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

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.

The Central Nervous System Contains ILC1s That Differ From NK Cells in the Response to Inflammation.

Innate lymphoid cells (ILCs) are tissue resident cells with organ-specific properties. Here, we show that the central nervous system (CNS) encompasses ILCs. In particular, CD3-NK1.1+ cells present in the murine CNS comprise natural killer (NK) cells, ILC1s, intermediate ILC1s (intILC1s) and ex-ILC3s. We investigated the properties of CNS-ILC1s in comparison with CNS-NK cells during steady state and experimental autoimmune encephalomyelitis (EAE). ILC1s characteristically express CXCR3, CXCR6, DNAM-1, TRAIL, and CD200R and display heightened TNF-α production upon stimulation. In addition, ILC1s express perforin and are able to degranulate, although in a lesser extent than NK cells. Within the CNS compartments, ILC1s are enriched in the choroid plexus where very few NK cells are present, and also reside in the brain parenchyma and meninges. During EAE, ILC1s maintain stable IFN-γ and TNF-α levels while in NK cells the production of these cytokines increases as EAE progresses. Moreover, the amount of ILC1s and intILC1s increase in the parenchyma during EAE, but in contrast to NK cells, they show no signs of local proliferation. The upregulation in the inflamed brain of chemokines involved in ILC1 migration, such as CXCL9, CXCL10, and CXCL16 may lead to a recruitment of ILC1s from meninges or choroid plexus into the brain parenchyma. In sum, CNS-ILC1 phenotype, distribution and moderate inflammatory response during EAE suggest that they may act as gatekeepers involved in the control of neuroinflammation.