Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

Zhou, Yebin; An, Ling–Ling; Chaerkady, Raghothama; Mittereder, Nanette; Clarke, Lori; Cohen, Taylor S.; Chen, Bin; Hess, Sonja; Sims, Gary P.; Mustelin, Tomas

doi:10.1038/s41598-018-33385-z

Cited by 71 publications

(55 citation statements)

References 65 publications

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“…The identified proteins include vimentin and myeloperoxidase (MPO), the latter also identified in the original report (1). Notably, citrullinated vimentin, /-tubulin, -actin, and NCF1 are known RA antigens (1,4,7,8). In addition, we identified S100-A8, plastin, coronin, moesin, and erzin, which are all linked to RA (9).…”

Section: Reassessment Of Citrullinome Cargo In Neutrophil Extracellulsupporting

confidence: 56%

Technical comment on “Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis”

et al. 2020

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Reassessment of citrullinome cargo in neutrophil extracellular traps confirms the presence of citrullinated peptides.In an article previously published in Science Immunology (1), we reported a previously unidentified mechanism of induction of pathogenic adaptive immunity against citrullinated antigens in rheumatoid arthritis (RA). This mechanism was mediated by an interaction between neutrophil extracellular traps (NETs) and synovial fibroblasts (FLS) that promote the presentation of citrullinated peptides identified in NETs by the FLS to the adaptive immune system. The identification of citrullinated peptides in NETs was performed by a variety of methods described in this and previous manuscripts and included the use of citrulline-specific probes, antibodies, and mass spectrometry (MS) (1, 2).Following this publication, we were contacted by an investigator with concerns about our MS analysis. Given the magnitude of the mass shift expected between a citrulline and an arginine residue (+0.984 Da), misannotation of citrulline-containing peptides can occur because of fragmentation of misassigned monoisotopic mass peaks. In addition, the +0.984-Da mass increase upon citrullination is identical to the mass increase observed upon protein deamination of asparagine and glutamine residues within a peptide, thereby confounding the correct assignment of citrullinated species. Last, most of the previously identified peptides contained a C-terminal citrulline. Whereas several endogenous proteases, including cathepsin B, cleave after citrulline, trypsin, the protease used in sample preparation, does not (3). These discrepancies prompted us to reevaluate the initial proteomic data more stringently. Upon reevaluation, the spectra were either not consistent with citrullination or not definitive enough to be consistent with a citrullinated residue. Therefore, we generated new samples for analysis. Herein, we report the identification of 58 sites of citrullination on 32 proteins that are present in NETs using a highly stringent data analysis workflow.In the analysis, healthy neutrophils were purified as described (1), resuspended in RPMI, seeded in 12-well plates, and incubated with 2.5 M calcium ionophore (A23187, Sigma-Aldrich) or 100 g of rheumatoid factor for 4 hours at 37°C. Supernatants were carefully removed, and NETs were harvested with micrococcal nuclease (MNase) (10 U/ml) in RPMI for 15 min at 37°C. Supernatant-containing NETs were isolated via centrifugation and stored at −80°C until analysis. Note that the proteomic analysis was performed on NETs and not from cells treated with ionophore or rheumatoid factor; hence, it is not possible to generate control proteomes.NETs (50 g) were precipitated with 20% trichloroacetic acid. The pellets were washed with acetone and then resuspended in urea (8 M). Ammonium bicarbonate (100 mM) was then added to the solution. Samples were reduced with dithiothreitol (15 mM) and alkylated with iodoacetamide (12.5 mM). Next, samples were diluted to 1.8 M urea with phosphate buffer (pH 7....

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Section: Reassessment Of Citrullinome Cargo In Neutrophil Extracellulsupporting

confidence: 56%

Technical comment on “Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis”

et al. 2020

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“…Some of the discrepancies on the role of PAD4 in NET formation may be explained by our limited understanding of the different functions that PAD4 may have in neutrophil biology. Per example, a recent study linked PAD4 to assembly and activation of NOX2 [100]. Although this function of PAD4 is citrullination-independent, it can be blocked by PAD4 inhibitors.…”

Section: Pathways Of Net Formationmentioning

confidence: 99%

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps

et al. 2019

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Since the discovery and definition of neutrophil extracellular traps (NETs) 14 years ago, numerous characteristics and physiological functions of NETs have been uncovered. Nowadays, the field continues to expand and novel mechanisms that orchestrate formation of NETs, their previously unknown properties, and novel implications in disease continue to emerge. The abundance of available data has also led to some confusion in the NET research community due to contradictory results and divergent scientific concepts, such as pro-and anti-inflammatory roles in pathologic conditions, demarcation from other forms of cell death, or the origin of the DNA that forms the NET scaffold. Here, we present prevailing concepts and state of the science in NET-related research and elaborate on open questions and areas of dispute.

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“…Neutrophils were cultured at a density of 200 000 cells per cm 2 for 8 hours on TCPS or modified Ti surfaces in the presence of 50 nM GSK484 (Cayman Chemical, Ann Arbor, MI), an inhibitor of PAD4 and consequently NETosis, or an equivalent concentration of ethanol vehicle 25 (n = 6 per variable). Then, CM was collected and spun at 2000g for 30 m to remove cellular debris and transferred to macrophages cultured on TCPS at a density of 10 000 cells per cm 2 .…”

Section: Macrophage Response To Neutrophil-conditioned Media (Cm)mentioning

confidence: 99%

Hydrophilic titanium surfaces reduce neutrophil inflammatory response and NETosis

et al. 2020

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Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils

Cited by 71 publications

References 65 publications

Technical comment on “Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis”

Technical comment on “Synovial fibroblast-neutrophil interactions promote pathogenic adaptive immunity in rheumatoid arthritis”

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps

Hydrophilic titanium surfaces reduce neutrophil inflammatory response and NETosis

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