Seizures and memory impairment induced by patient-derived anti-N-methyl-D-aspartate receptor antibodies in mice are attenuated by anakinra, an interleukin-1 receptor antagonist

Abstract

Objective: Neuroinflammation associated with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis may facilitate seizures. We previously showed that intraventricular administration of cerebrospinal fluid from patients with anti-NMDAR encephalitis to mice precipitates seizures, thereby confirming that antibodies are directly pathogenic. To determine whether interleukin (IL)-1-mediated inflammation exacerbates autoimmune seizures, we asked whether blocking the effects of IL-1 by anakinra, a selective IL-1 receptor antagonist, blunts antibody-induced seizures.

Methods: We infused C57BL/6 mice intraventricularly with purified serum IgG from patients with anti-NMDAR encephalitis or monoclonal anti-NMDAR IgG; subdural electroencephalogram was continuously recorded. After a 6-day interval, mice received anakinra (25 mg/kg sc, twice daily) or vehicle for 5 days. Following a 4-day washout period, we performed behavioral tests to assess motor function, anxiety, and memory, followed by hippocampus tissue analysis to assess astrocytic (glial fibrillary acidic protein [GFAP]) and microglial (ionized calcium-binding adapter molecule [Iba]-1) activation.

Results: Of 31 mice infused with purified patient NMDAR-IgG (n = 17) or monoclonal NMDAR-IgG (n = 14), 81% developed seizures. Median baseline daily seizure count during exposure to antibodies was 3.9; most seizures were electrographic. Median duration of seizures during the baseline was 82.5 s. Anakinra administration attenuated daily seizure frequency by 60% (p = .02). Anakinra reduced seizure duration; however, the effect was delayed and became apparent only after the cessation of treatment (p = .04). Anakinra improved novel object recognition in mice with antibody-induced seizures (p = .03) but did not alter other behaviors. Anakinra reduced the expression of GFAP and Iba-1 in the hippocampus of mice with seizures, indicating decreased astrocytic and microglial activation.

Significance: Our evidence supports a role for IL-1 in the pathogenesis of seizures in anti-NMDAR encephalitis. These data are consistent with therapeutic effects of anakinra in other severe autoimmune and inflammatory seizure syndromes. Targeting inflammation via blocking IL-1 receptor-mediated signaling may be promising for developing novel treatments for refractory autoimmune seizures.

Keywords: IL-1; anti-NMDA receptor encephalitis; autoantibodies; autoimmune seizures; cytokines; neuroinflammation.

Figure 1.

Experimental protocol to assess the role of anakinra in the persistence of seizures induced by anti-NMDAR antibodies, followed by behavioral and histological analysis.

Figure 2.

Administration of anakinra reduced seizures induced by anti-NMDAR antibodies. Data are daily medians and 25-75% interquartile ranges (IQR). Solid and dotted horizontal lines indicate median values and IQRs, respectively *, p < 0.05; **, p < 0.01, ANOVA with Sidak multiple comparisons tests. (A) Daily seizure counts in anakinra-treated mice (n = 12) were reduced during the treatment (orange bar) and post-treatment periods (green bar) compared to the corresponding baseline (yellow bar). The seizure counts were unchanged in the vehicle treated mice (n = 8). (B) Delayed effect of anakinra on seizure duration. The duration of seizures was decreased in the washout phase following anakinra. (C) Effects of anakinra on seizure counts were apparent 24-48 h following the initiation of treatment. Each time point represents the mean and SEM at the completion of a 24-h of recording. (D) Representative 60-min EEG recording and the corresponding spectrogram showing clusters of electrographic seizures in the parietal cortex of mice during the continuous intracerebroventicular (i.c.v.) infusion of anti-NMDAR antibodies. The vertical axis of the spectrogram represents frequency from 0 to 70 Hz and the horizontal axis represents time in min. The trace of one seizure is expanded to demonstrate the characteristic pattern of high amplitude sharp rhythmic activity.

Figure 3.

Behavioral phenotype of mice with autoimmune seizures treated with anakinra. (A) Anakinra rescued an ability to discriminate between familiar object (FO) and novel object (NO) in mice with seizures induced by anti-NMDAR antibodies. N = 9 (anakinra-treated), n = 7 (vehicle-treated). (B) Anakinra had no effect on locomotor activity in mice with seizures. N = 12 (anakinra- treated), n = 7 (vehicle-treated). (C) Anakinra did not affect the anxiety scores in mice with seizures. *, p<0.05, paired t-tests. Error bars represent mean ± SEM.

Figure 4.

Abundance of GFAP-positive astrocytes in the CA1 region of hippocampus of mice with autoimmune seizures. (A-D) Representative GFAP immunostaining of the CA1 region in vehicle-treated (upper panel) and anakinra-treated (lower panel) mice with seizures induced by continuous infusion of anti-NMDAR antibodies at 10 X (A, C) and 20 X (B, D). (E) Anakinra reduced the expression of GFAP in the CA1 region of hippocampus in mice with seizures. The abundance of GFAP labeling in the CA1 region was determined as the sum of the products of mean pixel intensity (grey scale units, gsu) and area of each event (μ2) in a fixed scan area. N = 7 (anakinra-treated), n = 6 (vehicle-treated). * p<0.05, Student’s t-test. Error bars represent mean ± SEM.

Figure 5.

Expression of Iba-1 immunoreactivity in the CA1 region of hippocampus in mice with autoimmune seizures. (A-D) Representative Iba-1 immunostaining images of the CA1 region of vehicle-treated (upper panel) and anakinra-treated (lower panel) mice with seizures induced by anti-NMDAR antibodies at 10 X (A, C) and 20 X (B, D). (E) Anakinra reduced the expression of Iba-1 in the CA1 region of hippocampus in mice with seizures. The abundance of Iba-1 labeling in the CA1 region was determined as the sum of the products of mean pixel intensity (gsu) and area of each event (μ2) in a fixed scan area. N=5 (anakinra-treated), n=5 (vehicle-treated). * p<0.05, Student’s t-test. Error bars represent mean ± SEM.