Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis

Our plain-English summary of Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis, by A.D. Gregory, C.R. Kliment, H.E. Metz, et al (Journal of Leukocyte Biology, 2015).

The study

• Authors: A.D. Gregory, C.R. Kliment, H.E. Metz, K.H. Kim, J. Kargl, B.A. Agostini, L.T. Crum, E.A. Oczypok, T.A. Oury, A.M. Houghton
• Journal: Journal of Leukocyte Biology
• Published: 2015;98(2):143-52
• Cited by: 186 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-β-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

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In plain English

This was a laboratory study in mice and in cultured lung cells, not a study of people. The authors placed 0.1 mg of crocidolite asbestos, or titanium dioxide (an inert control dust), into the windpipes of normal mice and of mice lacking neutrophil elastase, an enzyme released by a type of white blood cell. In normal mice, asbestos caused a significant rise in hydroxyproline, a measure of lung scarring (fibrosis), at day 14 but not at day 7. The authors described the elastase-deficient mice as protected completely. A scant amount of fibrosis was still seen in them. Their hydroxyproline did not differ significantly from that of control-dust mice, and their fibrosis scores were significantly lower than those of asbestos-treated normal mice. Asbestos-treated normal mice had about twice as many fibroblasts and myofibroblasts, the scar-forming cells, as asbestos-treated elastase-deficient mice. Lung inflammation rose with asbestos in both kinds of mice and did not differ between them. In cell cultures, elastase increased fibroblast growth and triggered changes typical of myofibroblasts. Normal mice given asbestos plus an elastase-blocking drug, ONO-5046, had less fibrosis. The authors concluded that blocking elastase holds promise as a potential treatment strategy for lung fibrosis.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Animal experiment in mice, combined with laboratory experiments on cultured lung fibroblasts
• Who was studied: Age- and sex-matched normal (wild-type) C57BL/6 mice and neutrophil elastase-deficient mice aged 8 to 10 weeks, studied at the University of Pittsburgh with 6 to 8 mice per group. The cell experiments used LL47 human lung fibroblasts and primary mouse lung fibroblasts.
• Exposure: A single dose of 0.1 mg crocidolite asbestos, or 0.1 mg titanium dioxide as an inert control, placed into the airway, with mice examined 7 or 14 days later. In separate experiments, asbestos-treated mice received the elastase inhibitor ONO-5046 (100 mg/kg daily by injection into the abdomen) or a vehicle control.
• Main results:
  • In normal mice, asbestos produced a statistically significant increase in lung hydroxyproline compared with titanium dioxide controls at day 14, but not at day 7.
  • The authors reported that elastase-deficient mice were completely protected from asbestos-induced lung fibrosis.
  • In elastase-deficient mice given asbestos, lung hydroxyproline was not statistically different from that of control-dust mice, although a scant amount of fibrosis was seen on tissue sections.
  • The fibrosis score was significantly lower in asbestos-treated elastase-deficient mice than in asbestos-treated normal mice.
  • Asbestos increased inflammatory cells in lung washings, with no difference between normal and elastase-deficient mice.
  • Asbestos-treated normal mice had double the fibroblast content of asbestos-treated elastase-deficient mice, a statistically significant difference.
  • By flow cytometry (7 mice per group), asbestos-treated normal mice had approximately twice as many myofibroblasts as asbestos-treated elastase-deficient mice. No significant differences were found in pericytes, smooth muscle cells or fibrocytes.
  • In cell cultures, elastase increased fibroblast proliferation at modest concentrations.
  • Elastase inactivated by heat or by the inhibitor PMSF did not reproduce the effects of active elastase: growth at low doses and toxicity at high doses.
  • One hour of elastase exposure caused complete loss of the signaling protein IRS-1 in fibroblasts.
  • Elastase significantly increased fibroblast contraction of collagen gels.
  • Elastase induced alpha smooth muscle actin, a myofibroblast marker. A TGF-beta receptor blocker did not stop this, although it did stop alpha smooth muscle actin production in the absence of elastase.
  • In asbestos-treated normal mice, the elastase inhibitor ONO-5046 reduced lung fibrosis, measured by hydroxyproline and fibrosis index. The hydroxyproline experiment used 8 mice per group.
• The authors' conclusion: The authors concluded that neutrophils and neutrophil elastase play a key role in the progression of lung fibrosis. They also concluded that approaches limiting fibroblast growth, such as elastase inhibition, hold promise as a potential therapeutic strategy for lung fibrosis patients.
• Limitations the authors state:
  • Immunofluorescence staining could not reliably count myofibroblasts in lung tissue, because of the method and because the cells were sparse.
  • The exact mechanism by which elastase promotes myofibroblast differentiation was not determined.

Topics

Medical terms in this study, explained in our Lexicon: Fibrosis, Fibroblast, Lung Cancer, Collagen.

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