst, Degranulation, Myeloperoxidase Detection, and NET Formation and Quantification

Neutrophils (4 × 105 per well) in HBSS were added to a black, clear-bottom 96-well tissue culture plate, simultaneously infected with exposed M. tuberculosis (MOI, 1:1 to 10:1), and then incubated at 37°C for up to 6 hours. Neutrophil respiratory burst, degranulation, and myeloperoxidase detection were assessed as described in the Supplementary Material and elsewhere [18–20].

Modulation of Resting Macrophages by Infected Neutrophils

Neutrophils infected with M. tuberculosis (4 hours; MOI, 10:1) in the absence of macrophages were washed and then incubated with resting macrophages obtained from the same donor as described elsewhere [2], at a ratio of 2:1 for 18 hours at 37°C and 5% CO2 [21]. Supernatants were collected, and levels of TNF, IL-6, IL-12, IL-1β, and IL-10 were determined with enzyme-linked immunosorbent assay, according to the manufacturer's instructions. Resting macrophages exposed to uninfected neutrophils were used as negative controls, and resting macrophages alone as background [2].

Determination of Apoptosis/Necrosis and Cytotoxicity in Infected Neutrophils

Human neutrophils were infected with exposed M. tuberculosis (MOI, 5:1), and apoptosis/necrosis and cytotoxicity were assessed as described in the Supplementary Material.

Statistical Analyses

Prism software (GraphPad 4.0) was used to determine the statistical significance of differences between the means for 2 experimental groups, using an unpaired, 2-tailed Student t test. Differences were considered significant at P < .05.

RESULTS

Association of ALF-Exposed M. tuberculosis With Neutrophils

To assess whether human ALF- or purified hydrolase-derived modifications of the M. tuberculosis cell wall alter bacterial association with neutrophils, neutrophils were cultured with exposed M. tuberculosis for 10 or 30 minutes. Figure 1A and 1B shows a significant increase in ALF- and Mix-exposed M. tuberculosis association with neutrophils compared with control NaCl-exposed M. tuberculosis. We confirmed that the increased association involved phagocytosis in experiments using cytochalasin D (which blocks actin polymerization), indicative of receptor-mediated recognition or uptake (Figure 1C). We concluded that M. tuberculosis cell wall modifications, as a result of M. tuberculosis exposure to human ALF and hydrolases, alter the recognition and uptake of M. tuberculosis by neutrophils.

Figure 1.

Figure 1.

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Association of alveolar lining fluid (ALF)–exposed Mycobacterium tuberculosis with human neutrophils. A, Neutrophil monolayers (1 × 106) were established on poly-l-lysine-coated glass coverslips. Exposed M. tuberculosis association studies with neutrophils were performed at a multiplicity of infection of 10:1 at 10 and 30 minutes after infection. Cumulative data show association assessed by phase-contrast microscopy after 10 minutes (n = 4) and 30 minutes (n = 5); all values represent means and standard errors of the mean. B, Fold increase (number of ALF- or Mix-exposed M. tuberculosis bacilli per 100 neutrophils vs 0.9% sodium chloride [NaCl]–exposed M. tuberculosis [control]) for both time points (n = 4 for 10 minutes; n = 5 for 30 minutes). C, To assess whether this increase in association was dependent on receptor-mediated phagocytosis, neutrophil monolayers were preincubated with cytochalasin D (actin-filament polymerization blocker) for 30 minutes and then cultured with exposed M. tuberculosis or beads for 30 minutes more (n = 3). Student t test was used to compare ALF- or Mix-exposed M. tuberculosis with NaCl-exposed M. tuberculosis (control). *P < .05; †P < .005; ‡P < .0005. For each n value, both ALF and neutrophils were obtained from different human donors. Abbreviation: DMSO, dimethyl sulfoxide.

