Heme scavenging reduces pulmonary endoplasmic reticulum stress, fibrosis, and emphysema

Abstract

Pulmonary fibrosis and emphysema are irreversible chronic events after inhalation injury. However, the mechanism(s) involved in their development remain poorly understood. Higher levels of plasma and lung heme have been recorded in acute lung injury associated with several insults. Here, we provide the molecular basis for heme-induced chronic lung injury. We found elevated plasma heme in chronic obstructive pulmonary disease (COPD) (GOLD stage 4) patients and also in a ferret model of COPD secondary to chronic cigarette smoke inhalation. Next, we developed a rodent model of chronic lung injury, where we exposed C57BL/6 mice to the halogen gas, bromine (Br2) (400 ppm, 30 minutes), and returned them to room air resulting in combined airway fibrosis and emphysematous phenotype, as indicated by high collagen deposition in the peribronchial spaces, increased lung hydroxyproline concentrations, and alveolar septal damage. These mice also had elevated pulmonary endoplasmic reticulum (ER) stress as seen in COPD patients; the pharmacological or genetic diminution of ER stress in mice attenuated Br2-induced lung changes. Finally, treating mice with the heme-scavenging protein, hemopexin, reduced plasma heme, ER stress, airway fibrosis, and emphysema. This is the first study to our knowledge to report elevated heme in COPD patients and establishes heme scavenging as a potential therapy after inhalation injury.

Keywords: COPD; Fibrosis; Pulmonology.

Figure 1. Plasma heme and ER stress levels are elevated in patients with very severe COPD and in ferrets exposed to cigarette smoke.

Total heme levels were measured in the plasma of COPD patients and their healthy counterparts. Although total heme levels were not significantly higher in COPD patients compared with the healthy individuals (P = 0.06) (n = 18–22) (A), the stratification of patients according to the severity of the disease revealed significantly elevated plasma heme in COPD patients with GOLD stage 4 disease compared with the healthy individuals or patients with the mild disease (n = 6–18) (B). Plasma heme levels were also significantly higher in ferrets exposed to cigarette smoke for 6 months, which induced emphysema and attenuated lung function (n = 6–8) (C). ELISA showed that GOLD stage 4 COPD patients had significantly higher levels of ER stress marker Grp78/Bip (n = 6–14) (D). Values are means ± SEM. *P < 0.05 versus healthy patients or air-exposed ferrets; ^†P < 0.05 versus COPD GOLD stage 2; ^‡P < 0.05 versus COPD GOLD stage 3, by unpaired t test for 2 groups or 1-way ANOVA followed by Tukey’s post hoc testing for more than 2 groups.

Figure 2. Lung injury and fibrosis in rodent model of inhalation injury.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and returned to room air. Plasma heme and acute and chronic lung injury parameters were measured in mice on days 1, 7, 14, or 21 after Br₂ exposure. Plasma levels of total heme were elevated in mice until 14 days after Br₂ inhalation (n = 6–11) (A). Bronchoalveolar lavage fluid (BALF) showed a significant increase in protein levels (n = 9–14) (B) and total cell count (n = 9–17) (C) on days 14 and 21 after Br₂ inhalation. Peripheral lung tissue staining for α-smooth muscle actin (α-SMA) (n = 5) (D) and with Masson’s trichrome stain (n = 5) (E) demonstrated an increased accumulation of α-SMA and thickening of the smooth muscle layer and collagen deposition (blue stain) primarily around airways on days 14 and 21 after Br₂. Characteristic images were obtained from the indicated number of lungs for each condition. Similarly, the quantification of collagen by measuring lung hydroxyproline levels showed significant increases at 14 and 21 days after Br₂ inhalation (F). Values are means ± SEM. *P < 0.05 versus air-exposed C57BL/6 mice by 1-way ANOVA followed by Tukey’s post hoc test. Scale bars are 100 µm.

Figure 3. Acute exposure to Br₂ induces lung emphysematous changes.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. On days 7, 14, or 21 after Br₂ exposure, mouse lung compliance was assessed by the slope of the deflation limbs of pressure-volume (PV) curves. Br₂ inhalation increased lung volumes as indicated by shifting up and left of PV curves on days 14 and 21 after exposure (n = 5–9) (A). The staining of peripheral lung tissue with hematoxylin and eosin (H&E) showed airspace enlargement (B) and increased alveolar mean linear intercept (L_m) (n = 5). Scale bars are 100 µm. (C) on days 14 and 21 after exposure. Characteristic images are shown. Finally, plasma elastase levels (n = 6–10) (D) and BALF elastase activity (n = 7–14) (E) were elevated in Br₂-exposed mice on days 14 and 21 after exposure. Values are means ± SEM. *P < 0.05 versus air-exposed C57BL/6 mice by 1-way ANOVA followed by Tukey’s post hoc test. PV curves were analyzed by 2-way ANOVA with Bonferroni’s post hoc test.

