Acute neuronal cell death and neuroinflammation per se do not trigger secondary autoimmune encephalitis

Acute neuronal cell death and neuroinflammation per se do not trigger secondary autoimmune encephalitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Acute neuronal cell death and neuroinflammation per se do not trigger secondary autoimmune encephalitis Justus Wilke, Antonios Ntolkeras, Vinicius Daguano Gastaldi, Kathrin Bobrowski, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6499111/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted 15 You are reading this latest preprint version Abstract Patients with virus encephalitis, such as herpes simplex encephalitis and Japanese encephalitis frequently relapse with autoimmune encephalitides associated with neural autoantibodies. It has been hypothesized that the infection-induced damage to the central nervous system results in shedding of neural autoantigens, their presentation to the peripheral immune system, and initiation of a secondary autoimmune encephalitis that targets these autoantigens. To test this hypothesis, we utilized a transgenic mouse model of virus-like but sterile encephalitis. After induction of acute neuronal death in the hippocampus, we monitored the mice for encephalitis-like symptoms for up to 10 months, evaluated the degree of neuroinflammation at several time points and screened their plasma for autoantibodies against 49 different autoimmune disease-associated brain autoantibodies. Throughout the study period, we did not detect any symptoms of severe autoimmune encephalitis, like hyperactivity, circling, seizures, lethargy. Evaluation of microglia numbers and morphology revealed pronounced microgliosis 1-week after initial encephalitis induction, which decreased over time. Scattered lymphocyte infiltration was present at all times in hippocampi of encephalitis mice, and did not increase over time. Perivascular cuffs were not detected. Infiltrating lymphocytes mainly consisted of CD8 + T cells. B cell infiltration was rare and did not differ from healthy control mice. High-parameter immunophenotyping of peripheral blood leukocytes did not reveal any changes associated with an autoimmune response. Testing all plasma samples (n = 30/group) at a dilution of 1:100 for autoantibodies against 49 neural autoantigens gave only two positive results, namely one healthy control with anti-CASPR2 autoantibodies (IgG) and one post-encephalitis mouse with anti-homer-3 autoantibodies (IgM). Overall, these findings suggest that acute neuronal cell death and neuroinflammation per se are not sufficient to trigger downstream autoimmune encephalitis relapses. Biological sciences/Immunology/Autoimmunity Health sciences/Molecular medicine Biological sciences/Immunology/Neuroimmunology Biological sciences/Neuroscience/Diseases of the nervous system/Encephalopathy acute encephalitis autoimmune relapse microglia autoimmunity autoantibodies Figures Figure 1 Figure 2 Figure 3 Figure 4 HIGHLIGHTS Acute neuronal cell death and neuroinflammation alone do not cause autoimmune encephalitis relapses. Microgliosis, seen fully developed at 1 week after initial encephalitis induction, decreases over time. Testing plasma of post-encephalitis mice and controls at a dilution of 1:100 for 49 neural autoantibodies yielded only one positive result in each group. INTRODUCTION Autoimmune-mediated clinical symptoms and autoantibodies against neuronal surface antigens are frequently observed in about one quarter of patients recovering from herpes simplex encephalitis (HSE) and Japanese encephalitis (JE) [ 1 – 15 ]. This secondary encephalitis is often referred to as autoimmune relapse or autoimmune encephalitis and typically occurs within 1–2 months after the primary virus encephalitis. It is characterized by an absence of the original pathogen, the presence of anti-neuronal autoantibodies, and autoimmune encephalitis-like symptoms that frequently improve after immunosuppressive treatment [ 7 , 10 , 12 , 13 ]. While most cases appear to be associated with autoantibodies against NMDA receptors, other anti-neuronal antibodies, including GABAAR, AMPAR, D2R-AB, are also detected in CSF of symptomatic and convalescent patients after HSE and JE [ 2 , 3 , 5 – 7 , 9 , 13 , 15 , 16 ]. It has been hypothesized that the virus-induced neuronal cell death, which is common in HSE and JE [ 14 , 17 , 18 ], results in a release of neuronal surface antigens. These antigens are supposedly drained to peripheral lymphoid organs, where they are presented to the adaptive immune system, eliciting an autoimmune response against neuronal autoantigens. Presumably, activated autoreactive B cells, T cells, and antibody secreting cells then traffic to the CNS and induce a second neuroinflammatory response [ 19 – 23 ]. This hypothesis is supported by the absence of anti-neuronal autoantibodies during the acute phase of JE and HSE [ 7 , 13 ], which indicates de novo autoantibody formation in response to encephalitis. The central nervous system (CNS) has long been considered an immune-privileged site, but accumulating evidence suggests that its cellular components can differentially shape immune responses. A landmark study by Traka et al. [ 24 ] demonstrated that targeted ablation of oligodendrocytes using a diphtheria toxin (DTA)-based approach in transgenic mice leads to delayed but sustained secondary autoimmune inflammation resembling multiple sclerosis. These findings suggest that oligodendrocyte death may not only be a consequence of CNS inflammation but also serve as a trigger for autoimmunity under certain conditions. To date, it has remained unclear whether the selective loss of other CNS cell types can initiate a similar cascade. In particular, the immunological consequences of targeted neuronal ablation have not been systematically explored. Given the widespread neuronal loss observed in HSE and JE, as well as various neurodegenerative and autoimmune diseases, this question bears significant clinical relevance. Here, we investigated whether selective ablation of neurons in adult mice using an analogous DTA-mediated system leads to the induction of CNS-targeted autoimmunity. In stark contrast to the oligodendrocyte ablation model, we found that neuroinflammation gradually subsided after targeted neuronal loss without detectable peripheral immune activation or relapse of CNS inflammation. Our findings suggest that the immunogenic potential of cell death within the CNS is not uniform across cell types and point to a unique role for oligodendrocytes in modulating neuroimmune interactions. Furthermore, the results suggest that additional factors, such as the prominent activation of the innate and adaptive immune system by virus/pathogen-associated molecular pattern pathways, are required to drive post-encephalitis autoimmune relapses. METHODS Mice : All animal experiments were approved by the local animal care and use committee (LAVES, Niedersaechsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany; license number 33.19-42502-04-18/2803) in accordance with the German animal protection law. The experiments were performed in accordance to approved protocols and reported following the ARRIVE guidelines. Mice were housed in groups of up to 16 in a temperature (~22°C) and humidity (~50%) controlled environment, 12h light/dark cycle with food (standard food, Sniff Spezialdiäten, Germany) and water ad libitum . Cages were equipped with wood-chip bedding and nesting material (Sizzle Nest, Datesand, United Kingdom). Experimental mice were weaned at postnatal day 21 and separated by sex. Mice were randomly allocated to treatment groups (tamoxifen versus corn oil) on a cage by cage basis. Investigators were unaware of group assignment (‘fully blinded’) throughout data acquisition and analysis. Sterile Encephalitis Model: Acute neurodegeneration in pyramidal neurons and subsequent neuroinflammation was induced in transgenic C57BL/6 mice double heterozygous for Neurod6 tm2.1(cre/ERT2)Kan (‘NexCreERT2’, [25]) and Gt(ROSA)26Sor tm1(DTA)Jpmb (‘Rosa26-eGFP-DTA’, [26]), at the age of 7-8 months via tamoxifen. Tamoxifen was administered intraperitoneally for 3 consecutive days at a dose of 100mg tamoxifen (CAS#10540-29-1 T5648, Sigma-Aldrich) per kilogram body weight per day. Tamoxifen was dissolved in corn oil (C8267, Sigma-Aldrich) at a dose of 10mg/mL. Control littermates were injected with equivalent corn oil volumes. Mouse monitoring: Groups of mice were inspected daily for signs of encephalitis-associated behavior (hyperactivity, circling, seizures & lethargy) in home cages. In addition, all mice were individually inspected for aberrant behavior on a weekly basis during cage changes. Blood Sampling: Mice were euthanized via intraperitoneal injection of 300-700mg/kg body weight 2,2,2,-tribromoethanol (T48402, Sigma) solubilized in water. Approximately 400uL blood was collected into syringes filled with 50uL of 80 mM EDTA solution by cardiac puncture prior to perfusion. For blood flow cytometry, 100uL whole blood were used immediately. Plasma was collected from the remaining blood samples after centrifugation at 1000 g for 10min at room temperature and stored at -80°C. Blood Flow Cytometry : Whole blood samples (100µL) were transferred into flow cytometry tubes and incubated with antibody master mix (supplementary table 1) for 15min at room temperature. Red blood cells were lysed by 15min incubation with 2mL ACK buffer (0.15 M NH 4 Cl, 10 mM KHCO 3 , 0.1 mM EDTA, pH 7.2-7.4) per sample. Samples were centrifuged at 300g for 5min and washed with 1mL ACK buffer. Lymphocytes were resuspended in 500µL FACS buffer (2% BSA in PBS) and measured on a FACSymphony S6 (BD). Data acquisition was stopped after recording 30,000 APC quantification beads per sample. Flow cytometry data was imported to FlowJo v.10.9.0 and compensated using single stained controls. An empty 540LP/35BP channel in the 445 nm Laser line was used to record autofluorescence. Manual clean up gating was performed on SSC-A versus FSC-A to separate cells and quantification beads. As eGFP is ubiquitously expressed in cells of heterozygous Rosa26-eGFP-DTA mice, lymphocytes were selected by gating of eGFP and CD45 double positive events. FSC-H versus FSC-A gating was used to exclude doublets. After individual clean-up gating, all single lymphocyte events from all samples were concatenated. Uniform hierarchical gating was performed on the concatenated sample and copied to individual samples for enumeration of cell types and subsets. First, granulocytes were identified based on high SSC-A and Ly6G. Non-granulocytes were gated for CD115 and NK1.1 to identify NK cells (NK1.1+) and monocytes (CD115+). Next, B cells were identified by gating for CD19 within the CD115 and NK1.1 negative cells, followed by gating for CD8a versus CD4 within CD19- events to identify CD8+ and CD4+ T cells respectively. Negative events were further subdivided into other myeloid cells (CD11b+, FcR+) and unclassified events (lineage negative). The quality of the hierarchical gating strategy was further evaluated qualitatively using t-SNE clustering. Major cell types were further divided into distinct subsets based on their expression of CD27, CD138, CD44, CD62L, and CD11b. The following immune cell subsets were identified: CD27+/CD11b+ M1 NK cells, CD27-/CD11b+ M2 NK cells, CD27+/CD11b- immature NK cells, and CD27-/CD11b- immature NK cells [27], CD27-/CD138- naïve B cells; CD27+/CD138- memory B cells; CD138+ antibody secreting cells [28]; CD62L+/CD44- naive T cells, CD62L-/CD44+ effector/ effector memory T cells, and CD62L+/CD44+ central memory T cells [29, 30]. For statistical analyses, the major immune cell types were normalized to the total amount of white blood cells measured per sample. To assess the differentiation state of individual cell types, immune cell subsets were normalized to their parent population (cell type of interest). Data was exported to GraphPad Prism (v 10.1.0). Data normality was assessed using Shapiro Wilk test. Groups with normally distributed data were compared using Welch’s corrected unpaired two-sided t-tests, whereas non-normal data was compared using the non-parametric Mann-Whitney tests. Autoantibody Testing : For serological analyses, mouse blood was diluted 1:100 and tested for autoantibodies using biochip mosaics (Euroimmun, Lübeck, Germany) as previously described [31]. Briefly, these mosaics contained unfixed, nitrogen-frozen tissue cryosections (4 µm; rat hippocampus, monkey cerebellum) alongside recombinant cell substrates of formalin- or acetone-fixed transfected HEK293 cells. The expression of recombinant autoantigens was confirmed using human or commercially available monospecific antibodies. The following 49 disease-associated neural antigens [19, 21, 23, 32-53] were evaluated: Myelin, NMDAR, AMPA-R1/R2, GABAR-B1/B2, LGI1, CASPR2, GAD65, mGluR1, mGluR5, Ma2, Recoverin, Zic-4, CARPVIII, Hu, Ri, CV2, Neurochondrin, Yo, ITPR1, Homer-3, Neurexin, ERC1, ARHGAP26, DNER, IgLON5, DPPX, AT1A3, GLRa1b, AQP4, GluRD2, MOG, GABA-a, Flotillin, KCNA1, MBP, DRD2, Contactin-2, KCNA2, Neurofascin 155, Neurofascin 186, Contactin-1, Sez6l2, AP3B2, PCA-2, AGNA, Amphiphysin, MAG, GFAP, ANNA-3. Autoantigen expression was validated by immunological methods employing monospecific control antibodies. To detect autoantibodies of the IgG, IgA and IgM isotypes, the following fluorescently labeled isotype-specific anti-mouse immunoglobulin antibodies were used: anti-mouse IgA (Cat. #62-6700, Thermo; Alexa Fluor 488 custom-labeled at Synaptic Systems), Alexa Fluor 488 labeled anti-mouse IgM (A21042, Thermo), Alexa Fluor 488 labeled anti-mouse IgG (A21202, Thermo). Histology : Mice were transcardially perfused with Ringer solution and 4% formaldehyde in PBS after anesthetic euthanasia by intraperitoneal injection of 300-700mg/kg body weight 2,2,2,-tribromoethanol (T48402, Sigma), solubilized in water. Brains were dissected and post-fixed overnight in 4% formaldehyde in PBS, dehydrated for two days in 30% sucrose solution, embedded in optimal cutting medium (Tissue-Tek, #4583, Sakura), frozen, cryosectioned, and stored in cryoprotectant solution (1x PBS with 25% ethylene glycol, 25% glycerol). Histological analyses were performed in 30-70µm coronal sections as previously described [54, 55]. Fluorojade C staining of degenerating neurons was performed as previously described (1, 2). Briefly, 30µm coronal sections were mounted on microscopy slides, dried overnight at room temperature and rehydrated. Rehydrated slides were incubated with 0.06% potassium permanganate solution for 10min, rinsed with water, and stained for 10min in a 0.0001% solution of Fluorojade C (AG325, Sigma) and 0.1µg/mL 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma), dissolved in 0.1% acetic acid. Lastly, slides were rinsed with ddH 2 O, dried at 60°C, and mounted. Images were acquired as tile scans with a pixel scaling of 1.04µm on a Zeiss LSM880 laser scanning confocal microscope equipped with a 10x air objective (0.45NA). Presence of neurodegeneration was evaluated qualitatively by a blinded investigator. Rating criteria was the presence or absence of at least 10 distinct Fluorojade C positive cell bodies within the hippocampus. Iba1 and CD45 double-labeling of microglia and infiltrating lymphocytes, was performed in 30µm coronal sections using standard free-floating immunofluorescence techniques with the following antibodies: rat anti-CD45 (1:250; clone 30-F11, 103101, Biolegend), rabbit anti-IBA-1 (1:250, 019-19741, Wako), Alexa Fluor-555 anti-rabbit (1:500, A-21428, Invitrogen), Alexa Fluor 647 anti-rat (1:500, A-31571, Thermo). Images of 2-4 hippocampi per mouse were acquired as tile scans with a pixel scaling of 0.52µm on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ [56]. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn's test for post-hoc pairwise comparisons. Quantification of CNS-infiltrating T cells and B cells were performed using triple-labeling of CD45, CD3, and CD19 in 30µm coronal brain sections. Standard free-floating immunofluorescence were used with the following antibodies: rat anti-CD45 (1:250; clone 30-F11, #103101, Biolegend), hamster anti-CD3 (1:250, clone 145-2C11, #557306, BD), rabbit anti-CD19 (1:250, clone D4V4B, #90176S, Cell Signaling), Alexa Fluor-488 anti-rat (1:500, #712-547-003, Jackson Immuno Research), Alexa Fluor-555 anti-rabbit (1:500, #A-21428, Invitrogen), Alexa Fluor-647 anti-hamster (1:500, #127-605-1685, Jackson Immuno Research). Images were acquired as tile scans with a pixel scaling of 0.69µm on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ [56]. CD3+/CD45+ lymphocytes were quantified as T cells while CD19+/CD45+ lymphocytes were classified as B cells. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn's test for post-hoc pairwise comparisons. CD8 and CD3 double labelingof CD8+ T cells was performed in 30µm coronal sections using standard free-floating immunofluorescence techniques with the following antibodies: hamster anti-CD3 (1:250, clone 145-2C11, #557306, BD), rat anti-CD8a (1:250, clone S18018A, #164702, Biolegend), Alexa Fluor-555 anti-hamster (1:500, A78964, Invitrogen), Alexa Fluor-647 anti-rat (1:500, A21247, Invitrogen). Images were acquired as tile scans with a pixel scaling of 0.83µm on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ [56]. CD3+/CD8+ double positive cells were counted as CD8+ T cells. