Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy

Manzenreiter, Reinhard; Kienberger, Ferry; Marcos, Verónica; Schilcher, Kurt; Krautgartner, Wolf‐Dietrich; Obermayer, Astrid; Huml, Marlene; Stoiber, Walter; Hector, Andreas; Griese, Matthias; Hannig, Matthias; Studnicka, Michael; Vitkov, Ljubomir; Hartl, Dominik

doi:10.1016/j.jcf.2011.09.008

Cited by 176 publications

(172 citation statements)

References 34 publications

(44 reference statements)

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“…Human DNase I, or dornase alfa, has been approved since 1994 for use in cystic fibrosis patients to solubilize dense DNA-containing cystic fibrosis sputum (26,39). Our work expands the applicability of recombinant human DNase by demonstrating that nuclease treatment can be used as an adjunctive treatment strategy during acute keratitis to expedite bacterial clearance.…”

Section: Figmentioning

confidence: 74%

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: Figmentioning

confidence: 74%

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

et al. 2014

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“…The transcription factor NF-kB is a key regulator of inflammation and therefore plays a pivotal role in a wide range of inflammatory diseases (Manzenreiter et al, 2012). Because the effects of high ASA concentrations are not only related to cyclo-oxygenase acetylation and include inhibition of NF-kB activation (Yin et al, 1998), we next investigated the role of NF-kB blockade on NET formation.…”

Section: Resultsmentioning

confidence: 99%

“…Excessive NET generation as a result of noninfectious inflammatory processes as well as a lack of DNAses to degrade them is associated with the pathogenesis of inflammatory and autoimmune diseases, including preeclampsia (Gupta et al, 2005), cystic fibrosis (Manzenreiter et al, 2012), and lupus (Hakkim et al, 2010). Moreover, the observation that NETs act as a scaffold for thrombus formation (Fuchs et al, 2010(Fuchs et al, , 2012von Bruhl et al, 2012) is increasingly being recognized as a critical phenomenon linking inflammation with venous thrombosis in both infectious and noninfectious clinical settings.…”

Section: Introductionmentioning

confidence: 99%

Regulation of Neutrophil Extracellular Trap Formation by Anti-Inflammatory Drugs

Lapponi

Carestia

Landoni

et al. 2013

J Pharmacol Exp Ther

159

133

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The formation of neutrophil extracellular traps (NETs) is a newly described phenomenon that increases the bacteria-killing ability and the inflammatory response of neutrophils. Because NET generation occurs in an inflammatory microenvironment, we examined its regulation by anti-inflammatory drugs. Treatment of neutrophils with dexamethasone had no effect, but acetylsalicylic acid (ASA) treatment prevented NET formation. NETosis was also abrogated by the presence of BAY 11-7082 [(E)-3-[4-methylphenylsulfonyl]-2-propenenitrile] and Ro 106-9920 [6-(phenylsulfinyl)tetrazolo [1,5-b]pyridazine], two structurally unrelated nuclear factor-kB (NF-kB) inhibitors. The decrease in NET formation mediated by ASA, BAY-11-7082, and Ro 106-9920 was correlated with a significant reduction in the phosphorylation of NF-kB p65 subunit, indicating that the activation of this transcription factor is a relevant signaling pathway involved in the generation of DNA traps. The inhibitory effect of these drugs was also observed when NET generation was induced under acidic or hyperthermic conditions, two stress signals of the inflammatory microenvironment. In a mouse peritonitis model, while pretreatment of animals with ASA or BAY 11-7082 resulted in a marked suppression of NET formation along with increased bacteremia, dexamethasone had no effect. Our results show that NETs have an important role in the local control of infection and that ASA and NF-kB blockade could be useful therapies to avoid undesired effect of persistent neutrophil activation.

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“…However, in human sites of infection, such as the CF lung, the eDNA detected is almost entirely derived from human polymorphonuclear leukocytes (PMNs) that are recruited heavily to quell infection (22). This abundance of neutrophil DNA likely results from a combination of the PMN immune response of NETosis and leukocyte lysis mediated by bacterial virulence factors (23)(24)(25)(26)(27)(28).…”

mentioning

confidence: 99%

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

Wilton

Charron-Mazenod

Moore

et al. 2016

Antimicrob Agents Chemother

186

188

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bBiofilms consist of surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, exopolysaccharides, and proteins. Extracellular DNA (eDNA) has a structural role in the formation of biofilms, can bind and shield biofilms from aminoglycosides, and induces antimicrobial peptide resistance mechanisms. Here, we provide evidence that eDNA is responsible for the acidification of Pseudomonas aeruginosa planktonic cultures and biofilms. Further, we show that acidic pH and acidification via eDNA constitute a signal that is perceived by P. aeruginosa to induce the expression of genes regulated by the PhoPQ and PmrAB two-component regulatory systems. Planktonic P. aeruginosa cultured in exogenous 0.2% DNA or under acidic conditions demonstrates a 2-to 8-fold increase in aminoglycoside resistance. This resistance phenotype requires the aminoarabinose modification of lipid A and the production of spermidine on the bacterial outer membrane, which likely reduce the entry of aminoglycosides. Interestingly, the additions of the basic amino acid L-arginine and sodium bicarbonate neutralize the pH and restore P. aeruginosa susceptibility to aminoglycosides, even in the presence of eDNA. These data illustrate that the accumulation of eDNA in biofilms and infection sites can acidify the local environment and that acidic pH promotes the P. aeruginosa antibiotic resistance phenotype.

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Ultrastructural characterization of cystic fibrosis sputum using atomic force and scanning electron microscopy

Abstract: These studies suggest that excessive NET formation represents the major factor underlying the gel-like structure of CF sputum and provide evidence that CF-NETs contain ectosome-like structures that could represent targets for future therapeutic approaches.

Cited by 176 publications

References 34 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

Regulation of Neutrophil Extracellular Trap Formation by Anti-Inflammatory Drugs

Extracellular DNA Acidifies Biofilms and Induces Aminoglycoside Resistance in Pseudomonas aeruginosa

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