Progranulin derivative attenuates lung neutrophilic infiltration from diesel exhaust particle exposure

Abstract

Background: Air pollutants, such as diesel exhaust particles (DEPs), induce respiratory disease exacerbation with neutrophilic infiltration. Progranulin (PGRN), an epithelial cell and macrophage-derived secretory protein, is associated with neutrophilic inflammation. PGRN is digested into various derivatives at inflammatory sites and is involved in several inflammatory processes. PGRN and its derivatives likely regulate responses to DEP exposure in allergic airway inflammation.

Aim: To investigate the role of PGRN and its derivatives in the regulation of responses to DEP exposure in allergic airway inflammation.

Methods: A murine model of allergic airway inflammation was generated in PGRN-deficient mice, and they were simultaneously exposed to DEP followed by intranasal administration of full-length recombinant PGRN (PGRN-FL) and a PGRN-derived fragment (FBAC). Inflammatory status was evaluated by bronchoalveolar lavage fluid and histopathologic analyses. Human bronchial epithelial cells were stimulated with DEPs and house dust mites (HDMs), and the effect of FBAC treatment was evaluated by assessing various intracellular signaling molecules, autophagy markers, inflammatory cytokines, and intracellular oxidative stress.

Results: DEP exposure exaggerated neutrophilic inflammation, enhanced IL-6 and CXCL15 secretions, and increased oxidative stress in the murine model; this effect was greater in PGRN-deficient mice than in wild-type mice. The DEP-exposed mice with PGRN-FL treatment revealed no change in neutrophil infiltration and higher oxidative stress status in the lungs. On the contrary, FBAC administration inhibited neutrophilic infiltration and reduced oxidative stress. In human bronchial epithelial cells, DEP and HDM exposure increased intracellular oxidative stress and IL-6 and IL-8 secretion. Decreased nuclear factor erythroid 2-related factor 2 (Nrf2) expression and increased phosphor-p62 and LC3B expression were also observed. FBAC treatment attenuated oxidative stress from DEP and HDM exposure.

Conclusions: FBAC reduced neutrophilic inflammation exaggerated by DEP exposure in a mouse model of allergic airway inflammation by reducing oxidative stress. PGRN and PGRN-derived proteins may be novel therapeutic agents in attenuating asthma exacerbation induced by air pollutant exposure.

Keywords: ROS; asthma; diesel exhaust particles; lung inflammation; progranulin.

FIGURE 1

DEP aggravates OVA‐induced lung inflammation. (A) Experimental design for generating a mouse model of allergic airway inflammation with or without nasal DEP administration. Mice are sensitized through an intraperitoneal injection of OVA/alum, followed by intranasal OVA and DEP challenges (n = 6–10 per group). (B, C) Total cell counts in BAL fluids from different groups and differential cell analysis in BAL fluids. (D) Hematoxylin and eosin (H&E) staining and inflammation score of lungs in the mouse model of allergic airway inflammation and in control mice. Original magnification, ×12.5, ×100. The graphs represent mean ± standard deviation and Mann–Whitney U test results between each experimental group. (E) Mouse IL‐6 and CXCL15 levels in BAL fluids. (F) Representative immunoblotting and quantification graphs of HO‐1, LC3A/B, and GAPDH in lung tissue from the allergic airway inflammation mouse model. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control group and each experimental groups are shown.*p < .05, **p < .01.

FIGURE 2

PGRN regulates airway inflammation in an asthma model exacerbated by DEP. (A) Experimental design of allergic airway inflammation in PGRN‐deficient mice. Mice are sensitized through an intraperitoneal injection of OVA/alum, followed by intranasal OVA and DEP challenges (n = 8–19 per group). (B, C) Total cell counts in BAL fluids from different groups and differential cell analysis in BAL fluids. (D) Hematoxylin and eosin (H&E) staining and inflammation score of lungs in the mouse model of allergic airway inflammation and in control mice. Original magnification, ×12.5, ×100. The graphs represent mean ± standard deviation and Mann–Whitney test results between each experimental group. (E) Mouse IL‐6 and CXCL15 levels in BAL fluids. (F) Representative immunoblotting and quantification graph of HO‐1 and GAPDH in lung tissue from the allergic airway inflammation mouse model. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control group and each experimental groups are shown. *p < .05, **p < .01.

FIGURE 3

DEP‐induced allergic airway inflammation is exacerbated by the administration of full‐length PGRN. (A) Experimental design for generating a mouse model of allergic airway inflammation by exposure to DEP and administration of full‐length PGRN. Mice are sensitized through an intraperitoneal injection of OVA/alum, followed by intranasal OVA and DEP challenges. The full length of PGRN is intranasally administered 6 h before DEP administration (n = 17–33 per group). (B, C) Total cell counts in BAL fluids from different groups and differential cell analysis in BAL fluids. (D) Hematoxylin and eosin (H&E) staining and inflammation score of lungs in the mouse model of allergic airway inflammation and in control mice. Original magnification, ×12.5, ×100. The graphs represent mean ± standard deviation and Mann–Whitney test results between each experimental group. (E) Mouse IL‐6 and CXCL15 levels in BAL fluids. (F) Lipid peroxidation assay for changes in oxidative stress. (G) Representative immunoblotting and quantification graphs of HO‐1, LC3A/B, and GAPDH in lung tissue from the allergic airway inflammation mouse model. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control group and each experimental groups are shown. *p < .05, **p < .01.

FIGURE 4

Recombinant PGRN (FBAC) attenuates allergic airway inflammation. (A) PGRN cleaved by human neutrophil elastase (NE). (B) Experimental design for inducing a mouse model of allergic airway inflammation by exposure to DEP and administration of recombinant PGRN (FBAC). Mice are sensitized through an intraperitoneal injection of OVA/alum, followed by intranasal OVA and DEP challenges. Recombinant PGRN (FBAC) is intranasally administered 6 h before DEP administration (n = 15–33 per group). (C, D) Total cell counts in BAL fluids from different groups and differential cell analysis in BAL fluids. (E) Mouse IL‐6 and CXCL15 levels in BAL fluids. (F) Lipid peroxidation assay for changes in oxidative stress. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control group and each experimental groups are shown. (G) Hematoxylin and eosin (H&E) staining and inflammation score of lungs in the mouse model of allergic airway inflammation and in control mice. Original magnification, ×12.5, ×100. The graphs represent mean ± standard deviation and Mann–Whitney test results between each experimental group. (H) Representative immunoblotting and quantification graphs of HO‐1, LC3A/B, and GAPDH in lung tissue from the allergic airway inflammation mouse model. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control group and each experimental groups are shown. *p < .05, **p < .01.

FIGURE 5

Progranulin regulates oxidative stress and inflammation induced by DEP in bronchial epithelial cells. (A) Cell viability of BEAS‐2B cells treated with DEP for 48 h. (B) Representative immunoblotting of HO‐1, LC3A/B, and GAPDH, and the phosphorylated and unphosphorylated forms of Nrf2 and p62 in BEAS‐2B cells treated with different concentrations of DEP and HDM for 48 h. (C) ROS analysis with DEP and HDM treatment and antioxidant effect of full‐length or fragmented PGRN treatment for 15 min in BEAS‐2B cells. (D, E) Human IL‐8 and IL‐6 levels in the supernatants of BEAS‐2B cells and HSAEpC cells treated with DEP, HDM, and full‐length or fragmented PGRN for 48 h. (F) TEER values (hm*cm²) in HSAEpC cells. The graphs show average ± standard deviation, and the paired Student's t‐test results between the control and experimental groups are shown.