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn's test for post-hoc pairwise comparisons. Morphological analysis of microglia was performed in 70µm coronal brain sections after staining with a standard free-floating immunofluorescence protocol using the following antibodies: rabbit anti-Iba1 (1:250, #019-19741, Wako), Alexa Fluor 555 labeled anti-rabbit (1:500, A-21428, Invitrogen). Nuclei were stained for 10 min at room temperature with 0.2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI, D9542, Sigma) in PBS. Per mouse, 1-3 Z stacks spanning 20µm (0.4µm step size) were acquired within the cornu ammonis (CA) with a pixel scaling of 0.2µm on a Zeiss LSM880 laser scanning confocal microscope equipped with a 40x oil objective (1.4 NA). All imaging settings including LASER intensities and pixel dwell times were kept constant throughout the experiment. Images were processed using the microglia morphology quantification tool (MMQT) developed by Heindl, Gesierich and colleagues [57]. Images with visible artifacts in the orthogonal views and images with poor spatial correlations in Z and XY dimensions were excluded from the analysis. Relevant parameters describing the 3D morphology of microglia were extracted and microglia were filtered using the following parameters: distance to X & Y borders >15µm; distance to Z borders >5µm; number of nuclei = 1; merged soma =0; shortest distance between microglia soma >15µm; number of branches >0. Linear mixed-effects models were used to compare morphological parameters between groups, with groups as a fixed effect and individual animals as a random effect to account for repeated measures within mice (multiple microglia per mouse). Models were fitted using the lmerTest package [58] and p-values for fixed (group) effects were extracted in R. Statistical Analysis : Statistical analyses were performed as described in respective method sections. Analyses were performed either in Prism 10.1.0 (GraphPad Software) or R v4.3.1 [59]. Statistical tests were selected based on experimental design and data distribution. Unless stated otherwise, results are presented as mean±standard deviations (SD). P-values <0.05 were considered statistically significant. RESULTS Longitudinal monitoring of mice after sterile induction of neuronal death did not reveal autoimmune encephalitis-like symptoms To test if the acute destruction of neurons in the absence of any virus is sufficient to induce an autoimmune relapse in mice, we utilized a previously characterized transgenic mouse model of virus-like but sterile encephalitis [ 55 ]. Acute neurodegeneration of hippocampal and cortical pyramidal neurons and subsequent neuroinflammation was induced in adult double heterozygous NexCreERT2 x Rosa26-eGFP-DTA mice via intraperitoneal tamoxifen injections (Fig. 1 A). A total of 64 mice (1:1 male/female ratio) were used for this study, of which half received tamoxifen whereas the other half received corn oil (healthy controls). To monitor disease progression histologically, 4 mice per group and timepoint were euthanized for tissue collection 1 week and 10 weeks after encephalitis induction (Fig. 1 ). The remaining 24 mice per group were monitored daily for autoimmune encephalitis-like symptoms, such as hyperactivity, circling, seizures, lethargy, and death [ 60 ] for 10 months. Two corn oil control mice died spontaneously 221 days and 305 days after injections, likely due to age-related reasons (14.7 and 17.5 months). None of the tamoxifen injected mice died and autoimmune encephalitis-like symptoms were absent in all mice throughout the study period. The presence of neurodegeneration and neuroinflammation were confirmed 1 week after tamoxifen injection during the acute phase of the primary encephalitis (n = 4/ group; Fig. 1 B and Fig. 2 A). By ten weeks after tamoxifen induction, degenerating neurons had been cleared from the hippocampus, as indicated by the absence of Fluorojade C positive cell bodies (n = 4/group; Fig. 1 B). Microglia profiling in the hippocampus points towards chronic neuroinflammation rather than relapse with acute autoimmune encephalitis While there is no clear consensus on the definition of neuroinflammation yet, it is generally accepted that a key feature of neuroinflammation is the response of microglia to pathogenic stimuli, such as degenerating neurons and DAMPs or CNS infiltrating leukocytes [ 61 ]. In neurodegenerative diseases and autoimmune encephalitis, this response is typically characterized by an increase in microglia numbers/density and morphological changes [ 62 – 68 ]. To longitudinally monitor the inflammatory status of microglia we quantified their density and morphology 1 week, 10 weeks and 10 months after induction of acute neuronal cell death (Fig. 2 A). During the acute phase, microgliosis was most prominent in the cornu ammonis , which contained the majority of degenerating neurons, but a significant increase of microglia numbers was also observed within the neighboring dentate gyrus (Fig. 2 B). Within the cornu ammonis , microglia numbers remained significantly higher in tamoxifen-induced mice versus corn oil controls but significantly decreased over time (Fig. 2 B). In the dentate gyrus, microglia numbers remained significantly elevated during the 10-weeks timepoint, but did not differ from control mice 10 months after tamoxifen induction. Further automated profiling of microglia 3D-morphology via MMQT [ 57 ] revealed significant morphological changes, such as increased sphericity and decreased branching that are consistent with an inflammatory phenotype (Fig. 2 C). While the microglia morphology of tamoxifen-induced mice remained significantly different from microglia in healthy control mice, we noticed a significant shift of morphological parameters towards a homeostatic morphology over time (Fig. 2 C). Characterization of CNS-infiltrating immune cells reveals an absence of B cell and presence of CD8 + T cell infiltration A hallmark of autoimmune encephalitis is the presence of CNS infiltrating lymphocytes, in particular B cells, T cells, and antibody secreting cells [ 19 , 63 , 66 ]. For this reason, we monitored the lymphocyte infiltration at discrete time points after primary encephalitis induction. Throughout the study period, mild to moderate significant lymphocyte infiltration was observed in the cornu ammonis (Fig. 3 A). No significant lymphocyte infiltration was observed in the adjacent dentate gyrus (Fig. 3 B). CD19 positive B cells were nearly absent in all hippocampal regions throughout the study period (Fig. 3 A-B) and were not detected in noticeable amounts in other brain regions. The majority of hippocampus infiltrating CD45 + lymphocytes in tamoxifen-induced mice were CD3 + T cells (82.5% ± 14.6%; mean ± SD). While the total amount of intrahippocampal CD3 + T cells did not significantly change overtime, we noticed a significant time-dependent increase in CD8 + T cells in tamoxifen-induced mice (Fig. 3 A-B). After primary encephalitis induction, the proportion of hippocampal CD8 + T cells amongst total hippocampal T cells increased from 25.0% ± 24.0% during the acute phase to 71.6% ± 29.8% during the recovery phase and 89.2% ± 14.8% after completion of the long-term follow-up. Perivascular cuffs or clusters of nuclei dense immune cell infiltrates, that are frequently present in brains of mice and humans with autoimmune encephalitis such as NMDAR encephalitis [ 60 , 63 , 66 ], were not observed. Blood flow cytometry indicates absence of a peripheral autoimmune response We performed high-parameter flow cytometry analyses of peripheral blood to test if mice recovering from a virus-like but sterile encephalitis show a peripheral immune response that would be indicative of an adaptive immune response and autoimmune encephalitis. Using classical cell surface markers and hierarchical gating, we quantified granulocytes, NK-cells, monocytes, B cells, CD4 + T cells and CD8 + T cells in blood samples of 11 mice tamoxifen-induced post-encephalitis mice and 10 healthy control mice after completion of the 10-months follow-up period (Fig. 4 A). Within the major immune cell populations, we quantified the proportions of distinct immune cell subtypes (Fig. 4 B). NK cell subsets were classified based on differential surface expression of CD11b and CD27 [ 27 ]. B cells were categorized as CD27-/CD138- naïve B cells, CD27+/CD138- memory B cells, and CD138 + antibody secreting cells [ 28 ]. T cells were divided into CD62L+/CD44- naïve T cells, CD62L+/CD44 + central memory T cells, and CD62L-/CD44 + effector memory T cells [ 29 , 30 ]. Dimensionality reduction and t-SNE clustering was used to confirm the quality of the hierarchical gating strategy (Fig. 4 C). At the time of follow-up completion, none of the major cell types were significantly altered in tamoxifen-induced post-encephalitis mice (Fig. 4 D). Similarly, neither the NK cell population, B cell population, or CD4 + T cell population showed significant changes in their composition (Fig. 4 E-G). However, the peripheral CD8 + T cell compartment of post-encephalitis mice showed a significant increase in naïve T cells that was accompanied by a reduction of effector memory cells (Fig. 4 H). Testing for 49 CNS-targeting autoantibodies did not find increased autoantibody production in mice weeks to months after sterile encephalitis CNS-directed autoantibodies are frequently observed in patients after HSE and JE [ 2 , 3 , 5 – 7 , 9 , 13 , 15 , 16 ] as well as in mice infected with HSV-1 [ 69 ]. To test if this autoantibody production occurs in response to acute neuronal cell death without additional co-stimulation of the immune system by virus/pathogen associated molecular patterns, we tested plasma samples of healthy mice and mice after induction of virus-like but sterile encephalitis for the presence of anti-CNS autoantibodies. Autoantibody titers against 49-disease associated antibodies were determined for IgG, IgA and IgM isotypes using commercially available cell-based assays developed for the in vitro diagnostic of autoantibodies in suspected autoimmune encephalitis patients (Euroimmun biochip mosaic IVD assays). Due to the limited amount of mouse plasma, autoantibody testing was performed with a cut-off titer of 1:100. Plasma samples of 60 mice (n = 30 per group) were tested. One healthy control had a titer of 1:320 for anti-CASPR2 autoantibodies of the IgG subtype and one post-encephalitis mouse had IgM autoantibodies against homer-3 at a titer of 1:1000 (supplementary table 2). Overall seroprevalence for all 49 autoantibodies was low (2/60 mice; 3.33%) and no difference was observed between groups (1/30 positive mice post-encephalitis versus 1/30 positive control mice; p > 0.9999, Chi-square test). DISCUSSION To test if the acute destruction of neurons in the absence of encephalitogenic virus is sufficient to induce a secondary autoimmune encephalitis in mice, we monitored mice for up to 10 months after sterile induction of cell death in hippocampal and pyramidal neurons. Throughout the study period, we observed no symptoms of severe autoimmune encephalitis, such as hyperactivity, circling, seizures, or lethargy. Microglia analysis revealed significant microgliosis one week after encephalitis induction, which diminished over time. Scattered lymphocyte infiltration persisted in the hippocampi of encephalitis mice but did not increase significantly. Perivascular cuffs were not detected, and B cell infiltration was rare and comparable to healthy controls. High-parameter immunophenotyping of peripheral blood leukocytes did not reveal an expansion of memory B cells or antibody secreting cells, that could be indicative of an antibody-associated autoimmune response. Concomitantly, autoantibody testing against 49 neural antigens in plasma samples of post-encephalitis mice yielded mostly negative results, similar to healthy controls. Overall, these findings suggest that acute neuronal cell death and neuroinflammation alone are insufficient to trigger relapses with autoimmune encephalitis. While we observed significant time-dependent recovery of microglial characteristics, that are typically associated with homeostatic functioning, hippocampal microglia from mice after encephalitis induction remained significantly different from healthy control microglia for up to 10 months. A potential reason for this incomplete recovery could be the accumulation of CD8 + T cells in the hippocampus of post-encephalitis mice in combination with their advanced age. In the peripheral nervous system, CD8 + memory T cells restrict axonal regeneration after spinal cord injury and mediate aging-dependent regenerative decline [ 70 ]. Within the central nervous system, CD8 + tissue-resident memory T-cells are commonly observed in several neuroinflammatory and neurodegenerative conditions [ 71 ]. While CD8 + tissue resident memory Τ cells are typically induced in response to CNS infections and associated with protective properties [ 72 ], autoreactive CD8 + tissue resident memory T cells can also drive autoinflammatory responses within the CNS [ 73 , 74 ]. In our model, we observed a partial time-dependent recovery of homeostatic-like characteristics in microglia as well as an absence of additional lymphocyte recruitment or encephalitis-like symptoms, pointing against a CD8 + T cell mediated autoimmune pathology. In this study we found a lack of autoimmune encephalitis-like pathology after the induction of neuronal cell death and sterile encephalitis. A probable reason could be an insufficient stimulation of the peripheral immune system due to the absence of virus/pathogen associated molecular patterns. Several histopathological analyses of ‘sporadic’ and NMDAR encephalitis-associated ovarian teratomas highlight the relevance of peripheral immune cell activation in the encephalitogenic process. In both patient subsets, neuronal tissue, that ectopically express NMDA receptors, was frequently observed. However, ovarian teratomas from patients who progressed to autoimmune encephalitis exhibited a higher density of lymphocytic infiltrates near neuronal tissue [ 65 , 75 – 78 ]. This suggests that co-stimulatory signals play a key role in determining whether exposure to neuronal antigens is tolerated or leads to autoimmune encephalitis. The role of peripheral immune activation as risk factor for autoimmune encephalitis after virus encephalitis was further highlighted by a recent prospective study by Armangué and colleagues who found that an elevated blood IFN response was the most important predictor of post-HSE autoimmune encephalitis [ 7 ]. In principle, the absence of an autoimmune relapse following encephalitis could also be explained by an insufficient presentation of autoantigens to the immune system. While shedding of neuronal antigens, such as NMDA and AMPA receptors, into the periphery via small vesicles has been shown to occur in human encephalitis patients [ 79 , 80 ], we did not assess this shedding process in the present mouse model. Interestingly, DTA-mediated ablation of oligodendrocytes in adult mice induced the generation of myelin oligodendrocyte glycoprotein (MOG)-specific T cells in peripheral lymphoid organs and resulted in an autoimmune relapse approximately 30 weeks after oligodendrocyte ablation [ 24 ]. The apparent discrepancy between the outcome after acute pyramidal cell and oligodendrocyte cell death suggest that factors such as the antigenicity of the released proteins as well as antigen-specific clearance and shedding may determine susceptibility to autoimmune relapses. Limitations A limitation of the utilized autoantibody screening is the use of commercially available cell-based assays, that were developed as in vitro diagnostics for the detection of autoantibodies in patients with (suspected) autoimmune encephalitis. While all of the assessed autoantigens are well conserved between human and mice (95 ± 5% sequence identity), we cannot rule out the presence of autoantibodies targeting mouse-specific epitopes. Similarly, low affinity autoantibodies or autoantibodies with a low plasma concentration might have been missed due to the stringent cut-off titer of 1:100. This would also explain the lower seroprevalence in comparison to our previous work, that focused on the NMDAR1-AB seroprevalence across mammals, in which samples were tested with 1:1 and 1:10 dilutions [ 81 , 82 ]. Another limitation is that we did not investigate cases with more massive and global destruction of neurons, which would likely increase the amount of neuronal autoantigens and damage associated molecular patterns that are drained to peripheral lymphoid organs, and may reach a threshold that is sufficient to induce autoimmune relapses. Another potential factor predisposing to pathogenic brain directed autoimmunity could be damage to glial cells and the blood brain barrier [ 83 ]. In fact, we previously observed an increase in NMDAR1-autoantibodies in mice in response to a small standardized cryolesion of the right parietal cortex [ 82 ], with local damage of all cells near the lesion site and blood-brain barrier dysfunction [ 84 ]. Conclusions While clinical observations suggest a solid link between autoimmune encephalitis and virus encephalitides, such as HSE and JE, the pathomechanisms behind the secondary autoimmune response have remained obscure. The present study on a sterile encephalitis model reveals that acute destruction of neurons alone is not sufficient to induce an autoimmune response against neuronal surface antigens, as previously associated with autoimmune encephalitides. This observation indicates that co-factors are required for the initiation of anti-neuronal autoimmune responses. Identifying these co-factors may ultimately enable targeted therapies. Declarations Ethics approval All animal experiments were approved by the local animal care and use committee (LAVES, Niedersaechsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany; license number 33.19-42502-04-18/2803) in accordance with the German animal protection law. Availability of data and materials The code used to analyze MMQT output files is available at https://github.com/vgastaldi/MMQT-longDTA. Further information and requests for data and resources should be directed to the lead contact, Prof. Dr. Dr. Hannelore Ehrenreich ( [email protected] ). Competing interests WS is holder of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. KB and BT are full-time employees of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. All other authors declare no competing financial or other interests. Funding This work has been funded by the DFG TRR-274/1 2020-408885537. Furthermore, the study has been fostered by the Max Planck Society and the Max Planck Förderstiftung. Research in the HE lab was funded by the European Research Council (ERC) Advanced Grant under the European Union’s Horizon Europe research and innovation programme (acronym BREPOCI; grant agreement No 101054369). KAN is supported by the Adelson Medical Research Foundation. VDG received backing from the IMPRS-Genome Science PhD program. For the publication fee we acknowledge financial support by Heidelberg University. Authors' contributions Supervision: HE, JBHW, Funding acquisition: HE, KAN Concept and design: HE, JBHW, KAN Data