Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD

Abstract

Background In neuromyelitis optica spectrum disorders (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), neutrophils are found in CNS lesions. We previously demonstrated that NMOSD neutrophils show functional deficiencies. Thus, we hypothesized that neutrophil accumulation in the CNS may be facilitated by impairments affecting mechanisms of neutrophil death. Objective To evaluate cell death in blood neutrophils from aquaporin-4 (AQP4)-IgG-seropositive NMOSD and MOGAD patients as well as matched healthy controls (HC) using in vitro assays. Methods Twenty-eight AQP4 + NMOSD and 19 MOGAD patients in stable disease phase as well as 45 age- and sex-matched HC were prospectively recruited. To induce cell death, isolated neutrophils were cultured with/without phorbol 12-myristate 13-acetate (PMA). Spontaneous and PMA-induced NETosis and apoptosis were analyzed using 7-AAD and annexin-V by flow cytometry. Caspase-3 was assessed by western blot. Myeloperoxidase-DNA complexes (MPO-DNA), MPO and elastase were evaluated by ELISA, and cell-free DNA (cfDNA) by a fluorescence-based assay. Reactive oxygen species (ROS) were evaluated by a dihydrorhodamine 123-based cytometric assay. Serum GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ and IL-10 were evaluated by multiplex assays, and neurofilament light chain (NfL) by single-molecule array assay. Results In response to PMA, neutrophils from AQP4 + NMOSD but not from MOGAD patients showed an increased survival, and subsequent reduced cell death (29.6% annexin V+ 7-AAD+) when compared to HC (44.7%, p = 0.0006). However, AQP4 + NMOSD also showed a mild increase in annexin V+ 7-AAD− early apoptotic neutrophils (24.5%) compared to HC (20.8%, p = 0.048). PMA-induced reduction of caspase-3 activation was more pronounced in HC (p = 0.020) than in AQP4 + NMOSD neutrophils (p = 0.052). No differences were observed in neutrophil-derived MPO-DNA or serum levels of MPO, elastase, IL-6, IL-8 and TNF-ɑ. IL-15 levels were increased in both groups of patients. In AQP4 + NMOSD, an increase in cfDNA, GM-CSF and IL-10 was found in serum. A positive correlation among cfDNA and NfL was found in AQP4 + NMOSD. Conclusions AQP4 + NMOSD neutrophils showed an increased survival capacity in response to PMA when compared to matched HC neutrophils. Although the data indicate that the apoptotic but not the NETotic response is altered in these neutrophils, additional evaluations are required to validate this observation.

Upregulated complement receptors correlate with Fc gamma receptor 3A-positive natural killer and natural killer-T cells in neuromyelitis optica spectrum disorder

“…The immunopathogenesis of neuromyelitis optica spectrum disorder (NMOSD) has been shown to involve many components of the adaptive immune system including T cells [ 1 , 2 ], B cells [ 3 ], monocytes [ 4 ], as well as the innate immune system, including complement [ 5 ], granulocytes [ 6 , 7 ], plasmablasts [ 8 ], and antibodies [ 9 ]. In a simplistic model, upstream communication among peripheral T cells, B cells and monocytes leads to a coordinated decision to attack the central nervous system.…”

Anti-aquaporin-4 immune complex stimulates complement-dependent Th17 cytokine release in neuromyelitis optica spectrum disorders

“…Principal Component Analysis revealed that IL-8 was the top factor separating the NMOSD group from healthy controls. While a comprehensive analysis of cytokine levels in cerebrospinal fluid of NMOSD patients showed that IL-8 was significantly elevated compared to multiple sclerosis and controls 21 , another study using AQP4-IgG-positive NMOSD sera showed no significant elevation between healthy controls 22 . IL-8 is known www.nature.com/scientificreports/ as a chemotactic factor for granulocytes, mainly neutrophils, and also functions as a promoter of phagocytosis induction and angiogenesis 23 .…”

Construction of a Multi-Indicator Model for Abscess Prediction in Granulomatous Lobular Mastitis Using Inflammatory Indicators

“… 38 , 47 Phagocytosis of apoptotic neutrophils is upregulated with the release of IL-10, and as they disintegrate due to their own apoptosis, they release a variety of lysosomal enzymes that lyse the surrounding tissue and form an abscess. 48 , 49 In addition to its anti-inflammatory effects, IL-10 has pro-inflammatory properties that can characterise aggressive inflammation by switching the function of regulatory B cells from inducing CD8 + T cell tolerance to inducing inflammatory CD4 + T cell responses. 50 In our study, IL-10 is a risk factor for GLM abscess formation, and the mechanism may be related to the induction of neutrophil apoptosis and increased inflammatory aggressiveness.…”