Persistent neutrophilic inflammation is associated with delayed toxicity of phenylarsine oxide in lungs

Abstract

Phenyl arsine oxide (PAO) is a vesicant, similar to Lewisite, a potential chemical warfare agent and an environmental contaminant. PAO-induced skin burns can trigger acute organ injury, including lungs. We have recently demonstrated that PAO burns can have delayed toxicity, although the specific mechanism/s remain to be determined. A single cutaneous exposure to PAO resulted in inflammatory acute lung injury at 6 and 24 h. While acute injury subsiding by 1 week, we observed a significant airway remodeling at 10 weeks post-PAO exposure. The mechanism of prolonged PAO toxicity was associated with the influx of neutrophils that produced harmful neutrophil extracellular traps (NETs). We demonstrated that the crosstalk between NET deployments and expression of IL-33, a pro-remodeling mediator was associated with the development of peribronchial fibrosis. In summary, these results suggest that a single cutaneous exposure to PAO causes the acute inflammatory phase followed by NETs/IL-33 feed forward signaling implicated for the persistent neutrophil influx and NETs formation resulting in airway remodeling.

Keywords: Airway remodeling; Arsenicals; IL-33; Neutrophil extracellular traps; Persistent inflammation; Phenyl Arsine oxide.

Fig. 1

Cutaneous PAO induced lung injury in mice: Mice (n = 5 per group) were cutaneously exposed to PAO (0 or 150 µM) for 6 h, 24 h, 1 week, and 10 weeks. (a) Representative images of H&E stained lung sections from indicated groups of mice. Scale bar = 100 μm. (b) Lung injury Score. The graph is presented as mean ± SEM, n = 5. * indicates statistically significant differences compared to the control group. One-way ANOVA (****P < 0.0001).

Fig. 2

Cutaneous PAO-induced neutrophilic inflammation in the lungs: (a) Immunohistochemistry of neutrophil elastase (NE) in control and PAO exposure groups. Scale bar = 100 μm. (b) Representative immunoblot analysis of the whole lung lysates and (c) quantitative analysis for NE and GAPDH. The densitometry for NE, normalized with the loading control GAPDH. Each lane represents individual mice, and quantitative analysis were performed in 5 mice/group. (d) Percent of neutrophils counted in BALF (e) Digested lung of controls and PAO-exposed 6 h, 24 h, 1 week and 10 weeks mice groups by flow cytometry with gate (F480-CD11b + Ly-6G+). All data are presented as mean ± SEM, n = 5. * Indicates statistically significant differences compared to the control group. One-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (f–j) ELISAs for cytokines in BALF for (f) TNF-α, (g) IL-1β, (h) IL-6, (i) MIP-2, and (j) KC. All data are presented as mean ± SEM, for controls, 6 h, 24 h, 1 week and 10 weeks (n = 5). One-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 3

PAO-exposed mice have persistent presence of Netosis in lungs: (a) Immunohistochemistry of Netosis marker; citrullinated histones 3 (Cit-H3) in control and PAO exposure groups. Scale bar = 100 μm. (b) ELISA for Cit-H3 in BALF and (c) whole lung lysates of mice exposed to PAO after 6 h, 24 h, 1 week and 10 weeks. (d) Immunohistochemistry of IL-33 in mouse lung sections after PAO treatment. Scale bar 100 µm. (e) ELISA for IL-33 in whole lung lysates of mice exposed to PAO. All data are presented as mean ± SEM, * indicates statistically significant differences compared to the control group (n = 5 / group). One-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001).

Fig. 4

A single cutaneous exposure to PAO causes delayed airway remodeling. (a) Representative images show lung sections stained with Picro-Sirius. (b) Graph represents the percentage of Picro Sirius-stained area of the total tissue-stained area. (c) Levels of α-SMA positive cells within the airways of PAO-treated mice. Scale bar 100 μm. AW = airway. (d) Graph represents the percentage of tissue area positive for DAB intensity for the α-SMA immunostaining around the airways. Here * indicates statistically significant differences compared to the control group. One-way ANOVA (*P < 0.05****P < 0.0001).

Fig. 5

PAO-induced production of IL-33 in primary human airway epithelial cells. BEAS-2B cells were treated with PAO (0, 20, 50, 100 nM) for 24 h. (a, b) Immunoblot and quantitative analysis for the expression of IL-33. (c) Representative immunoblot (d) quantitative analysis for the expression of pSMAD2/3, SMAD2/3, and GAPDH. Experiments were conducted in triplicate. Data are presented as mean ± SEM, *P < 0.05, ANOVA, as compared control (vehicle) to PAO-treated cells.

Fig. 6

NETs-induced expression of IL-33 and pro-fibrotic SMAD2/3 signaling in BEAS-2B cells. BEAS-2B cells were treated with NETs (100 and 200 ng/ml) for different time points as shown. Representative immunoblot and densitometry analyses of (a, b) IL-33; (c, d) pSMAD2/3, (c–e) SMAD2/3. GAPDH was used as loading control.; and (f–g) for vimentin in NETs treated BEAS-2B cell lysates. β-actin used as loading control. All quantitative analysis is from 3 experiments. All data are presented as mean ± SEM. One-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.01).