Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Abstract

ObjectivesLow-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE.MethodsProteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively.ResultsProteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM.ConclusionsModulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

Activated low-density granulocytes in peripheral and intervillous blood and neutrophil inflammation in placentas from SLE pregnancies

“…The reason for this can only be speculated on, but the active labour process itself may lead to increased granulocyte activation and degranulation in the placenta. 33 34 Whether activated LDG are associated with placental pathology in SLE pregnancy remains to be examined, but SLE LDG may be retained in microvasculature networks 35 and are associated with vascular inflammation. 36 37 Furthermore, SLE LDG spontaneously secrete proinflammatory cytokines and the neutrophil-attracting and activating chemokine CXCL8 12 and spontaneously form NETs that may lead to endothelial apoptosis and vascular dysfunction.…”

Neutrophils in the Pathogenesis of Rheumatoid Arthritis and Systemic Lupus Erythematosus: Same Foe Different M.O.

“…This phenomenon is also observed in chronic granulomatous disease LDGs, which lack functional NOX2 but can produce mtROS and NETs (122). SLE LDGs have stiffer biomechanical properties and are slower to migrate through microvascular mimetics in vitro (204). This may explain why LDGs are not found in affected tissues in SLE (284).…”

“…Some success in stratifying interferon-high patients to anti-IFNα therapy (anifrolumab) is evident (201), although the largest clinical trials of anifrolumab (TULIP-1 and TULIP-2) found no significant differences in the response to antiinterferon therapy between the interferon-high and interferonlow patient groups (202,203). Expression of neutrophil genes within SLE whole blood has been attributed to the increase in the population of low density granulocytes (LDGs), discussed below (198), although proteomics analysis of SLE neutrophils and LDGs suggests that protein levels of MPO and other granule proteins is higher in SLE neutrophils (204). The expression of neutrophil granule protein genes in SLE whole blood is strongly associated with lupus nephritis (197,205), vascular inflammation and cardiovascular involvement (206).…”

“…Recent work has revealed that the SLE LDG population is heterogeneous (mature CD10 + or immature CD10 − ), with significant differences in transcriptomic and epigenomic regulation of function and phenotype that correlates with clinical manifestations of the disease (187,284). Mature CD10 + SLE LDGs express high amounts of mRNA and protein for interferon-regulated genes, whereas immature CD10 − SLE LDGs express high amounts of mRNA for cell cycle genes (187,204). CD10 + SLE LDGs undergo phagocytosis, chemotaxis and NETosis at higher levels than CD10 − LDGs, which release MPO at higher amounts than CD10 + LDGs and normal density SLE neutrophils (187).…”

Novel Therapeutic Strategy Based on Neutrophil Subset and Its Function in Autoimmune Disease

“…During the past decade, NETs in patients with SLE occur predominantly in the LDGs subset, which displays morphologically and genetically immature characteristics and is involved in SLE development ( Mistry et al, 2019 ). The human SLE-LDGs impair phagocytic function; instead, they show pro-inflammatory properties and injure the endothelium, indicating the involvement of cardiovascular disease development in SLE ( Denny et al, 2010 ; Rahman et al, 2019 ; Bashant et al, 2021 ). Furthermore, the human LDGs comprise two subpopulations of CD10-negative immature and CD10-positive intermediate mature types based on transcriptomic and epigenetic analyses.…”