Intracellular Killing of ALF-Exposed M. tuberculosis by Neutrophils

To evaluate the effect of ALF and purified hydrolase exposure on the survival of M. tuberculosis on contact with neutrophils, we infected resting neutrophil monolayers with exposed M. tuberculosis over time without washing. At each time point, total colony-forming unit (CFU) counts were determined. Resting neutrophils had significantly better control of ALF-exposed M. tuberculosis 30 minutes after infection; this capacity was maintained over time (Figure 2A). Moreover, TNF preactivated neutrophils (which activate oxidative killing [22]) were also capable of superior control of ALF-exposed M. tuberculosis; however, this was delayed until 120 minutes relative to NaCl-exposed M. tuberculosis (control) (Figure 2B). Independent of the length of infection (30–360 minutes), there was failure to control Mix-exposed M. tuberculosis in resting or TNF-preactivated neutrophils compared with control NaCl-exposed M. tuberculosis (Figure 2A and 2B).

Figure 2.

Figure 2.

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Survival of exposed Mycobacterium tuberculosis on incubation with neutrophils. Resting or preactivated (with 10 ng/mL human tumor necrosis factor [TNF] for 30 minutes) neutrophils (4 × 105) were infected with single-cell suspensions of exposed M. tuberculosis (multiplicity of infection [MOI], 1:1) for different lengths of time (30, 60, 120, 180, and 360 minutes), leaving the M. tuberculosis inoculum in the culture at each time point. Total killing (extracellular and intracellular) was assessed for resting (A) or TNF-preactivated (B) neutrophils. Cumulative data are shown as means and standard errors of the mean from triplicate experiments (each n = 4). Student t test was used to compare alveolar lining fluid (ALF)–exposed (A) or Mix-exposed (M) M. tuberculosis with 0.9% sodium chloride–exposed (N) M. tuberculosis (control). *P < .05, ‡P < .0005. For each n value, both ALF and neutrophils were obtained from different human donors. Abbreviation: CFUs, colony-forming units.

Because we observed an increase in killing of ALF-exposed M. tuberculosis in the total neutrophil killing assay (extracellular plus intracellular), we next determined whether killing was primarily related to an intracellular killing mechanism. Neutrophil monolayers were infected with M. tuberculosis for 30 minutes, and then intracellular killing was assessed after washing of unbound M. tuberculosis and killing of bound but nonphagocytosed M. tuberculosis with gentamicin. Figure. 3A shows that neutrophil intracellular killing of ALF- and Mix-exposed M. tuberculosis was enhanced compared with the control NaCl-exposed M. tuberculosis, a difference reaching significance 180 minutes after infection. Combined with data in Figure 2, our results provide evidence that neutrophils are better able to control the intracellular growth of ALF-exposed M. tuberculosis .

Figure 3.

Figure 3.