acquisition/generation: AN, JBHW, BT, KB, WS Data analyses/interpretation: JBHW, VDG, AN, FL Drafting the manuscript: JBHW, HE Drafting display items: JBHW, AN, HE Critical input, review & editing: AN, JBHW, VDG, KB, BT, WS, FL, KAN, HE All authors read and approved the final version of the manuscript. Acknowledgements The authors thank Roman Schröder for his assistance and critical input in flow cytometry experiments and Anja Ronnenberg, Nadine Barnkothe, and Viktoria Bonet for outstanding technical support. References Pruss H, Finke C, Holtje M, Hofmann J, Klingbeil C, Probst C, Borowski K, Ahnert-Hilger G, Harms L, Schwab JM, Ploner CJ, Komorowski L, Stoecker W, Dalmau J, Wandinger KP: N-methyl-D-aspartate receptor antibodies in herpes simplex encephalitis. Ann Neurol 2012, 72: 902-911. Linnoila JJ, Binnicker MJ, Majed M, Klein CJ, McKeon A: CSF herpes virus and autoantibody profiles in the evaluation of encephalitis. Neurol Neuroimmunol Neuroinflamm 2016, 3: e245. Armangue T, Leypoldt F, Malaga I, Raspall-Chaure M, Marti I, Nichter C, Pugh J, Vicente-Rasoamalala M, Lafuente-Hidalgo M, Macaya A, Ke M, Titulaer MJ, Hoftberger R, Sheriff H, Glaser C, Dalmau J: Herpes simplex virus encephalitis is a trigger of brain autoimmunity. Ann Neurol 2014, 75: 317-323. Hacohen Y, Deiva K, Pettingill P, Waters P, Siddiqui A, Chretien P, Menson E, Lin JP, Tardieu M, Vincent A, Lim MJ: N-methyl-D-aspartate receptor antibodies in post-herpes simplex virus encephalitis neurological relapse. Mov Disord 2014, 29: 90-96. Mohammad SS, Sinclair K, Pillai S, Merheb V, Aumann TD, Gill D, Dale RC, Brilot F: Herpes simplex encephalitis relapse with chorea is associated with autoantibodies to N-Methyl-D-aspartate receptor or dopamine-2 receptor. Mov Disord 2014, 29: 117-122. Armangue T, Moris G, Cantarin-Extremera V, Conde CE, Rostasy K, Erro ME, Portilla-Cuenca JC, Turon-Vinas E, Malaga I, Munoz-Cabello B, Torres-Torres C, Llufriu S, Gonzalez-Gutierrez-Solana L, Gonzalez G, Casado-Naranjo I, Rosenfeld M, Graus F, Dalmau J, Spanish Prospective Multicentric Study of Autoimmunity in Herpes Simplex E: Autoimmune post-herpes simplex encephalitis of adults and teenagers. Neurology 2015, 85: 1736-1743. Armangue T, Olive-Cirera G, Martinez-Hernandez E, Rodes M, Peris-Sempere V, Guasp M, Ruiz R, Palou E, Gonzalez A, Marcos MA, Erro ME, Bataller L, Corral-Corral I, Planaguma J, Caballero E, Vlagea A, Chen J, Bastard P, Materna M, Marchal A, Abel L, Cobat A, Alsina L, Fortuny C, Saiz A, Mignot E, Vanderver A, Casanova JL, Zhang SY, Dalmau J: Neurologic complications in herpes simplex encephalitis: clinical, immunological and genetic studies. Brain 2023, 146: 4306-4319. Sutcu M, Akturk H, Somer A, Tatli B, Torun SH, Yildiz EP, Sik G, Citak A, Agacfidan A, Salman N: Role of Autoantibodies to N-Methyl-d-Aspartate (NMDA) Receptor in Relapsing Herpes Simplex Encephalitis: A Retrospective, One-Center Experience. J Child Neurol 2016, 31: 345-350. Pruss H: Postviral autoimmune encephalitis: manifestations in children and adults. Curr Opin Neurol 2017, 30: 327-333. Nosadini M, Mohammad SS, Corazza F, Ruga EM, Kothur K, Perilongo G, Frigo AC, Toldo I, Dale RC, Sartori S: Herpes simplex virus-induced anti-N-methyl-d-aspartate receptor encephalitis: a systematic literature review with analysis of 43 cases. Dev Med Child Neurol 2017, 59: 796-805. Ma J, Zhang T, Jiang L: Japanese encephalitis can trigger anti-N-methyl-D-aspartate receptor encephalitis. J Neurol 2017, 264: 1127-1131. Ma J, Han W, Jiang L: Japanese encephalitis-induced anti-N-methyl-d-aspartate receptor encephalitis: A hospital-based prospective study. Brain Dev 2020, 42: 179-184. Liu B, Liu J, Sun H, Xie M, Yang C, Pan Y, Huang D, Cheng L, Chen H, Ma J, Jiang L: Autoimmune encephalitis after Japanese encephalitis in children: A prospective study. J Neurol Sci 2021, 424: 117394. Ashraf U, Ding Z, Deng S, Ye J, Cao S, Chen Z: Pathogenicity and virulence of Japanese encephalitis virus: Neuroinflammation and neuronal cell damage. Virulence 2021, 12: 968-980. Armangue T, Spatola M, Vlagea A, Mattozzi S, Carceles-Cordon M, Martinez-Heras E, Llufriu S, Muchart J, Erro ME, Abraira L, Moris G, Monros-Gimenez L, Corral-Corral I, Montejo C, Toledo M, Bataller L, Secondi G, Arino H, Martinez-Hernandez E, Juan M, Marcos MA, Alsina L, Saiz A, Rosenfeld MR, Graus F, Dalmau J, Spanish Herpes Simplex Encephalitis Study G: Frequency, symptoms, risk factors, and outcomes of autoimmune encephalitis after herpes simplex encephalitis: a prospective observational study and retrospective analysis. Lancet Neurol 2018, 17: 760-772. Graus F, Titulaer MJ, Balu R, Benseler S, Bien CG, Cellucci T, Cortese I, Dale RC, Gelfand JM, Geschwind M, Glaser CA, Honnorat J, Hoftberger R, Iizuka T, Irani SR, Lancaster E, Leypoldt F, Pruss H, Rae-Grant A, Reindl M, Rosenfeld MR, Rostasy K, Saiz A, Venkatesan A, Vincent A, Wandinger KP, Waters P, Dalmau J: A clinical approach to diagnosis of autoimmune encephalitis. Lancet Neurol 2016, 15: 391-404. Whitley RJ: Herpes simplex encephalitis: adolescents and adults. Antiviral Res 2006, 71: 141-148. Kennedy PG, Chaudhuri A: Herpes simplex encephalitis. J Neurol Neurosurg Psychiatry 2002, 73: 237-238. Dalmau J, Graus F: Antibody-Mediated Encephalitis. N Engl J Med 2018, 378: 840-851. Sun B, Ramberger M, O'Connor KC, Bashford-Rogers RJM, Irani SR: The B cell immunobiology that underlies CNS autoantibody-mediated diseases. Nat Rev Neurol 2020, 16: 481-492. Sabatino JJ, Jr., Probstel AK, Zamvil SS: B cells in autoimmune and neurodegenerative central nervous system diseases. Nat Rev Neurosci 2019, 20: 728-745. Gibson LL, McKeever A, Coutinho E, Finke C, Pollak TA: Cognitive impact of neuronal antibodies: encephalitis and beyond. Transl Psychiatry 2020, 10: 304. Pruss H: Autoantibodies in neurological disease. Nat Rev Immunol 2021, 21: 798-813. Traka M, Podojil JR, McCarthy DP, Miller SD, Popko B: Oligodendrocyte death results in immune-mediated CNS demyelination. Nat Neurosci 2016, 19: 65-74. Agarwal A, Dibaj P, Kassmann CM, Goebbels S, Nave KA, Schwab MH: In vivo imaging and noninvasive ablation of pyramidal neurons in adult NEX-CreERT2 mice. Cereb Cortex 2012, 22: 1473-1486. Ivanova A, Signore M, Caro N, Greene ND, Copp AJ, Martinez-Barbera JP: In vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A. Genesis 2005, 43: 129-135. Goh W, Huntington ND: Regulation of Murine Natural Killer Cell Development. Front Immunol 2017, 8: 130. Brynjolfsson SF, Persson Berg L, Olsen Ekerhult T, Rimkute I, Wick MJ, Martensson IL, Grimsholm O: Long-Lived Plasma Cells in Mice and Men. Front Immunol 2018, 9: 2673. Gerberick GF, Cruse LW, Miller CM, Sikorski EE, Ridder GM: Selective modulation of T cell memory markers CD62L and CD44 on murine draining lymph node cells following allergen and irritant treatment. Toxicol Appl Pharmacol 1997, 146: 1-10. Sckisel GD, Mirsoian A, Minnar CM, Crittenden M, Curti B, Chen JQ, Blazar BR, Borowsky AD, Monjazeb AM, Murphy WJ: Differential phenotypes of memory CD4 and CD8 T cells in the spleen and peripheral tissues following immunostimulatory therapy. J Immunother Cancer 2017, 5: 33. Daguano Gastaldi V, Bh Wilke J, Weidinger CA, Walter C, Barnkothe N, Teegen B, Luessi F, Stocker W, Luhder F, Begemann M, Zipp F, Nave KA, Ehrenreich H: Factors predisposing to humoral autoimmunity against brain-antigens in health and disease: Analysis of 49 autoantibodies in over 7000 subjects. Brain Behav Immun 2023, 108: 135-147. Dalmau J, Tuzun E, Wu HY, Masjuan J, Rossi JE, Voloschin A, Baehring JM, Shimazaki H, Koide R, King D, Mason W, Sansing LH, Dichter MA, Rosenfeld MR, Lynch DR: Paraneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma. Ann Neurol 2007, 61: 25-36. Hart IK, Waters C, Vincent A, Newland C, Beeson D, Pongs O, Morris C, Newsom-Davis J: Autoantibodies detected to expressed K+ channels are implicated in neuromyotonia. Ann Neurol 1997, 41: 238-246. Jarius S, Wildemann B: 'Medusa head ataxia': the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook. J Neuroinflammation 2015, 12: 168. Abboud H, Probasco JC, Irani S, Ances B, Benavides DR, Bradshaw M, Christo PP, Dale RC, Fernandez-Fournier M, Flanagan EP, Gadoth A, George P, Grebenciucova E, Jammoul A, Lee ST, Li Y, Matiello M, Morse AM, Rae-Grant A, Rojas G, Rossman I, Schmitt S, Venkatesan A, Vernino S, Pittock SJ, Titulaer MJ, Autoimmune Encephalitis Alliance Clinicians N: Autoimmune encephalitis: proposed best practice recommendations for diagnosis and acute management. J Neurol Neurosurg Psychiatry 2021, 92: 757-768. Fang B, McKeon A, Hinson SR, Kryzer TJ, Pittock SJ, Aksamit AJ, Lennon VA: Autoimmune Glial Fibrillary Acidic Protein Astrocytopathy: A Novel Meningoencephalomyelitis. JAMA Neurol 2016, 73: 1297-1307. Hoftberger R, Sabater L, Ortega A, Dalmau J, Graus F: Patient with homer-3 antibodies and cerebellitis. JAMA Neurol 2013, 70: 506-509. Hoftberger R, van Sonderen A, Leypoldt F, Houghton D, Geschwind M, Gelfand J, Paredes M, Sabater L, Saiz A, Titulaer MJ, Graus F, Dalmau J: Encephalitis and AMPA receptor antibodies: Novel findings in a case series of 22 patients. Neurology 2015, 84: 2403-2412. Tanaka J, Nakamura K, Takeda M, Tada K, Suzuki H, Morita H, Okado T, Hariguchi S, Nishimura T: Enzyme-linked immunosorbent assay for human autoantibody to glial fibrillary acidic protein: higher titer of the antibody is detected in serum of patients with Alzheimer's disease. Acta Neurol Scand 1989, 80: 554-560. De Camilli P, Thomas A, Cofiell R, Folli F, Lichte B, Piccolo G, Meinck HM, Austoni M, Fassetta G, Bottazzo G, Bates D, Cartlidge N, Solimena M, Kilimann MW, et al.: The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer. J Exp Med 1993, 178: 2219-2223. Irani SR, Alexander S, Waters P, Kleopa KA, Pettingill P, Zuliani L, Peles E, Buckley C, Lang B, Vincent A: Antibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan's syndrome and acquired neuromyotonia. Brain 2010, 133: 2734-2748. Xiao BG, Linington C, Link H: Antibodies to myelin-oligodendrocyte glycoprotein in cerebrospinal fluid from patients with multiple sclerosis and controls. J Neuroimmunol 1991, 31: 91-96. Zuliani L, Sabater L, Saiz A, Baiges JJ, Giometto B, Graus F: Homer 3 autoimmunity in subacute idiopathic cerebellar ataxia. Neurology 2007, 68: 239-240. Hutchinson M, Waters P, McHugh J, Gorman G, O'Riordan S, Connolly S, Hager H, Yu P, Becker CM, Vincent A: Progressive encephalomyelitis, rigidity, and myoclonus: a novel glycine receptor antibody. Neurology 2008, 71: 1291-1292. Swayne A, Tjoa L, Broadley S, Dionisio S, Gillis D, Jacobson L, Woodhall MR, McNabb A, Schweitzer D, Tsang B, Vincent A, Irani SR, Wong R, Waters P, Blum S: Antiglycine receptor antibody related disease: a case series and literature review. Eur J Neurol 2018, 25: 1290-1298. Solimena M, Folli F, Denis-Donini S, Comi GC, Pozza G, De Camilli P, Vicari AM: Autoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type I diabetes mellitus. N Engl J Med 1988, 318: 1012-1020. Mathey EK, Derfuss T, Storch MK, Williams KR, Hales K, Woolley DR, Al-Hayani A, Davies SN, Rasband MN, Olsson T, Moldenhauer A, Velhin S, Hohlfeld R, Meinl E, Linington C: Neurofascin as a novel target for autoantibody-mediated axonal injury. J Exp Med 2007, 204: 2363-2372. Honorat JA, Lopez-Chiriboga AS, Kryzer TJ, Komorowski L, Scharf M, Hinson SR, Lennon VA, Pittock SJ, Klein CJ, McKeon A: Autoimmune gait disturbance accompanying adaptor protein-3B2-IgG. Neurology 2019, 93: e954-e963. Peterson K, Rosenblum MK, Kotanides H, Posner JB: Paraneoplastic cerebellar degeneration. I. A clinical analysis of 55 anti-Yo antibody-positive patients. Neurology 1992, 42: 1931-1937. Gelpi E, Hoftberger R, Graus F, Ling H, Holton JL, Dawson T, Popovic M, Pretnar-Oblak J, Hogl B, Schmutzhard E, Poewe W, Ricken G, Santamaria J, Dalmau J, Budka H, Revesz T, Kovacs GG: Neuropathological criteria of anti-IgLON5-related tauopathy. Acta Neuropathol 2016, 132: 531-543. Gelpi E, Reinecke R, Gaig C, Iranzo A, Sabater L, Molina-Porcel L, Aldecoa I, Endmayr V, Hogl B, Schmutzhard E, Poewe W, Pfausler B, Popovic M, Pretnar-Oblak J, Leypoldt F, Matschke J, Glatzel M, Erro EM, Jerico I, Caballero MC, Zelaya MV, Mariotto S, Heidbreder A, Kalev O, Weis S, Macher S, Berger-Sieczkowski E, Ferrari J, Reisinger C, Klupp N, Tienari P, Rautila O, Niemela M, Yilmazer-Hanke D, Guasp M, Bloem B, Van Gaalen J, Kusters B, Titulaer M, Fransen NL, Santamaria J, Dawson T, Holton JL, Ling H, Revesz T, Myllykangas L, Budka H, Kovacs GG, Lewerenz J, Dalmau J, Graus F, Koneczny I, Hoftberger R: Neuropathological spectrum of anti-IgLON5 disease and stages of brainstem tau pathology: updated neuropathological research criteria of the disease-related tauopathy. Acta Neuropathol 2024, 148: 53. Graus F, Vogrig A, Muniz-Castrillo S, Antoine JG, Desestret V, Dubey D, Giometto B, Irani SR, Joubert B, Leypoldt F, McKeon A, Pruss H, Psimaras D, Thomas L, Titulaer MJ, Vedeler CA, Verschuuren JJ, Dalmau J, Honnorat J: Updated Diagnostic Criteria for Paraneoplastic Neurologic Syndromes. Neurol Neuroimmunol Neuroinflamm 2021, 8: e1014. Pollak TA, Lennox BR, Muller S, Benros ME, Pruss H, Tebartz van Elst L, Klein H, Steiner J, Frodl T, Bogerts B, Tian L, Groc L, Hasan A, Baune BT, Endres D, Haroon E, Yolken R, Benedetti F, Halaris A, Meyer JH, Stassen H, Leboyer M, Fuchs D, Otto M, Brown DA, Vincent A, Najjar S, Bechter K: Autoimmune psychosis: an international consensus on an approach to the diagnosis and management of psychosis of suspected autoimmune origin. Lancet Psychiatry 2020, 7: 93-108. Wilke JBH, Hindermann M, Berghoff SA, Zihsler S, Arinrad S, Ronnenberg A, Barnkothe N, Steixner-Kumar AA, Roglin S, Stocker W, Hollmann M, Nave KA, Luhder F, Ehrenreich H: Autoantibodies against NMDA receptor 1 modify rather than cause encephalitis. Mol Psychiatry 2021, 26: 7746-7759. Wilke JBH, Hindermann M, Moussavi A, Butt UJ, Dadarwal R, Berghoff SA, Sarcheshmeh AK, Ronnenberg A, Zihsler S, Arinrad S, Hardeland R, Seidel J, Luhder F, Nave KA, Boretius S, Ehrenreich H: Inducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis. Acta Neuropathol Commun 2021, 9: 121. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: Fiji: an open-source platform for biological-image analysis. Nat Methods 2012, 9: 676-682. Heindl S, Gesierich B, Benakis C, Llovera G, Duering M, Liesz A: Automated Morphological Analysis of Microglia After Stroke. Front Cell Neurosci 2018, 12: 106. Kuznetsova A, Brockhoff PB, Christensen RHB: lmerTest Package: Tests in Linear Mixed Effects Models. Journal of Statistical Software 2017, 82 . R Development Core Team: R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2020. Jones BE, Tovar KR, Goehring A, Jalali-Yazdi F, Okada NJ, Gouaux E, Westbrook GL: Autoimmune receptor encephalitis in mice induced by active immunization with conformationally stabilized holoreceptors. Sci Transl Med 2019, 11 . Paolicelli RC, Sierra A, Stevens B, Tremblay ME, Aguzzi A, Ajami B, Amit I, Audinat E, Bechmann I, Bennett M, Bennett F, Bessis A, Biber K, Bilbo S, Blurton-Jones M, Boddeke E, Brites D, Brone B, Brown GC, Butovsky O, Carson MJ, Castellano B, Colonna M, Cowley SA, Cunningham C, Davalos D, De Jager PL, de Strooper B, Denes A, Eggen BJL, Eyo U, Galea E, Garel S, Ginhoux F, Glass CK, Gokce O, Gomez-Nicola D, Gonzalez B, Gordon S, Graeber MB, Greenhalgh AD, Gressens P, Greter M, Gutmann DH, Haass C, Heneka MT, Heppner FL, Hong S, Hume DA, Jung S, Kettenmann H, Kipnis J, Koyama R, Lemke G, Lynch M, Majewska A, Malcangio M, Malm T, Mancuso R, Masuda T, Matteoli M, McColl BW, Miron VE, Molofsky AV, Monje M, Mracsko E, Nadjar A, Neher JJ, Neniskyte U, Neumann H, Noda M, Peng B, Peri F, Perry VH, Popovich PG, Pridans C, Priller J, Prinz M, Ragozzino D, Ransohoff RM, Salter MW, Schaefer A, Schafer DP, Schwartz M, Simons M, Smith CJ, Streit WJ, Tay TL, Tsai LH, Verkhratsky A, von Bernhardi R, Wake H, Wittamer V, Wolf SA, Wu LJ, Wyss-Coray T: Microglia states and nomenclature: A field at its crossroads. Neuron 2022, 110: 3458-3483. Leng F, Edison P: Neuroinflammation and microglial activation in Alzheimer disease: where do we go from here? Nat Rev Neurol 2021, 17: 157-172. Zrzavy T, Endmayr V, Bauer J, Macher S, Mossaheb N, Schwaiger C, Ricken G, Winklehner M, Glatter S, Breu M, Wimmer I, Kovacs GG, Risser DU, Klupp N, Simonitsch-Klupp I, Roetzer T, Rommer P, Berger T, Gelpi E, Lassmann H, Graus F, Dalmau J, Hoftberger R: Neuropathological Variability within a Spectrum of NMDAR-Encephalitis. Ann Neurol 2021, 90: 725-737. Nauen DW: Extra-central nervous system target for assessment and treatment in refractory anti-N-methyl-d-aspartate receptor encephalitis. J Crit Care 2017, 37: 234-236. Tuzun E, Zhou L, Baehring JM, Bannykh S, Rosenfeld MR, Dalmau J: Evidence for antibody-mediated pathogenesis in anti-NMDAR encephalitis associated with ovarian teratoma. Acta Neuropathol 2009, 118: 737-743. Bien CG, Vincent A, Barnett MH, Becker AJ, Blumcke I, Graus F, Jellinger KA, Reuss DE, Ribalta T, Schlegel J, Sutton I, Lassmann H, Bauer J: Immunopathology of autoantibody-associated encephalitides: clues for pathogenesis. Brain 2012, 135: 1622-1638. Filatenkov A, Richardson TE, Daoud E, Johnson-Welch SF, Ramirez DM, Torrealba J, Greenberg B, Monson NL, Rajaram V: Persistence of parenchymal and perivascular T-cells in treatment-refractory anti-N-methyl-D-aspartate receptor encephalitis. Neuroreport 2017, 28: 890-895. Li Q, Barres BA: Microglia and macrophages in brain homeostasis and disease. Nat Rev Immunol 2018, 18: 225-242. Linnoila J, Pulli B, Armangue T, Planaguma J, Narsimhan R, Schob S, Zeller MWG, Dalmau J, Chen J: Mouse model of anti-NMDA receptor post-herpes simplex encephalitis. Neurol Neuroimmunol Neuroinflamm 2019, 6: e529. Zhou L, Kong G, Palmisano I, Cencioni MT, Danzi M, De Virgiliis F, Chadwick JS, Crawford G, Yu Z, De Winter F, Lemmon V, Bixby J, Puttagunta R, Verhaagen J, Pospori C, Lo Celso C, Strid J, Botto M, Di Giovanni S: Reversible CD8 T cell-neuron cross-talk causes aging-dependent neuronal regenerative decline. Science 2022, 376: eabd5926. Merkler D, Vincenti I, Masson F, Liblau RS: Tissue-resident CD8 T cells in central nervous system inflammatory diseases: present at the crime scene and ...guilty. Curr Opin Immunol 2022, 77: 102211. Urban SL, Jensen IJ, Shan Q, Pewe LL, Xue HH, Badovinac VP, Harty JT: Peripherally induced brain tissue-resident memory CD8(+) T cells mediate protection