Cerebrospinal Fluid Extracellular Vesicles with Distinct Properties in Autoimmune Encephalitis and Herpes Simplex Encephalitis

Abstract

Encephalitis mediated by autoantibodies against neuronal antigens and herpes simplex encephalitis (HSE) are seemingly separate causes of encephalopathy in adults. Autoimmune encephalitis (AE) is autoimmune in origin, and herpes simplex encephalitis is infectious. The purpose of this study was to examine the role of cerebrospinal fluid (CSF) exosomes from patients with antibody-positive AE and HSE. Towards this, exosomes were isolated from CSF from 13 patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis, 11 patients with anti-gamma-aminobutyric acid-B (GABAB) receptor encephalitis, 9 patients with anti-leucine-rich glioma-inactivated 1 (LGI1) encephalitis, and 8 patients with anti-contactin-associated protein-like 2 (CASPR2) encephalitis, and 12 control individuals negative of antibodies against neuronal autoantigens. There were ten miRNAs highly expressed in patients with anti-NMDAR encephalitis compared to those in control subjects. Eight miRNAs were found to be lower expressed in anti-NMDAR encephalitis CSF-derived exosomes. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched by AE differential expressed exosomic miRNAs demonstrated that AE-related exosomic miRNAs may participate as a feedback regulation in cancer development. In addition, the exosome concentration in CSF of 9 HSE patients was significantly higher compared to those from 9 HSV( -) patients. This observation was consistent with the results that exosome concentration was found to be higher in the animal model which was inoculated intranasally with HSV-1 compared to controls. Furthermore, western blot demonstrated that the subunits of NMDAR, GABA_BR, and AMPAR were detected highly expressed in exosomes derived from sera of HSV-1-treated animal model compared to controls. More importantly, exosomes isolated from CSF of HSE patients contained higher expression levels of two miRNAs encoded by HSV, miR-H2-3p, and miR-H4-3p compared to those from HSV( -) patients. In summary, HSV may trigger brain autoimmunity in HSE by presentation of surface autoantigens via exosomes.

Keywords: Autoimmune encephalitis (AE); Exosomes; Herpes simplex encephalitis (HSE); microRNAs.

Fig. 1

Exosome characterization. Characterization of exosome-like vesicles released from cerebrospinal fluid by A transmission electron microscopy of cerebrospinal fluid–derived exosomes from patients with anti-NMDA receptor encephalitis, patients with anti-GABA_B receptor encephalitis, patients with anti-LGI1 encephalitis, patients with anti-CASPR2 encephalitis, herpes simplex encephalitis patients, and control subjects. The scale bar indicates 100 nm. B Western blotting. Presence of exosomal markers, CD63 and CD9, in protein lysates from cerebrospinal fluid–derived exosomes from antibody-positive autoimmune encephalitis patients, herpes simplex encephalitis patients, and control subjects

Fig. 2

Cancer-associated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in anti-NMDAR autoimmune encephalitis-associated dysregulated miRNAs. Graphical representation of anti-NMDAR‐mediated targeted pathways generated using DIANA‐miRPath (y‐axis: p values; x‐axis: miRNA‐associated pathways). p values for pathway analysis were obtained by Fisher’s exact test as enrichment analysis method and the false discovery rate (FDR) was estimated using the Benjamini and Hochberg method

Fig. 3

Validation of miRNA expression. TaqMan real-time PCR to validate the expression levels of nine miRNAs (miR-301a-5p, miR-21-5p, miR-128-5p, miR-155-5p, miR-34a-5p, miR-326-5p, miR-132-5p, miR-29b-5p, and miR-20a-5p) selected for further validation using an independent cohort of 6 patients with anti-NMDA receptor encephalitis, 6 patients with anti-GABA receptor encephalitis, 6 patients with anti-LGI1 encephalitis, 6 patients with anti-CASPR2 encephalitis, and 6 control individuals. Data shown are as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 4

Exosome was induced for secretion into CSF in HSE compared to HSV( −) patients. A The electron micrographs of the microvesicles. B Microvesicle concentration was measured by nanoparticle tracking analysis (NTA). C Comparison of exosome concentration between 9 HSE patients compared to 9 HSV( −) patients

Fig. 5

Exosome was induced in mouse model of HSE. A The electron micrographs of the exosomes derived from sera of mouse model of HSE. B Nanoparticle tracking analysis (NTA) to show size distribution of exosomes derived from sera of mouse model of herpes simplex encephalitis. C Comparison of the difference of exosome concentration in HSV-treated mice (n = 6) compared to control group (n = 6)

Fig. 6

Exosome expressed higher NMDAR, GABAR, and AMPAR subunits in HSV-induced animal model. Western blot to show the protein level of NR2B subunits of NMDAR and GABA_B1R, and the GluR1 subunits of AMPAR on the pooled sera exosomes from 6 HSV-treated mice compared to those in control group (n = 6). Protein quantification was performed by bandscan and densitometry analysis with optical density for NR2B, GABAb1R, GluR1, TSG101, and CD9. TSG101 and CD9 were detected as the exosome markers and loading control

Fig. 7

Exosomal miR-H2-3p and miR-H4-3p miRNAs expressed by HSV. miR-H2-3p and miR-H4-3p were compared for expression from cerebrospinal fluid exosomes isolated from 5 HSE patients compared to 5 HSV( −) patients. Relative expression of miR-H2-3p and miR-H4-3p was calculated and observed statistically differentially expressed compared to the control group. *p < 0.05, **p < 0.01