Neutrophil extracellular traps activate IL-8 and IL-1 expression in human bronchial epithelia

Abstract

Neutrophil extracellular traps (NETs) provide host defense but can contribute to the pathobiology of diverse human diseases. We sought to determine the extent and mechanism by which NETs contribute to human airway cell inflammation. Primary normal human bronchial epithelial cells (HBEs) grown at air-liquid interface and wild-type (wt)CFBE41o- cells (expressing wtCFTR) were exposed to cell-free NETs from unrelated healthy volunteers for 18 h in vitro. Cytokines were measured in the apical supernatant by Luminex, and the effect on the HBE transcriptome was assessed by RNA sequencing. NETs consistently stimulated IL-8, TNF-α, and IL-1α secretion by HBEs from multiple donors, with variable effects on other cytokines (IL-6, G-CSF, and GM-CSF). Expression of HBE RNAs encoding IL-1 family cytokines, particularly IL-36 subfamily members, was increased in response to NETs. NET exposure in the presence of anakinra [recombinant human IL-1 receptor antagonist (rhIL-1RA)] dampened NET-induced changes in IL-8 and TNF-α proteins as well as IL-36α RNA. rhIL-36RA limited the increase in expression of proinflammatory cytokine RNAs in HBEs exposed to NETs. NETs selectively upregulate an IL-1 family cytokine response in HBEs, which enhances IL-8 production and is limited by rhIL-1RA. The present findings describe a unique mechanism by which NETs may contribute to inflammation in human lung disease in vivo. NET-driven IL-1 signaling may represent a novel target for modulating inflammation in diseases characterized by a substantial NET burden.

Keywords: IL-1; IL-36; IL-8; airway inflammation; neutrophil extracellular trap.

Fig. 1.

Characterization of cell-free human neutrophil extracellular traps (NETs) and experimental design. A–F: fluorescence microscopy of neutrophils stimulated to form NETs or isolated NETs. Representative images depicting colocalization of DNA (Hoechst) and NET-related protein neutrophil elastase (Alexa Fluor 488). Freshly harvested neutrophils were stimulated with 500 nM PMA for 4 h to induce NET formation directly on a slide (A–C) or in a dish, and then cell-free NETs were isolated (D–F). G: schema illustrating the experimental design. NETs in PBS were added to the apical surface of human bronchial epithelial cells (HBEs) grown at air-liquid interface (ALI). H and I: HBEs were grown at ALI and exposed to PBS, 5 µg/mL NETs, 5 µg/mL NETs + 0.5 µg/mL dornase alfa (DA), or 0.5 µg/mL DA for 18 h. NETs alone were simultaneously incubated for 18 h without epithelial cells. Enzymatic activity of myeloperoxidase (MPO) and neutrophil elastase (NE) was measured in the supernatant and analyzed by one-way ANOVA. Each symbol represents NETs isolated from a different donor that were exposed to unrelated HBE donor cells in triplicate wells. (HBE donors = 4–5, NET donors = 3–4.) ****P < 0.0001.

Fig. 2.

Human neutrophil extracellular traps (NETs) alter cytokine secretion by human bronchial epithelial cells (HBEs). HBEs were grown at air-liquid interface and exposed to PBS, 5 μg/mL NETs, 5 μg/mL NETs + 0.5 μg/mL dornase alfa (DA), or 0.5 μg/mL DA for 18 h. NETs alone were simultaneously incubated for 18 h without epithelial cells. Cytokine concentrations were measured in the apical supernatant by Luminex, and IL-8 and IL-1 receptor antagonist (IL-1RA) concentrations were confirmed by ELISA. Results were analyzed by one-way ANOVA. (HBE donors = 6, NET donors = 5.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Human neutrophil extracellular traps (NETs) alter cytokine secretion by wild-type (wt)CFBE41o- cells. wtCFBE41o- cells were exposed to PBS, 5 μg/mL NETs, 5 μg/mL NETs + 0.5 μg/mL dornase alfa (DA), or 0.5 μg/mL DA for 18 h in triplicate wells. NETs alone were simultaneously incubated for 18 h without epithelial cells. Cytokine concentrations were measured in triplicate wells of apical supernatant by Luminex, and IL-8 concentration was confirmed by ELISA. Data were analyzed by one-way ANOVA. Each symbol represents NETs isolated from a different donor. IL-1RA, IL-1 receptor antagonist. (NET donors = 4.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4.