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Intracellular killing of exposed Mycobacterium tuberculosis in neutrophils. Resting neutrophil monolayers (4 × 105) were infected with single-cell suspensions of exposed M. tuberculosis (multiplicity of infection [MOI], 1:1) for 30 minutes, washed to remove unbound bacteria, and treated with gentamicin for 30 minutes to kill extracellularly bound, nonphagocytosed bacteria before assessment of M. tuberculosis intracellular neutrophil killing. Infected cells were lysed at the specific time points studied, and lysates plated to determine colony-forming unit (CFU) counts. A, Cumulative data are shown as means and standard errors of the mean from triplicate experiments (each n = 3). Intracellular killing was assessed at different time points after 30 minutes of infection followed by 30 minutes of gentamicin treatment (60, 90, 150, 210, and 390 minutes). A, M, and N, respectively, represent alveolar lining fluid (ALF)–, Mix-, and 0.9% sodium chloride (NaCl)–exposed M. tuberculosis. B, Neutrophil (4 × 105) monolayers were preincubated for 30 minutes with 10 mmol/L ammonium chloride (NH4Cl; phagosome-lysosome fusion blocker), infected for 30 minutes with single-cell suspensions of exposed green fluorescent protein (GFP)–M. tuberculosis (MOI, 1:1) in the presence of NH4Cl, washed, treated with gentamicin (30 minutes), and lysed, and lysates were plated to determine CFU counts. C, Neutrophils (1 × 106) were adhered to poly-l-lysine-treated glass coverslips, then infected with single-cell suspensions of exposed M. tuberculosis (MOI, 10:1) for 30 minutes, washed, fixed, permeabilized, and labeled for CD63 (lysosomal maker) or LC3 (autophagosome marker). For the Lyso-Tracker assay (acidification marker), neutrophils on coverslips were preincubated with 75 mmol/L Lyso-Tracker for 30 minutes, infected with exposed GFP–M. tuberculosis, washed, and fixed. In merged images, CD63 and Lyso-Tracker–positive compartments are red, LC3-positive compartments are orange (white arrows), exposed GFP–M. tuberculosis bacilli are green, and bacteria colocalized with CD63, Lyso-Tracker or LC3 are yellow (original magnification, ×600 for LC3 and Lyso-Tracker and ×300 for CD63). Phagosome-lysosome fusion and autophagosome formation events were examined and enumerated with confocal microscopy, counting >50 events per coverslip, in triplicate. Cumulative data show the fold increase in phagosome-lysosome fusion of ALF- or Mix-exposed M. tuberculosis compared with NaCl-exposed M. tuberculosis, (n = 4 for CD63, n = 3 for LC3, and n = 4 for Lyso-Tracker). Student t test was used to compare ALF- or Mix-exposed M. tuberculosis with NaCl-exposed M. tuberculosis (control). *P < .05. For each n value, both ALF and neutrophils were obtained from different human donors. Abbreviations: HBSS, Hank's buffered salt solution; iA, heat-inactivated ALF-exposed M. tuberculosis (boiled at 80°C for 2 hours).

We next assessed whether neutrophil intracellular killing of ALF- and Mix-exposed M. tuberculosis was directly related to an increase in phagolysosomal fusion, using NH4Cl as a phagolysosomal fusion blocker [15] (Figure 3B). The presence of NH4Cl reversed the enhanced killing seen with exposed M. tuberculosis, consistent with this mechanism. We confirmed increased phagolysosomal fusion using lysosomal (CD63) and phagosome acidification (Lyso-Tracker) markers. Results 30 minutes after infection showed an increase in phagolysosomal fusion and subsequent compartment acidification for ALF-exposed (1.84- and 0.74-fold increases, respectively) and Mix-exposed (0.63 and 0.63-fold increases) M. tuberculosis compared with control (Figure 3C). This increase in phagolysosomal fusion was driven by protein components present in ALF (ie, hydrolases altering the M. tuberculosis cell wall [2]), because neutrophils lost their ability to kill intracellular M. tuberculosis exposed to heat-inactivated ALF (Figure 3B; NaCl- vs heat-inactivated ALF-exposed M. tuberculosis in the absence of NH4Cl).

Autophagy is considered another intracellular killing mechanism to control M. tuberculosis infection [23]; however, our results indicate that neither ALF-exposed nor Mix-exposed M. tuberculosis infection results in increased autophagosome formation compared with control (Figure 3C).

IL-8 and Extracellular Mechanisms of Neutrophil Killing in Response to ALF-Exposed M. tuberculosis

Neutrophils are capable of secreting inflammatory mediators in response to M. tuberculosis infection [24]. We determined whether ALF-exposed M. tuberculosis was able to induce neutrophil TNF, IL-6, and IL-8, 3 major immunomodulators involved in the neutrophil response during M. tuberculosis infection [25]. Results show that ALF- or Mix-exposed M. tuberculosis increased neutrophil expression of TNF ( approximately 1-fold) and IL-8 (>2 fold) compared with control NaCl-exposed M. tuberculosis (Figure 4A). IL-6 gene expression up-regulation was not observed (not shown).

Figure 4.

Figure 4.

