Osteopontin modulates inflammation, mucin production, and gene expression signatures after inhalation of asbestos in a murine model of fibrosis - PubMed (original) (raw)

Osteopontin modulates inflammation, mucin production, and gene expression signatures after inhalation of asbestos in a murine model of fibrosis

Tara Sabo-Attwood et al. Am J Pathol. 2011 May.

Abstract

Inflammation and lung remodeling are hallmarks of asbestos-induced fibrosis, but the molecular mechanisms that control these events are unclear. Using laser capture microdissection (LCM) of distal bronchioles in a murine asbestos inhalation model, we show that osteopontin (OPN) is up-regulated by bronchiolar epithelial cells after chrysotile asbestos exposures. In contrast to OPN wild-type mice (OPN(+/+)) inhaling asbestos, OPN null mice (OPN(-/-)) exposed to asbestos showed less eosinophilia in bronchoalveolar lavage fluids, diminished lung inflammation, and decreased mucin production. Bronchoalveolar lavage fluid concentrations of inflammatory cytokines (IL-1β, IL-4, IL-6, IL-12 subunit p40, MIP1α, MIP1β, and eotaxin) also were significantly less in asbestos-exposed OPN(-/-) mice. Microarrays performed on lung tissues from asbestos-exposed OPN(+/+) and OPN(-/-) mice showed that OPN modulated the expression of a number of genes (Col1a2, Timp1, Tnc, Eln, and Col3a1) linked to fibrosis via initiation and cross talk between IL-1β and epidermal growth factor receptor-related signaling pathways. Novel targets of OPN identified include genes involved in cell signaling, immune system/defense, extracellular matrix remodeling, and cell cycle regulation. Although it is unclear whether the present findings are specific to chrysotile asbestos or would be observed after inhalation of other fibers in general, these results highlight new potential mechanisms and therapeutic targets for asbestosis and other diseases (asthma, smoking-related interstitial lung diseases) linked to OPN overexpression.

Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1

OPN mRNA expression is increased in lung epithelial cells exposed to asbestos. C57BL/6 mice were exposed to chrysotile asbestos (8 mg/m3 per day) for 3, 9, and 40 days. Lung tissue was collected and RNA was purified and analyzed for gene expression changes using microarrays and expression validated by qPCR. A: The transcriptional profile for OPN shows increased expression at all time points tested, compared with air-exposed animals (ie, clean air-exposed). B: Lungs were sectioned from mice exposed to asbestos for 3 days and 9 days, and LCM was performed on epithelial cells. Isolated RNA was examined for gene expression using GEArray analysis. Genes that were significantly altered by asbestos-exposed versus air-exposed animals are plotted. *P ≤ 0.05, air versus asbestos exposure.

Figure 2

Figure 2

General asbestos-induced injury and immune cell profiles measured in BALF. OPN+/+ and OPN−/− mice were exposed to chrysotile asbestos (8 mg/m3 per day) for 9 days. The mice were lavaged and BALF was collected and processed for lactate dehydrogenase (A), total cell counts (B), and differential cell counts (C). *P ≤ 0.05, asbestos versus air exposure; **P ≤ 0.05, asbestos-exposed OPN+/+ versus OPN−/−. Cell types: eosinophils (Eosin), polymorphonuclear cells (PMN), lymphocytes (Lymph), and macrophages (Macs).

Figure 3

Figure 3

Decreased severity of asbestos-induced inflammation and mucin in OPN−/− mice. OPN+/+ and OPN−/− mice were exposed to chrysotile asbestos (8 mg/m3 per day) for 9 days. Lung tissue sections were stained with H&E or Alcian Blue-PAS and were scored for inflammation or mucin production, respectively. A: Severity of inflammation scored by assessing influx of macrophages and polymorphonuclear cells in H&E-stained tissue sections from asbestos-exposed mice. No detectable inflammation was observed in any air-exposed animals. B: A representative histological section of mucin staining with Alcian Blue-PAS for each treatment group. Arrowheads indicate examples of positive staining (pinkish-purple). C: Percentage of bronchioles affected with mucin in Alcian Blue-PAS stained tissue sections from asbestos-exposed mice. No detectable mucin was observed in any air-exposed animals. *P ≤ 0.05, asbestos versus air exposure; **P ≤ 0.05, asbestos-exposed OPN+/+ versus OPN−/−.