against CNS infection. Nat Immunol 2020, 21: 938-949. Frieser D, Pignata A, Khajavi L, Shlesinger D, Gonzalez-Fierro C, Nguyen XH, Yermanos A, Merkler D, Hoftberger R, Desestret V, Mair KM, Bauer J, Masson F, Liblau RS: Tissue-resident CD8(+) T cells drive compartmentalized and chronic autoimmune damage against CNS neurons. Sci Transl Med 2022, 14: eabl6157. Vincenti I, Page N, Steinbach K, Yermanos A, Lemeille S, Nunez N, Kreutzfeldt M, Klimek B, Di Liberto G, Egervari K, Piccinno M, Shammas G, Mariotte A, Fonta N, Liaudet N, Shlesinger D, Liuzzi AR, Wagner I, Saadi C, Stadelmann C, Reddy S, Becher B, Merkler D: Tissue-resident memory CD8(+) T cells cooperate with CD4(+) T cells to drive compartmentalized immunopathology in the CNS. Sci Transl Med 2022, 14: eabl6058. Day GS, Laiq S, Tang-Wai DF, Munoz DG: Abnormal neurons in teratomas in NMDAR encephalitis. JAMA Neurol 2014, 71: 717-724. Tabata E, Masuda M, Eriguchi M, Yokoyama M, Takahashi Y, Tanaka K, Yukitake M, Horikawa E, Hara H: Immunopathological significance of ovarian teratoma in patients with anti-N-methyl-d-aspartate receptor encephalitis. Eur Neurol 2014, 71: 42-48. Iemura Y, Yamada Y, Hirata M, Kataoka TR, Minamiguchi S, Haga H: Histopathological characterization of the neuroglial tissue in ovarian teratoma associated with anti-N-methyl-D-aspartate (NMDA) receptor encephalitis. Pathol Int 2018, 68: 677-684. Chefdeville A, Treilleux I, Mayeur ME, Couillault C, Picard G, Bost C, Mokhtari K, Vasiljevic A, Meyronet D, Rogemond V, Psimaras D, Dubois B, Honnorat J, Desestret V: Immunopathological characterization of ovarian teratomas associated with anti-N-methyl-D-aspartate receptor encephalitis. Acta Neuropathol Commun 2019, 7: 38. Gu J, Jin T, Li Z, Chen H, Xia H, Xu X, Gui Y: Exosomes expressing neuronal autoantigens induced immune response in antibody-positive autoimmune encephalitis. Mol Immunol 2021, 131: 164-170. Li Y, Gu J, Mao Y, Wang X, Li Z, Xu X, Chen H, Gui Y: Cerebrospinal Fluid Extracellular Vesicles with Distinct Properties in Autoimmune Encephalitis and Herpes Simplex Encephalitis. Mol Neurobiol 2022, 59: 2441-2455. Pan H, Oliveira B, Saher G, Dere E, Tapken D, Mitjans M, Seidel J, Wesolowski J, Wakhloo D, Klein-Schmidt C, Ronnenberg A, Schwabe K, Trippe R, Matz-Rensing K, Berghoff S, Al-Krinawe Y, Martens H, Begemann M, Stocker W, Kaup FJ, Mischke R, Boretius S, Nave KA, Krauss JK, Hollmann M, Luhder F, Ehrenreich H: Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model. Mol Psychiatry 2019, 24: 1489-1501. Pan H, Steixner-Kumar AA, Seelbach A, Deutsch N, Ronnenberg A, Tapken D, von Ahsen N, Mitjans M, Worthmann H, Trippe R, Klein-Schmidt C, Schopf N, Rentzsch K, Begemann M, Wienands J, Stocker W, Weissenborn K, Hollmann M, Nave KA, Luhder F, Ehrenreich H: Multiple inducers and novel roles of autoantibodies against the obligatory NMDAR subunit NR1: a translational study from chronic life stress to brain injury. Mol Psychiatry 2021, 26: 2471-2482. Levin EC, Acharya NK, Han M, Zavareh SB, Sedeyn JC, Venkataraman V, Nagele RG: Brain-reactive autoantibodies are nearly ubiquitous in human sera and may be linked to pathology in the context of blood–brain barrier breakdown. Brain Research 2010, 1345: 221-232. Siren AL, Radyushkin K, Boretius S, Kammer D, Riechers CC, Natt O, Sargin D, Watanabe T, Sperling S, Michaelis T, Price J, Meyer B, Frahm J, Ehrenreich H: Global brain atrophy after unilateral parietal lesion and its prevention by erythropoietin. Brain 2006, 129: 480-489. Additional Declarations Competing interest reported. WS is holder of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. KB and BT are full-time employees of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. All other authors declare no competing financial or other interests. Supplementary Files WilkeNtolkerasSupplement.docx Cite Share Download PDF Status: Published Journal Publication published 27 Jun, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 23 May, 2025 Reviews received at journal 22 May, 2025 Reviews received at journal 16 May, 2025 Reviews received at journal 13 May, 2025 Reviewers agreed at journal 12 May, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers agreed at journal 09 May, 2025 Reviewers invited by journal 09 May, 2025 Editor assigned by journal 09 May, 2025 Editor invited by journal 09 May, 2025 Submission checks completed at journal 07 May, 2025 First submitted to journal 21 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6499111","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":455433695,"identity":"596554c5-052c-4027-8370-f15f902ae58a","order_by":0,"name":"Justus Wilke","email":"","orcid":"","institution":"Max Planck Institute for Multidisciplinary Sciences","correspondingAuthor":false,"prefix":"","firstName":"Justus","middleName":"","lastName":"Wilke","suffix":""},{"id":455433696,"identity":"90056fd6-8845-4f64-8c0f-6360cfa2a5dd","order_by":1,"name":"Antonios Ntolkeras","email":"","orcid":"","institution":"Max Planck Institute for Multidisciplinary Sciences","correspondingAuthor":false,"prefix":"","firstName":"Antonios","middleName":"","lastName":"Ntolkeras","suffix":""},{"id":455433697,"identity":"b7620a0c-8890-4b49-91ad-36ee2a674406","order_by":2,"name":"Vinicius Daguano Gastaldi","email":"","orcid":"","institution":"Max Planck Institute for Multidisciplinary Sciences","correspondingAuthor":false,"prefix":"","firstName":"Vinicius","middleName":"Daguano","lastName":"Gastaldi","suffix":""},{"id":455433698,"identity":"20a8f242-b3d5-48e8-bb87-45f313fa6831","order_by":3,"name":"Kathrin Bobrowski","email":"","orcid":"","institution":"Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker Groß Grönau","correspondingAuthor":false,"prefix":"","firstName":"Kathrin","middleName":"","lastName":"Bobrowski","suffix":""},{"id":455433699,"identity":"7a247146-a121-4609-96ee-a060cc9c4ee8","order_by":4,"name":"Bianca Teegen","email":"","orcid":"","institution":"Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker Groß Grönau","correspondingAuthor":false,"prefix":"","firstName":"Bianca","middleName":"","lastName":"Teegen","suffix":""},{"id":455433700,"identity":"60800094-0239-486a-9369-4c932837eccc","order_by":5,"name":"Winfried Stöcker","email":"","orcid":"","institution":"Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker Groß Grönau","correspondingAuthor":false,"prefix":"","firstName":"Winfried","middleName":"","lastName":"Stöcker","suffix":""},{"id":455433701,"identity":"ee8785e3-f395-498a-b271-8eafb17fc9e1","order_by":6,"name":"Fred Lühder","email":"","orcid":"","institution":"University Medical Center of the Georg August University","correspondingAuthor":false,"prefix":"","firstName":"Fred","middleName":"","lastName":"Lühder","suffix":""},{"id":455433702,"identity":"956ff713-7b79-45a1-b3a7-348ecd9227d7","order_by":7,"name":"Klaus-Armin Nave","email":"","orcid":"","institution":"Max Planck Institute for Multidisciplinary Sciences","correspondingAuthor":false,"prefix":"","firstName":"Klaus-Armin","middleName":"","lastName":"Nave","suffix":""},{"id":455433703,"identity":"c0c6bd8e-e4c5-422f-a547-0225327b6e7e","order_by":8,"name":"Hannelore Ehrenreich","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFklEQVRIie2QsWrDMBRFFQTyItBq4Z94oaBgcNtfaTE4i2MXsnjIYBNIl5SuyY94Ngg8uXTN0CEQ0JTBU8lQ0soOdAhKabcOOojH5aHDFULIYvmHQD8f+klOK6xDi9Bdfy4qcKYMVn9TdMD0B2XkvNTbFoKUIVzvsuwtZQ5Wu+tFkCAnrEyKv0zGwxVE/jon42HTKH89J6OrySKaIqqMNVDFwqMgASoqeLHQQVLiTUp5n7sxGJXXvfA+tHJbsXdeHHXoFL/81EraGpWNbkFdC6KEF7kOWCuDsupazD+22Qu+hAhcSQTPa3kKT8dwSqi68LBYuIcsAPY4VzyfSWDPUrmH5iZhTrg11nyDzxfEdMtisVgsv+ILlrpZqjfFjUcAAAAASUVORK5CYII=","orcid":"","institution":"Central Institute of Mental Health, Heidelberg University","correspondingAuthor":true,"prefix":"","firstName":"Hannelore","middleName":"","lastName":"Ehrenreich","suffix":""}],"badges":[],"createdAt":"2025-04-22 00:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6499111/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6499111/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-08035-w","type":"published","date":"2025-06-27T15:57:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82715787,"identity":"1132ef06-a288-4eaa-8e96-3abc86ba6526","added_by":"auto","created_at":"2025-05-14 12:08:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1413492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term monitoring for autoimmune relapse in mice after sterile induction of acute neuronal cell death. \u003c/strong\u003eA) Experimental outline. B) Fluorojade C staining of degenerating neurons, demonstrating the pronounced degeneration of neurons in the \u003cem\u003ecornu ammonis\u003c/em\u003eregion 1 week after tamoxifen-induced DTA expression. Neurodegeneration was absent during the recovery phase at 10 weeks post induction. Representative images of n=4 mice per group and time point.\u003c/p\u003e","description":"","filename":"WilkeNtolkerasetatFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/b5acde72e3eefb7e4d4cb373.png"},{"id":82715795,"identity":"db5aea4b-5a96-4e6b-8362-6ceb3fe1804b","added_by":"auto","created_at":"2025-05-14 12:08:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1948773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMonitoring of microglia activity at 1 week, 10 weeks, and 10 months after tamoxifen-induced neurodegeneration. \u003c/strong\u003eA) Representative maximum intensity projections of Z-stacks within the CA1 of control versus tamoxifen-induced mice at several time points. B) Quantification of Iba1+ cells (microglia/macrophages) in \u003cem\u003ecornu ammonis\u003c/em\u003e and dentate gyrus. Data from 4-12 mice/group and time points presented as mean±SD. Statistical analysis was performed in Prism 10.1.0 using Kruskal-Wallis test and for pairwise comparisons the post-hoc uncorrected Dunn's test. C) Morphological analysis of microglia within the \u003cem\u003ecornu ammonis\u003c/em\u003eof tamoxifen-induced (TAM) versus control (Ctrl) mice. Data from 273-1016 microglia of 4-12 mice/group were analyzed using linear mixed-effects models to compare morphological parameters between groups, with groups as a fixed effect and individual animals as a random effect to account for repeated measures within mice (multiple microglia per mouse).\u003c/p\u003e","description":"","filename":"WilkeNtolkerasetatFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/38f830371655c8949f161bcb.png"},{"id":82715789,"identity":"5263ee11-e60f-411d-9fb8-f12b078eef76","added_by":"auto","created_at":"2025-05-14 12:08:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":55869,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacterization of lymphocytes \u003c/strong\u003ewithin the cornu ammonis (A) and dentate gyrus (B) at different time points after tamoxifen-induced neurodegeneration (TAM, T) versus healthy control mice (Ctrl, C). t1, 1 week; t2, 10 weeks; t3, 10 months after primary encephalitis induction. Data from 4-12 mice/group and time point presented as mean±SD. Statistical analysis performed in Prism 10.1.0 using Kruskal-Wallis and pairwise comparisons post-hoc uncorrected Dunn's test.\u003c/p\u003e","description":"","filename":"WilkeNtolkerasetatFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/ca09a2d5368394874bfa037e.png"},{"id":82715745,"identity":"f521a736-3501-44b3-a04d-e35e6e3784a4","added_by":"auto","created_at":"2025-05-14 12:08:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":710364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFlow cytometry of leukocytes in blood, 10 months after encephalitis induction via tamoxifen (TAM) versus healthy control (Ctrl) mice. \u003c/strong\u003eA) Gating strategy of major immune cell types. Presented data includes all live single CD45+ cells of all mice. B) Immunophenotyping of major immune cell types. C) Dimensionality reduction of flow cytometry parameters showing clusters of major immune cell types. D) Quantification of major immune cell types. E-H) Quantification of immune cell subsets. Data from 10-11 mice per group are presented as mean±SD. Statistical analysis was performed in Prism 10.1.0, data normality tested using Shapiro-Wilk test. Normally distributed data was analyzed using Welch's corrected t-tests, non-normal data using the Mann-Whitney test.\u003c/p\u003e","description":"","filename":"WilkeNtolkerasetatFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/88c41b6185054c23f234a8fb.png"},{"id":85686175,"identity":"caa245e6-be85-45ea-9171-1018ee455bcf","added_by":"auto","created_at":"2025-06-30 16:04:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8753367,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/c0562b3f-1906-43e6-acf3-376c763df4a7.pdf"},{"id":82715740,"identity":"59004195-8c7d-40b4-ae6b-80275aaff46b","added_by":"auto","created_at":"2025-05-14 12:08:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":35739,"visible":true,"origin":"","legend":"","description":"","filename":"WilkeNtolkerasSupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-6499111/v1/5fe64f3dbb09f7d142c238f3.docx"}],"financialInterests":"Competing interest reported. WS is holder of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. KB and BT are full-time employees of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried Stöcker. All other authors declare no competing financial or other interests.","formattedTitle":"Acute neuronal cell death and neuroinflammation per se do not trigger secondary autoimmune encephalitis","fulltext":[{"header":" HIGHLIGHTS","content":"\u003cul\u003e\n \u003cli\u003eAcute neuronal cell death and neuroinflammation alone do not cause autoimmune encephalitis relapses.\u003c/li\u003e\n \u003cli\u003eMicrogliosis, seen fully developed at 1\u0026nbsp;week after initial encephalitis induction, decreases over time.\u003c/li\u003e\n \u003cli\u003eTesting plasma of post-encephalitis mice and controls at a dilution of 1:100 for 49 neural autoantibodies yielded only one positive result in each group.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eAutoimmune-mediated clinical symptoms and autoantibodies against neuronal surface antigens are frequently observed in about one quarter of patients recovering from herpes simplex encephalitis (HSE) and Japanese encephalitis (JE) [\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12 CR13 CR14\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This secondary encephalitis is often referred to as autoimmune relapse or autoimmune encephalitis and typically occurs within 1\u0026ndash;2 months after the primary virus encephalitis. It is characterized by an absence of the original pathogen, the presence of anti-neuronal autoantibodies, and autoimmune encephalitis-like symptoms that frequently improve after immunosuppressive treatment [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While most cases appear to be associated with autoantibodies against NMDA receptors, other anti-neuronal antibodies, including GABAAR, AMPAR, D2R-AB, are also detected in CSF of symptomatic and convalescent patients after HSE and JE [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. It has been hypothesized that the virus-induced neuronal cell death, which is common in HSE and JE [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], results in a release of neuronal surface antigens. These antigens are supposedly drained to peripheral lymphoid organs, where they are presented to the adaptive immune system, eliciting an autoimmune response against neuronal autoantigens. Presumably, activated autoreactive B cells, T cells, and antibody secreting cells then traffic to the CNS and induce a second neuroinflammatory response [\u003cspan additionalcitationids=\"CR20 CR21 CR22\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. This hypothesis is supported by the absence of anti-neuronal autoantibodies during the acute phase of JE and HSE [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which indicates \u003cem\u003ede novo\u003c/em\u003e autoantibody formation in response to encephalitis.\u003c/p\u003e \u003cp\u003eThe central nervous system (CNS) has long been considered an immune-privileged site, but accumulating evidence suggests that its cellular components can differentially shape immune responses. A landmark study by Traka et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] demonstrated that targeted ablation of oligodendrocytes using a diphtheria toxin (DTA)-based approach in transgenic mice leads to delayed but sustained secondary autoimmune inflammation resembling multiple sclerosis. These findings suggest that oligodendrocyte death may not only be a consequence of CNS inflammation but also serve as a trigger for autoimmunity under certain conditions.\u003c/p\u003e \u003cp\u003eTo date, it has remained unclear whether the selective loss of other CNS cell types can initiate a similar cascade. In particular, the immunological consequences of targeted neuronal ablation have not been systematically explored. Given the widespread neuronal loss observed in HSE and JE, as well as various neurodegenerative and autoimmune diseases, this question bears significant clinical relevance.\u003c/p\u003e \u003cp\u003eHere, we investigated whether selective ablation of neurons in adult mice using an analogous DTA-mediated system leads to the induction of CNS-targeted autoimmunity. In stark contrast to the oligodendrocyte ablation model, we found that neuroinflammation gradually subsided after targeted neuronal loss without detectable peripheral immune activation or relapse of CNS inflammation. Our findings suggest that the immunogenic potential of cell death within the CNS is not uniform across cell types and point to a unique role for oligodendrocytes in modulating neuroimmune interactions. Furthermore, the results suggest that additional factors, such as the prominent activation of the innate and adaptive immune system by virus/pathogen-associated molecular pattern pathways, are required to drive post-encephalitis autoimmune relapses.