Repetitive sulfur dioxide exposure in mice models post-deployment respiratory syndrome

Abstract

Soldiers deployed to Iraq and Afghanistan have a higher prevalence of respiratory symptoms than nondeployed military personnel and some have been shown to have a constellation of findings on lung biopsy termed post-deployment respiratory syndrome (PDRS). Since many of the subjects in this cohort reported exposure to sulfur dioxide (SO₂), we developed a model of repetitive exposure to SO₂ in mice that phenocopies many aspects of PDRS, including adaptive immune activation, airway wall remodeling, and pulmonary vascular (PV) disease. Although abnormalities in small airways were not sufficient to alter lung mechanics, PV remodeling resulted in the development of pulmonary hypertension and reduced exercise tolerance in SO₂-exposed mice. SO₂ exposure led to increased formation of isolevuglandins (isoLGs) adducts and superoxide dismutase 2 (SOD2) acetylation in endothelial cells, which were attenuated by treatment with the isoLG scavenger 2-hydroxybenzylamine acetate (2-HOBA). In addition, 2-HOBA treatment or Siruin-3 overexpression in a transgenic mouse model prevented vascular remodeling following SO₂ exposure. In summary, our results indicate that repetitive SO₂ exposure recapitulates many aspects of PDRS and that oxidative stress appears to mediate PV remodeling in this model. Together, these findings provide new insights regarding the critical mechanisms underlying PDRS.NEW & NOTEWORTHY We developed a mice model of "post-deployment respiratory syndrome" (PDRS), a condition in Veterans with unexplained exertional dyspnea. Our model successfully recapitulates many of the pathological and physiological features of the syndrome, revealing involvement of the ROS-isoLGs-Sirt3-SOD2 pathway in pulmonary vasculature pathology. Our study provides additional knowledge about effects and long-term consequences of sulfur dioxide exposure on the respiratory system, serving as a valuable tool for future PDRS research.

Keywords: constrictive bronchiolitis; experimental animal models; oxidative stress; pulmonary hypertension; sulfur dioxide.

Graphical abstract

Figure 1.

SO₂ exposure model optimization and validation. A: normal-appearing distal airway in untreated WT mice (left) and epithelial sloughing and epithelial cell agglomerate in distal airway lumen of 125-ppm SO₂-exposed mice for 4 h (right). Periodic acid-Schiff (PAS) staining. Scale bars = 50 µm. B: epithelial damage score after 2 and 4 h of 125 ppm SO₂ exposure. Groups were compared using Student’s t test and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with untreated mice, †P < 0.05 compared with untreated and SO₂-exposed (for 2 h) mice. n = 4 mice/group. C: normal-appearing distal airway in untreated WT mice (left) and collagen deposition in distal airway wall of 125 ppm SO₂-exposed mice for 4 h at 4 wk after the last exposure (right). Picrosirius red staining. Scale bars = 50 µm. D: collagen content in distal airway wall increased exponentially over postexposure time. Data are presented as means (red dots) ± SD (whiskers). Groups were compared using Student’s t test and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with untreated mice. n = 3–12 mice/time point. E: normal-appearing distal airway in untreated WT mice (left) and distal airway wall thickening after 10 days of 125 ppm SO₂-exposed mice for 4 h and 4 wk of postexposure time (right). Red lines outline the subepithelial layer. PAS staining. Scale bars = 50 µm. F: dose-dependent distal airway wall thickening. V_S(se,rbm), subepithelium thickness. Groups were compared using Student’s t test and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with untreated mice, †P < 0.05 compared with untreated, single and three times SO₂-exposed mice. n = 12 mice/study group. SO₂, sulfur dioxide; WT, wild type.

Figure 2.

SO₂ inhalation exposure leads to distal airway remodeling and chronic inflammatory cell influx. A: strategy and timeline of SO₂ exposure for reproduction of PDRS-associated pathological lesions in mice. B: normal-appearing bronchovascular bundle in untreated WT mice; increased collagen deposition in distal small airways in WT mice exposed to 125 ppm SO₂ for 4 h a day, 5 days per week for 2 wk, followed by 28 days postexposure period (red arrow). Picrosirius red staining. Scale bars = 50 µm. C: collagen deposition in distal airways. Groups were compared using Student’s t test. *P < 0.001 compared with untreated mice. n = 12 mice/study group. D–F: increased numbers of CD4⁺ and CD8⁺ T cells, conventional type 1 (cDC1) and 2 (cDC2) and monocyte-derived dendritic cells (moDCs), and alveolar macrophages (AMs) in SO₂-exposed mice compared with unexposed age-matched mice. Ne, neutrophils. Groups were compared using Student’s t test. *P < 0.01 compared with untreated mice. n = 6 mice/study group. PDRS, post-deployment respiratory syndrome; SO₂, sulfur dioxide; WT, wild type.

Figure 3.

Effects of SO₂ inhalation exposure on pulmonary vasculature. A: normal-appearing bronchovascular bundle in untreated WT mice; increased medial thickness (red arrow) and fibrosis of adventitia in pulmonary artery in WT mice exposed to SO₂ (4 wk after exposure). Verhoeff-Van Gieson staining. Scale bars = 50 µm. B: bar plot showing tunica media thickness, V_S(media,iem), in adjacent to distal airway pulmonary arteries. Groups were compared using Student’s t test. *P < 0.001 compared with untreated mice. n = 12 mice/study group. C: images illustrating the examples of nonmuscularized, partially muscularized (<50% vessel circumference with smooth muscle), and muscularized (>50% vessel circumference with smooth muscle) blood vessels with size <50 µm. Double immunofluorescence with anti-CD31 (red) and anti-α-smooth muscle actin (green) antibodies. Scale bars = 25 µm. The 2-D pie charts illustrate the distribution of nonmuscularized, partially muscularized, and muscularized intra-acinar blood vessels in untreated and SO₂-exposed mice. P < 0.01 for group at 4 wk after SO₂ exposure compared with untreated control mice by chi-square test. n = 12 mice/study group. 2-D, two-dimensional; SO₂, sulfur dioxide; WT, wild type.

