Abstract
BackgroundNeutrophil extracellular traps (NETs) from patients with systemic lupus erythematosus (SLE) are characterized by lower ubiquitylation and myeloperoxidase (MPO) as a substrate. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown.MethodsTo assess the expression and functional role of PTMs in NETs of patients with SLE; reactivation, proliferation and cytokine production was evaluated by flow cytometry using co-cultures with dendritic cells (DC) and CD4+ from SLE patients and healthy controls. The impact of ubiquitylation on MPO was assessed by molecular dynamics. The expression of ISG15 in NETs was evaluated by immunofluorescence and Western Blot.ResultsFifteen patients with SLE and ten healthy controls were included. In the co-cultures of CD4+ lymphocytes with DC stimulated with ubiquitylated MPO or recombinant MPO, a higher expression of IFNγ and IL-17A was found in CD4+ from SLE patients (p < 0.05). Furthermore, with DC stimulated with ubiquitylated MPO a trend towards increased expression of CD25 and Ki67 was found in lupus CD4+ lymphocytes, while the opposite was documented in controls (p < 0.05). Through molecular dynamics we found the K129-K488-K505 residues of MPO as susceptible to ubiquitylation. Ubiquitylation affects the hydration status of the HEME group depending on the residue to which it is conjugated. R239 was found near by the HEME group when the ubiquitin was in K488-K505. In addition, we found greater expression of ISG15 in the SLE NETs vs controls (p < 0.05), colocalization with H2B (r = 0.81) only in SLE samples and increased production of IFNγ in PBMCs stimulated with lupus NETs compared to healthy controls NETs.ConclusionThe ubiquitylated MPO has a differential effect on the induction of reactivation of CD4+ lymphocytes in patients with SLE, which may be related to structural changes by ubiquitylation at the catalytic site of MPO. Besides a lower ubiquitylation pattern, NETs of patients with SLE are characterized by the expression of ISG15, and the induction of IFNγ by Th1 cells.
Highlights
Neutrophil extracellular traps (NETs) play a pathogenic role in diverse autoimmune diseases, including systemic lupus erythematosus (SLE) [1]
Petretto A, et al [4] showed by proteomic analysis a differential protein cargo and post-translational modifications, dependent upon the induction stimuli for NETosis; interestingly the majority of modified peptides are derived from myeloperoxidase (MPO), which might be responsible for diverse biologic effects [4]
The trend towards increased proliferative responses of CD4+ T lymphocytes from patients with SLE, as well as the polarization towards Th1 and Th17 subpopulations producing IFNγ or IL-17A in the presence of components of NETs such as recombinant MPO and ubiquitylated MPO is consistent with a previous study in which CD4+ and CD8+ T lymphocytes from controls stimulated with supernatants of NETs induced in vitro, had higher levels of CD25, CD69, phosphorylation of ZAP70, secretion of IFNγ and IL-17A; there was no increase in the percentage of proliferation with ethinyl deoxyuridine (EdU) [40]
Summary
Neutrophil extracellular traps (NETs) play a pathogenic role in diverse autoimmune diseases, including systemic lupus erythematosus (SLE) [1] These NETs are fibrillar mesh structures decorated by nuclear chromatin and neutrophil granular peptides (i.e. myeloperoxidase, elastase, lactoferrin and LL-37) and are one of the most potent tools to combat microorganisms [2]. Petretto A, et al [4] showed by proteomic analysis a differential protein cargo and post-translational modifications, dependent upon the induction stimuli for NETosis; interestingly the majority of modified peptides are derived from myeloperoxidase (MPO), which might be responsible for diverse biologic effects [4]. The structural and functional effect of such modification and if there are additional post-translational modifications (PTMs) are unknown
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