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Alveolar lining fluid (ALF)– or Mix-exposed Mycobacterium tuberculosis up-regulates interleukin 8 (IL-8) but diminishes the production of reactive oxygen species (ROS) and does not mediate granule release by neutrophils. A, Neutrophils (5 × 106) were infected with single-cell suspensions of exposed M. tuberculosis (multiplicity of infection [MOI], 5:1) for 6 hours and lysed. Expression of tumor necrosis factor (TNF) or IL-8 was measured with reverse-transcription polymerase chain reaction. Data represent means and standard deviations from representative experiment in triplicate (n = 3). A and M represent ALF- and Mix-exposed M. tuberculosis, respectively; NaCl, 0.9% sodium chloride. B, Kinetics of ROS release by neutrophils (4 × 105) infected with single-cell suspensions of exposed M. tuberculosis (MOI, 10:1). The relative amount of ROS generated by neutrophils over time was detected by measuring the conversion of 2′,7′-dichlorodihydrofluorescein diacetate to the hihgly fluorescent 2′,7′-dichlorofluorescein using a Spectramax GEMENI-EM fluorescent reader. Cumulative data are shown as means and standard errors of the mean in triplicate (each n = 3). C, Neutrophils (1 × 106) were infected with exposed M. tuberculosis (MOI, 10:1), and neutrophil degranulation was assessed with flow cytometry according to increased surface expression of CD63 (marker for primary/azurophilic granules), CD66b (marker for secondary/specific granules), and CD35 (marker for tertiary/secretory granules) in infected neutrophils. In all experiments medium (Hank's buffered salt solution [HBSS]) control values were subtracted out as background. Student t test was used to compare ALF- or Mix-exposed M. tuberculosis with NaCl-exposed M. tuberculosis (N; control). Phorbol myristate acetate was used as a positive control (PC) and induced a significant increase in all granule markers indicative of extensive neutrophil degranulation it was also used as a PC for ROS production (not shown). *P < .05. For each n value, both ALF and neutrophils were obtained from different human donors. Abbreviations: ISO, isotype control; MFI, mean fluorescence intensity; RFUs, relative fluorescence units.

Our data provide evidence that control of intracellular growth of ALF-exposed M. tuberculosis in resting neutrophils occurs primarily by increased phagosome-lysosome fusion. We next sought to determine whether ALF-exposed M. tuberculosis could also induce neutrophil extracellular killing mechanisms. Because M. tuberculosis can induce the oxidative burst of human neutrophils [26], we tested whether ALF-induced M. tuberculosis cell envelope alterations [2] changed the neutrophil capacity to generate extracellular reactive oxygen species (ROS). Results in Figure 4B show that ALF-exposed M. tuberculosis significantly reduced neutrophil ROS production compared with control NaCl-exposed M. tuberculosis. Mix-exposed M. tuberculosis showed similar patterns (Figure 4B).

We then assessed whether M. tuberculosis exposed to human ALF was capable of inducing neutrophil degranulation [5]. Figure 4C shows that exposed M. tuberculosis failed to induce degranulation in infected neutrophils, as indicated by the lack of up-regulation of surface expressed CD63 (primary/azurophilic), CD66b (secondary/specific), and CD35 (tertiary/secretory) granule markers compared with noninfected neutrophils. The inability of exposed M. tuberculosis to induce neutrophil primary degranulation was confirmed by the lack of myeloperoxidase (primary granule enzyme) release in supernatants from exposed M. tuberculosis–infected neutrophils 90 and 120 minutes after infection (not shown). Moreover, we also examined the possibility that exposed M. tuberculosis could induce the release of NETs by neutrophils, as described elsewhere [19]. However, production of NETs by ALF-exposed M. tuberculosis was limited and similar to that in our controls (Figure 5A and 5B).

Figure 5.

Figure 5.

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NETosis and cross-talk activation of resting macrophages by alveolar lining fluid (ALF)–exposed Mycobacterium tuberculosis. Neutrophils (1 × 106) monolayers on poly-l-lysine-treated glass coverslips were incubated with exposed M. tuberculosis for 4 hours at a multiplicity of infection (MOI) of 10:1. A, Representative confocal photomicrographs are shown for uninfected neutrophils (Hank's buffered salt solution [HBSS]) or neutrophils infected with green fluorescent protein–M. tuberculosis (green) exposed to 0.9% sodium chloride (NaCl) or human ALF. DNA (DAPI; blue), elastase (indicator of degranulation; orange), and colocalization (pink) are indicative of neutrophil extracellular trap (NET) release. B, Quantification of NETs with confocal microscopy (≥300 events per coverslip) or direct DNA quantification; data represent means and standard errors of the mean for triplicate experiments (each at least n = 3). A and N, ALF-exposed and NaCl-exposed (control) M. tuberculosis, respectively; PC, positive control (neutrophils exposed to phorbol myristate acetate). Resting neutrophils in medium HBSS control values were subtracted out as background. ‡P < .0005. C, Infected neutrophils (1 × 105; MOI, 10:1; 4 hours) were directly incubated with human resting macrophages isolated from the same donor at a ratio of 2:1 for 18 hours, and cytokine production indicative of macrophage activation was assessed. Data represent means and standard errors of the mean for triplicate experiments (n = 3). Student t test was used to compare ALF- and NaCl-exposed M. tuberculosis. For each n value, both ALF and neutrophils (and also macrophages in C) were obtained from different human donors. Abbreviations: DAPI, 4′,6′-diamino-2-phenylindole; IL-1β, interleukin 1β; IL-6, interleukin 6; IL-12, interleukin 12; MØ, macrophages; TNF, tumor necrosis factor; U, uninfected.

We next assessed whether ALF-exposed M. tuberculosis infected neutrophils could modulate responses in resting macrophages. Our results indicate that neutrophils infected with ALF-exposed M. tuberculosis were unable to induce the production of IL-12 in resting macrophages by direct contact (Figure 5C). Furthermore, for all tested groups, secreted products of infected neutrophils were also unable to modulate the cytokine response in resting macrophages (not shown).

Neutrophil Apoptosis in Response to ALF-Exposed M. tuberculosis

Mycobacterium tuberculosis can induce apoptosis and necrosis in neutrophils [13]. We evaluated whether exposed M. tuberculosis was capable of inducing early apoptosis (annexin V–positive cells), which precedes loss of membrane integrity that accompanies the later stages of cell death resulting from either apoptotic or necrotic processes (annexin V– and 7-aminoactinomycin D–positive cells). None of the tested groups, including ALF- or Mix-exposed M. tuberculosis, induced any increase in apoptosis or necrosis when compared with uninfected neutrophils resting in minimal medium, HBSS (Figure 6A and 6B). Contrary to studies showing that M. tuberculosis induces apoptosis, our control NaCl-exposed M. tuberculosis did not induce apoptosis of neutrophils in minimal medium without serum. Our results show a baseline between 20% and 25% apoptotic cells. Moreover, we did not observe a significant induction of necrosis. These results were confirmed by measuring the release of lactate dehydrogenase, indicative of disruption of the neutrophil plasma membrane, where exposed M. tuberculosis induced a cytotoxic effect equivalent to that observed in resting neutrophils in HBSS (Figure 6C).

Figure 6.

Figure 6.

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Alveolar lining fluid (ALF)–exposed or Mix-exposed Mycobacterium tuberculosis does not induce neutrophil death. A, Resting neutrophils (1 × 106) were infected with exposed M. tuberculosis at a multiplicity of infection (MOI) of 5:1 for 30–360 minutes, washed, fixed and stained for annexin V (apoptosis) and 7-aminoactinomycin D (for live/dead necrosis). A representative experiment 60 minutes after infection is shown, with early apoptosis (bottom right), late apoptosis (top right), and necrosis (top left). B, Cumulative data are shown as means and standard errors of the mean from duplicate experiments at different time points after infection (each n = 2). A, M, and N, respectively, represent ALF-, Mix-, and NaCl-exposed M. tuberculosis; C, control (HBSS medium as background). C, Neutrophil monolayers (4 × 105) were infected with exposed M. tuberculosis (MOI, 1:1) and the release of lactate dehydrogenase indicative of cytotoxicity was measured at the indicated time points after infection. Cumulative cytotoxicity data are shown for experiments in triplicate (n = 3). For each n value, both ALF and neutrophils were obtained from different human donors. Abbreviations: HBSS, Hank's buffered salt solution; NaCl, 0.9% sodium chloride.

