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

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: alveolar lining fluid; innate immunity; lung surfactant; neutrophil; tuberculosis.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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).

Figure 4.

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.

Figure 5.

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.

Figure 6.

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.