Neutrophil proteases degrade autoepitopes of NET-associated proteins

Abstract

Neutrophils can form neutrophil extracellular traps (NETs) to capture microbes and facilitate their clearance. NETs consist of decondensed chromatin decorated with anti-microbial proteins. Here, we describe the effect of neutrophil proteases on the protein content of NETs. We show that the neutrophil serine proteases degrade several neutrophil proteins associated with NETs. Interestingly, the anti-bacterial proteins associated with NETs, such as myeloperoxidase, calgranulin B and neutrophil elastase (NE), seem to be less susceptible to proteolytic degradation than other NET proteins, such as actin and MNDA. NETs have been proposed to play a role in autoimmune reactions. Our data demonstrate that a large number of the autoepitopes of NET proteins that are recognized by autoantibodies produced by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients are also removed by the proteases. In conclusion, neutrophil serine proteases have a major impact on the NET proteome and the proteolytic changes of NET-associated proteins may counteract autoimmune reactions to NET components.

Keywords: NETosis; autoepitopes; neutrophil proteases; rheumatoid arthritis; systemic lupus erythematosus.

Figure 1

Schematic representation of the experimental set‐up. Starting 2 h after stimulation of the neutrophils, phenylmethylsulfonyl fluoride (PMSF) was added every 15 min (red arrows; the chemical structure represents PMSF) to inhibit neutrophil proteases. Also, the MNase solution used for harvesting of the neutrophil extracellular traps (NETs) contained PMSF.

Figure 2

Neutrophil extracellular trap (NET) formation in the absence and presence of phenylmethylsulfonyl fluoride (PMSF). (a) NET proteins harvested from NETs produced by neutrophils stimulated with phorbol myristate acetate (PMA) and by non‐stimulated neutrophils as a control, and produced with (+) or without (–) PMSF were separated by sodium dodecyl sulphate‐polymerase gel electrophoresis (SDS‐PAGE) and stained with Coomassie brilliant blue. The position of molecular weight markers (kDa) is indicated on the left. (b) NET quantification by Sytox green (DNA) of non‐stimulated cells and neutrophils stimulated with PMA in the presence or absence of PMSF. a.u. = arbitrary units. Mean plus standard error of the mean of at least 10 biological replicates. *P < 0·05, **P < 0·001, paired Student’s t‐test.

Figure 3

Effect of proteases on neutrophil extracellular trap (NET) proteome. (a) Western blots containing NET proteins were incubated with antibodies to the indicated proteins. Cal‐B = calgranulin‐B; H2B = histone 2B; H3 = histone 3; H3cit = citrullinated histone 3; H4 = histone 4; MNDA = myeloid nuclear differentiation antigen; MPO = myeloperoxidase; NE = neutrophil elastase. The presence (+) or absence (–) of phenylmethylsulfonyl fluoride (PMSF) during NET production is indicated. Arrows mark the positions of the full‐length histones; asterisks indicate stable degradation products. (b) Relative reduction of the full‐length NET‐associated proteins in the absence of PMSF compared to NETs treated with PMSF. Signal intensity was quantified using Image Studio Lite software (version 5.2.5, Licor). Mean plus standard deviation of at least three biological replicates.

Figure 4

Neutrophil proteases degrade autoepitopes that are recognized by systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) sera. (a) Reactivity of SLE and RA sera with proteins from neutrophil extracellular traps (NETs) produced with (upper panel) or without phenylmethylsulfonyl fluoride (PMSF) (lower panel) and analysed by immunoblotting. *Reactivity with polypeptides of approximately 10–15 kDa. Representative results of three independent analyses are shown. (b) Reactivity of SLE and RA sera with NET proteins, analysed by enzyme‐linked immunosorbent assay (ELISA). The dotted line represents the cut‐off value based on the mean plus two times the standard deviation of healthy control sera in the same assay with NETs produced in the presence of PMSF. *P < 0·05, **P < 0·001, Student’s t‐test.