Figure 4

Figure 4

Inhibition of asbestos-induced production of immune-related proteins in BALF in OPN−/− mice. OPN+/+ and OPN−/− mice were exposed to chrysotile asbestos (8 mg/m3 per day) for 9 days. Bio-Plex analysis of BALF was performed. Chemokines and cytokines that were altered by asbestos in OPN+/+ and differentially produced in OPN−/− mice are shown. All values are presented as pg/mL BALF. Open bars: air-exposed animals; black bars: asbestos-exposed animals. *P ≤ 0.05, asbestos versus air exposure; **P ≤ 0.05, asbestos-exposed OPN+/+ versus OPN−/−.

Figure 5

Figure 5

Gene profiling in OPN−/− mice exposed to asbestos. OPN+/+ and OPN−/− mice were exposed to chrysotile asbestos (8 mg/m3 per day) for 9 days. Total RNA was isolated from lung tissue and processed using microarrays. A: Total number of significant gene changes in lung tissue of OPN+/+ and OPN−/− asbestos-exposed mice and OPN−/− air-exposed mice, compared with OPN+/+ air-exposed mice. B: The number of genes that were significantly altered in lung tissue (P ≤ 0.05 and ≥2-fold compared with air-exposed OPN+/+ animals) and between asbestos-exposed OPN+/+ and OPN−/− mice, categorized by biological function. C: Validation of Plunc and Areg expression by qPCR in whole lung tissue from mice exposed to asbestos for 9 days. Data are reported as fold-change in expression relative to OPN+/+ air-exposed animals of each target, normalized to the expression of the housekeeping gene Hprt. Open bars: air-exposed animals; black bars: asbestos-exposed animals. *P ≤ 0.05, asbestos versus air exposure; **P ≤ 0.05, asbestos-exposed OPN+/+ versus OPN−/−.

Figure 6

Figure 6

Expression profiles of select genes, by functional category. Data are reported as fold change relative to OPN+/+ air-exposed animals. A: Extracellular matrix. B: Cytoskeleton/muscle contraction. C: Cell signaling. D: Biotransformation. E: Cell cycle. F: Immune system/defense. *P ≤ 0.05, asbestos versus air exposure; **P ≤ 0.05, asbestos-exposed OPN+/+ versus OPN−/−.

Figure 7

Figure 7

Schematic of plausible signaling networks affected by asbestos, and the role of OPN in regulating genes involved in inflammation and ECM remodeling. Solid arrows represent a positive relationship between two entities, through either expression or activation; double-headed arrows represent positive relationships that occur in both directions. Negative relationships are indicated by T-junction arrows. Shapes indicate protein or molecule type (as in legend). Filled shapes (gray) indicate likely down-regulation by OPN; open shapes (white) indicate up-regulation by OPN. Initial signaling by asbestos occurs through NLRP3-IL-1β-AREG, which activates EGFR and OPN, which then converge on AP-1. OPN acts via CD44 and integrin receptors to trigger the AP-1 transcription factor (and other pathways) that stimulate signaling pathways to regulate downstream genes or proteins related to extracellular matrix (ECM) remodeling and inflammation.

References

    1. Mossman B.T., Bignon J., Corn M., Seaton A., Gee J.B. Asbestos: scientific developments and implications for public policy. Science. 1990;247:294–301. - PubMed
    1. Mossman B.T., Churg A. Mechanisms in the pathogenesis of asbestosis and silicosis. Am J Respir Crit Care Med. 1998;157:1666–1680. - PubMed
    1. O'Reilly K.M., McLaughlin A.M., Beckett W.S., Sime P.J. Asbestos-related lung disease. Am Fam Physician. 2007;75:683–688. - PubMed
    1. O'Regan A. The role of osteopontin in lung disease. Cytokine Growth Factor Rev. 2003;14:479–488. - PubMed
    1. Pardo A., Gibson K., Cisneros J., Richards T.J., Yang Y., Becerril C., Yousem S., Herrera I., Ruiz V., Selman M., Kaminski N. Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis. PLoS Med. 2005;2:e251. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources