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CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation

A Retraction to this article was published on 07 July 2011

This article has been updated

Abstract

Upon activation, neutrophils release DNA fibers decorated with antimicrobial proteins, forming neutrophil extracellular traps (NETs)1,2,3. Although NETs are bactericidal and contribute to innate host defense, excessive NET formation has been linked to the pathogenesis of autoinflammatory diseases4,5. However, the mechanisms regulating NET formation, particularly during chronic inflammation, are poorly understood. Here we show that the G protein–coupled receptor (GPCR) CXCR2 mediates NET formation. Downstream analyses showed that CXCR2-mediated NET formation was independent of NADPH oxidase and involved Src family kinases. We show the pathophysiological relevance of this mechanism in cystic fibrosis lung disease, characterized by chronic neutrophilic inflammation6,7. We found abundant NETs in airway fluids of individuals with cystic fibrosis and mouse cystic fibrosis lung disease, and NET amounts correlated with impaired obstructive lung function. Pulmonary blockade of CXCR2 by intra-airway delivery of small-molecule antagonists inhibited NET formation and improved lung function in vivo without affecting neutrophil recruitment, proteolytic activity or antibacterial host defense. These studies establish CXCR2 as a receptor mediating NADPH oxidase–independent NET formation and provide evidence that this GPCR pathway is operative and druggable in cystic fibrosis lung disease.

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Figure 1: CXCR2 mediates NETosis.
Figure 2: CXCR2-mediated NET formation is NADPH oxidase independent.
Figure 3: NET formation in cystic fibrosis lung disease.
Figure 4: CXCR2 mediates increased NET formation in mouse cystic fibrosis lung disease in vivo.

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Change history

  • 15 June 2011

     In the version of this article initially published, we reported that CXCL8 could efficiently induce neutrophil extracellular trap (NET) formation in vitro. When we followed up on the effect of recombinant CXCL8 (IL-8) on NET formation by comparing different cell culture conditions and extending our studies to neutrophils obtained from a larger number of healthy blood donors, we found that the CXCL8 effect was donor dependent and was less robust than we previously thought. In investigating the underlying factors, we observed that the CXCL8 effect that we initially observed was favored by our cell culture conditions (CXCL8-72aa (CXCL8 that is 72 amino acids in length) at 100 nM; RPMI-1640 medium; absence of albumin, buffers or serum; supplementation with L-glutamine; and precoating of culture plates with poly-D-lysine 30–70 kDa). We had initially chosen these conditions because we felt that they resembled the pulmonary microenvironment. On the basis of our recent observations, however, we conclude that these culture conditions are unstable and allow nonspecific neutrophil activation and autocrine/paracrine CXCL8 release. In light of these results, we revise our conclusions to state that the effect of recombinant CXCL8 on NET formation is less efficient than we previously reported, donor dependent and less robust compared to the effect of phorbol 12-myristate 13-acetate. Thus, we wish to retract the paper. We did not use this in vitro methodology in the ex vivo and in vivo studies of human and mouse cystic fibrosis lung disease. Accordingly, we continue to endorse our NETosis studies in the context of cystic fibrosis lung disease.

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Acknowledgements

We thank B. Lange-Sperandio and F. Beitinger for lung sample preparations. We thank O. Gires and L. Mays for manuscript discussions. We thank M. Klappacher, A. Obermayer, T. Stöger, B. Ritter, S. Stritzke and M. Franz for assistance in experimental workup and S. Hirtz for breeding and genotyping of experimental mice. Supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, Emmy Noether Programme HA 5274/3-1 to D.H., MA 2081/3-3 and MA 2081/4-1 to M.A.M.), the Alexander von Humboldt Foundation (B.K.), the German Society of Pediatric Pneumology (D.H.), PINAe.V. (D.H.), the Novartis Foundation (D.H.) and the Ernest-Solvay-Foundation (D.H.).

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Contributions

V.M. and E.M.W. performed the in vitro and ex vivo experiments. Z.Z. performed mouse experiments. A.Ö.Y. and A.B. performed pulmonary function tests. L.V. contributed to data analysis and discussion. A.H. provided patient material and clinical data and contributed to data analysis and discussion. W.D.K., G.D. and W. S. contributed to data analysis and discussion. B.H.B. and N.R. provided material from human subjects and clinical data. B.K. performed CXCL2 assays and contributed to data analysis and discussion. D.R. contributed to data analysis and discussion. G.D., M.K. and A.R. contributed to data analysis, discussion and manuscript preparation. M.G. provided material from human subjects and clinical data and contributed to discussion. O.E. designed pulmonary function experiments, contributed to discussion and wrote the manuscript. M.A.M. designed animal experiments, contributed to discussion and wrote the manuscript. D.H. designed and supervised the study, analyzed data, provided funding and wrote the manuscript.

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Correspondence to Dominik Hartl.

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The authors declare no competing financial interests.

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Supplementary Figs. 1–10, Supplementary Tables 1–3 and Supplementary Methods (PDF 1113 kb)

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Marcos, V., Zhou, Z., Yildirim, A. et al. CXCR2 mediates NADPH oxidase–independent neutrophil extracellular trap formation in cystic fibrosis airway inflammation. Nat Med 16, 1018–1023 (2010). https://doi.org/10.1038/nm.2209

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