Microglial phenotypes in the human epileptic temporal lobe

Abstract

Microglia, the immune cells of the brain, are highly plastic and possess multiple functional phenotypes. Differences in phenotype in different regions and different states of epileptic human brain have been little studied. Here we use transcriptomics, anatomy, imaging of living cells and ELISA measurements of cytokine release to examine microglia from patients with temporal lobe epilepsies. Two distinct microglial phenotypes were explored. First we asked how microglial phenotype differs between regions of high and low neuronal loss in the same brain. Second, we asked how microglial phenotype is changed by a recent seizure. In sclerotic areas with few neurons, microglia have an amoeboid rather than ramified shape, express activation markers and respond faster to purinergic stimuli. The repairing interleukin, IL-10, regulates the basal phenotype of microglia in the CA1 and CA3 regions with neuronal loss and gliosis. To understand changes in phenotype induced by a seizure, we estimated the delay from the last seizure until tissue collection from changes in reads for immediate early gene transcripts. Pseudotime ordering of these data was validated by comparison with results from kainate-treated mice. It revealed a local and transient phenotype in which microglia secrete the human interleukin CXCL8, IL-1B and other cytokines. This secretory response is mediated in part via the NRLP3 inflammasome.

Figure 1. Diverse microglia shape and distribution.

(A) Diverse forms (stars) of IBA1+ microglia in the DG. (B) Cluster analysis of microglia shape (n=283 from CA1, CA3, SUB and DG of 9 patients) based on total length, L, number of primary ramifications, PR, and secondary ramifications, SR. Unsupervised clustering (Euclidean distance, Ward method) separated produced four groups, A1, A2, B1 and B2. The scale bar indicates normalized values for L, PR and SR. A typical reconstructed cell of each cluster is shown below. Scale bar: 10μm. (C) Interleaved box and whiskers plot showing the minimum and maximum % of microglia in clusters A1-B2 in CA1, CA3, DG and Sub. Differences were significant at p<0.005 (***) or p=0.05 (*), 2-way ANOVA with Tukey’s multiple comparison. Lines show a mean of 30 microglia per patient (n=15). (D) Neuronal and (E) microglia densities (cells/mm²) in CA1, CA3, DG and Sub (n=13 patients). The type of hippocampal sclerosis was 1, blue; 2, green or 3, red. Bar is mean ± SEM. (F) Microglia and neuronal densities were well correlated in CA1 (r=0.75 p=0.006, n=12 patients) and CA3 (r=0.72, p=0.001, n=12 patients) but less well correlated (G) in DG (r=0.24, p=0.4, n=13 patients) and SUB (r=0.19, p=0.5, n=14 patients).Spearman correlation, two tailed p-value. (H) Amoeboid microglia of cluster A1 (blue) were associated with regions of low neuronal density (r=-0.49, p=0.006, n=44; Spearman correlation) and ramified cells (cluster B1) with regions of higher neuronal density (r=-0.35, p=0.01, n=44; Spearman correlation, 2-tailed p).

Figure 2. Correlation of microglia phenotype marker with differences in form.

(A) Immunostaining for MHCII (purple) in microglia of clusters A1/A2 and B1/B2. Counterstained for IBA1+ (green) and DAPI (blue). Insets below, show MHCII+ in soma (A1/A2) or processes (B1/B2). Scale 10 μm. (B) Density of MCHII+ microglia for cells of clusters A1, A2, B1 and B2 and for (C) cells of the CA1, CA3, DG and subicular regions. Each point is a mean of 15 microglia (n=10 or 11 patients). Bar is ± SEM. (D) Immunostaining for CD68 (purple) in microglia, counterstained for IBA1 (green) and DAPI (blue) of clusters A1/A2 and B1/B2. Insets below, show CD68+ vesicles in microglia processes. Scale 10μm, 5μm for inset. (E) Density of CD68+ microglia for cells of clusters A1, A2, B1 and B2 and for (F) cells of the CA1, CA3, DG and subiculum. Fewer microglia of cluster B2 were CD68+, but little difference between regions. Each point is a mean of 15 microglia (n=10 or 11 patients). Bar shows mean +/- SEM. No significant differences were evident between areas. (G) Immunostaining shows CD68 (blue), Lamp1 (green), and Cathepsin D (red) colocalization in the phago-lysosome of an IBA1+ microglia (white). Scale bar 10μm. (H) CD32a (purple) staining, with IBA1 (green) and DAPI (blue) counterstain confirms the phagocytic phenotype. Scale bar 10μm. Stars in B, C, E, F show significance (Mann-Whitney 2-tailed test at p<0.01, ** and at p<0.05, *).