DISCUSSION

Little is known about how the human lung environment dictates the interaction of M. tuberculosis with host cells and how this determines the pathway for establishment and outcome of infection. The exposure of M. tuberculosis to ALF occurs in 3 scenarios: during primary infection, on release from dying macrophages, and, importantly, during active tuberculosis and transmission, when M. tuberculosis can be extracellular and located within necrotic tissue pockets called cavities. Neutrophils are thought to be important in these cavities during active and disseminated tuberculosis [9]. However, the neutrophil innate response against M. tuberculosis infection, even at the first M. tuberculosis encounter with host cells, is still not clearly defined.

Arcos et al [2] showed that on exposure to ALF the M. tuberculosis cell envelope is altered by ALF homeostatic hydrolases, and as a result of these cell envelope alterations there is a significant effect on M. tuberculosis association with and intracellular survival within human macrophages. This was due to hydrolases, because the mix of 3 most bioactive hydrolases (Mix) gave the same results. In neutrophils, our results now show that ALF exposure with subsequent hydrolase modifications of the M. tuberculosis cell envelope [2] alter early interactions with neutrophils by limiting the extracellular oxidative response, inflammation, and tissue damage while enhancing intracellular M. tuberculosis killing through increased phagosome-lysosome fusion and acidification, thus influencing the neutrophil's acute inflammatory response to infection. Moreover, neutrophils infected by ALF-exposed M. tuberculosis can modulate the inflammatory response of resting macrophages by limiting their IL-12 production, an important mediator of both innate and adaptive immune responses against M. tuberculosis infection [25, 27].

In human ALF, neutrophils normally represent <2% of all cells; however, during inflammation a massive influx of neutrophils occurs [28, 29], especially in lung cavities during active tuberculosis, in which M. tuberculosis is extracellular and exposed to ALF in the presence of large numbers of neutrophils [4]. In this regard, neutrophils have been shown to have a life span of up to 5 days under these conditions [30]. The importance of neutrophils in vivo was highlighted in a neutrophil depletion study, in which depletion of neutrophils resulted in an increased severity of M. tuberculosis infection [31]. Earlier studies showed that human neutrophils could control the total growth of M. tuberculosis [32] but did not discern between extracellular or intracellular killing and did not account for the influence of the lung environment on M. tuberculosis–neutrophil interactions.

In the current study, we show that M. tuberculosis exposure to human ALF allows neutrophils to control intracellular growth of M. tuberculosis while not readily activating neutrophil extracellular mechanisms of killing. Our results are akin to those of another published study showing that, in the absence or presence of serum, M. tuberculosis is resistant to neutrophil oxidative mediators; thus, neutrophils do not kill M. tuberculosis via the oxygen metabolic burst [33]. Supporting this explanation, cytokine-activated neutrophils with enhanced oxidative responses are capable of killing Staphylococcus aureus but not M. tuberculosis [34]. Similarly, our results show that TNF-preactivated neutrophils can initially control ALF-exposed M. tuberculosis growth better; however this control wanes over time.

Other studies have shown that on M. tuberculosis internalization, activated neutrophils kill intracellular M. tuberculosis exclusively via a reduced nicotinamide adenine dinucleotide phosphate oxidase-dependent mechanism by ROS and p38 activation [35]. This oxidative response is correlated with the induction of neutrophil apoptosis 3 hours after infection [36]. Our data, however, provide evidence that ALF hydrolase-derived modifications of the M. tuberculosis cell wall, a process that limits the oxidative response, do not induce apoptosis or necrosis but instead induce increased phagosome-lysosome fusion, TNF, and IL-8 [37] for the length of time studied (up to 6 hours) in our in vitro system.

