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Impairment of Apoptotic Cell Engulfment by Pyocyanin, a Toxic Metabolite of Pseudomonas aeruginosa

¹ Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; ² Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado; ³ Academic Unit of Cardiovascular Research, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom; ⁴ Department of Medicine, Hampstead Campus, Royal Free and University College School of Medicine, London, United Kingdom; and ⁵ Academic Unit of Infectious Diseases, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, United Kingdom

Correspondence and requests for reprints should be addressed to Prof. Moira Whyte, F.R.C.P., Academic Unit of Respiratory Medicine, School of Medicine and Biomedical Sciences, M Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, UK. E-mail: m.k.whyte{at}sheffield.ac.uk

Rationale: Cystic fibrosis lung disease is characterized by accumulation of apoptotic neutrophils, indicating impaired clearance of dying cells. Pseudomonas aeruginosa, the principal microbial pathogen in cystic fibrosis, manipulates apoptosis induction via production of toxic metabolites. Whether these metabolites, particularly pyocyanin, can also modulate apoptotic cell engulfment is unknown.

Objectives: To assess the effects of pyocyanin on apoptotic cell engulfment by macrophages in vitro and in vivo and to investigate potential mechanisms of the observed effects.

Methods: Human monocyte–derived macrophages were treated with pyocyanin before challenge with apoptotic neutrophils, apoptotic Jurkat cells, or latex beads, and phagocytosis was assessed by light microscopy and flow cytometry. Effects of pyocyanin production on apoptotic cell clearance in vivo were assessed in a murine model, comparing infection by wild-type or pyocyanin-deficient P. aeruginosa. Oxidant production was investigated using fluorescent probes and pharmacologic inhibition and Rho GTPase signaling by immunoblotting and inhibitor studies.

Measurements and Main Results: Pyocyanin treatment impaired macrophage engulfment of apoptotic cells in vitro, without inducing significant macrophage apoptosis, whereas latex bead uptake was preserved. Macrophage ingestion of apoptotic cells was reduced and late apoptotic/necrotic cells were increased in mice infected with pyocyanin-producing P. aeruginosa compared with the pyocyanin-deficient strain. Inhibition of apoptotic cell uptake involved intracellular generation of reactive oxygen species (ROS) and effects on Rho GTPase signaling. Antioxidants or blockade of Rho signaling substantially restored apoptotic cell engulfment.

Conclusions: These studies demonstrate that P. aeruginosa can manipulate the inflammatory microenvironment through inhibition of apoptotic cell engulfment, and suggest potential strategies to limit pulmonary inflammation in cystic fibrosis.

Key Words: macrophages • phagocytosis • apoptosis • inflammation • cystic fibrosis

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject Engulfment of apoptotic cells plays a crucial role in the resolution of inflammation and infection. What This Study Adds to the Field This study reports that a bacterial toxin modulates the ingestion of apoptotic cells, and identifies an additional mechanism by which pathogens subvert the host response to favor their own survival.