Figure 3. Microglia of different shapes respond differently to purinergic stimuli.

(A) changes in form of an amoeboid microglia from the CA1 region and (B) a ramified microglia of the subiculum induced by 2 mM ADP. Two-photon imaging of cells stained with a fluorescent tomato lectin. At left, a microglia imaged at t=0 and towards the right 3D reconstructions from t=0, 5, 12, 30 min after ADP stimulus onset. Scale bar 10μm. Light blue in 3D images, differences between successive reconstructions, shows ruffling of amoeboid and ramified cells. Yellow arrows for ramified microglia point to process retraction between 0 and 5, and between 5 and 12 min. (C) Time course of changes in mean somatic area (circles), arborization area (diamonds) and bleb area (triangles) for amoeboid microglia (n=7, 3 patients) and for (D) ramified microglia (n=7, 2 patients). (E) A longer delay from ADP application to membrane ruffling for ramified than for amoeboid microglia (2-tailed t test, t = -4,554 -, p< 0.001, n=14, 5 patients). (F) Correlation between cellular morphological index and the latency to ruffling. (Pearson correlation, r = 0.9; p=9.0e^-6). Amoeboid microglia open circles, ramified microglia filled circles.

Figure 4. Transcriptomic profile for regions of high or low neuronal death.

(A) Venn diagram of transcripts differentially expressed between CA1 or CA2/CA3 and SUB (n=7313, tissue from 12 patients, not including the type III sclerosis tissue, p-adj < 0.05). 169 microglia associated transcripts were differentially expressed. (B) Top five enriched cellular functions (IPA analysis; -log(p-value); z-score>2) deduced from all differentially expressed transcripts. (C) Top 5 enriched functions (–log p-value) from microglia associated transcripts (PSEA, Supplementary material 2). (D) Predicted network of links to cytokine regulators (IPA). The anti-inflammatory cytokine IL-10 was predicted from transcripts upregulated in CA1 and CA3. IL-10 acts inversely to the cytokine IFNG on 5 out of 7 downstream molecules (direct effect, solid line; z-score>2). Significant upregulation, red; activation of regulators, orange (z-score>2). (E) Heatmap of log2 fold changes for differentially regulated and IL-10 related, transcripts (p-adj <0.05). Microglia-associated in bold. (F) IL-10 (purple) is more highly expressed in CA1-CA3 than in SUB. Insets right, immunostaining for IL- 10 (purple), IBA1+ (white) and CD68 (green). (G) CD163 (red) immunostaining near blood vessels (BV) in CA1-3, but not SUB. Inset right CD163 staining (purple) in IBA1+ cell (green). (H) SOCS3 (purple) is more strongly expressed in IBA1+ (green) cells of CA1-3 than SUB. Inset right, SOCS3 signal in IBA1+ microglia. Scale bar 10μm. DAPI (blue) nuclear counterstain.

Figure 5. Transcriptomic and anatomical evidence for a recent, local seizure

(A) Ordered reads for immediate early genes (ARC, EGR1, FOS, FOSB, FOSL1, IER2, JUN, JUNB, NR4A1 and NR4A3) in tissue from 13 patients. Mean of 10 (red line) and values for each transcript (black lines). Highest value from any area (CA1, CA3, DG, SUB) is shown for each patient. Reads from all transcripts normalized to 1 over all samples. (B) Mean reads for the same immediate early genes in mouse CA1 region tissue at defined delays after kainic-acid (KA) treatment. Transcript differences between KA (n=3) or NaCl (n=3) injected animals at 6 hrs to 12 days after KA-injection. Red line mean of 10 transcripts. (C) Variation in mean reads for 10 immediate early genes from CA1, CA3, DG and SUB region of patients ordered to ptimes1, 2 and 11. The region (arrow) with the highest mean value of immediate early gene reads was defined as seizure-associated area for ptimes 1 and 2. (D) Immunostaining for PTGS2 (brown) in a seizure-associated and a non-seizure associated region at ptime1 tissue and ptime10 tissue. Scale, 20μm. (E) Immunostaining for MAP2 (purple) and Fos B (green) in seizure-associated and non-seizure associated regions from ptime1 and ptime4 tissue. Scale, 20μm. (Fi-iv) Data from all samples: 13 patients, ptimes1–13, 1-4 regions per patient, seizure-associated regions in red. Significance at p=0.05, dotted line. (Fi) –log p-values for enrichment of the function ‘epileptic seizure’ (IPA, 190 transcripts) from each sample (log2FC > ±1.5, p-adj<0.05), red point is the seizure-associated area. (Fii) -log p-value for differential expression of the 10 immediate early genes (Fiii) -log p-value for transcripts enriched in microglia (623 transcripts, PSEA list). (Fiv) Patients 1-13. High reported seizure frequency, red; low reported seizure frequency, blue. Numbers are clinical estimates of the latency from the last seizure to tissue collection

