Neutrophil dysfunction in cystic fibrosis

Abstract

Background: Excessive neutrophil inflammation is the hallmark of cystic fibrosis (CF) airway disease. Novel technologies for characterizing neutrophil dysfunction may provide insight into the nature of these abnormalities, revealing a greater mechanistic understanding and new avenues for CF therapies that target these mechanisms.

Methods: Blood was collected from individuals with CF in the outpatient clinic, CF individuals hospitalized for a pulmonary exacerbation, and non-CF controls. Using microfluidic assays and advanced imaging technologies, we characterized 1) spontaneous neutrophil migration using microfluidic motility mazes, 2) neutrophil migration to and phagocytosis of Staphylococcal aureus particles in a microfluidic arena, 3) neutrophil swarming on Candida albicans clusters, and 4) Pseudomonas aeruginosa-induced neutrophil transepithelial migration using micro-optical coherence technology (µOCT).

Results: Participants included 44 individuals: 16 Outpatient CF, 13 Hospitalized CF, and 15 Non-CF individuals. While no differences were seen with spontaneous migration, CF neutrophils migrated towards S. aureus particles more quickly than non-CF neutrophils (p < 0.05). CF neutrophils, especially Hospitalized CF neutrophils, generated significantly larger aggregates around S. aureus particles over time. Hospitalized CF neutrophils were more likely to have dysfunctional swarming (p < 0.01) and less efficient clearing of C. albicans (p < 0.0001). When comparing trans-epithelial migration towards Pseudomonas aeruginosa epithelial infection, Outpatient CF neutrophils displayed an increase in the magnitude of transmigration and adherence to the epithelium (p < 0.05).

Conclusions: Advanced technologies for characterizing CF neutrophil function reveal significantly altered migratory responses, cell-to-cell clustering, and microbe containment. Future investigations will probe mechanistic basis for abnormal responses in CF to identify potential avenues for novel anti-inflammatory therapeutics.

Keywords: Cystic fibrosis; Inflammation; Micro-fluidics; Micro-optical coherence tomography; Neutrophil.

Figure 1:. CF neutrophils do not exhibit increased spontaneous migration in whole blood.

A drop of diluted blood was loaded into a microfluidic device composed of 8 migratory mazes filled with media. Spontaneous neutrophil migration parameters were studied in the absence of chemoattractants. (A) Schematic representation of the device showing spontaneous neutrophil migration from the whole blood loading chamber to the migratory maze composed of red blood cell (RBC) filters, migration channels, and a maze. (B) The average velocity of neutrophils in non-CF (N=13), CF-Outpatient (N=13), and CF-Hospitalized (N=12) were comparable (non-parametric Kruskal-Wallis with Dunn’s test). (C) Percentages of maze coverage in non-CF, CF-Outpatient, and CF-Hospitalized were similar (parametric ANOVA with Tukey’s test). (D) The relationship between NSM score and maze coverage was highly linear (R²=0.81), with no differences between neutrophils from the three different groups. NSM= neutrophil spontaneous migration

Figure 2:. CF neutrophils display differences in microbe-like particle phagocytosis:

S. aureus bio-particles with fMLP were loaded into microfluidic chambers. Buffy coat-containing neutrophils stained with Hoechst were loaded around these chambers to observe host-pathogen interactions, specifically neutrophil recruitment to and phagocytosis of these particles. (A) A panel of images taken at 1, 3 and 5 hours showing the distribution of neutrophils (Hoechst, blue) and phagocytosis behaviors in response to S. aureus particles (FITC, green), which are a faint green, becoming brighter with increased aggregation. (B) A panel (left to right) showing small areas of phagocytosed particles formed by individual neutrophils to larger aggregates of S. aureus particles formed by multiple neutrophils. Phagocytosed S. aureus particles are shown in the FITC channel, while neutrophils are shown in the BF/DAPI channel. (C) Non-CF neutrophils take longer on average to reach 50% of the maximum number of neutrophils recruited at 5 hours compared to CF-outpatient and CF-hospitalized individuals. (D) The distribution of phagocytosed particles and aggregate sizes formed after 5 hours from Non-CF and CF individuals. Comparisons were made between each group for the percent of accumulated, phagocytoses S. aureus particles that were (E) <15µm or (F) >100µm in size at 5 hours. (G) Average phagocytosed particle sizes measured over time. (H) When large aggregates (>500µm) were formed, average size of aggregate was compared between groups. An ordinary one-way ANOVA with Tukey’s multiple comparison test was used to test for significance. *P<0.05

Figure 3.. CF-hospitalized neutrophils display dysfunctional swarming dynamics and function:

Live C. albicans were patterned in 100 um diameter clusters on poly-l-lysine/Zetag arrays. Purified human neutrophils were stained with Hoechst, then added to the arrays to observe host-pathogen interactions, particularly swarming responses. (A) A panel of images showing a typical swarming response of neutrophils (Hoechst, blue) to a cluster of C. albicans (pink) is shown. (B) The area covered by individual neutrophil swarms was measured at specific timepoints for non-CF and CF-individuals. N=77 non-CF and 143 CF swarms. (C) The CF population was split into CF-outpatient and CF-hospitalized populations. CF-hospitalized displayed two aberrant swarming phenotypes, one with significantly smaller swarms than non-CF or CF-outpatient and the other with significantly larger swarms than non-CF or CF-outpatient. N=77 for non-CF and 71 for CF-outpatient swarms. N=24 for the “small” CF-hospitalized phenotype and N=48 for the CF-hospitalized “large” phenotype. (D) In parallel, neutrophils were isolated using the gelatin/RBC lysis method, then stimulated with DMSO or A23187. LTB4 was quantified by ELISA (E) The amount of C. albicans growth was quantified at 16 hours. CF-hospitalized neutrophils displayed a significant defect in restricting fungal growth compared to non-CF or CF-outpatient populations. (F) The fungal growth allowed by swarms from each donor is also displayed. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 by Kruskal-Wallis with Dunn’s post-test. Scale bar represents 100 µm.

Figure 4.. Neutrophil transepithelial migration across a lung epithelial monolayer.

(A) Schematic illustrating µOCT imaging of the neutrophil transepithelial migration assay. (B) Aggregation and adherence of migrated neutrophils, as well as subsequent detachment of individual neutrophils are shown in representative 3D µOCT images captured at various time points after initiation of transepithelial migration. Using the 60-minute time point to compare paired experiments, we found that there was (C) a significantly greater number of CF-outpatient neutrophils that migrated and (D) adhered to the apical side of the lung-epithelial layer. (E) En face views taken about at 10 µm below the epithelial monolayer revealed that the mean area of adherent CF-outpatient neutrophil columns was significantly larger than that of non-CF neutrophils. (F) Maximal neutrophil migration occurred more rapidly in the CF-outpatient group, and G) unstimulated, unmigrated CF-outpatient neutrophils released greater MPO (as quantified by OD405) per 500,000 neutrophils than healthy controls. N = 7 non-CF, N = 7 CF-outpatient, paired-samples on the same day. * P < 0.05; ** P < 0.01. MPO = myeloperoxidase