Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor

Dwyer, Markryan; Shan, Qiang; D'Ortona, Samantha; Maurer, Rie; Mitchell, Robert A.; Olesen, Hanne Vebert; Thiel, Steffen; Hüebner, Johannes; Gadjeva, Mihaela

doi:10.1159/000363242

Cited by 176 publications

(180 citation statements)

References 39 publications

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“…Twenty-eight proteins were shared between the PAO1 strain and the PA14 strain (see Table S1 in the supplemental material). Many of these proteins were identical to previously reported NET-associated proteins, confirming the ability of PA14 to induce NETosis (13,24,25). To functionally prove that the cytotoxic PA14 strain induced NETosis, separate experiments were carried out with differentiated HL-60 cells (used as a model system for neutrophil responses) exposed to P. aeruginosa strain PA14 (MOI, 10) in the presence or absence of DNase I.…”

Section: Resultssupporting

confidence: 80%

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Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

et al. 2014

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Ocular bacterial keratitis, often associated with Pseudomonas aeruginosa bacterial infection, commonly occurs in contact lens wearers and may lead to vision impairment. In this study, we analyzed the contribution of neutrophil extracellular traps (NETs) to the mediation of protection during ocular keratitis. Both invasive and cytotoxic P. aeruginosa clinical isolates induced NET release by neutrophils. NETs carried the characteristic histone proteins, elastase, lysozyme, myeloperoxidase, and metabolic enzymes. While the invasive P. aeruginosa strains PAO1 (serogroup O5) and 6294 (serogroup O6) were trapped by NETs, the cytotoxic P. aeruginosa strains 6077, 6206 (serogroup O11), and PA14 (serogroup 010) were less sensitive to NET capture. The mechanism of escape by the cytotoxic strains from adhesion to NETs involved the shedding of outer membrane vesicles (OMVs) that outcompeted the cytotoxic P. aeruginosa strains for NET binding. When ocular infection was caused by an invasive strain in vivo, NETs were released at the ocular surface to capture bacteria, limiting their spread. Treatment with MNase I had a dose-dependent effect, with low doses of MNase speeding up bacterial clearance and high doses of MNase having toxic consequences. Cumulatively, our data suggest that NET-mediated immunity is a two-step process. Initially, pathogens attach to NET fragments; subsequently, upon nuclease activity, active serine proteases, which proteolytically degrade NET-associated proteins and promote DNase activity, are released. Therefore, a balance between NET production and NET degradation is needed to achieve maximal NET immunity. Infections caused by Pseudomonas aeruginosa are frequently associated with bacterial ocular keratitis, a condition that carries the risk of vision impairment and occurs in contact lens wearers or after ocular trauma. The majority of bacterial clinical isolates derived from corneal ulcers arising during keratitis can be divided into two functionally distinct groups: invasive and cytotoxic isolates. These two groups have distinct phenotypes; the cytotoxic strains cause rapid cell lysis, whereas the invasive strains replicate in the target cells (1, 2). These differences translate into various severities of disease; in mouse keratitis studies where infection is induced by cytotoxic strains, disease pathology is worse than that caused by invasive strains (3). Consistent with these findings, patients infected with cytotoxic strains have experienced more severe pathology and, consequently, suffered from greater visual impairment after recovery from infection (4). The immune response to both invasive and cytotoxic strains is dominated by dense neutrophil infiltrates, yet the infiltration pattern and responses of polymorphonuclear neutrophils (PMNs) differ depending on the type of strain. Due to the recently described ability of neutrophils to release DNA in the form of neutrophil extracellular traps (NETs) (5) and the presence of extracellular DNA during cytotoxic infections, we questioned whether the cyt...

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Section: Resultssupporting

confidence: 80%

“…Whether NETs trap and kill P. aeruginosa is controversial. We, along with others, reported that the nonmucoid cystic fibrosis-derived P. aeruginosa strains were sensitive to NET trapping while the mucoid strains were resistant (13)(14)(15). In contrast, Parker et al reported that P. aeruginosa was not sensitive to NET-mediated killing (16).…”

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confidence: 59%

Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

et al. 2014

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“…The CF lung is highly enriched in DNA derived primarily from neutrophils, including the presence of NETs that trap and kill bacteria (47,49,57). It was recently shown that P. aeruginosa lysates induce the H1-T6SS of kin cells but that the signal is unknown (51).…”

Section: Resultsmentioning

confidence: 99%

Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa

et al. 2016

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e Pseudomonas aeruginosa employs its type VI secretion system (T6SS) as a highly effective and tightly regulated weapon to deliver toxic molecules to target cells. T6SS-secreted proteins of P. aeruginosa can be detected in the sputum of cystic fibrosis (CF) patients, who typically present a chronic and polymicrobial lung infection. However, the mechanism of T6SS activation in the CF lung is not fully understood. Here we demonstrate that extracellular DNA (eDNA), abundant within the CF airways, stimulates the dynamics of the H1-T6SS cluster apparatus in Pseudomonas aeruginosa PAO1. Addition of Mg 2؉ or DNase with eDNA abolished such activation, while treatment with EDTA mimicked the eDNA effect, suggesting that the eDNA-mediated effect is due to chelation of outer membrane-bound cations. DNA-activated H1-T6SS enables P. aeruginosa to nonselectively attack neighboring species regardless of whether or not it was provoked. Because of the importance of the T6SS in interspecies interactions and the prevalence of eDNA in the environments that P. aeruginosa inhabits, our report reveals an important adaptation strategy that likely contributes to the competitive fitness of P. aeruginosa in polymicrobial communities.

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“…Given the abundance of nucleic acids in NETs, the induction of autoantibodies by NETosis is likely to be facilitated by TLR 7/9-mediated B cell activation (27). While formation of NETs in the CF lung has been appreciated as both an antibacterial defense mechanism and a contributor to proteaseinduced lung damage (10,(28)(29)(30), the role of NETosis in CF autoimmunity has not been studied.…”

Section: Introductionmentioning

confidence: 99%

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

Skopelja¹,

Hamilton²,

Jones³

et al. 2016

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Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor

Cited by 176 publications

References 39 publications

Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

Chelation of Membrane-Bound Cations by Extracellular DNA Activates the Type VI Secretion System in Pseudomonas aeruginosa

The role for neutrophil extracellular traps in cystic fibrosis autoimmunity

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