Figure 4.

Long-term physiological effects of SO₂ exposure. A–C: total respiratory system resistance (A), compliance (B), and elastance (C) measurements by the flexiVent “Snapshot model.” Groups were compared using Student’s t test. n = 11 or 12 mice/study group. D: incremental treadmill exercise test showing reduced maximal running speed in SO₂-exposed mice (red) compared with unexposed mice (green). *P < 0.05 compared with untreated mice (Student’s t test). E and F: increase of RV systolic pressure (RVSP) and Fulton index in SO₂-exposed mice compared with unexposed mice. Pulmonary hemodynamics were analyzed via the jugular vein using 1.4-F Mikro-Tip catheter. All experiments are conducted 6 mo after the last exposure. Groups were compared using Student’s t test. *P < 0.05 compared with untreated mice. n = 17 or 18 mice/study group. SO₂, sulfur dioxide.

Figure 5.

ROS and isoLGs accumulation with downstream inactivation of Sirt3 and SOD2 after SO₂ exposure. A and B: plots showing increased 2OH-ethidium (marker of superoxide) normalized to protein content (2OH-Et/Pr) in lung tissue of 1 mm thick section (A) and endothelial cells isolated from the lungs (B). Groups were compared using Mann–Whitney U test (A) or Student’s t test (B). n = 4–6 mice/study group. C: ROS accumulation in endothelial cells after SO₂ exposure compared with vehicle. Dihydroethidium (DHE) stained in red. Scale bars = 25 µm. Note that data in A–C were obtained immediately after the last SO₂ exposure. D: representative Western blots for proteins with isoLG adducts and SOD2 acetylation in isolated endothelial cells. Endothelial cells were isolated from lungs of untreated or SO₂-exposed mice with or without supplementation of isoLG scavenger 2-HOBA (1 g/L in drinking water). E and F: densitometry graphs showing the accumulation of isoLG protein adducts (E) and SOD2 hyperacetylation (F) in SO₂-exposed mice and effect mitigation by 2-HOBA supplementation. Groups were compared using Mann–Whitney U test (E) or Student’s t test (F) and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with unexposed mice, †P < 0.05 compared with SO₂-exposed mice. n = 6 mice/study group. Measurements in D–F were performed 4 wk after SO₂ exposure. 2-HOBA, 2-hydroxybenzylamine acetate; isoLGs, isolevuglandins; ROS, reactive oxygen species; SO₂, sulfur dioxide.

Figure 6.

2-HOBA supplementation prevents vascular remodeling after SO₂ exposure. A and B: graphs showing the distal airway wall thickening (A) and collagen deposition (B) in WT mice exposed to SO₂ with or without 2-HOBA supplementation. V_S(se,rbm), subepithelium thickness. C: graph showing the protective effect of 2-HOBA on increased medial thickness in pulmonary arteries adjacent to distal airways in WT mice exposed to SO₂. V_S(media,iem), tunica media thickness. Groups were compared using Mann–Whitney U test (A and C) or Student’s t test (B) and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with untreated mice, †P < 0.05 compared with SO₂-exposed mice. n = 6–12 mice/study group. D: 2-D pie charts illustrate distribution of nonmuscularized, partially muscularized, and muscularized intra-acinar blood vessels (<25 μm in size) in WT untreated or exposed to SO₂ mice with or without 2-HOBA supplementation. P < 0.01 for group exposed to SO₂ without 2-HOBA supplementation compared with unexposed groups by chi-square test. P < 0.05 for group exposed to SO₂ with 2-HOBA supplementation compared with group exposed to SO₂ without 2-HOBA supplementation. n = 6–12 mice/study group. All measurements were performed 4 wk after SO₂ exposure. 2-HOBA, 2-hydroxybenzylamine acetate; SO₂, sulfur dioxide; WT, wild type.

Figure 7.

Sirt3 overexpression prevents vascular remodeling after SO₂ exposure. A and B: graphs showing the distal airway wall thickening (A) and collagen deposition (B) in WT or Sirt3 overexpressing (OX) mice exposed to SO₂. V_S(se,rbm), subepithelium thickness. C: graph showing the protective effect of Sirt3 overexpression on increased medial thickness in pulmonary arteries adjacent to distal airways in WT mice exposed to SO₂. V_S(media,iem), tunica media thickness. Groups were compared using Mann–Whitney U test (A and C) or Student’s t test (B) and P values were Bonferroni-adjusted for multiple-group comparisons. *P < 0.05 compared with untreated mice, †P < 0.05 compared with SO₂-exposed mice. n = 5–11 mice/study group. D: 2-D pie charts illustrate distribution of nonmuscularized, partially muscularized, and muscularized intra-acinar blood vessels (<25 μm in size) in WT and Sirt3 OX untreated or exposed to SO₂ mice. P < 0.01 for group of WT mice exposed to SO₂ compared with unexposed groups by chi-square test. P < 0.05 for group of Sirt3 OX mice exposed to SO₂ compared with WT mice exposed to SO₂. n = 5–12 mice/study group. All measurements were performed 4 wk after SO₂ exposure. 2-D, two-dimensional; SO₂, sulfur dioxide; Sirt3, sirtuin-3; WT, wild type.