Figure 4. ER stress is increased in Br₂-exposed mice.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. On days 7, 14, or 21 after exposure, lungs were harvested and immunoblotted for ER stress markers Grp78/Bip (n = 6–10) (A), phospho-PERK (n = 10) (B), phospho-IRE1α (n = 8) (C), and ATF6 (n = 8) (D). Br₂ exposure increased the lung expression of Grp78, phopho-PERK and the downstream transcriptional effectors of the PERK pathway ATF4 (n = 7) (E) and CHOP (n = 5–8) (F), 14 days after exposure. Values are means ± SEM. *P < 0.05 versus air-exposed C57BL/6 mice by 1-way ANOVA followed by Tukey’s post hoc test.

Figure 5. Attenuation of ER stress abrogates lung injury and fibrosis.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. Some Br₂-exposed mice received an intraperitoneal injection of the ER stress inhibitor salubrinal (1 mg/kg BW), starting at 1 hour after Br₂ exposure, and then daily for 13 consecutive days. Air-exposed and some Br₂-exposed mice received DMSO (vehicle) as a control. Fourteen days after Br₂ exposure, immunoblot of lung tissue showed that salubrinal increased phospho-eIF2α levels (n = 7–8) (A) but decreased lung CHOP levels (n = 12–13) (B) after Br₂ exposure. Salubrinal also attenuated BALF protein (n = 5–6) (C) and total cell count (n = 5–6) (D) in Br₂-exposed mice. Masson’s trichrome staining (n = 5) (E) and quantification of lung hydroxyproline levels (n = 5–6). Scale bars are 100 µm. (F) showed a decrease in collagen levels and lung fibrotic changes 14 days after Br₂ exposure in salubrinal-treated mice. Values are means ± SEM. *P < 0.05 versus air + DMSO–treated mice and ^†P < 0.05 versus Br₂ + DMSO–treated mice by 1-way ANOVA followed by Tukey’s post hoc test.

Figure 6. Attenuation of ER stress reduces lung emphysematous changes.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. Some Br₂-exposed mice were given an intraperitoneal injection of the ER stress inhibitor salubrinal (1 mg/kg BW), starting at 1 hour after Br₂ exposure, and then daily for 13 consecutive days. Air-exposed and some Br₂-exposed mice received DMSO (vehicle) as a control. Fourteen days after Br₂ exposure, the lung volumes were increased in mice as indicated by the shifting up and left of the pressure-volume (PV) curves. (n = 5–6) (A). Staining of peripheral lung tissue with hematoxylin and eosin (H&E) showed airspace enlargement (n = 5) (B), which was quantified by measuring alveolar mean linear intercept (L_m) (n = 5). Scale bars 100 µm. (C). Treatment with salubrinal attenuated these Br₂-induced emphysematous changes in mouse lung. Salubrinal also reduced plasma elastase levels (n = 10–16) (D) and BALF elastase activity (n = 5–7) (E) in Br₂-exposed mice. Values are means ± SEM. All animals were males. *P < 0.05 versus air + DMSO–treated mice and ^†P < 0.05 versus Br₂ + DMSO–treated mice by 1-way ANOVA followed by Tukey’s post hoc test. PV curves were analyzed by 2-way ANOVA with Bonferroni’s post hoc test.

Figure 7. ATF4-haplodeficient mice (ATF4^+/–) mice are protected against inhalation injury.