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the local animal care and use committee (LAVES, Niedersaechsisches Landesamt f\u0026uuml;r Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany; license number 33.19-42502-04-18/2803) in accordance with the German animal protection law. The experiments were performed in accordance to approved protocols and reported following the ARRIVE guidelines. Mice were housed in groups of up to 16 in a temperature (~22\u0026deg;C) and humidity (~50%) controlled environment, 12h light/dark cycle with food (standard food, Sniff Spezialdi\u0026auml;ten, Germany) and water \u003cem\u003ead libitum\u003c/em\u003e. Cages were equipped with wood-chip bedding and nesting material (Sizzle Nest, Datesand, United Kingdom). Experimental mice were weaned at postnatal day 21 and separated by sex. Mice were randomly allocated to treatment groups (tamoxifen versus corn oil) on a cage by cage basis. Investigators were unaware of group assignment (\u0026lsquo;fully blinded\u0026rsquo;) throughout data acquisition and analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSterile Encephalitis Model:\u003c/strong\u003e Acute neurodegeneration in pyramidal neurons and subsequent neuroinflammation was induced in transgenic C57BL/6 mice double heterozygous for Neurod6\u003csup\u003etm2.1(cre/ERT2)Kan\u003c/sup\u003e (\u0026lsquo;NexCreERT2\u0026rsquo;, [25]) and Gt(ROSA)26Sor\u003csup\u003etm1(DTA)Jpmb\u003c/sup\u003e (\u0026lsquo;Rosa26-eGFP-DTA\u0026rsquo;, [26]), at the age of 7-8 months via tamoxifen. Tamoxifen was administered intraperitoneally for 3 consecutive days at a dose of 100mg tamoxifen (CAS#10540-29-1 T5648, Sigma-Aldrich) per kilogram body weight per day. Tamoxifen was dissolved in corn oil (C8267, Sigma-Aldrich) at a dose of 10mg/mL. Control littermates were injected with equivalent corn oil volumes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse monitoring:\u003c/strong\u003e Groups of mice were inspected daily for signs of encephalitis-associated behavior (hyperactivity, circling, seizures \u0026amp; lethargy) in home cages. In addition, all mice were individually inspected for aberrant behavior on a weekly basis during cage changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Sampling:\u0026nbsp;\u003c/strong\u003eMice were euthanized via intraperitoneal injection of 300-700mg/kg body weight 2,2,2,-tribromoethanol (T48402, Sigma) solubilized in water. Approximately 400uL blood was collected into syringes filled with 50uL of 80\u0026nbsp;mM EDTA solution by cardiac puncture prior to perfusion. For blood flow cytometry, 100uL whole blood were used immediately. Plasma was collected from the remaining blood samples after centrifugation at 1000\u003cem\u003eg\u003c/em\u003e for 10min at room temperature and stored at -80\u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Flow Cytometry\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole blood samples (100\u0026micro;L) were transferred into flow cytometry tubes and incubated with antibody master mix (supplementary table 1) for 15min at room temperature. Red blood cells were lysed by 15min incubation with 2mL ACK buffer (0.15\u0026nbsp;M NH\u003csub\u003e4\u003c/sub\u003eCl, 10\u0026nbsp;mM KHCO\u003csub\u003e3\u003c/sub\u003e, 0.1 mM EDTA, pH 7.2-7.4) per sample. Samples were centrifuged at 300g for 5min and washed with 1mL ACK buffer. Lymphocytes were resuspended in 500\u0026micro;L FACS buffer (2% BSA in PBS) and measured on a\u0026nbsp;FACSymphony S6 (BD). Data acquisition was stopped after recording 30,000 APC quantification beads per sample.\u003c/p\u003e\n\u003cp\u003eFlow cytometry data was imported to FlowJo v.10.9.0 and compensated using single stained controls. An empty 540LP/35BP channel in the 445 nm Laser line was used to record autofluorescence. Manual clean up gating was performed on SSC-A versus FSC-A to separate cells and quantification beads. As eGFP is ubiquitously expressed in cells of heterozygous Rosa26-eGFP-DTA mice, lymphocytes were selected by gating of eGFP and CD45 double positive events. FSC-H versus FSC-A gating was used to exclude doublets. After individual clean-up gating, all single lymphocyte events from all samples were concatenated. Uniform hierarchical gating was performed on the concatenated sample and copied to individual samples for enumeration of cell types and subsets. First, granulocytes were identified based on high SSC-A and Ly6G. Non-granulocytes were gated for CD115 and NK1.1 to identify NK cells (NK1.1+) and monocytes (CD115+). Next, B cells were identified by gating for CD19 within the CD115 and NK1.1 negative cells, followed by gating for CD8a versus CD4 within CD19- events to identify CD8+ and CD4+ T cells respectively. Negative events were further subdivided into other myeloid cells (CD11b+, FcR+) and unclassified events (lineage negative). The quality of the hierarchical gating strategy was further evaluated qualitatively using t-SNE clustering. Major cell types were further divided into distinct subsets based on their expression of CD27, CD138, CD44, CD62L, and CD11b. The following immune cell subsets were identified: CD27+/CD11b+ M1 NK cells, CD27-/CD11b+ M2 NK cells, CD27+/CD11b- immature NK cells, and CD27-/CD11b- immature NK cells [27], CD27-/CD138- na\u0026iuml;ve B cells; CD27+/CD138- memory B cells; CD138+ antibody secreting cells [28]; CD62L+/CD44- naive T cells, CD62L-/CD44+ effector/ effector memory T cells, and CD62L+/CD44+ central memory T cells [29, 30]. For statistical analyses, the major immune cell types were normalized to the total amount of white blood cells measured per sample. To assess the differentiation state of individual cell types, immune cell subsets were normalized to their parent population (cell type of interest). Data was exported to GraphPad Prism (v 10.1.0). Data normality was assessed using Shapiro Wilk test. Groups with normally distributed data were compared using Welch\u0026rsquo;s corrected unpaired two-sided t-tests, whereas non-normal data was compared using the non-parametric Mann-Whitney tests. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAutoantibody Testing\u003c/strong\u003e\u003cstrong\u003e:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor serological analyses, mouse blood was diluted 1:100 and tested for autoantibodies using biochip mosaics (Euroimmun, L\u0026uuml;beck, Germany) as previously described [31]. Briefly, these mosaics contained unfixed, nitrogen-frozen tissue cryosections (4 \u0026micro;m; rat hippocampus, monkey cerebellum) alongside recombinant cell substrates of formalin- or acetone-fixed transfected HEK293 cells. The expression of recombinant autoantigens was confirmed using human or commercially available monospecific antibodies. The following 49 disease-associated neural antigens [19, 21, 23, 32-53] were evaluated: Myelin, NMDAR, AMPA-R1/R2, GABAR-B1/B2, LGI1, CASPR2, GAD65, mGluR1, mGluR5, Ma2, Recoverin, Zic-4, CARPVIII, Hu, Ri, CV2, Neurochondrin, Yo, ITPR1, Homer-3, Neurexin, ERC1, ARHGAP26, DNER, IgLON5, DPPX, AT1A3, GLRa1b, AQP4, GluRD2, MOG, GABA-a, Flotillin, KCNA1, MBP, DRD2, Contactin-2, KCNA2, Neurofascin 155, Neurofascin 186, Contactin-1, Sez6l2, AP3B2, PCA-2, AGNA, Amphiphysin, MAG, GFAP, ANNA-3. Autoantigen expression was validated by immunological methods employing monospecific control antibodies. To detect autoantibodies of the IgG, IgA and IgM isotypes, the following fluorescently labeled isotype-specific anti-mouse immunoglobulin antibodies were used: anti-mouse IgA (Cat. #62-6700, Thermo; Alexa Fluor 488 custom-labeled at Synaptic Systems), Alexa Fluor 488 labeled anti-mouse IgM (A21042, Thermo), Alexa Fluor 488 labeled anti-mouse IgG (A21202, Thermo).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were transcardially perfused with Ringer solution and 4% formaldehyde in PBS after anesthetic euthanasia by intraperitoneal injection of 300-700mg/kg body weight 2,2,2,-tribromoethanol (T48402, Sigma), solubilized in water. Brains were dissected and post-fixed overnight in 4% formaldehyde in PBS, dehydrated for two days in 30% sucrose solution, embedded in optimal cutting medium (Tissue-Tek, #4583, Sakura), frozen, cryosectioned, and stored in cryoprotectant solution (1x PBS with 25% ethylene glycol, 25% glycerol). Histological analyses were performed in 30-70\u0026micro;m coronal sections as previously described [54, 55].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorojade C staining of degenerating neurons\u003c/strong\u003e was performed as previously described (1, 2). Briefly, 30\u0026micro;m coronal sections were mounted on microscopy slides, dried overnight at room temperature and rehydrated. Rehydrated slides were incubated with 0.06% potassium permanganate solution for 10min, rinsed with water, and stained for 10min in a 0.0001% solution of Fluorojade C (AG325, Sigma) and 0.1\u0026micro;g/mL 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, D9542, Sigma), dissolved in 0.1% acetic acid. Lastly, slides were rinsed with ddH\u003csub\u003e2\u003c/sub\u003eO, dried at 60\u0026deg;C, and mounted. Images were acquired as tile scans with a pixel scaling of 1.04\u0026micro;m on a Zeiss LSM880 laser scanning confocal microscope equipped with a 10x air objective (0.45NA). Presence of neurodegeneration was evaluated qualitatively by a blinded investigator. Rating criteria was the presence or absence of at least 10 distinct Fluorojade C positive cell bodies within the hippocampus.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIba1 and CD45\u003c/strong\u003e double-labeling of microglia and infiltrating lymphocytes, was performed in 30\u0026micro;m coronal sections using standard free-floating immunofluorescence techniques with the following antibodies:\u0026nbsp;rat anti-CD45 (1:250; clone 30-F11, 103101, Biolegend), rabbit anti-IBA-1 (1:250, 019-19741, Wako), Alexa Fluor-555 anti-rabbit (1:500, A-21428, Invitrogen), Alexa Fluor 647 anti-rat (1:500, A-31571, Thermo). Images of 2-4 hippocampi per mouse were acquired as tile scans with a pixel scaling of 0.52\u0026micro;m on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ\u0026nbsp;[56]. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn\u0026apos;s test for post-hoc pairwise comparisons. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantification of CNS-infiltrating T\u0026nbsp;cells and B cells\u003c/strong\u003e were performed using triple-labeling of CD45, CD3, and CD19 in 30\u0026micro;m coronal brain sections. Standard free-floating immunofluorescence were used with the following antibodies:\u0026nbsp;rat anti-CD45 (1:250; clone 30-F11, #103101, Biolegend), hamster anti-CD3 (1:250, clone 145-2C11, #557306, BD), rabbit anti-CD19 (1:250, clone D4V4B, #90176S, Cell Signaling), Alexa Fluor-488 anti-rat (1:500, #712-547-003, Jackson Immuno Research), Alexa Fluor-555 anti-rabbit (1:500, #A-21428, Invitrogen), Alexa Fluor-647 anti-hamster (1:500, #127-605-1685, Jackson Immuno Research). Images were acquired as tile scans with a pixel scaling of 0.69\u0026micro;m on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ [56]. CD3+/CD45+ lymphocytes were quantified as T cells while CD19+/CD45+ lymphocytes were classified as B cells. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn\u0026apos;s test for post-hoc pairwise comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD8 and CD3\u0026nbsp;\u003c/strong\u003edouble labelingof CD8+ T cells was performed in 30\u0026micro;m coronal sections using standard free-floating immunofluorescence techniques with the following antibodies: hamster anti-CD3 (1:250, clone 145-2C11,\u0026nbsp;#557306, BD), rat anti-CD8a (1:250, clone S18018A, #164702, Biolegend), Alexa Fluor-555 anti-hamster (1:500, A78964, Invitrogen), Alexa Fluor-647 anti-rat (1:500, A21247, Invitrogen). Images were acquired as tile scans with a pixel scaling of 0.83\u0026micro;m on a Zeiss LSM880 laser scanning confocal microscope equipped with a 20x air objective (0.8NA). Image segmentation and quantification was performed manually with FIJI-ImageJ [56]. CD3+/CD8+ double positive cells were counted as CD8+ T\u0026nbsp;cells. Data from multiple hippocampi per mouse were averaged and groups were compared in Prism 10.1.0 using the Kruskal-Wallis test and the uncorrected Dunn\u0026apos;s test for post-hoc pairwise comparisons.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMorphological analysis\u003c/strong\u003e \u003cstrong\u003eof microglia\u003c/strong\u003e was performed in 70\u0026micro;m coronal brain sections after staining with a standard free-floating immunofluorescence protocol using the following antibodies: rabbit anti-Iba1 (1:250, #019-19741, Wako), Alexa Fluor 555 labeled anti-rabbit (1:500, A-21428, Invitrogen). Nuclei were stained for 10 min at room temperature with 0.2 \u0026micro;g/mL 4\u0026prime;,6-diamidino-2-phenylindole (DAPI, D9542, Sigma) in PBS. Per mouse, 1-3 Z stacks spanning 20\u0026micro;m (0.4\u0026micro;m step size) were acquired within the \u003cem\u003ecornu ammonis\u003c/em\u003e (CA) with a pixel scaling of 0.2\u0026micro;m on a Zeiss LSM880 laser scanning confocal microscope equipped with a 40x oil objective (1.4 NA). All imaging settings including LASER intensities and pixel dwell times were kept constant throughout the experiment. Images were processed using the microglia morphology quantification tool (MMQT) developed by Heindl, Gesierich and colleagues [57]. Images with visible artifacts in the orthogonal views and images with poor spatial correlations in Z and XY dimensions were excluded from the analysis.\u0026nbsp;Relevant parameters describing the 3D morphology of microglia were extracted and microglia were filtered using the following parameters: distance to X \u0026amp; Y borders \u0026gt;15\u0026micro;m; distance to Z borders \u0026gt;5\u0026micro;m; number of nuclei = 1; merged soma =0; shortest distance between microglia soma \u0026gt;15\u0026micro;m; number of branches \u0026gt;0. Linear mixed-effects models were used to compare morphological parameters between groups, with groups as a fixed effect and individual animals as a random effect to account for repeated measures within mice (multiple microglia per mouse). Models were fitted using the lmerTest package [58] and p-values for fixed (group) effects were extracted in R.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed as described in respective method sections. Analyses were performed either in Prism 10.1.0 (GraphPad Software) or R v4.3.1 [59]. Statistical tests were selected based on experimental design and data distribution. Unless stated otherwise, results are presented as mean\u0026plusmn;standard deviations (SD). P-values \u0026lt;0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eLongitudinal monitoring of mice after sterile induction of neuronal death did not reveal autoimmune encephalitis-like symptoms\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo test if the acute destruction of neurons in the absence of any virus is sufficient to induce an autoimmune relapse in mice, we utilized a previously characterized transgenic mouse model of virus-like but sterile encephalitis [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Acute neurodegeneration of hippocampal and cortical pyramidal neurons and subsequent neuroinflammation was induced in adult double heterozygous NexCreERT2 x Rosa26-eGFP-DTA mice via intraperitoneal tamoxifen injections (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). A total of 64 mice (1:1 male/female ratio) were used for this study, of which half received tamoxifen whereas the other half received corn oil (healthy controls). To monitor disease progression histologically, 4 mice per group and timepoint were euthanized for tissue collection 1 week and 10 weeks after encephalitis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The remaining 24 mice per group were monitored daily for autoimmune encephalitis-like symptoms, such as hyperactivity, circling, seizures, lethargy, and death [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e] for 10 months. Two corn oil control mice died spontaneously 221 days and 305 days after injections, likely due to age-related reasons (14.7 and 17.5 months). None of the tamoxifen injected mice died and autoimmune encephalitis-like symptoms were absent in all mice throughout the study period. The presence of neurodegeneration and neuroinflammation were confirmed 1 week after tamoxifen injection during the acute phase of the primary encephalitis (n\u0026thinsp;=\u0026thinsp;4/ group; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). By ten weeks after tamoxifen induction, degenerating neurons had been cleared from the hippocampus, as indicated by the absence of Fluorojade C positive cell bodies (n\u0026thinsp;=\u0026thinsp;4/group; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eMicroglia profiling in the hippocampus points towards chronic neuroinflammation rather than relapse with acute autoimmune encephalitis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWhile there is no clear consensus on the definition of neuroinflammation yet, it is generally accepted that a key feature of neuroinflammation is the response of microglia to pathogenic stimuli, such as degenerating neurons and DAMPs or CNS infiltrating leukocytes [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. In neurodegenerative diseases and autoimmune encephalitis, this response is typically characterized by an increase in microglia numbers/density and morphological changes [\u003cspan additionalcitationids=\"CR63 CR64 CR65 CR66 CR67\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. To longitudinally monitor the inflammatory status of microglia we quantified their density and morphology 1 week, 10 weeks and 10 months after induction of acute neuronal cell death (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). During the acute phase, microgliosis was most prominent in the \u003cem\u003ecornu ammonis\u003c/em\u003e, which contained the majority of degenerating neurons, but a significant increase of microglia numbers was also observed within the neighboring dentate gyrus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Within the \u003cem\u003ecornu ammonis\u003c/em\u003e, microglia numbers remained significantly higher in tamoxifen-induced mice versus corn oil controls but significantly decreased over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the dentate gyrus, microglia numbers remained significantly elevated during the 10-weeks timepoint, but did not differ from control mice 10 months after tamoxifen induction. Further automated profiling of microglia 3D-morphology via MMQT [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] revealed significant morphological changes, such as increased sphericity and decreased branching that are consistent with an inflammatory phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). While the microglia morphology of tamoxifen-induced mice remained significantly different from microglia in healthy control mice, we noticed a significant shift of morphological parameters towards a homeostatic morphology over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCharacterization of CNS-infiltrating immune cells reveals an absence of B cell and presence of CD8\u0026thinsp;+\u0026thinsp;T cell infiltration\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA hallmark of autoimmune encephalitis is the presence of CNS infiltrating lymphocytes, in particular B cells, T cells, and antibody secreting cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. For this reason, we monitored the lymphocyte infiltration at discrete time points after primary encephalitis induction. Throughout the study period, mild to moderate significant lymphocyte infiltration was observed in the \u003cem\u003ecornu ammonis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). No significant lymphocyte infiltration was observed in the adjacent dentate gyrus (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). CD19 positive B cells were nearly absent in all hippocampal regions throughout the study period (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B) and were not detected in noticeable amounts in other brain regions. The majority of hippocampus infiltrating CD45\u0026thinsp;+\u0026thinsp;lymphocytes in tamoxifen-induced mice were CD3\u0026thinsp;+\u0026thinsp;T cells (82.5% \u0026plusmn; 14.6%; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). While the total amount of intrahippocampal CD3\u0026thinsp;+\u0026thinsp;T cells did not significantly change overtime, we noticed a significant time-dependent increase in CD8\u0026thinsp;+\u0026thinsp;T cells in tamoxifen-induced mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). After primary encephalitis induction, the proportion of hippocampal CD8\u0026thinsp;+\u0026thinsp;T cells amongst total hippocampal T cells increased from 25.0% \u0026plusmn; 24.0% during the acute phase to 71.6% \u0026plusmn; 29.8% during the recovery phase and 89.2% \u0026plusmn; 14.8% after completion of the long-term follow-up. Perivascular cuffs or clusters of nuclei dense immune cell infiltrates, that are frequently present in brains of mice and humans with autoimmune encephalitis such as NMDAR encephalitis [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], were not observed.