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Sci Rep
2022 Oct 26;121:17956. doi: 10.1038/s41598-022-22806-9.
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GABAA receptor function is enhanced by Interleukin-10 in human epileptogenic gangliogliomas and its effect is counteracted by Interleukin-1β.
Ruffolo G
,
Alfano V
,
Romagnolo A
,
Zimmer T
,
Mills JD
,
Cifelli P
,
Gaeta A
,
Morano A
,
Anink J
,
Mühlebner A
,
Vezzani A
,
Aronica E
,
Palma E
.
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Gangliogliomas (GGs) are low-grade brain tumours that cause intractable focal epilepsy in children and adults. In GG, as in epileptogenic focal malformations (i.e., tuberous sclerosis complex, TSC), there is evidence of sustained neuroinflammation with involvement of the pro-inflammatory cytokine IL-1β. On the other hand, anti-inflammatory mediators are less studied but bear relevance for understanding seizure mechanisms. Therefore, we investigated the effect of the key anti-inflammatory cytokine IL-10 on GABAergic neurotransmission in GG. We assessed the IL-10 dependent signaling by transcriptomic analysis, immunohistochemistry and performed voltage-clamp recordings on Xenopus oocytes microtransplanted with cell membranes from brain specimens, to overcome the limited availability of acute GG slices. We report that IL-10-related mRNAs were up-regulated in GG and slightly in TSC. Moreover, we found IL-10 receptors are expressed by neurons and astroglia. Furthermore, GABA currents were potentiated significantly by IL-10 in GG. This effect was time and dose-dependent and inhibited by blockade of IL-10 signaling. Notably, in the same tissue, IL-1β reduced GABA current amplitude and prevented the IL-10 effect. These results suggest that in epileptogenic tissue, pro-inflammatory mechanisms of hyperexcitability prevail over key anti-inflammatory pathways enhancing GABAergic inhibition. Hence, boosting the effects of specific anti-inflammatory molecules could resolve inflammation and reduce intractable seizures.
Figure 1. Expression of genes of interest in controls, GG and TSC. RNAseq data indicate significant up-regulation (adjusted p value = 0.05) of IL-10Rα, IL-10Rβ, IL-1Ra, IL-1β and STAT3 in both GG and TSC. In addition, there is a significant upregulation of IL-10 and JAK1 in GG. IL-10 downstream signaling protein such as TIK1, phosphokinases (PIK3CA, PIK3CB, PIK3CD) and IL1-R1 did not show significant changes in either GG or TSC vs controls. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. A linear model was fit for each gene and moderated t-statistic was calculated after applying an empirical Bayes smoothing to the standard errors. Those genes with a Benjamini–Hochberg adjusted p value < 0.05 were considered significant. Differential expression analysis compared 21 TSC patients and 15 matched control cortices; 37 GG patients and 15 matched controls cortices.
Figure 2. Cellular expression pattern of IL-10 receptor in GG and TSC. Representative photomicrographs of immunocytochemical staining for IL-10Rα. Sections are counterstained with haematoxylin. Panels (A–C): IL-10Rα immunoreactivity (IR) in control brain. No detectable neuronal or glial labelling is observed in normal cortex (A–B; arrows indicate neurons) and white matter (C). Panels (D–E): IL-10Rα in gangliogliomas (GG). IL-10Rα IR was observed in dysplastic neurons (arrows, D and insert-a in E); insert in D shows expression of IL-10Rα (green) in a NeuN positive (red) dysplastic neuron; insert-b in E shows co-localization of IL-10Rα (blue) with pJAK (red). IL-10Rα IR was observed in scattered astroglial cells (arrowheads in E); insert-c in E shows co-localization of IL-10Rα (green) with GFAP (red). Panels (F–H): IL-10Rα in tuberous sclerosis complex (TSC). IL-10Rα IR was observed in dysmorphic neurons (arrows and inserts in F and H); IR was also observed in astrocytes (arrowheads; insert in H-a) and few giant cells (asterisks) within the dysplastic area. Insert in (F) shows expression of IL-10Rα (green) in a NeuN positive (red) dysmorphic neuron. Insert in (G) shows co-localization of IL-10Rα (green) with MAP2 (red). Insert (a) in (H) shows co-localization of IL-10Rα (green) with GFAP (red). Insert (b) in (H) shows co-localization of IL-10Rα (blue) with pS6 (red) in a dysmorphic neuron. Scale bar: (A, D): 100 µm; (F, G): 80 µm; (B, C, E): 40 µm; (H): 30 µm. Details on the cohort used are reported in Supplementary Information.