Male wild-type (WT, C57BL/6) and ATF4^+/– mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. Fourteen days later, mouse lungs were harvested. Immunoblot analyses showed that ATF4^+/– mice had lower lung ATF4 (n = 6) (A) and CHOP levels (n = 6) (B) compared with WT mice exposed to Br₂. Masson’s trichrome staining (n = 5) (C) and quantification of lung hydroxyproline levels (n = 6–10). Scale bars are 100 µm. (D) showed increased collagen deposition primarily around airways in the WT mice compared with the ATF4^+/– mice. Assessment of lung pressure-volume (PV) curves demonstrated that Br₂ exposure increased lung volumes as indicated by shifting up and left of PV curve in the WT but not the ATF4^+/– mice (n = 4 for WT + air and n = 5 for others) (E). Staining of peripheral lung tissue with hematoxylin and eosin (H&E) showed airspace enlargement after Br₂ exposure (n = 5–6) in WT but not ATF4^+/– mice (C), which was quantified by measuring alveolar mean linear intercept (L_m) (n = 5–6) (F). Plasma elastase levels (n = 6–13) (G) and BALF elastase activity (n = 6–7) (H) were significantly higher in the WT compared with the ATF4^+/– mice after Br₂ exposure. Values are means ± SEM. *P < 0.05 versus WT + air, ^†P < 0.05 versus ATF^+/– + air, and ‡P < 0.05 versus WT + Br₂ for A and B; ^†P < 0.05 versus WT + Br₂ for D–H by 1-way ANOVA followed by Tukey’s post hoc test. PV curves were analyzed by 2-way ANOVA with Bonferroni’s post hoc test.

Figure 8. Heme scavenging attenuates ER stress.

Immunoblot analysis showed that the incubation of the human bronchial epithelial cells with hemin (a form of heme, 25 μM), increased the ER stress markers ATF4 and CHOP (n = 3) (A) at 6 and 24 hours after hemin challenge. In addition, male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. Some Br₂-exposed mice were given an intraperitoneal injection of purified human hemopexin (Hx) (4 μg/g BW) 1 hour after Br₂ exposure. All air-exposed mice and some Br₂-exposed mice received saline injection as an appropriate control. Hx attenuated plasma total heme levels in Br₂-exposed mice (n = 9–24) (B). Immunoblotting showed that Hx lowered Br₂-induced ER stress markers, ATF4 (n = 10) (C) and CHOP (n = 11–15) (D), in mouse lungs 14 days after Br₂ exposure. Similarly, immunohistochemical staining of lung sections showed an increased accumulation of ATF4 and CHOP (n = 4–5) (E) (arrows showing brown stain) lining bronchioles and in the lung parenchyma in Br₂-exposed mice 14 days after exposure. Hx lowered ATF4 and CHOP levels. Values are mean ± SEM. All animals were males. Scale bars are 200 µm. For A, *P < 0.05 versus air + saline, ^†P < 0.05 versus Br₂ + saline (1 day after), and ^‡P < 0.05 versus Br₂ + saline (14 days after); for B and C, ^†P < 0.05 versus Br₂ + saline (14 days after) by 1-way ANOVA followed by Tukey’s post hoc test.

Figure 9. Hemopexin attenuates lung injury, airway fibrosis, and lung emphysema.

Male C57BL/6 mice were exposed to air or Br₂ gas (400 ppm, 30 minutes) and then returned to room air. Some Br₂-exposed mice were given an intraperitoneal injection of purified human hemopexin (Hx) (4 μg/g BW) 1 hour or 5 days after Br₂ exposure. All air-exposed mice and some Br₂-exposed mice received saline injection as an appropriate control. Fourteen days after Br₂ exposure, mouse BALF protein levels (n = 5–9) (A) and total cell count (n = 5–7) (B) were elevated in saline-treated mice but were significantly lower in Hx-treated mice. Hx-treated mice had decreased lung deposition of collagen on Masson’s trichrome staining (n = 5–8) (C) and lower lung hydroxyproline levels (n = 5–8) (D) compared with saline-treated mice, 14 days after Br₂ inhalation. Assessment of mouse lung pressure-volume (P-V) curves demonstrated that Br₂ exposure increased lung volumes, as indicated by the shifting up and left of PV curve in the saline-treated mice but not in the Hx-treated mice (n = 5–10) (F). Hematoxylin and eosin (H&E) staining of lungs showed that Hx prevented Br₂-induced alveolar septa damage (n = 5–8) (C) and reduced alveolar L_m (n = 5–8). Scale bars 100 µm. (E). In addition, Hx lowered plasma elastase levels (n = 14–16) (G) and BALF elastase activity (n = 10–13) (H) in Br₂-exposed mice. The Kaplan-Meier curve demonstrated that Hx reduced mortality after Br₂ exposure (n = 42 for Br₂ + saline; n = 20 for Br₂ + Hx [1 hour after]; n = 17 for Br₂ + Hx [5 days after]) (I). *P < 0.05 versus air + saline and ^†P < 0.05 versus Br₂ + saline by 1-way ANOVA followed by Tukey’s post hoc test. PV curves were analyzed by 2-way ANOVA with Bonferroni’s post hoc test. Overall survival was analyzed by the Kaplan-Meier method. Differences in survival were tested for statistical significance by the log-rank test.