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eBlood flow cytometry indicates absence of a peripheral autoimmune response\u003c/h3\u003e\n\u003cp\u003eWe performed high-parameter flow cytometry analyses of peripheral blood to test if mice recovering from a virus-like but sterile encephalitis show a peripheral immune response that would be indicative of an adaptive immune response and autoimmune encephalitis. Using classical cell surface markers and hierarchical gating, we quantified granulocytes, NK-cells, monocytes, B cells, CD4\u0026thinsp;+\u0026thinsp;T cells and CD8\u0026thinsp;+\u0026thinsp;T cells in blood samples of 11 mice tamoxifen-induced post-encephalitis mice and 10 healthy control mice after completion of the 10-months follow-up period (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Within the major immune cell populations, we quantified the proportions of distinct immune cell subtypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). NK cell subsets were classified based on differential surface expression of CD11b and CD27 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. B cells were categorized as CD27-/CD138- na\u0026iuml;ve B cells, CD27+/CD138- memory B cells, and CD138\u0026thinsp;+\u0026thinsp;antibody secreting cells [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. T cells were divided into CD62L+/CD44- na\u0026iuml;ve T cells, CD62L+/CD44\u0026thinsp;+\u0026thinsp;central memory T cells, and CD62L-/CD44\u0026thinsp;+\u0026thinsp;effector memory T cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Dimensionality reduction and t-SNE clustering was used to confirm the quality of the hierarchical gating strategy (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At the time of follow-up completion, none of the major cell types were significantly altered in tamoxifen-induced post-encephalitis mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Similarly, neither the NK cell population, B cell population, or CD4\u0026thinsp;+\u0026thinsp;T cell population showed significant changes in their composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). However, the peripheral CD8\u0026thinsp;+\u0026thinsp;T cell compartment of post-encephalitis mice showed a significant increase in na\u0026iuml;ve T cells that was accompanied by a reduction of effector memory cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTesting for 49 CNS-targeting autoantibodies did not find increased autoantibody production in mice weeks to months after sterile encephalitis\u003c/b\u003e \u003c/p\u003e \u003cp\u003eCNS-directed autoantibodies are frequently observed in patients after HSE and JE [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] as well as in mice infected with HSV-1 [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. To test if this autoantibody production occurs in response to acute neuronal cell death without additional co-stimulation of the immune system by virus/pathogen associated molecular patterns, we tested plasma samples of healthy mice and mice after induction of virus-like but sterile encephalitis for the presence of anti-CNS autoantibodies. Autoantibody titers against 49-disease associated antibodies were determined for IgG, IgA and IgM isotypes using commercially available cell-based assays developed for the \u003cem\u003ein vitro\u003c/em\u003e diagnostic of autoantibodies in suspected autoimmune encephalitis patients (Euroimmun biochip mosaic IVD assays). Due to the limited amount of mouse plasma, autoantibody testing was performed with a cut-off titer of 1:100. Plasma samples of 60 mice (n\u0026thinsp;=\u0026thinsp;30 per group) were tested. One healthy control had a titer of 1:320 for anti-CASPR2 autoantibodies of the IgG subtype and one post-encephalitis mouse had IgM autoantibodies against homer-3 at a titer of 1:1000 (supplementary table 2). Overall seroprevalence for all 49 autoantibodies was low (2/60 mice; 3.33%) and no difference was observed between groups (1/30 positive mice post-encephalitis versus 1/30 positive control mice; p\u0026thinsp;\u0026gt;\u0026thinsp;0.9999, Chi-square test).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eTo test if the acute destruction of neurons in the absence of encephalitogenic virus is sufficient to induce a secondary autoimmune encephalitis in mice, we monitored mice for up to 10 months after sterile induction of cell death in hippocampal and pyramidal neurons. Throughout the study period, we observed no symptoms of severe autoimmune encephalitis, such as hyperactivity, circling, seizures, or lethargy. Microglia analysis revealed significant microgliosis one week after encephalitis induction, which diminished over time. Scattered lymphocyte infiltration persisted in the hippocampi of encephalitis mice but did not increase significantly. Perivascular cuffs were not detected, and B cell infiltration was rare and comparable to healthy controls. High-parameter immunophenotyping of peripheral blood leukocytes did not reveal an expansion of memory B cells or antibody secreting cells, that could be indicative of an antibody-associated autoimmune response. Concomitantly, autoantibody testing against 49 neural antigens in plasma samples of post-encephalitis mice yielded mostly negative results, similar to healthy controls. Overall, these findings suggest that acute neuronal cell death and neuroinflammation alone are insufficient to trigger relapses with autoimmune encephalitis.\u003c/p\u003e \u003cp\u003eWhile we observed significant time-dependent recovery of microglial characteristics, that are typically associated with homeostatic functioning, hippocampal microglia from mice after encephalitis induction remained significantly different from healthy control microglia for up to 10 months. A potential reason for this incomplete recovery could be the accumulation of CD8\u0026thinsp;+\u0026thinsp;T cells in the hippocampus of post-encephalitis mice in combination with their advanced age. In the peripheral nervous system, CD8\u0026thinsp;+\u0026thinsp;memory T cells restrict axonal regeneration after spinal cord injury and mediate aging-dependent regenerative decline [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Within the central nervous system, CD8\u0026thinsp;+\u0026thinsp;tissue-resident memory T-cells are commonly observed in several neuroinflammatory and neurodegenerative conditions [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. While CD8\u0026thinsp;+\u0026thinsp;tissue resident memory Τ cells are typically induced in response to CNS infections and associated with protective properties [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e], autoreactive CD8\u0026thinsp;+\u0026thinsp;tissue resident memory T cells can also drive autoinflammatory responses within the CNS [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. In our model, we observed a partial time-dependent recovery of homeostatic-like characteristics in microglia as well as an absence of additional lymphocyte recruitment or encephalitis-like symptoms, pointing against a CD8\u0026thinsp;+\u0026thinsp;T cell mediated autoimmune pathology.\u003c/p\u003e \u003cp\u003eIn this study we found a lack of autoimmune encephalitis-like pathology after the induction of neuronal cell death and sterile encephalitis. A probable reason could be an insufficient stimulation of the peripheral immune system due to the absence of virus/pathogen associated molecular patterns. Several histopathological analyses of \u0026lsquo;sporadic\u0026rsquo; and NMDAR encephalitis-associated ovarian teratomas highlight the relevance of peripheral immune cell activation in the encephalitogenic process. In both patient subsets, neuronal tissue, that ectopically express NMDA receptors, was frequently observed. However, ovarian teratomas from patients who progressed to autoimmune encephalitis exhibited a higher density of lymphocytic infiltrates near neuronal tissue [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e, \u003cspan additionalcitationids=\"CR76 CR77\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. This suggests that co-stimulatory signals play a key role in determining whether exposure to neuronal antigens is tolerated or leads to autoimmune encephalitis. The role of peripheral immune activation as risk factor for autoimmune encephalitis after virus encephalitis was further highlighted by a recent prospective study by Armangu\u0026eacute; and colleagues who found that an elevated blood IFN response was the most important predictor of post-HSE autoimmune encephalitis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn principle, the absence of an autoimmune relapse following encephalitis could also be explained by an insufficient presentation of autoantigens to the immune system. While shedding of neuronal antigens, such as NMDA and AMPA receptors, into the periphery via small vesicles has been shown to occur in human encephalitis patients [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], we did not assess this shedding process in the present mouse model. Interestingly, DTA-mediated ablation of oligodendrocytes in adult mice induced the generation of myelin oligodendrocyte glycoprotein (MOG)-specific T cells in peripheral lymphoid organs and resulted in an autoimmune relapse approximately 30 weeks after oligodendrocyte ablation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The apparent discrepancy between the outcome after acute pyramidal cell and oligodendrocyte cell death suggest that factors such as the antigenicity of the released proteins as well as antigen-specific clearance and shedding may determine susceptibility to autoimmune relapses.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLimitations\u003c/h2\u003e \u003cp\u003eA limitation of the utilized autoantibody screening is the use of commercially available cell-based assays, that were developed as \u003cem\u003ein vitro\u003c/em\u003e diagnostics for the detection of autoantibodies in patients with (suspected) autoimmune encephalitis. While all of the assessed autoantigens are well conserved between human and mice (95\u0026thinsp;\u0026plusmn;\u0026thinsp;5% sequence identity), we cannot rule out the presence of autoantibodies targeting mouse-specific epitopes. Similarly, low affinity autoantibodies or autoantibodies with a low plasma concentration might have been missed due to the stringent cut-off titer of 1:100. This would also explain the lower seroprevalence in comparison to our previous work, that focused on the NMDAR1-AB seroprevalence across mammals, in which samples were tested with 1:1 and 1:10 dilutions [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother limitation is that we did not investigate cases with more massive and global destruction of neurons, which would likely increase the amount of neuronal autoantigens and damage associated molecular patterns that are drained to peripheral lymphoid organs, and may reach a threshold that is sufficient to induce autoimmune relapses. Another potential factor predisposing to pathogenic brain directed autoimmunity could be damage to glial cells and the blood brain barrier [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. In fact, we previously observed an increase in NMDAR1-autoantibodies in mice in response to a small standardized cryolesion of the right parietal cortex [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e], with local damage of all cells near the lesion site and blood-brain barrier dysfunction [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eWhile clinical observations suggest a solid link between autoimmune encephalitis and virus encephalitides, such as HSE and JE, the pathomechanisms behind the secondary autoimmune response have remained obscure. The present study on a sterile encephalitis model reveals that acute destruction of neurons alone is not sufficient to induce an autoimmune response against neuronal surface antigens, as previously associated with autoimmune encephalitides. This observation indicates that co-factors are required for the initiation of anti-neuronal autoimmune responses. Identifying these co-factors may ultimately enable targeted therapies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were approved by the local animal care and use committee (LAVES, Niedersaechsisches Landesamt f\u0026uuml;r Verbraucherschutz und Lebensmittelsicherheit, Oldenburg, Germany; license number 33.19-42502-04-18/2803) in accordance with the German animal protection law.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe code used to analyze MMQT output files is available at https://github.com/vgastaldi/MMQT-longDTA. Further information and requests for data and resources should be directed to the lead contact, Prof. Dr. Dr. Hannelore Ehrenreich ([email protected]).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWS is holder of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried St\u0026ouml;cker. KB and BT are full-time employees of Clinical Immunological Laboratory Prof. h.c. (RCH) Winfried St\u0026ouml;cker. All other authors declare no competing financial or other interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work has been funded by the DFG TRR-274/1 2020-408885537. Furthermore, the study has been fostered by the Max Planck Society and the Max Planck F\u0026ouml;rderstiftung. Research in the HE lab was funded by the European Research Council (ERC) Advanced Grant under the European Union\u0026rsquo;s Horizon Europe research and innovation programme (acronym BREPOCI; grant agreement No 101054369). KAN is supported by the Adelson Medical Research Foundation. VDG received backing from the IMPRS-Genome Science PhD program. For the publication fee we acknowledge financial support by Heidelberg University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupervision: HE, JBHW,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding acquisition: HE, KAN\u003c/p\u003e\n\u003cp\u003eConcept and design: HE, JBHW, KAN\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData acquisition/generation: AN, JBHW, BT, KB, WS\u003c/p\u003e\n\u003cp\u003eData analyses/interpretation: JBHW, VDG, AN, FL\u003c/p\u003e\n\u003cp\u003eDrafting the manuscript: JBHW, HE\u003c/p\u003e\n\u003cp\u003eDrafting display items: JBHW, AN, HE\u003c/p\u003e\n\u003cp\u003eCritical input,\u0026nbsp;review \u0026amp; editing: AN, JBHW, VDG, KB, BT, WS, FL, KAN, HE\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAll authors read and approved the final version of the manuscript.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Roman Schr\u0026ouml;der for his assistance and critical input in flow cytometry experiments and Anja Ronnenberg, Nadine Barnkothe, and Viktoria Bonet for outstanding technical support.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePruss H, Finke C, Holtje M, Hofmann J, Klingbeil C, Probst C, Borowski K, Ahnert-Hilger G, Harms L, Schwab JM, Ploner CJ, Komorowski L, Stoecker W, Dalmau J, Wandinger KP: \u003cstrong\u003eN-methyl-D-aspartate receptor antibodies in herpes simplex encephalitis.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e2012, \u003cstrong\u003e72:\u003c/strong\u003e902-911.\u003c/li\u003e\n\u003cli\u003eLinnoila JJ, Binnicker MJ, Majed M, Klein CJ, McKeon A: \u003cstrong\u003eCSF herpes virus and autoantibody profiles in the evaluation of encephalitis.\u003c/strong\u003e \u003cem\u003eNeurol Neuroimmunol Neuroinflamm \u003c/em\u003e2016, \u003cstrong\u003e3:\u003c/strong\u003ee245.\u003c/li\u003e\n\u003cli\u003eArmangue T, Leypoldt F, Malaga I, Raspall-Chaure M, Marti I, Nichter C, Pugh J, Vicente-Rasoamalala M, Lafuente-Hidalgo M, Macaya A, Ke M, Titulaer MJ, Hoftberger R, Sheriff H, Glaser C, Dalmau J: \u003cstrong\u003eHerpes simplex virus encephalitis is a trigger of brain autoimmunity.