Figure 6. Pseudotime kinetics of the response to a seizure.

(A) variation in the top 5 reads for cytokine-related transcripts, (B) immune-related transcription factors and for (C) other molecules from ptimes1 -13. Values for the seizure-associated region of each patient. Each inset highlights the top 5 transcripts on plots of reads of all transcripts from the seizure-associated region at ptime1 against mean reads for all other samples. (D) Field records of seizure-like activity generated by a naïve human temporal lobe slice (unknown delay from last seizure) exposed to high excitability solution. (E) Cytokine release (CXCL8, IL-1A, IL-6, TFNA and IL-1B, multiplex-ELISA) from superfusate of acute slice after seizure-like activity (red) or after exposure to a control solution (blue). Bars show mean ± SEM. Stars, differences significant at ** p<0.01, or at * p<0.05 (n=4-6 patients, Mann-Whitney, 2-tailed p-value). (F) Fold enrichment of reads for the same cytokines in the seizure-associated area (defined from immediate early genes) at ptime1 compared to ptimes5-13 (all areas, all patients). (G) Changes in the proportion of CXCL8 immunopositive microglia (purple), numbers of extracellular CXCL8+ vesicles at less than 10μm from IBA1+ microglia (green) for ptimes1, 4 and 6. Stars show differences significant at **p<0.01 - (n=25-30 cells per patient, Mann Whitney, 2-tailed p-value). (H) CXCL8 (green) and IBA1 (white) immunostaining in tissue from ptimes1 and 6. Purple indicates CXCL8 colocalization with IBA1 (white arrow). Green shows extracellular CXCL8 vesicles (yellow arrow). Insets show co-localization at higher resolution. CXCL8+ microglia increased from ptime1-6. Extracellular CXCL8+ vesicles were detected only at ptimes1-4. Scale bar, 10μm.

Figure 7. NLRP3 inflammasome and cytokine secretion.

(A) Inflammasome linked transcripts predicted as key regulators in seizure-associated tissue at ptimes1-4. Heatmap of –log (p-value) for inflammasome components in seizure-associated regions (red box) at ptimes1, 3 and 10. (B) Pathway for P2RX7 receptor, inflammasome activation and cytokine secretion (from IPA). Upregulated transcripts from ptimes1-4, red. (C) Cytokine secretion (multiplex-ELISA) from superfusate of temporal lobe slices exposed to 2 mM ADP (red) or control solution (blue). Stars show differences significant at * p<0.05 (n=4-6, Mann-Whitney, 2-tailed p-value). (D) Immuno-staining for NLRP3 (green) and PYCARD (purple) inflammasome components in IBA1+ cells (blue) of tissue from ptime4 and (F) ptime 10. NLRP3 and PYCARD colocalize in microglia at ptime4 but not ptime10. Scale bar 10μm. (E) Proportion of microglia expressing NLRP3 and PYCARD (green) or only PYCARD (purple) at ptime1, 4, 5, 10 and 12 (mean ± SEM for 20 microglia per patient in all areas). PYCARD was present at all ptimes, but colocalized with NLRP3 only at ptimes1-4 when (G) reads for NLRP3 were elevated. (H) Immunostaining of PYCARD (Red) and IBA1 (white) at ptime1 and (G) ptime12. Blue indicates IBA1 and PYCARD colocalization (Blue arrow). (I) As perisomatic NLRP3 was reduced, PYCARD migrated to microglia ramifications. Data from 20 microglia at ptimes 1–12.