Neuronal pSTAT1 hallmarks synaptic pathology in autoimmune encephalitis against intracellular antigens

Abstract

Autoimmune encephalitis (AE) is an inflammatory syndrome of the central nervous system (CNS) triggered by aberrant immune responses against neuronal intracellular (IC-AE) or surface (NS-AE) autoantigens. The resulting neuronal alterations and clinical trajectories differ, with IC-AE often leading to fatal outcomes. Unfortunately, the scarce availability of tissue from AE cases has hampered systematic analyses that would allow an understanding of the pathogenesis underlying neuronal alterations in T cell-mediated AE syndromes. Here, we assembled a cohort comprising both NS-AE (n = 8) and IC-AE (n = 12) from multiple institutions to delineate key histopathological features that distinguish neuronal pathology between IC-AE and NS-AE. In contrast to NS-AE, IC-AE lesions present a prominent neuronal pSTAT1 signature, accompanied by a high proportion of brain-resident memory CD8 + T cells and neurodegenerative GPNMB + phagocytes which show synaptic engulfment with little C3-complement deposition. Our findings highlight distinct histopathological features of IC-AE compared to NS-AE, providing actionable biomarkers for diagnostics and treatment strategies.

Keywords: Neurodegeneration; Neuroinflammation; Phagocytes; Resident memory T cells; Synapses.

Fig. 1

Neuronal pSTAT1 signature in autoimmune encephalitis. a Venn diagram showing the overlap between top 400 upregulated transcripts from the neuronal translatome data of the viral déjà vu murine model of T cell-mediated viral encephalitis [14] and the top 400 upregulated cerebrospinal fluid (CSF) proteins from the comparison of autoimmune encephalitis with antibodies against intracellular antigens (IC-AE) and autoimmune encephalitis with antibodies against neuronal surface antigens (NS-AE) [35]. b The top five pathways enriched by the shared signature, as identified from the Human Molecular Signatures Database (MSigDB), are presented in descending order based on the −log10 transformation of their p-values. c Heatmap displaying the z-score transformed expression levels of proteins from the shared signature in CSF for IC-AE (Yo, Ma2, and AK5) and NS-AE (LGI1 and CASPR2)[35]. d–f Representative immunostainings for pSTAT1(red), neurons (NeuN or HuC/D, cyan), phagocytes (IBA1, yellow) and nuclei (DAPI, white) in tissue sections of NS-AE (d), IC-AE (e) and non-neurological disease (NND) controls (f). Scale bar = 25 µm. g Quantification of neuronal pSTAT1 expression. Data are shown on a base 10 logarithmic scale. Lines indicate the median. Symbols represent individual samples. ****P < 0.0001, ns not significant by Kruskal–Wallis test with Dunn’s correction for multiple comparisons

Fig. 2

CD8 + T cell infiltration and tissue resident memory signature in autoimmune encephalitis. a, b Representative immunostainings and quantification of CD8 + T cells (red) in tissue sections with DAPI (white) nuclear counterstaining of NS-AE, IC-AE and NND controls. Regions: temporal cortex (TC), hippocampus (HC), frontal cortex (FC), caudate nucleus (CN), dorsal root ganglion (DRG). c, d Representative immunostainings and quantification of tissue resident memory (T_RM) cells co-expressing CD8 (red), CD103 (yellow), CD69 (light blue), GZMB (green) and BCL2 (purple) with DAPI (white) nuclear counterstaining. The proportion of CD103 + GZMB + of CD8 + T cells is shown for NS-AE and IC-AE. Data are shown on a base 10 logarithmic scale (b) or standard scale (d). Lines indicate the median. Symbols represent individual samples. ****P < 0.0001, ns not significant by Kruskal–Wallis test (B) with Dunn’s correction for multiple comparisons and unpaired Student’s t-test (D). Scale bar = 50 µm

Fig. 3

Neurodegenerative phagocytes engulf synapses in autoimmune encephalitis. a–c Representative immunostainings and quantification of IBA1 + GPNMB + phagocytes in tissue sections with DAPI (white) nuclear counterstaining of NS-AE, IC-AE and NND controls. IBA1 (light blue), GPNMB (red). Data are shown on a base 10 logarithmic scale for tissue densities of IBA1 + phagocytes (b) and IBA1 + GPNMB + phagocytes (c). Lines indicate the median. Symbols represent individual samples. Regions: temporal cortex (TC), hippocampus (HC), frontal cortex (FC), caudate nucleus (CN), dorsal root ganglion (DRG). d 3D cell reconstruction of confocal immunostainings of IBA1 + phagocytes (white) exhibiting synptophysin positive (SYP, red) inclusion overlapping with CD68 + phagosomes (yellow) in NS-AE, IC-AE and NND controls. Nuclei are indicated with DAPI (blue). e Quantification of engulfed synaptic terminals (SYP) localized in the phagosomal compartment (CD68) of CNS phagocytes (IBA1) in brain sections of NS-AE (n = 7) and IC-AE patients (n = 11) and NDD (n = 14). Number of SYP + CD68 + phagocytes per mm² are shown. Lines indicate the median. Symbols represent individual samples. ****P < 0.0001, *P < 0.05; ns = not significant by Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Scale bars are 50 µm (A) and 5 µm (D). Regions: temporal cortex (TC), hippocampus (HC), frontal cortex (FC), caudate nucleus (CN)