Neutrophil extracellular traps (NETs) influence gene expression in human bronchial epithelial cells (HBEs). HBEs grown at air-liquid interface were exposed to PBS, 5 μg/mL NETs, 5 μg/mL NETs + 0.5 μg/mL dornase alfa (DA), or 0.5 μg/mL DA for 18 h in triplicate wells. Differential gene expression was assessed by RNA sequencing. A: expression of 150 RNAs was increased, whereas expression of 97 RNAs was decreased, after exposure to 5 μg/mL NETs compared with PBS (P < 0.01 and a fold change >2.5). B: the most highly induced RNAs in response to NETs, particularly IL-1 family members (IL-36α, IL-36γ, etc.) and GM-CSF. C and D: HBEs were exposed to the above conditions as well as unstimulated human neutrophils, 0.5 µg/mL genomic DNA (gDNA) + 500 nM PMA, 0.5 µg/mL gDNA, or media control for 18 h. IL-36α and IL-36γ gene expression was assessed by RT-PCR, normalized to 18S, and analyzed by one-way ANOVA comparing each condition relative to RNA expression in HBEs exposed to PBS. Measured in biologic triplicate wells and run in experimental triplicates. E: functional enrichment analysis using ToppGene indicated that IL-1 cytokines and their corresponding antagonists were increased from all three HBE donors. F: schematic representation of the upstream regulators (orange) predicted by Ingenuity Pathway Analysis to influence the transcriptional changes seen in HBEs exposed to NETs. Pink indicates increased expression of RNAs, and aqua indicates decreased expression of RNAs. (HBE donors = 3, NET donors = 3.) *P < 0.05.

Fig. 5.

Recombinant human IL-1 receptor antagonist (rhIL-1RA) blunts neutrophil extracellular trap (NET)-induced cytokine changes in human bronchial epithelial cells (HBEs). HBEs grown at air-liquid interface were pretreated for 1 h with 100 ng/mL rhIL-1RA or PBS and then exposed to 5 μg/mL NETs +/− 100 ng/mL rhIL-1RA for 18 h in triplicate or more wells. A–E: supernatant IL-8 and IL-1RA concentrations were measured by ELISA, and the remaining cytokine concentrations were measured by Luminex and analyzed by one-way ANOVA. F: RNA expression of cytokine genes was measured by RT-PCR, and fold changes were calculated relative to HBEs exposed to PBS and analyzed by one-way ANOVA. (HBE donors = 3, NET donors = 3.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 6.

Recombinant human IL-36 receptor antagonist (rhIL-36RA) decreases expression of proinflammatory cytokine RNAs in human bronchial epithelial cells (HBEs) exposed to neutrophil extracellular traps (NETs). HBEs grown at air-liquid interface were exposed to PBS, NETs, NETs + 1 μg/mL activated rhIL-36RA, or rhIL-36RA alone for 18 h in triplicate wells. A–D: cytokine concentrations in apical supernatant of HBEs were measured by Luminex, and IL-8 concentration was confirmed by ELISA and analyzed by one-way ANOVA. E: RNA expression of cytokine genes was measured by RT-PCR, and fold changes were calculated relative to HBEs exposed to PBS and analyzed by one-way ANOVA. (HBE donors = 3, NET donors = 3.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 7.

Neutrophil elastase (NE) in neutrophil extracellular traps (NETs) contributes to cytokine changes in human bronchial epithelial cells (HBEs). A: neutrophil elastase activity was measured in NETs in the absence and presence of HBE, both with and without the NE inhibitor sivelestat 100 μg/mL for 18 h. B–E: HBEs grown at air-liquid interface were exposed to PBS, NETs, or NETs + 100 μg/mL sivelestat for 18 h. Cytokine concentrations in apical supernatant of HBEs were measured by Luminex, and IL-8 concentration was confirmed by ELISA and analyzed by Student’s t test. F: RNA expression of cytokine genes was measured by RT-PCR, and fold changes were calculated relative to HBEs exposed to PBS and analyzed by one-way ANOVA. (HBE donors = 2, NET donors = 2.) *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 8.

Neutrophil extracellular traps (NETs) increase inflammatory cytokine levels in human airway epithelium. Schematic illustration summarizing present findings. Neutrophils release NETs that contain cytokines [IL-8, IL-1α, and IL-1 receptor antagonist (IL-1RA)] and enzymatically active neutrophil elastase (NE) and myeloperoxidase (MPO). Inset: NETs induce airway epithelia to release increased levels of IL-1α, which binds to IL-1R1 and activates IL-1 signaling to increase airway IL-8 levels. Enhanced IL-1 signaling also increases expression of IL-36α RNA, which further drives IL-8 expression. The addition of the competitive inhibitor rhIL-1RA (anakinra) dampens NET-induced IL-1 signaling, which leads to decreases in levels of IL-8 protein and IL-36α and IL-36RN RNAs.