Neutrophil Extracellular Traps Increase Airway Mucus Viscoelasticity and Slow Mucus Particle Transit

Abstract

Mucus obstruction is a key feature of many inflammatory airway diseases. Neutrophil extracellular traps (NETs) are released upon neutrophil stimulation and consist of extracellular chromatin networks studded with cytotoxic proteins. When released in the airways, these NETs can become part of the airway mucus. We hypothesized that the extracellular DNA and/or oxidative stress (e.g., by the release of reactive oxygen species and myeloperoxidase during NETs formation in the airways) would increase mucus viscoelasticity. We collected human airway mucus from endotracheal tubes of healthy patients admitted for elective surgery and coincubated these samples with NETs from phorbol 12-myristate 13-acetate-stimulated neutrophils. Unstimulated neutrophils served as controls, and blocking experiments were performed with dornase alfa for extracellular DNA and the free radical scavenger dimethylthiourea for oxidation. Compared with controls, the coincubation of mucus with NETs resulted in 1) significantly increased mucus viscoelasticity (macrorheology) and 2) significantly decreased mesh pore size of the mucus and decreased movement of muco-inert nanoparticles through the mucus (microrheology), but 3) NETs did not cause visible changes in the microstructure of the mucus by scanning EM. Incubation with either dornase alfa or dimethylthiourea attenuated the observed changes in macrorheology and microrheology. This suggests that the release of NETs may contribute to airway mucus obstruction by increasing mucus viscoelasticity and that this effect is not solely due to the release of DNA but may in part be due to oxidative stress.

Keywords: macrorheology; microrheology; muco-inert nanoparticles; multiple particle tracking; respiratory tract diseases.

Figure 1.

Schematic workflow of methods. Neutrophils were cultured in the presence of 50 nM PMA (to induce NETosis) or without the presence of PMA (unstimulated neutrophils). The culture media (with or without PMA) was discarded, and neutrophil extracellular traps were washed three times with PBS. The neutrophil extracellular traps were then collected by vigorous pipetting in PBS and transferred to tubes. The tubes were spun, and the neutrophils pelleted down were discarded while the supernatant was collected. The supernatant was then added to mucus with or without the presence of either dimethylthiourea or dornase alfa. DMTU = dimethylthiourea; NETs =neutrophil extracellular traps.

Figure 2.

(A and B) Elastic modulus (A) and viscous modulus (B) of mucus after 6- and 24-hour exposure to NETs. Control mucus (control) was treated with the supernatant of unstimulated neutrophils (squares), and experimental mucus was treated with NETs (triangles). Paired t tests (n = 5; samples obtained from different mucus donors) to compare NETs and control mucus after 6 hours showed no significant differences between NETs mucus (7.55 ± 3.08) and control mucus (6.13 ± 3.11) (t = 1.84 [4]; P = 0.14) for the elastic or viscous moduli of NETs mucus (103.5 ± 16.39) compared with control mucus (92.5 ± 15.91) (t = 1.54 [4]; P = 0.20). Paired t tests (n = 14; samples obtained from different mucus donors) to compare NETs and control mucus after 24 hours had a significantly higher elastic modulus for NETs mucus (32.76 ± 37.23) compared with control mucus (7.62 ± 7.77) (t = 2.74 [13]; *P = 0.02) as well as for the viscous modulus of the NETs mucus (182.40 ± 125.40) and control mucus (103.90 ± 47.62) (t = 2.74 [13]; *P = 0.02). (C and D) Elastic modulus (C) and viscous modulus (D) for mucus exposed to NETs and blocked with DMTU or dornase alfa (n = 6 donors). Mucus was treated with NETs, NETs + DMTU, and NETs + dornase alfa, respectively. The control mucus was treated with the supernatant of unstimulated neutrophils (control), control + DMTU, and control + dornase alfa. In C and D, the same experiment outcomes as presented in A and B for control mucus and NETs mucus are presented. Each data point represents the mean value from the in triplicate–performed experiment for one mucus donor. Mean values for each group are represented in Table E2.

Figure 3.

Diffusion of muco-inert nanoparticles in human airway mucus. Ensemble-averaged geometric mean square displacement (MSD) as a function of time scale (2 s) for (A) 100-nm and (B) 500-nm muco-inert nanoparticles in mucus after experimental exposures. Distribution of individual particles’ log₁₀[MSD_1s] and representative trajectory traced 2 seconds (inserts) of a single particle for (C) 100-nm and (D) 500-nm muco-inert nanoparticles. The red dashed lines in C and D indicate the median values of log₁₀[MSD_1s]. *P < 0.05 and **P < 0.01. n = 4 donors.

Figure 4.

Microrheology (elastic and viscous moduli) of human airway mucus under different experimental conditions. (A and B) Submicron viscoelastic properties of each treated mucus were probed by 100-nm (A) and 500-nm (B) muco-inert nanoparticles. Local elastic (solid lines) and viscous (dashed lines) moduli as a function of frequency. Time lag was first transformed to frequency (Hz) and then multiplied by 2π to calculate the final frequency (rad/s). n = 4 donors. G′ = elastic; G′′ = viscus.

Figure 5.

Microrheology (phase angle and microviscosity) of human airway mucus under different experimental conditions. (A–D) Phase angle (δ) and microviscosity (η*) at a frequency of 2π rad/s for 100-nm (A and C) and 500-nm (B and D) muco-inert nanoparticles in mucus at the same frequency (mean ± SEM). The phase angle is 90° for an ideal Newtonian liquid and is 0° for an ideal Hookian solid. Comparations between the groups were performed by a Kruskal-Wallis test. *P < 0.05. n = 4 donors.

Figure 6.

Estimated mesh pore size of mucus. (A) Box–whisker plots of mesh pore size of each mucus sample measured by 100-nm muco-inert nanoparticles. (B) Mean mesh pore size of each experimental group (mean ± SEM). Comparations between the groups were performed by a Kruskal-Wallis test. *P < 0.05. n = 4 donors. Plotted samples correspond with samples used for the trajectory movies.

Figure 7.

Schematic figure of possible mucus changes and mechanisms measured by probing the mucus with muco-inert nanoparticles. In airway mucus, NETs may (A) increase the mesh pore size by NETs adherence to the mucin, enlarging the mesh pore size nearby and making 500-nm muco-inert nanoparticles permeable (denoted by the arrowhead); (B) reduce the mesh pore size by NETs entanglement; or (C) reduce the mesh pore size by increasing disulfide bonds because of oxidative stress. The 500-nm particle experiment outcomes suggests this to be unlikely.