Abstracts

Rationale: Seizures frequently occur in Autoimmune Encephalitis (AE), but the factors underlying their development have not been fully identified. Auto-Abs, indeed, can account for the synaptic dysfunction, but only incompletely explain the generation of seizures that do not invariably occur in all the AE patients sharing the same auto-Ab, and can occur even in AE-seronegative cases (Bien, 2017; PMID: 28684941; Dalmau and Graus, 2018; PMID: 29490181). Moreover, there is growing evidence that inflammatory mediators could contribute to seizure development in epilepsy (Vezzani, 2019; PMID: 31263255; Librizzi, 2021; PMID:34061984). In this study we investigated the specific role of peripheral blood mononuclear cells (PBMCs) in initiating brain inflammation and BBB damage, either autonomously or in cooperation with blood-borne molecules and microglial cells, to induce seizure-like events (SLEs). We proposed to identify novel mechanisms of seizure generation and new immunological markers specific to AE subtypes.

Methods: We will use the in vitro guinea pig brain (de Curtis, 2016; PMID: 25843067), to evaluate AE-derived immuno-inflammatory mediators (PBMC -derived cyto-chemokines) on seizure occurrence and on blood-brain barrier (BBB) integrity. Plasma samples were collected from healthy volunteers and from AE patients. HPBMCs were cultured in a standard medium. At t0 and at t24, supernatants were analyzed by Bio-Plex 200 multiplex array system for the human cytokine 27-plex panel (BioRad; Librizzi, 2021). Healthy volunteers and AE patient-derived hPBMCs (5-10x 10⁶) and their supernatant, were perfused intra-arterially in the isolated brain preparation. LPS (100 ng/ml) and human albumin (4 mg/mL) were previously perfused into the arterial stream to respectively favor and mimic a mild BBB impairment (Librizzi 2021). The specific ability of the experimental treatment to induce seizure-like events (SLEs) was investigated. Morphological analysis and reconstruction of GFAP- and Iba1-positive glial cells were also performed.

Results: The application of this protocol evoked seizure activity and glial cell activation when perfused in the isolated in vitro brain preparation. Bioplex immunoassay revealed an increased level of inflammatory mediators such as IL-1b, IL-1ra, TNFa, IL-8, and IL-9 in AE patient-derived hPBMC supernatants.

Conclusions: Activated PBMCs and their secreted molecules seem to have an impact on the neurovascular compartment and brain excitability. The present findings are preliminary to further in vitro studies on the pathogenic role of PBMC-mediated inflammation on ictogenesis. Our experiments might shed light on identifying key mediators involved in seizure generation in order to identify novel treatments in epilepsies due to inflammatory and dysimmune causes.

Funding: Fondazione Epilessia; European Union’s Horizon 2020; Research and Innovation Programme under the Marie Sklodowska-Curie grant agreement no. 722053 (EU-GliaPhD),Italian Health Ministry, EPICARE project of the Associazione Paolo Zorzi per le Neuroscienze.