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Published on: August 21, 2017
Impaired response of blood neutrophils to cell-death stimulus differentiates AQP4-IgG-seropositive NMOSD from MOGAD
Maria Schroeder-Castagno1,2,3, Alba Del Rio-Serrato1,2,3, Andreas Wilhelm4
1Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität Zu Berlin, ECRC Experimental and Clinical Research Center, a Cooperation Between the Max Delbrück Center for Molecular Medicine in the Helmholtz Association and Charité-Universitätsmedizin Berlin, Lindenberger Weg 80, 13125, Berlin, Germany.
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.
Insights
Neutrophils from aquaporin-4 (AQP4)-IgG-seropositive neuromyelitis optica spectrum disorder (NMOSD) patients exhibit enhanced survival. This suggests altered apoptosis, not NETosis, may contribute to NMOSD pathogenesis.
Area of Science:
- Neuroimmunology
- Cell Biology
- Autoimmune Diseases
Background:
- Neutrophils infiltrate central nervous system (CNS) lesions in neuromyelitis optica spectrum disorders (NMOSD) and MOGAD.
- Previous studies indicated functional deficiencies in NMOSD neutrophils.
- This study investigates if impaired neutrophil death mechanisms contribute to CNS neutrophil accumulation in NMOSD.
Purpose of the Study:
- To evaluate in vitro cell death (apoptosis and NETosis) in blood neutrophils from AQP4-IgG-seropositive NMOSD and MOGAD patients.
- To compare these findings with age- and sex-matched healthy controls (HC).
Main Methods:
- Prospective recruitment of 28 AQP4+ NMOSD, 19 MOGAD patients, and 45 HC.
- Neutrophil isolation and culture with/without PMA to induce cell death.
- Analysis of apoptosis and NETosis via flow cytometry (7-AAD, annexin-V).
- Assessment of caspase-3, MPO-DNA complexes, cfDNA, ROS, and serum cytokine/biomarker levels (GM-CSF, IL-6, IL-8, IL-15, TNF-ɑ, IL-10, NfL).
Main Results:
- Neutrophils from AQP4+ NMOSD patients showed significantly increased survival and reduced cell death (29.6%) compared to HC (44.7%) after PMA stimulation.
- A mild increase in early apoptotic neutrophils (annexin V+ 7-AAD-) was observed in AQP4+ NMOSD patients.
- Reduced caspase-3 activation and increased serum cfDNA, GM-CSF, and IL-10 were noted in AQP4+ NMOSD patients; cfDNA correlated positively with NfL.
Conclusions:
- Neutrophils from AQP4+ NMOSD patients exhibit enhanced survival capacity in response to PMA.
- The findings suggest altered apoptotic, but not NETotic, responses in these neutrophils.
- Further research is needed to validate these observations and their implications in NMOSD pathogenesis.
