Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus

Abstract

Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.

Neutrophils Mediate Kidney Inflammation Following Acute Skin Exposure to UVB Light

“…In addition, subclinical kidney injury may readily be missed until consecutive exposures to UV light compound the effects. While we found that neutrophil-mediated kidney inflammation in response to UV light does not cause clinical disease in healthy mice, such a mechanism may contribute to LN flares in photosensitive lupus patients in multiple ways: Fc receptor engagement by immune complexes could enhance neutrophil recruitment resulting in ROS and protease release 105,106 ; the heightened capacity of lupus neutrophils and LDGs to produce NETs, which in SLE patients are not cleared efficiently 107,108 , could lead to release of tissue-damaging proteases 109,110 , propagation of the IFN-I response 85,86 , or direct damage to the kidney endothelium by creating vascular damage and leakage 14,111 . Moreover, the underlying differences in lupus skin, such as enhanced IFN-I signaling 7,112,113 and defects in protective Langerhans cell population 114 could inform the extent and nature of neutrophil-mediated systemic responses.…”

Association Between Neutrophils and Renal Impairment of Rheumatoid Arthritis: A Retrospective Cross-sectional Study

“…The property of neutrophils to release extracellular traps was not conducive to the clinical development of RA [19]. It is well-known that a large number of neutrophils accumulate in the synovial tissue of RA, which spontaneously released neutrophil extracellular traps (NETs) [20]. NETs, a network structure consisted of DNA and granulose, could indirectly impair the endothelial function,promote blood vessel and glomerular damage, thus it would induce renal failure and even death [21].…”

Neutrophil Extracellular Traps (NETs) Take the Central Stage in Driving Autoimmune Responses

“…The protein cargo of NETs induced by different stimuli is heterogenous, making comparing research and drawing conclusions challenging. Due to this, there is an ongoing discussion about the precise mechanisms involved in NET formation, their composition and, thereby, their functional profile specifically their inflammatory/antimicrobial properties [6,9,10]. Recently, there have been new insights about how molecular mechanisms of NET formation may differ in a species specific manner [11,12] but, also based on the location of neutrophils in the blood stream or tissue, as well as local environmental alkaline or oxygen conditions [13].…”