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e2014, \u003cstrong\u003e75:\u003c/strong\u003e317-323.\u003c/li\u003e\n\u003cli\u003eHacohen Y, Deiva K, Pettingill P, Waters P, Siddiqui A, Chretien P, Menson E, Lin JP, Tardieu M, Vincent A, Lim MJ: \u003cstrong\u003eN-methyl-D-aspartate receptor antibodies in post-herpes simplex virus encephalitis neurological relapse.\u003c/strong\u003e \u003cem\u003eMov Disord \u003c/em\u003e2014, \u003cstrong\u003e29:\u003c/strong\u003e90-96.\u003c/li\u003e\n\u003cli\u003eMohammad SS, Sinclair K, Pillai S, Merheb V, Aumann TD, Gill D, Dale RC, Brilot F: \u003cstrong\u003eHerpes simplex encephalitis relapse with chorea is associated with autoantibodies to N-Methyl-D-aspartate receptor or dopamine-2 receptor.\u003c/strong\u003e \u003cem\u003eMov Disord \u003c/em\u003e2014, \u003cstrong\u003e29:\u003c/strong\u003e117-122.\u003c/li\u003e\n\u003cli\u003eArmangue T, Moris G, Cantarin-Extremera V, Conde CE, Rostasy K, Erro ME, Portilla-Cuenca JC, Turon-Vinas E, Malaga I, Munoz-Cabello B, Torres-Torres C, Llufriu S, Gonzalez-Gutierrez-Solana L, Gonzalez G, Casado-Naranjo I, Rosenfeld M, Graus F, Dalmau J, Spanish Prospective Multicentric Study of Autoimmunity in Herpes Simplex E: \u003cstrong\u003eAutoimmune post-herpes simplex encephalitis of adults and teenagers.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e2015, \u003cstrong\u003e85:\u003c/strong\u003e1736-1743.\u003c/li\u003e\n\u003cli\u003eArmangue T, Olive-Cirera G, Martinez-Hernandez E, Rodes M, Peris-Sempere V, Guasp M, Ruiz R, Palou E, Gonzalez A, Marcos MA, Erro ME, Bataller L, Corral-Corral I, Planaguma J, Caballero E, Vlagea A, Chen J, Bastard P, Materna M, Marchal A, Abel L, Cobat A, Alsina L, Fortuny C, Saiz A, Mignot E, Vanderver A, Casanova JL, Zhang SY, Dalmau J: \u003cstrong\u003eNeurologic complications in herpes simplex encephalitis: clinical, immunological and genetic studies.\u003c/strong\u003e \u003cem\u003eBrain \u003c/em\u003e2023, \u003cstrong\u003e146:\u003c/strong\u003e4306-4319.\u003c/li\u003e\n\u003cli\u003eSutcu M, Akturk H, Somer A, Tatli B, Torun SH, Yildiz EP, Sik G, Citak A, Agacfidan A, Salman N: \u003cstrong\u003eRole of Autoantibodies to N-Methyl-d-Aspartate (NMDA) Receptor in Relapsing Herpes Simplex Encephalitis: A Retrospective, One-Center Experience.\u003c/strong\u003e \u003cem\u003eJ Child Neurol \u003c/em\u003e2016, \u003cstrong\u003e31:\u003c/strong\u003e345-350.\u003c/li\u003e\n\u003cli\u003ePruss H: \u003cstrong\u003ePostviral autoimmune encephalitis: manifestations in children and adults.\u003c/strong\u003e \u003cem\u003eCurr Opin Neurol \u003c/em\u003e2017, \u003cstrong\u003e30:\u003c/strong\u003e327-333.\u003c/li\u003e\n\u003cli\u003eNosadini M, Mohammad SS, Corazza F, Ruga EM, Kothur K, Perilongo G, Frigo AC, Toldo I, Dale RC, Sartori S: \u003cstrong\u003eHerpes simplex virus-induced anti-N-methyl-d-aspartate receptor encephalitis: a systematic literature review with analysis of 43 cases.\u003c/strong\u003e \u003cem\u003eDev Med Child Neurol \u003c/em\u003e2017, \u003cstrong\u003e59:\u003c/strong\u003e796-805.\u003c/li\u003e\n\u003cli\u003eMa J, Zhang T, Jiang L: \u003cstrong\u003eJapanese encephalitis can trigger anti-N-methyl-D-aspartate receptor encephalitis.\u003c/strong\u003e \u003cem\u003eJ Neurol \u003c/em\u003e2017, \u003cstrong\u003e264:\u003c/strong\u003e1127-1131.\u003c/li\u003e\n\u003cli\u003eMa J, Han W, Jiang L: \u003cstrong\u003eJapanese encephalitis-induced anti-N-methyl-d-aspartate receptor encephalitis: A hospital-based prospective study.\u003c/strong\u003e \u003cem\u003eBrain Dev \u003c/em\u003e2020, \u003cstrong\u003e42:\u003c/strong\u003e179-184.\u003c/li\u003e\n\u003cli\u003eLiu B, Liu J, Sun H, Xie M, Yang C, Pan Y, Huang D, Cheng L, Chen H, Ma J, Jiang L: \u003cstrong\u003eAutoimmune encephalitis after Japanese encephalitis in children: A prospective study.\u003c/strong\u003e \u003cem\u003eJ Neurol Sci \u003c/em\u003e2021, \u003cstrong\u003e424:\u003c/strong\u003e117394.\u003c/li\u003e\n\u003cli\u003eAshraf U, Ding Z, Deng S, Ye J, Cao S, Chen Z: \u003cstrong\u003ePathogenicity and virulence of Japanese encephalitis virus: Neuroinflammation and neuronal cell damage.\u003c/strong\u003e \u003cem\u003eVirulence \u003c/em\u003e2021, \u003cstrong\u003e12:\u003c/strong\u003e968-980.\u003c/li\u003e\n\u003cli\u003eArmangue T, Spatola M, Vlagea A, Mattozzi S, Carceles-Cordon M, Martinez-Heras E, Llufriu S, Muchart J, Erro ME, Abraira L, Moris G, Monros-Gimenez L, Corral-Corral I, Montejo C, Toledo M, Bataller L, Secondi G, Arino H, Martinez-Hernandez E, Juan M, Marcos MA, Alsina L, Saiz A, Rosenfeld MR, Graus F, Dalmau J, Spanish Herpes Simplex Encephalitis Study G: \u003cstrong\u003eFrequency, symptoms, risk factors, and outcomes of autoimmune encephalitis after herpes simplex encephalitis: a prospective observational study and retrospective analysis.\u003c/strong\u003e \u003cem\u003eLancet Neurol \u003c/em\u003e2018, \u003cstrong\u003e17:\u003c/strong\u003e760-772.\u003c/li\u003e\n\u003cli\u003eGraus F, Titulaer MJ, Balu R, Benseler S, Bien CG, Cellucci T, Cortese I, Dale RC, Gelfand JM, Geschwind M, Glaser CA, Honnorat J, Hoftberger R, Iizuka T, Irani SR, Lancaster E, Leypoldt F, Pruss H, Rae-Grant A, Reindl M, Rosenfeld MR, Rostasy K, Saiz A, Venkatesan A, Vincent A, Wandinger KP, Waters P, Dalmau J: \u003cstrong\u003eA clinical approach to diagnosis of autoimmune encephalitis.\u003c/strong\u003e \u003cem\u003eLancet Neurol \u003c/em\u003e2016, \u003cstrong\u003e15:\u003c/strong\u003e391-404.\u003c/li\u003e\n\u003cli\u003eWhitley RJ: \u003cstrong\u003eHerpes simplex encephalitis: adolescents and adults.\u003c/strong\u003e \u003cem\u003eAntiviral Res \u003c/em\u003e2006, \u003cstrong\u003e71:\u003c/strong\u003e141-148.\u003c/li\u003e\n\u003cli\u003eKennedy PG, Chaudhuri A: \u003cstrong\u003eHerpes simplex encephalitis.\u003c/strong\u003e \u003cem\u003eJ Neurol Neurosurg Psychiatry \u003c/em\u003e2002, \u003cstrong\u003e73:\u003c/strong\u003e237-238.\u003c/li\u003e\n\u003cli\u003eDalmau J, Graus F: \u003cstrong\u003eAntibody-Mediated Encephalitis.\u003c/strong\u003e \u003cem\u003eN Engl J Med \u003c/em\u003e2018, \u003cstrong\u003e378:\u003c/strong\u003e840-851.\u003c/li\u003e\n\u003cli\u003eSun B, Ramberger M, O\u0026apos;Connor KC, Bashford-Rogers RJM, Irani SR: \u003cstrong\u003eThe B cell immunobiology that underlies CNS autoantibody-mediated diseases.\u003c/strong\u003e \u003cem\u003eNat Rev Neurol \u003c/em\u003e2020, \u003cstrong\u003e16:\u003c/strong\u003e481-492.\u003c/li\u003e\n\u003cli\u003eSabatino JJ, Jr., Probstel AK, Zamvil SS: \u003cstrong\u003eB cells in autoimmune and neurodegenerative central nervous system diseases.\u003c/strong\u003e \u003cem\u003eNat Rev Neurosci \u003c/em\u003e2019, \u003cstrong\u003e20:\u003c/strong\u003e728-745.\u003c/li\u003e\n\u003cli\u003eGibson LL, McKeever A, Coutinho E, Finke C, Pollak TA: \u003cstrong\u003eCognitive impact of neuronal antibodies: encephalitis and beyond.\u003c/strong\u003e \u003cem\u003eTransl Psychiatry \u003c/em\u003e2020, \u003cstrong\u003e10:\u003c/strong\u003e304.\u003c/li\u003e\n\u003cli\u003ePruss H: \u003cstrong\u003eAutoantibodies in neurological disease.\u003c/strong\u003e \u003cem\u003eNat Rev Immunol \u003c/em\u003e2021, \u003cstrong\u003e21:\u003c/strong\u003e798-813.\u003c/li\u003e\n\u003cli\u003eTraka M, Podojil JR, McCarthy DP, Miller SD, Popko B: \u003cstrong\u003eOligodendrocyte death results in immune-mediated CNS demyelination.\u003c/strong\u003e \u003cem\u003eNat Neurosci \u003c/em\u003e2016, \u003cstrong\u003e19:\u003c/strong\u003e65-74.\u003c/li\u003e\n\u003cli\u003eAgarwal A, Dibaj P, Kassmann CM, Goebbels S, Nave KA, Schwab MH: \u003cstrong\u003eIn vivo imaging and noninvasive ablation of pyramidal neurons in adult NEX-CreERT2 mice.\u003c/strong\u003e \u003cem\u003eCereb Cortex \u003c/em\u003e2012, \u003cstrong\u003e22:\u003c/strong\u003e1473-1486.\u003c/li\u003e\n\u003cli\u003eIvanova A, Signore M, Caro N, Greene ND, Copp AJ, Martinez-Barbera JP: \u003cstrong\u003eIn vivo genetic ablation by Cre-mediated expression of diphtheria toxin fragment A.\u003c/strong\u003e \u003cem\u003eGenesis \u003c/em\u003e2005, \u003cstrong\u003e43:\u003c/strong\u003e129-135.\u003c/li\u003e\n\u003cli\u003eGoh W, Huntington ND: \u003cstrong\u003eRegulation of Murine Natural Killer Cell Development.\u003c/strong\u003e \u003cem\u003eFront Immunol \u003c/em\u003e2017, \u003cstrong\u003e8:\u003c/strong\u003e130.\u003c/li\u003e\n\u003cli\u003eBrynjolfsson SF, Persson Berg L, Olsen Ekerhult T, Rimkute I, Wick MJ, Martensson IL, Grimsholm O: \u003cstrong\u003eLong-Lived Plasma Cells in Mice and Men.\u003c/strong\u003e \u003cem\u003eFront Immunol \u003c/em\u003e2018, \u003cstrong\u003e9:\u003c/strong\u003e2673.\u003c/li\u003e\n\u003cli\u003eGerberick GF, Cruse LW, Miller CM, Sikorski EE, Ridder GM: \u003cstrong\u003eSelective modulation of T cell memory markers CD62L and CD44 on murine draining lymph node cells following allergen and irritant treatment.\u003c/strong\u003e \u003cem\u003eToxicol Appl Pharmacol \u003c/em\u003e1997, \u003cstrong\u003e146:\u003c/strong\u003e1-10.\u003c/li\u003e\n\u003cli\u003eSckisel GD, Mirsoian A, Minnar CM, Crittenden M, Curti B, Chen JQ, Blazar BR, Borowsky AD, Monjazeb AM, Murphy WJ: \u003cstrong\u003eDifferential phenotypes of memory CD4 and CD8 T cells in the spleen and peripheral tissues following immunostimulatory therapy.\u003c/strong\u003e \u003cem\u003eJ Immunother Cancer \u003c/em\u003e2017, \u003cstrong\u003e5:\u003c/strong\u003e33.\u003c/li\u003e\n\u003cli\u003eDaguano Gastaldi V, Bh Wilke J, Weidinger CA, Walter C, Barnkothe N, Teegen B, Luessi F, Stocker W, Luhder F, Begemann M, Zipp F, Nave KA, Ehrenreich H: \u003cstrong\u003eFactors predisposing to humoral autoimmunity against brain-antigens in health and disease: Analysis of 49 autoantibodies in over 7000 subjects.\u003c/strong\u003e \u003cem\u003eBrain Behav Immun \u003c/em\u003e2023, \u003cstrong\u003e108:\u003c/strong\u003e135-147.\u003c/li\u003e\n\u003cli\u003eDalmau J, Tuzun E, Wu HY, Masjuan J, Rossi JE, Voloschin A, Baehring JM, Shimazaki H, Koide R, King D, Mason W, Sansing LH, Dichter MA, Rosenfeld MR, Lynch DR: \u003cstrong\u003eParaneoplastic anti-N-methyl-D-aspartate receptor encephalitis associated with ovarian teratoma.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e2007, \u003cstrong\u003e61:\u003c/strong\u003e25-36.\u003c/li\u003e\n\u003cli\u003eHart IK, Waters C, Vincent A, Newland C, Beeson D, Pongs O, Morris C, Newsom-Davis J: \u003cstrong\u003eAutoantibodies detected to expressed K+ channels are implicated in neuromyotonia.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e1997, \u003cstrong\u003e41:\u003c/strong\u003e238-246.\u003c/li\u003e\n\u003cli\u003eJarius S, Wildemann B: \u003cstrong\u003e\u0026apos;Medusa head ataxia\u0026apos;: the expanding spectrum of Purkinje cell antibodies in autoimmune cerebellar ataxia. Part 3: Anti-Yo/CDR2, anti-Nb/AP3B2, PCA-2, anti-Tr/DNER, other antibodies, diagnostic pitfalls, summary and outlook.\u003c/strong\u003e \u003cem\u003eJ Neuroinflammation \u003c/em\u003e2015, \u003cstrong\u003e12:\u003c/strong\u003e168.\u003c/li\u003e\n\u003cli\u003eAbboud H, Probasco JC, Irani S, Ances B, Benavides DR, Bradshaw M, Christo PP, Dale RC, Fernandez-Fournier M, Flanagan EP, Gadoth A, George P, Grebenciucova E, Jammoul A, Lee ST, Li Y, Matiello M, Morse AM, Rae-Grant A, Rojas G, Rossman I, Schmitt S, Venkatesan A, Vernino S, Pittock SJ, Titulaer MJ, Autoimmune Encephalitis Alliance Clinicians N: \u003cstrong\u003eAutoimmune encephalitis: proposed best practice recommendations for diagnosis and acute management.\u003c/strong\u003e \u003cem\u003eJ Neurol Neurosurg Psychiatry \u003c/em\u003e2021, \u003cstrong\u003e92:\u003c/strong\u003e757-768.\u003c/li\u003e\n\u003cli\u003eFang B, McKeon A, Hinson SR, Kryzer TJ, Pittock SJ, Aksamit AJ, Lennon VA: \u003cstrong\u003eAutoimmune Glial Fibrillary Acidic Protein Astrocytopathy: A Novel Meningoencephalomyelitis.\u003c/strong\u003e \u003cem\u003eJAMA Neurol \u003c/em\u003e2016, \u003cstrong\u003e73:\u003c/strong\u003e1297-1307.\u003c/li\u003e\n\u003cli\u003eHoftberger R, Sabater L, Ortega A, Dalmau J, Graus F: \u003cstrong\u003ePatient with homer-3 antibodies and cerebellitis.\u003c/strong\u003e \u003cem\u003eJAMA Neurol \u003c/em\u003e2013, \u003cstrong\u003e70:\u003c/strong\u003e506-509.\u003c/li\u003e\n\u003cli\u003eHoftberger R, van Sonderen A, Leypoldt F, Houghton D, Geschwind M, Gelfand J, Paredes M, Sabater L, Saiz A, Titulaer MJ, Graus F, Dalmau J: \u003cstrong\u003eEncephalitis and AMPA receptor antibodies: Novel findings in a case series of 22 patients.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e2015, \u003cstrong\u003e84:\u003c/strong\u003e2403-2412.\u003c/li\u003e\n\u003cli\u003eTanaka J, Nakamura K, Takeda M, Tada K, Suzuki H, Morita H, Okado T, Hariguchi S, Nishimura T: \u003cstrong\u003eEnzyme-linked immunosorbent assay for human autoantibody to glial fibrillary acidic protein: higher titer of the antibody is detected in serum of patients with Alzheimer\u0026apos;s disease.\u003c/strong\u003e \u003cem\u003eActa Neurol Scand \u003c/em\u003e1989, \u003cstrong\u003e80:\u003c/strong\u003e554-560.\u003c/li\u003e\n\u003cli\u003eDe Camilli P, Thomas A, Cofiell R, Folli F, Lichte B, Piccolo G, Meinck HM, Austoni M, Fassetta G, Bottazzo G, Bates D, Cartlidge N, Solimena M, Kilimann MW, et al.: \u003cstrong\u003eThe synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer.\u003c/strong\u003e \u003cem\u003eJ Exp Med \u003c/em\u003e1993, \u003cstrong\u003e178:\u003c/strong\u003e2219-2223.\u003c/li\u003e\n\u003cli\u003eIrani SR, Alexander S, Waters P, Kleopa KA, Pettingill P, Zuliani L, Peles E, Buckley C, Lang B, Vincent A: \u003cstrong\u003eAntibodies to Kv1 potassium channel-complex proteins leucine-rich, glioma inactivated 1 protein and contactin-associated protein-2 in limbic encephalitis, Morvan\u0026apos;s syndrome and acquired neuromyotonia.\u003c/strong\u003e \u003cem\u003eBrain \u003c/em\u003e2010, \u003cstrong\u003e133:\u003c/strong\u003e2734-2748.\u003c/li\u003e\n\u003cli\u003eXiao BG, Linington C, Link H: \u003cstrong\u003eAntibodies to myelin-oligodendrocyte glycoprotein in cerebrospinal fluid from patients with multiple sclerosis and controls.\u003c/strong\u003e \u003cem\u003eJ Neuroimmunol \u003c/em\u003e1991, \u003cstrong\u003e31:\u003c/strong\u003e91-96.\u003c/li\u003e\n\u003cli\u003eZuliani L, Sabater L, Saiz A, Baiges JJ, Giometto B, Graus F: \u003cstrong\u003eHomer 3 autoimmunity in subacute idiopathic cerebellar ataxia.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e2007, \u003cstrong\u003e68:\u003c/strong\u003e239-240.\u003c/li\u003e\n\u003cli\u003eHutchinson M, Waters P, McHugh J, Gorman G, O\u0026apos;Riordan S, Connolly S, Hager H, Yu P, Becker CM, Vincent A: \u003cstrong\u003eProgressive encephalomyelitis, rigidity, and myoclonus: a novel glycine receptor antibody.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e2008, \u003cstrong\u003e71:\u003c/strong\u003e1291-1292.\u003c/li\u003e\n\u003cli\u003eSwayne A, Tjoa L, Broadley S, Dionisio S, Gillis D, Jacobson L, Woodhall MR, McNabb A, Schweitzer D, Tsang B, Vincent A, Irani SR, Wong R, Waters P, Blum S: \u003cstrong\u003eAntiglycine receptor antibody related disease: a case series and literature review.\u003c/strong\u003e \u003cem\u003eEur J Neurol \u003c/em\u003e2018, \u003cstrong\u003e25:\u003c/strong\u003e1290-1298.\u003c/li\u003e\n\u003cli\u003eSolimena M, Folli F, Denis-Donini S, Comi GC, Pozza G, De Camilli P, Vicari AM: \u003cstrong\u003eAutoantibodies to glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type I diabetes mellitus.\u003c/strong\u003e \u003cem\u003eN Engl J Med \u003c/em\u003e1988, \u003cstrong\u003e318:\u003c/strong\u003e1012-1020.\u003c/li\u003e\n\u003cli\u003eMathey EK, Derfuss T, Storch MK, Williams KR, Hales K, Woolley DR, Al-Hayani A, Davies SN, Rasband MN, Olsson T, Moldenhauer A, Velhin S, Hohlfeld R, Meinl E, Linington C: \u003cstrong\u003eNeurofascin as a novel target for autoantibody-mediated axonal injury.\u003c/strong\u003e \u003cem\u003eJ Exp Med \u003c/em\u003e2007, \u003cstrong\u003e204:\u003c/strong\u003e2363-2372.\u003c/li\u003e\n\u003cli\u003eHonorat JA, Lopez-Chiriboga AS, Kryzer TJ, Komorowski L, Scharf M, Hinson SR, Lennon VA, Pittock SJ, Klein CJ, McKeon A: \u003cstrong\u003eAutoimmune gait disturbance accompanying adaptor protein-3B2-IgG.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e2019, \u003cstrong\u003e93:\u003c/strong\u003ee954-e963.\u003c/li\u003e\n\u003cli\u003ePeterson K, Rosenblum MK, Kotanides H, Posner JB: \u003cstrong\u003eParaneoplastic cerebellar degeneration. I. A clinical analysis of 55 anti-Yo antibody-positive patients.\u003c/strong\u003e \u003cem\u003eNeurology \u003c/em\u003e1992, \u003cstrong\u003e42:\u003c/strong\u003e1931-1937.\u003c/li\u003e\n\u003cli\u003eGelpi E, Hoftberger R, Graus F, Ling H, Holton JL, Dawson T, Popovic M, Pretnar-Oblak J, Hogl B, Schmutzhard E, Poewe W, Ricken G, Santamaria J, Dalmau J, Budka H, Revesz T, Kovacs GG: \u003cstrong\u003eNeuropathological criteria of anti-IgLON5-related tauopathy.\u003c/strong\u003e \u003cem\u003eActa Neuropathol \u003c/em\u003e2016, \u003cstrong\u003e132:\u003c/strong\u003e531-543.\u003c/li\u003e\n\u003cli\u003eGelpi E, Reinecke R, Gaig C, Iranzo A, Sabater L, Molina-Porcel L, Aldecoa I, Endmayr V, Hogl B, Schmutzhard E, Poewe W, Pfausler B, Popovic M, Pretnar-Oblak J, Leypoldt F, Matschke J, Glatzel M, Erro EM, Jerico I, Caballero MC, Zelaya MV, Mariotto S, Heidbreder A, Kalev O, Weis S, Macher S, Berger-Sieczkowski E, Ferrari J, Reisinger C, Klupp N, Tienari P, Rautila O, Niemela M, Yilmazer-Hanke D, Guasp M, Bloem B, Van Gaalen J, Kusters B, Titulaer M, Fransen NL, Santamaria J, Dawson T, Holton JL, Ling H, Revesz T, Myllykangas L, Budka H, Kovacs GG, Lewerenz J, Dalmau J, Graus F, Koneczny I, Hoftberger R: \u003cstrong\u003eNeuropathological spectrum of anti-IgLON5 disease and stages of brainstem tau pathology: updated neuropathological research criteria of the disease-related tauopathy.\u003c/strong\u003e \u003cem\u003eActa Neuropathol \u003c/em\u003e2024, \u003cstrong\u003e148:\u003c/strong\u003e53.