Fig. 4

Complement C3 deposition at synaptic terminals and engulfment of C3-tagged synapses in autoimmune encephalitis. a 3D reconstruction of confocal immunostainings of synaptophysin (SYP, cyan) and complement C3 colocalization with synaptic terminals (C3, red) in brain sections of NS-AE, IC-AE and NND controls. Yellow circles highlight C3 deposition at synapses. b Ratio of C3-tagged synapses indicated as the proportion of the colocalized C3 volume over the total SYP volume for NS-AE (n = 7), IC-AE (n = 11) and NND controls (n = 14). Data are shown on a base 10 logarithmic scale, symbols represent individual samples. NMDAR-antibody AE is indicated by a diamond shape. c 3D cell reconstruction of confocal immunostainings of IBA1 + phagocytes (white) exhibiting C3-tagged synapses (C3-SYP, purple) inclusion in NS-AE, IC-AE and NND controls. Nuclei are indicated with DAPI (blue). d Quantification of engulfed C3-tagged synaptic terminals (SYP) of CNS phagocytes (IBA1) in brain sections of NS-AE (n = 7) and IC-AE patients (n = 11) and NDD (n = 14). Number of engulfed C3-SYP + terminals per phagocytes (mm³) are shown. NMDAR-antibody AE is indicated by a diamond shape. ***P < 0.001, **P < 0.01, *P < 0.05; ns = not significant by Kruskal–Wallis test with Dunn’s correction for multiple comparisons. Scale bar = 5 µm. Regions: temporal cortex (TC), hippocampus (HC), frontal cortex (FC), caudate nucleus (CN)

Fig. 5

Pathological insights in post-infectious autoimmune Encephalitis. a Representative immunohistochemistry for HSV1 (DAB, brown) in PI NMDAR-antibody encephalitis indicating the absence of infection. b Representative immunostaining for pSTAT1(red), neurons (HuC/D, cyan), phagocytes (IBA1, yellow) and nuclei (DAPI, white) in tissue sections of PI NMDAR-antibody encephalitis. c Representative immunostaining of CD8 + T cells (red) with DAPI (white) nuclear counterstaining in tissue sections of PI NMDAR-antibody encephalitis. d Representative immunostainings of IBA1 + GPNMB + phagocytes with DAPI nuclear (white) counterstaining of post-infectious NMDAR-antibody encephalitis. IBA1 (light blue), GPNMB (red). e Representative immunostainings of tissue resident memory (T_RM) cells co-expressing CD8 (red), CD103 (yellow), CD69 (light blue), GZMB (green) and BCL2 (purple) with DAPI (white) nuclear counterstaining in PI NMDAR-encephalitis. f 3D cell reconstruction of confocal immunostainings of IBA1 + phagocytes (white) exhibiting synaptophysin positive (SYP, red) inclusion overlapping with CD68 + phagosomes (yellow) in PI anti-NMDAR encephalitis. g 3D reconstruction of confocal immunostainings of synaptophysin (SYP, cyan) and complement C3 colocalization with synaptic terminals (C3, red) in brain sections of PI NMDAR-antibody encephalitis. h 3D cell reconstruction of confocal immunostainings of IBA1 + phagocytes (white) showing the absence of inclusions of C3-tagged synapses (C3-SYP, purple) in PI NMDAR-antibody encephalitis. i Heatmap and dendrogram analysis of CD8 + T_RM cells, neuronal pSTAT1 levels, GPNMB + phagocyte densities, and synaptic engulfment different autoimmune encephalitis samples. PI NMDAR and fulminant AMPAR-antibody AE (AMPAR*) cluster with IC-AE. The color scale represents minimum and maximum values, which are determined after scaling each variable by columns. For pSTAT1, a signal absence is assigned a value of −2. Positive values range from 0.01 to 2 after scaling. Scale bars: 20 µm (a–e, g) and 5 µm (f, h)