A Role for Receptor-Interacting Protein Kinase-1 in Neutrophil Extracellular Trap Formation in Patients with Systemic Lupus Erythematosus: a Preliminary Study

Abstract

Background/Aims: Neutrophil extracellular traps (NETs) are known to play an important role in systemic lupus erythematosus (SLE) by triggering innate and adaptive immune responses. The molecular mechanisms responsible for their formation in SLE are still unclear. In this study, we aim to characterize the role of the receptor-interacting protein kinase-1 (RIPK1), a homologous serine/threonine kinase previously implicated in the regulation of necroptosis and tissue injury, in decreasing neutrophil death and formation of NETs, and to investigate the clinical implications of RIPK1 in SLE. Methods: Patients with SLE (n = 50) and healthy donors (n = 35) were enrolled in in vitro studies. Management of SLE patients was evaluated using the SLE disease activity index 2000 (SLEDAI-2K) score and laboratory variables. The mRNA level of RIPKs was measured by quantitative polymerase chain reaction (qPCR). Intracellular RIPK1 and RIPK3 production by peripheral blood leukocytes was detected by four-color flow cytometry and confirmed by automatic western blotting. TNF-α, IFN-γ, IL-1β, IL-2, IL-8, IL-18, and RIPK1 were measured by enzyme-linked immunosorbent assay. Cell death was assayed by Sytox green dye from peripheral neutrophils stimulated by RIPK-1-stabilizer necrostatin-1 (nec-1) and phorbol 12-myristate 13-acetate (PMA). Immunofluorescence staining and confocal microscopy were used to detect NET formation ex vivo. Quantification of NETs was determined by fluorescence spectrometry. Results: IFN-γ, IL-1β, IL-8, and IL-18 levels in serum were increased in SLE patients compared to controls. However, the expression of TNF-α, IL-2, and RIPK1 were decreased. In addition, we observed significant differences in the expression of RIPK1 in peripheral blood leukocytes. Of all the leukocytes, RIPK1 expression was significantly lower in neutrophils. Furthermore, we studied NETs formation in neutrophils of SLE with decreased RIPK1 expression, and these show increased susceptibility to NETosis, when stimulated with PMA and/or nec-1. Importantly, RIPK1 expression in neutrophils negatively correlated with ESR, CRP, 24-hour urine total protein, and the disease activity index in SLE. Conclusion: These data represent the first report of decreased RIPK1 expression in neutrophils of SLE patients and imply that RIPK1 may be involved in neutrophil death and NET formation. We suggest that RIPK1 is a potential biomarker to predict disease activity.

Neutrophil-to-lymphocyte ratio in relation to the risk of all-cause mortality and cardiovascular events in patients with chronic kidney disease: a systematic review and meta-analysis

“…They can kill pathogens by chemotaxis, phagocytosis, and direct sterilization [30]. In recent years, neutrophil extracellular trap (NET), a new neutrophil antibacterial method, has been discovered, which can not only capture and kill pathogens by releasing nucleic acid substances and granulocyte proteins into a network structure but also is related to multiple pathophysiological processes of human body, such as inflammation, tumor cell migration, ischemia/ reperfusion injury, autoimmunity [31][32][33], etc. It is noteworthy that neutrophils mediate the inflammatory response in kidney injury by various biochemical mechanisms, leading to further tissue damage [34].…”

Lymphocyte mass cytometry identifies a CD3-CD4+ cells subset with a potential role in psoriasis

“…Flow cytometry validation. Established panels (Supplemental Table 1) and protocols were used as described previously (53). Briefly, cells were blocked with Fc block in staining buffer for 10 minutes at room temperature, incubated with antibodies in staining buffer for 30 minutes at 4°C, and then washed twice with staining buffer.…”

“…qPCR. Protocols were employed as described previously (53). Total RNA from human PBMCs was isolated using TRIzol according to the manufacturer's instructions (Invitrogen).…”

Neutrophil Extracellular Traps: Signaling Properties and Disease Relevance

“…In addition, antiribonucleoprotein antibodies in SLE patients can also induce neutrophils in SLE patients to secrete NETs, further activating pDCs to release proinflammatory factors, such as IFN-α and IL-6 [111]. Guo et al [112] further confirmed that neutrophils derived from SLE patients were more likely to form NETs when stimulated by external stimuli, and SLE patients have a higher levels of IFN-α. In addition, neutrophil intracellular MMP-9 is externalized during NET formation, and MMP-9 induces endothelial dysfunction by activating MMP-2, which participates in the SLE disease process [18].…”