Figure 3. IL-10 effect on GABA current amplitude in oocytes injected with human α1β2γ2 cDNA. The bar-graph represents the mean and ± s.e.m. of the IGABA amplitudes evoked from oocytes intranuclearly injected with α1β2γ2 cDNAs before (black) and after (red) the incubation with IL-10 (200 ng/mL, 3 h; n = 8; p > 0.05 by paired t-test).The IGABA amplitudes recorded after the IL-10 incubation were normalized to the response obtained before exposure to IL-10 for each cell (range of current amplitudes: from 247.5 to 1119.0 nA), then averaged and expressed as a percent variation. Traces depict representative currents measured after 4 s application of GABA (white bar, 50 μM) in oocytes injected with α1β2γ2 cDNAs before (black trace) and after (red trace) IL-10 incubation (for 3 h). Grey bar on the right trace represents the block by 100 μM bicuculline (representative of 4 experiments).
Figure 4. Time-course effect of IL-10 on GABA current amplitude in oocytes microinjected with GG tissue. GABA alone was applied at 250 μM at the beginning of each experiment (time zero) and at various time points after treatment with 100 ng/ml IL-10. Data (mean ± s.e.m.; n = 6–12 oocytes/time point; patients #8–12 in Table 1) represent the percentage increase of the peak amplitude induced by IL-10. Data were normalized to the mean current amplitude recorded at time zero (23.7 ± 8.6 nA, n = 12). Inset: Traces depict representative GABA currents at the indicated times. * = p < 0.05 by Wilcoxon signed rank test; ** = p < 0.01 by paired t-test.
Figure 5. Characterization and blockade of IL-10 effect on GABA current amplitude. (A) Bar-graph of IL-10 effect on IGABA current amplitude in oocytes microtransplanted with control tissue (n = 17), TSC (n = 24) and GG (n = 80) tissues. Data are expressed as mean ± s.e.m. Inset: IGABA amplitude is expressed as percent increase above baseline (before IL-10 incubation, ranges of current amplitudes: control tissue, from 3.8 to 53.7 nA; TSC, from 7.7 to 73.7 nA; GG, from 5.5 to 84.0 nA). ** = p < 0.01 by paired t-test. (B) Representative superimposed current traces (GABA 250 μM, white bars) of control-, TSC- and GG-injected oocytes before (black trace) and after (red trace) incubation with IL-10 (100 ng/mL for 3 h). Grey bars represent the block by 100 μM bicuculline (representative of 3 experiments for each tissue) (C) Bar-graph shows the effect of incubation of K252a (2 μM, a broad-spectrum protein kinases inhibitor) or baricitinib (Bar 0.5 μM, a selective JAK1 and JAK2 inhibitor) with IL-10 (100 ng/ml). Black bar-graph represents the mean current value (nA) before incubation with IL-10 alone (red, n = 18) or in combination with the two blockers (blue, n = 8 for each blocker). ** = p < 0.01 by paired t-test (D) Dose–response curves of GABA (1 μM–1 mM) before (black curve) and after (red curve) incubation with IL-10 (100 ng/ml for 3 h) in oocytes microinjected with GG tissues (Patients # 8–10, Table 1). Averaged EC50 were 107.0 ± 9.7 μM, nH = 1.4 ± 0.10, before IL-10 and 67.0 ± 3.79 μM, nH = 1.77 ± 0.16; n = 16; statistics for the dose–response experiments: p < 0.05 by paired t-test.
Figure 6. Effect of IL-1β on GABA current in the absence or presence of IL-10. Bar-graphs show the % variation in IGABA amplitude induced by IL-10 (red bar and trace), IL-1β (green bar and trace) or their combination (grey bar and trace) in oocytes injected with GG tissues (Patients # 8–10; Table 1). Data are expressed as a % variation of the mean current amplitude after the incubation with each cytokine singularly or in combination. Mean current variation was + 31.0 ± 2.6% after IL-10 incubation (100 ng/ml, n = 10), − 19.6 ± 3.15% after incubation with IL-1β (25 ng/ml, n = 10) and − 15.6 ± 3.5% after co-incubation with IL-10 + IL-1β (n = 10) as described in the text. ** p < 0.01 by paired t-test.
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