Engineering in vitro models of cystic fibrosis lung disease using neutrophil extracellular trap inspired biomaterials

Abstract

Cystic fibrosis (CF) is a muco-obstructive lung disease where inflammatory responses due to chronic infection result in the accumulation of neutrophil extracellular traps (NETs) in the airways. NETs are web-like complexes comprised mainly of decondensed chromatin that function to capture and kill bacteria. Prior studies have established excess release of NETs in CF airways increases viscoelasticity of mucus secretions and reduces mucociliary clearance. Despite the pivotal role of NETs in CF disease pathogenesis, current in vitro models of this disease do not account for their contribution. Motivated by this, we developed a new approach to study the pathobiological effects of NETs in CF by combining synthetic NET-like biomaterials, composed of DNA and histones, with an in vitro human airway epithelial cell culture model. To determine the impact of synthetic NETs on airway clearance function, we incorporated synthetic NETs into mucin hydrogels and cell culture derived airway mucus to assess their rheological and transport properties. We found that the addition of synthetic NETs significantly increases mucin hydrogel viscoelasticity. As a result, mucociliary transport in vitro was significantly reduced with the addition of mucus containing synthetic NETs. Given the prevalence of bacterial infection in the CF lung, we also evaluated the growth of Pseudomonas aeruginosa in mucus with or without synthetic NETs. We found mucus containing synthetic NETs promoted microcolony growth and prolonged bacterial survival. Together, this work establishes a new biomaterial enabled approach to study innate immunity mediated airway dysfunction in CF.

Figure 1.. Synthetic NET (sNET) formulation.

(A) Schematic of sNET formation by combining 50 wt% DNA and 50 wt% histones dissolved separately in buffer. The DNA and histone will electrostatically interact to form large complexes that can be sonicated to create a suspension of sNETs. (B) Fluorescence microscopy image of the DNA structure of an sNET, stained with DAPI. Scale bar, 25 μm.

Figure 2.. sNETs Alter Synthetic Mucus Microstructure.

(A) Schematic of synthetic mucus hydrogel preparation with various additives (sNETs, DNA, histones, or additional PGM). (B) Representative trajectories of PEGylated 100 nm nanoparticle diffusing within each hydrogel type for 1.5 seconds. Scale bar, 200 nm. Box and whisker plots of (C) the logarithm based 10 of the measured MSD at 1 second (log₁₀[MSD_1s]) of PEGylated 100 nm nanoparticles in each hydrogel type (n = 3 replicates per condition; 5 randomly selected regions of each individual gel were imaged), and (D) the estimated pore size of each hydrogel type calculated based on measured MSD. (E) Box and whisker plot of the log₁₀[MSD_1s] of 100 nm PEGylated nanoparticles measured for each hydrogel type with and without 7 μg/ml DNase I treatment for 1 hour at 37°C (n = 3 replicates per condition, with 5 different randomly chosen regions of each individual gel imaged). For (C–E), statistical significance determined by Kruskall Wallis test with Dunn’s multiple comparisons test (**** = p < 0.0001).

Figure 3.. sNETs Increase Synthetic Mucus Viscoelasticity.

(A) Mean elastic moduli, (B) viscous moduli, and (C) complex viscosity (G′, G″, η*, respectively) at ω = 1 rad/s for SM and sNET-SM hydrogels (n = 3 replicates per condition). Statistical significance determined by unpaired two tailed t-test (** = p < 0.01).

Figure 4.. sNETs Decrease Mucociliary Transport In Vitro.

(A) Schematic of the process of washing cultures to harvest HAE mucus, then incorporating sNETs into the fresh HAE mucus (sNET+ HAE mucus), and overlaying the sNET+ HAE mucus back onto the surface of the cultures to measure MCT using fluorescent microspheres. (B) Median transport rates of 2 μm microspheres, (C) representative trajectories of the microspheres being transported across the mucosal surface of cultures over the course of 10 seconds, and (D) mean cilia beat frequency of cultures overlaid with control and sNET+ HAE mucus (n = 3 replicates per condition, with 3 randomly chosen regions of each individual culture imaged), Scale bar, 100 μm. Statistical significance determined by unpaired two tailed t-tests (Ns = p > 0.05, * = p < 0.05).

Figure 5.. DNase Treatment of sNET+ Mucus Enhances Mucociliary Transport.

(A) Median transport rates, (B) trajectories of microspheres over the course of 10 seconds, and (C) mean cilia beat frequency measured from the same set of cultures with either control or sNET+ HAE mucus overlaid before (pretreatment) and after (DNase treated) application of DNase I at a concentration of 7 μg/ml for 30 minutes at 37°C (n = 3 replicates per condition, with 3 randomly chosen regions of each individual culture imaged). Trajectories are displayed from the same individual culture pretreatment and with DNase treatment. Scale bar, 100 μm. Statistical significance determined by two-way repeated measures ANOVA tests (Ns = p > 0.05, * = p < 0.05).

Figure 6.. sNETs Alter Bacterial Growth Patterns and Sustain Survival in Mucus.

Fluorescence microscopy images of GFP-expressing PAO1 (GFP PAO1) bacteria grown for 24 hours at 37°C in (A) control or (B) sNET+ HAE mucus. Scale bar, 100 μm. GFP PAO1 were grown in (C) control or (D) sNET+ HAE mucus for 24 hours and the GFP fluorescence of the bacteria was measured at 0, 3, 6, and 24 hours. Values are plotted as percentage of the baseline fluorescence at 0 h with background fluorescence subtracted from wells containing either control or sNET+ HAE mucus with no bacteria. Statistical significance determined by repeated measures ANOVA tests with Tukey’s multiple comparisons tests (Ns = p > 0.05, *=p<0.05, **** = p < 0.0001).