Investigation of Neutrophil Extracellular Trap (NET) Induction in Patients with Chronic Idiopathic Neutropenia Bearing Mutations in the Mediterranean Fever (MEFV) Gene

Introduction: Chronic idiopathic neutropenia (CIN) is a disorder characterized by the prolonged and unexplained reduction in the number of circulating neutrophils. The pathophysiology of CIN has been associated with an inflammatory bone marrow microenvironment consisting of activated T-cells with an oligoclonal/monoclonal profile and intermediate CD14^bright/CD16⁺ monocytes that induce the apoptotic death of the granulocytic progenitor cells. To probe further the basis of immune cell activation in CIN we have recently investigated by next generation sequencing (NGS) the frequency of mutations in the MEFV gene encoding pyrin, in a cohort of 50 CIN patients without any signs/symptoms of Familial Mediterranean Fever (FMF). We have found that 9/50 patients displayed variants associated with typical or atypical FMF phenotype in the Greek population implicating exon 10 and/or exon 2 (Skendros P et al. Blood, 138, Sup.1, p3124;2021).

Aim:MEFV mutations have been associated with increased neutrophil activation in FMF and other inflammatory diseases, neutrophil entry in autophagy and extrusion of Neutrophil Extracellular Traps (NETs) decorated with the pro-inflammatory cytokine IL-1β. The aim of the current study is to investigate the association between MEFV mutations and NET formation in neutrophils of CIN patients.

Patients-Methods: We studied 20 CIN patients (17 females and 3 males) aged 27 to 78 years. Six CIN patients harbored at least one MEFV mutation with potential clinical significance (A744S homo/R202Q homo, I720M hetero, R202Q homo, K965R hetero, A744S hetero, M694V hetero) and 14 were negative for MEFV pathogenic mutations. The MEFV R202Q mutation is located in exon 2 whereas all the rest mutations are located in exon 10. We isolated neutrophils from healthy subjects (n=5), age- and sex- matched with the patients, and co-cultured them with serum from CIN patients. We used the supernatants of the cultures to quantify NETs with Sytox green staining for extracellular DNA and/or ELISA for dsDNA/MPO complexes. We also used the serum-treated neutrophils for the evaluation of NETosis by immunostaining using confocal microscopy. In addition, neutrophils isolated from CIN patients were used to detect their direct response to spontaneous NETosis induction. Statistical analysis was performed with one-way ANOVA adjusting for multiple comparisons.

Results: By co-culturing patients' sera with healthy neutrophils obtained from five different healthy donors, we did not identify statistically significant differences in NETosis evaluation between the three study groups i.e. CIN patients with MEFV mutations, CIN patients negative for pathogenic MEFV mutations, and healthy individuals. On an individual basis, the patient harboring the double homozygous MEFV mutations (A744S/R202Q) displayed increased NETosis in immunostaining, Sytox and ELISA evaluation, as compared to MEFV unmutated CIN patients and healthy individuals. In addition, ex vivo culture of patients' neutrophils and microscopic observation gave us the indication that CIN patients, regardless of their MEFV gene variants, present increased NETosis compared to healthy individuals, suggesting an overactivation status of neutrophils in CIN.

Conclusions: The neutrophils of CIN patients display increased NETosis irrespectively of the MEFV mutation status and may thus contribute to the inflammatory processes associated with the disease, a field that has not been investigated so far. Although in our study MEFV mutations did not appear to contribute significantly to inflammatory processes through the NETosis mechanism in CIN patients, their involvement in other aspects of immune deregulation such as through the mTOR/autophagy pathway remains to be further explored.

No relevant conflicts of interest to declare.