\u003c/li\u003e\n\u003cli\u003eGraus F, Vogrig A, Muniz-Castrillo S, Antoine JG, Desestret V, Dubey D, Giometto B, Irani SR, Joubert B, Leypoldt F, McKeon A, Pruss H, Psimaras D, Thomas L, Titulaer MJ, Vedeler CA, Verschuuren JJ, Dalmau J, Honnorat J: \u003cstrong\u003eUpdated Diagnostic Criteria for Paraneoplastic Neurologic Syndromes.\u003c/strong\u003e \u003cem\u003eNeurol Neuroimmunol Neuroinflamm \u003c/em\u003e2021, \u003cstrong\u003e8:\u003c/strong\u003ee1014.\u003c/li\u003e\n\u003cli\u003ePollak TA, Lennox BR, Muller S, Benros ME, Pruss H, Tebartz van Elst L, Klein H, Steiner J, Frodl T, Bogerts B, Tian L, Groc L, Hasan A, Baune BT, Endres D, Haroon E, Yolken R, Benedetti F, Halaris A, Meyer JH, Stassen H, Leboyer M, Fuchs D, Otto M, Brown DA, Vincent A, Najjar S, Bechter K: \u003cstrong\u003eAutoimmune psychosis: an international consensus on an approach to the diagnosis and management of psychosis of suspected autoimmune origin.\u003c/strong\u003e \u003cem\u003eLancet Psychiatry \u003c/em\u003e2020, \u003cstrong\u003e7:\u003c/strong\u003e93-108.\u003c/li\u003e\n\u003cli\u003eWilke JBH, Hindermann M, Berghoff SA, Zihsler S, Arinrad S, Ronnenberg A, Barnkothe N, Steixner-Kumar AA, Roglin S, Stocker W, Hollmann M, Nave KA, Luhder F, Ehrenreich H: \u003cstrong\u003eAutoantibodies against NMDA receptor 1 modify rather than cause encephalitis.\u003c/strong\u003e \u003cem\u003eMol Psychiatry \u003c/em\u003e2021, \u003cstrong\u003e26:\u003c/strong\u003e7746-7759.\u003c/li\u003e\n\u003cli\u003eWilke JBH, Hindermann M, Moussavi A, Butt UJ, Dadarwal R, Berghoff SA, Sarcheshmeh AK, Ronnenberg A, Zihsler S, Arinrad S, Hardeland R, Seidel J, Luhder F, Nave KA, Boretius S, Ehrenreich H: \u003cstrong\u003eInducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis.\u003c/strong\u003e \u003cem\u003eActa Neuropathol Commun \u003c/em\u003e2021, \u003cstrong\u003e9:\u003c/strong\u003e121.\u003c/li\u003e\n\u003cli\u003eSchindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A: \u003cstrong\u003eFiji: an open-source platform for biological-image analysis.\u003c/strong\u003e \u003cem\u003eNat Methods \u003c/em\u003e2012, \u003cstrong\u003e9:\u003c/strong\u003e676-682.\u003c/li\u003e\n\u003cli\u003eHeindl S, Gesierich B, Benakis C, Llovera G, Duering M, Liesz A: \u003cstrong\u003eAutomated Morphological Analysis of Microglia After Stroke.\u003c/strong\u003e \u003cem\u003eFront Cell Neurosci \u003c/em\u003e2018, \u003cstrong\u003e12:\u003c/strong\u003e106.\u003c/li\u003e\n\u003cli\u003eKuznetsova A, Brockhoff PB, Christensen RHB: \u003cstrong\u003elmerTest Package: Tests in Linear Mixed Effects Models.\u003c/strong\u003e \u003cem\u003eJournal of Statistical Software \u003c/em\u003e2017, \u003cstrong\u003e82\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eR Development Core Team: \u003cstrong\u003eR: A language and environment for statistical computing.\u003c/strong\u003e Vienna, Austria: R Foundation for Statistical Computing; 2020.\u003c/li\u003e\n\u003cli\u003eJones BE, Tovar KR, Goehring A, Jalali-Yazdi F, Okada NJ, Gouaux E, Westbrook GL: \u003cstrong\u003eAutoimmune receptor encephalitis in mice induced by active immunization with conformationally stabilized holoreceptors.\u003c/strong\u003e \u003cem\u003eSci Transl Med \u003c/em\u003e2019, \u003cstrong\u003e11\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003ePaolicelli RC, Sierra A, Stevens B, Tremblay ME, Aguzzi A, Ajami B, Amit I, Audinat E, Bechmann I, Bennett M, Bennett F, Bessis A, Biber K, Bilbo S, Blurton-Jones M, Boddeke E, Brites D, Brone B, Brown GC, Butovsky O, Carson MJ, Castellano B, Colonna M, Cowley SA, Cunningham C, Davalos D, De Jager PL, de Strooper B, Denes A, Eggen BJL, Eyo U, Galea E, Garel S, Ginhoux F, Glass CK, Gokce O, Gomez-Nicola D, Gonzalez B, Gordon S, Graeber MB, Greenhalgh AD, Gressens P, Greter M, Gutmann DH, Haass C, Heneka MT, Heppner FL, Hong S, Hume DA, Jung S, Kettenmann H, Kipnis J, Koyama R, Lemke G, Lynch M, Majewska A, Malcangio M, Malm T, Mancuso R, Masuda T, Matteoli M, McColl BW, Miron VE, Molofsky AV, Monje M, Mracsko E, Nadjar A, Neher JJ, Neniskyte U, Neumann H, Noda M, Peng B, Peri F, Perry VH, Popovich PG, Pridans C, Priller J, Prinz M, Ragozzino D, Ransohoff RM, Salter MW, Schaefer A, Schafer DP, Schwartz M, Simons M, Smith CJ, Streit WJ, Tay TL, Tsai LH, Verkhratsky A, von Bernhardi R, Wake H, Wittamer V, Wolf SA, Wu LJ, Wyss-Coray T: \u003cstrong\u003eMicroglia states and nomenclature: A field at its crossroads.\u003c/strong\u003e \u003cem\u003eNeuron \u003c/em\u003e2022, \u003cstrong\u003e110:\u003c/strong\u003e3458-3483.\u003c/li\u003e\n\u003cli\u003eLeng F, Edison P: \u003cstrong\u003eNeuroinflammation and microglial activation in Alzheimer disease: where do we go from here?\u003c/strong\u003e \u003cem\u003eNat Rev Neurol \u003c/em\u003e2021, \u003cstrong\u003e17:\u003c/strong\u003e157-172.\u003c/li\u003e\n\u003cli\u003eZrzavy T, Endmayr V, Bauer J, Macher S, Mossaheb N, Schwaiger C, Ricken G, Winklehner M, Glatter S, Breu M, Wimmer I, Kovacs GG, Risser DU, Klupp N, Simonitsch-Klupp I, Roetzer T, Rommer P, Berger T, Gelpi E, Lassmann H, Graus F, Dalmau J, Hoftberger R: \u003cstrong\u003eNeuropathological Variability within a Spectrum of NMDAR-Encephalitis.\u003c/strong\u003e \u003cem\u003eAnn Neurol \u003c/em\u003e2021, \u003cstrong\u003e90:\u003c/strong\u003e725-737.\u003c/li\u003e\n\u003cli\u003eNauen DW: \u003cstrong\u003eExtra-central nervous system target for assessment and treatment in refractory anti-N-methyl-d-aspartate receptor encephalitis.\u003c/strong\u003e \u003cem\u003eJ Crit Care \u003c/em\u003e2017, \u003cstrong\u003e37:\u003c/strong\u003e234-236.\u003c/li\u003e\n\u003cli\u003eTuzun E, Zhou L, Baehring JM, Bannykh S, Rosenfeld MR, Dalmau J: \u003cstrong\u003eEvidence for antibody-mediated pathogenesis in anti-NMDAR encephalitis associated with ovarian teratoma.\u003c/strong\u003e \u003cem\u003eActa Neuropathol \u003c/em\u003e2009, \u003cstrong\u003e118:\u003c/strong\u003e737-743.\u003c/li\u003e\n\u003cli\u003eBien CG, Vincent A, Barnett MH, Becker AJ, Blumcke I, Graus F, Jellinger KA, Reuss DE, Ribalta T, Schlegel J, Sutton I, Lassmann H, Bauer J: \u003cstrong\u003eImmunopathology of autoantibody-associated encephalitides: clues for pathogenesis.\u003c/strong\u003e \u003cem\u003eBrain \u003c/em\u003e2012, \u003cstrong\u003e135:\u003c/strong\u003e1622-1638.\u003c/li\u003e\n\u003cli\u003eFilatenkov A, Richardson TE, Daoud E, Johnson-Welch SF, Ramirez DM, Torrealba J, Greenberg B, Monson NL, Rajaram V: \u003cstrong\u003ePersistence of parenchymal and perivascular T-cells in treatment-refractory anti-N-methyl-D-aspartate receptor encephalitis.\u003c/strong\u003e \u003cem\u003eNeuroreport \u003c/em\u003e2017, \u003cstrong\u003e28:\u003c/strong\u003e890-895.\u003c/li\u003e\n\u003cli\u003eLi Q, Barres BA: \u003cstrong\u003eMicroglia and macrophages in brain homeostasis and disease.\u003c/strong\u003e \u003cem\u003eNat Rev Immunol \u003c/em\u003e2018, \u003cstrong\u003e18:\u003c/strong\u003e225-242.\u003c/li\u003e\n\u003cli\u003eLinnoila J, Pulli B, Armangue T, Planaguma J, Narsimhan R, Schob S, Zeller MWG, Dalmau J, Chen J: \u003cstrong\u003eMouse model of anti-NMDA receptor post-herpes simplex encephalitis.\u003c/strong\u003e \u003cem\u003eNeurol Neuroimmunol Neuroinflamm \u003c/em\u003e2019, \u003cstrong\u003e6:\u003c/strong\u003ee529.\u003c/li\u003e\n\u003cli\u003eZhou L, Kong G, Palmisano I, Cencioni MT, Danzi M, De Virgiliis F, Chadwick JS, Crawford G, Yu Z, De Winter F, Lemmon V, Bixby J, Puttagunta R, Verhaagen J, Pospori C, Lo Celso C, Strid J, Botto M, Di Giovanni S: \u003cstrong\u003eReversible CD8 T cell-neuron cross-talk causes aging-dependent neuronal regenerative decline.\u003c/strong\u003e \u003cem\u003eScience \u003c/em\u003e2022, \u003cstrong\u003e376:\u003c/strong\u003eeabd5926.\u003c/li\u003e\n\u003cli\u003eMerkler D, Vincenti I, Masson F, Liblau RS: \u003cstrong\u003eTissue-resident CD8 T cells in central nervous system inflammatory diseases: present at the crime scene and ...guilty.\u003c/strong\u003e \u003cem\u003eCurr Opin Immunol \u003c/em\u003e2022, \u003cstrong\u003e77:\u003c/strong\u003e102211.\u003c/li\u003e\n\u003cli\u003eUrban SL, Jensen IJ, Shan Q, Pewe LL, Xue HH, Badovinac VP, Harty JT: \u003cstrong\u003ePeripherally induced brain tissue-resident memory CD8(+) T cells mediate protection against CNS infection.\u003c/strong\u003e \u003cem\u003eNat Immunol \u003c/em\u003e2020, \u003cstrong\u003e21:\u003c/strong\u003e938-949.\u003c/li\u003e\n\u003cli\u003eFrieser D, Pignata A, Khajavi L, Shlesinger D, Gonzalez-Fierro C, Nguyen XH, Yermanos A, Merkler D, Hoftberger R, Desestret V, Mair KM, Bauer J, Masson F, Liblau RS: \u003cstrong\u003eTissue-resident CD8(+) T cells drive compartmentalized and chronic autoimmune damage against CNS neurons.\u003c/strong\u003e \u003cem\u003eSci Transl Med \u003c/em\u003e2022, \u003cstrong\u003e14:\u003c/strong\u003eeabl6157.\u003c/li\u003e\n\u003cli\u003eVincenti I, Page N, Steinbach K, Yermanos A, Lemeille S, Nunez N, Kreutzfeldt M, Klimek B, Di Liberto G, Egervari K, Piccinno M, Shammas G, Mariotte A, Fonta N, Liaudet N, Shlesinger D, Liuzzi AR, Wagner I, Saadi C, Stadelmann C, Reddy S, Becher B, Merkler D: \u003cstrong\u003eTissue-resident memory CD8(+) T cells cooperate with CD4(+) T cells to drive compartmentalized immunopathology in the CNS.\u003c/strong\u003e \u003cem\u003eSci Transl Med \u003c/em\u003e2022, \u003cstrong\u003e14:\u003c/strong\u003eeabl6058.\u003c/li\u003e\n\u003cli\u003eDay GS, Laiq S, Tang-Wai DF, Munoz DG: \u003cstrong\u003eAbnormal neurons in teratomas in NMDAR encephalitis.\u003c/strong\u003e \u003cem\u003eJAMA Neurol \u003c/em\u003e2014, \u003cstrong\u003e71:\u003c/strong\u003e717-724.\u003c/li\u003e\n\u003cli\u003eTabata E, Masuda M, Eriguchi M, Yokoyama M, Takahashi Y, Tanaka K, Yukitake M, Horikawa E, Hara H: \u003cstrong\u003eImmunopathological significance of ovarian teratoma in patients with anti-N-methyl-d-aspartate receptor encephalitis.\u003c/strong\u003e \u003cem\u003eEur Neurol \u003c/em\u003e2014, \u003cstrong\u003e71:\u003c/strong\u003e42-48.\u003c/li\u003e\n\u003cli\u003eIemura Y, Yamada Y, Hirata M, Kataoka TR, Minamiguchi S, Haga H: \u003cstrong\u003eHistopathological characterization of the neuroglial tissue in ovarian teratoma associated with anti-N-methyl-D-aspartate (NMDA) receptor encephalitis.\u003c/strong\u003e \u003cem\u003ePathol Int \u003c/em\u003e2018, \u003cstrong\u003e68:\u003c/strong\u003e677-684.\u003c/li\u003e\n\u003cli\u003eChefdeville A, Treilleux I, Mayeur ME, Couillault C, Picard G, Bost C, Mokhtari K, Vasiljevic A, Meyronet D, Rogemond V, Psimaras D, Dubois B, Honnorat J, Desestret V: \u003cstrong\u003eImmunopathological characterization of ovarian teratomas associated with anti-N-methyl-D-aspartate receptor encephalitis.\u003c/strong\u003e \u003cem\u003eActa Neuropathol Commun \u003c/em\u003e2019, \u003cstrong\u003e7:\u003c/strong\u003e38.\u003c/li\u003e\n\u003cli\u003eGu J, Jin T, Li Z, Chen H, Xia H, Xu X, Gui Y: \u003cstrong\u003eExosomes expressing neuronal autoantigens induced immune response in antibody-positive autoimmune encephalitis.\u003c/strong\u003e \u003cem\u003eMol Immunol \u003c/em\u003e2021, \u003cstrong\u003e131:\u003c/strong\u003e164-170.\u003c/li\u003e\n\u003cli\u003eLi Y, Gu J, Mao Y, Wang X, Li Z, Xu X, Chen H, Gui Y: \u003cstrong\u003eCerebrospinal Fluid Extracellular Vesicles with Distinct Properties in Autoimmune Encephalitis and Herpes Simplex Encephalitis.\u003c/strong\u003e \u003cem\u003eMol Neurobiol \u003c/em\u003e2022, \u003cstrong\u003e59:\u003c/strong\u003e2441-2455.\u003c/li\u003e\n\u003cli\u003ePan H, Oliveira B, Saher G, Dere E, Tapken D, Mitjans M, Seidel J, Wesolowski J, Wakhloo D, Klein-Schmidt C, Ronnenberg A, Schwabe K, Trippe R, Matz-Rensing K, Berghoff S, Al-Krinawe Y, Martens H, Begemann M, Stocker W, Kaup FJ, Mischke R, Boretius S, Nave KA, Krauss JK, Hollmann M, Luhder F, Ehrenreich H: \u003cstrong\u003eUncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model.\u003c/strong\u003e \u003cem\u003eMol Psychiatry \u003c/em\u003e2019, \u003cstrong\u003e24:\u003c/strong\u003e1489-1501.\u003c/li\u003e\n\u003cli\u003ePan H, Steixner-Kumar AA, Seelbach A, Deutsch N, Ronnenberg A, Tapken D, von Ahsen N, Mitjans M, Worthmann H, Trippe R, Klein-Schmidt C, Schopf N, Rentzsch K, Begemann M, Wienands J, Stocker W, Weissenborn K, Hollmann M, Nave KA, Luhder F, Ehrenreich H: \u003cstrong\u003eMultiple inducers and novel roles of autoantibodies against the obligatory NMDAR subunit NR1: a translational study from chronic life stress to brain injury.\u003c/strong\u003e \u003cem\u003eMol Psychiatry \u003c/em\u003e2021, \u003cstrong\u003e26:\u003c/strong\u003e2471-2482.\u003c/li\u003e\n\u003cli\u003eLevin EC, Acharya NK, Han M, Zavareh SB, Sedeyn JC, Venkataraman V, Nagele RG: \u003cstrong\u003eBrain-reactive autoantibodies are nearly ubiquitous in human sera and may be linked to pathology in the context of blood\u0026ndash;brain barrier breakdown.\u003c/strong\u003e \u003cem\u003eBrain Research \u003c/em\u003e2010, \u003cstrong\u003e1345:\u003c/strong\u003e221-232.\u003c/li\u003e\n\u003cli\u003eSiren AL, Radyushkin K, Boretius S, Kammer D, Riechers CC, Natt O, Sargin D, Watanabe T, Sperling S, Michaelis T, Price J, Meyer B, Frahm J, Ehrenreich H: \u003cstrong\u003eGlobal brain atrophy after unilateral parietal lesion and its prevention by erythropoietin.\u003c/strong\u003e \u003cem\u003eBrain \u003c/em\u003e2006, \u003cstrong\u003e129:\u003c/strong\u003e480-489.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acute encephalitis, autoimmune relapse, microglia, autoimmunity, autoantibodies","lastPublishedDoi":"10.21203/rs.3.rs-6499111/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6499111/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePatients with virus encephalitis, such as herpes simplex encephalitis and Japanese encephalitis frequently relapse with autoimmune encephalitides associated with neural autoantibodies. It has been hypothesized that the infection-induced damage to the central nervous system results in shedding of neural autoantigens, their presentation to the peripheral immune system, and initiation of a secondary autoimmune encephalitis that targets these autoantigens. To test this hypothesis, we utilized a transgenic mouse model of virus-like but sterile encephalitis. After induction of acute neuronal death in the hippocampus, we monitored the mice for encephalitis-like symptoms for up to 10 months, evaluated the degree of neuroinflammation at several time points and screened their plasma for autoantibodies against 49 different autoimmune disease-associated brain autoantibodies. Throughout the study period, we did not detect any symptoms of severe autoimmune encephalitis, like hyperactivity, circling, seizures, lethargy. Evaluation of microglia numbers and morphology revealed pronounced microgliosis 1-week after initial encephalitis induction, which decreased over time. Scattered lymphocyte infiltration was present at all times in hippocampi of encephalitis mice, and did not increase over time. Perivascular cuffs were not detected. Infiltrating lymphocytes mainly consisted of CD8\u0026thinsp;+\u0026thinsp;T cells. B cell infiltration was rare and did not differ from healthy control mice. High-parameter immunophenotyping of peripheral blood leukocytes did not reveal any changes associated with an autoimmune response. Testing all plasma samples (n\u0026thinsp;=\u0026thinsp;30/group) at a dilution of 1:100 for autoantibodies against 49 neural autoantigens gave only two positive results, namely one healthy control with anti-CASPR2 autoantibodies (IgG) and one post-encephalitis mouse with anti-homer-3 autoantibodies (IgM). Overall, these findings suggest that acute neuronal cell death and neuroinflammation \u003cem\u003eper se\u003c/em\u003e are not sufficient to trigger downstream autoimmune encephalitis relapses.\u003c/p\u003e","manuscriptTitle":"Acute neuronal cell death and neuroinflammation per se do not trigger secondary autoimmune encephalitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-14 12:08:32","doi":"10.21203/rs.3.rs-6499111/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-23T17:04:04+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-22T11:26:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-16T13:54:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-13T21:46:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"912147808641633249848591408398505860","date":"2025-05-12T14:22:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114028919886930058383302680201000635484","date":"2025-05-11T18:38:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"236064080479505992966036729061672859882","date":"2025-05-11T15:36:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"106260823032336074799415484452518357443","date":"2025-05-11T10:56:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251127673988535943075225936037847625970","date":"2025-05-11T08:12:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"229056262380888689518714405957043846767","date":"2025-05-09T07:50:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T07:37:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-09T07:34:49+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-09T04:23:47+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-07T11:39:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-22T00:39:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6335dd15-e4ea-4729-80f6-3f7454523f3a","owner":[],"postedDate":"May 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":48411296,"name":"Biological sciences/Immunology/Autoimmunity"},{"id":48411297,"name":"Health sciences/Molecular medicine"},{"id":48411298,"name":"Biological sciences/Immunology/Neuroimmunology"},{"id":48411299,"name":"Biological sciences/Neuroscience/Diseases of the nervous system/Encephalopathy"}],"tags":[],"updatedAt":"2025-06-30T16:00:57+00:00","versionOfRecord":{"articleIdentity":"rs-6499111","link":"https://doi.org/10.1038/s41598-025-08035-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-06-27 15:57:38","publishedOnDateReadable":"June 27th, 2025"},"versionCreatedAt":"2025-05-14 12:08:32","video":"","vorDoi":"10.1038/s41598-025-08035-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-08035-w","workflowStages":[]},"version":"v1","identity":"rs-6499111","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6499111","identity":"rs-6499111","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}