It is thought that release of antimicrobial granule contents by neutrophils into the surrounding environment will kill extracellular M. tuberculosis and also provides signals to attract T cells and immature dendritic cells to the site of infection in initiating the adaptive immune response [4]. However, we did not observe a release of neutrophil granules or their contents (ie, myeloperoxidase) to the environment in ALF-exposed M. tuberculosis–infected neutrophils. Moreover, ALF-exposed M. tuberculosis did not trigger an increase in the formation of elastase-containing NETs by neutrophils and/or NETosis of neutrophils [38], where NETosis differs from classic apoptosis or necrosis [38, 39]. The neutrophil serine protease elastase in NETs has been shown to exhibit bactericidal activity [7]; however, although we observed few elastase-containing NET-trapped M. tuberculosis (data not shown), our extracellular killing data indicate that released NETs are unable to efficiently kill M. tuberculosis, as described elsewhere [19].

An explanation for why ALF-exposed M. tuberculosis has limited ability to induce NET formation may be the limited expression of mannose-capped lipoarabinomannan and trehalose dimycolate on the surface of ALF-exposed M. tuberculosis [2], 2 pathogen-associated molecular patterns thought to be involved in triggered NET release by neutrophils [4]. Another plausible explanation is the limited neutrophil oxidative burst and autophagome formation induced by ALF-exposed M. tuberculosis, because both superoxide production and autophagy have been linked to NETosis [40] as well as being required in apoptotic cell death [41], the latter also not induced by ALF-exposed M. tuberculosis.

The studies published to date show contradictory results regarding neutrophil responsiveness to M. tuberculosis. This may be because they are based on in vitro studies using rich medium (ie, Roswell Park Memorial Institute 1640 medium) and bovine calf serum, a combination that activates neutrophils over time [42, 43]. In this regard, studies have shown that neutrophils suspended in Roswell Park Memorial Institute 1640 medium containing bovine calf serum alone undergo significant phenotypic and functional changes in response to extracellular acidification of the medium [42]. Our studies used minimal medium, thus minimizing this effect.

In summary, in vitro human models and in vivo animal models to assess the role of neutrophils in M. tuberculosis infection have not accounted for the impact of the human lung mucosa in determining M. tuberculosis–host interactions. We and others have shown that some components of human ALF (surfactant protein A and D, homeostatic hydrolases, surfactant lipids, and the complement system) are critical elements of the innate immune system during M. tuberculosis infection [2, 44–47] and play important roles in M. tuberculosis–phagocyte encounters [48, 49]. The role of ALF components during active pulmonary tuberculosis in humans is not known.

Overall, our results provide evidence that the lung mucosal environment can play an important role in the normal resolution of inflammation by limiting the extracellular killing potential of the neutrophil, which in the short term may contribute to the successful but limited establishment of infection, a hypothesis that we are currently evaluating in vivo.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online (http://jid.oxfordjournals.org). Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.

Supplementary Data
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Notes

Acknowledgments. We thank Dr Larry S. Schlesinger for his careful review of this manuscript and the Campus Microscopy and Imaging Facility at The Ohio State University (OSU) for their services. We also acknowledge the facilities and programmatic support of the OSU Biosafety Level 3 Program.

Financial support. This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (NIH) (grants K99/R00-AI073856 and R01-AI093570 to J. B. T.), the American Federation for Aging Research (Julie Martin Mid-Career award to J. T.), the OSU College of Medicine Systems in Integrative Biology Training Program Fellowship supported by the National Institute of General Medical Sciences, NIH (grant T32-GM-068412 to J. M. S. and J. I. M.), and the National Institute of Allergy and Infectious Diseases Diversity Fellowship (grant AI093570-S1 to J. I. M.).

Potential conflicts of interest. All authors: No potential conflicts of interest.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

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