Stem-like intestinal Th17 cells give rise to pathogenic effector T cells during autoimmunity.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1+ IL-17+ SLAMF6+ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF+ IFN-γ+ CXCR6+ T cells. Our study defines a direct in vivo relationship between IL-17+ non-pathogenic and GM-CSF+ and IFN-γ+ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

Indoleamine 2,3-dioxygenase 1 activation in mature cDC1 promotes tolerogenic education of inflammatory cDC2 via metabolic communication.

Conventional dendritic cells (cDCs), cDC1 and cDC2, act both to initiate immunity and maintain self-tolerance. The tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) is used by cDCs in maintaining tolerance, but its role in different subsets remains unclear. At homeostasis, only mature CCR7+ cDC1 expressed IDO1 that was dependent on IRF8. Lipopolysaccharide treatment induced maturation and IDO1-dependent tolerogenic activity in isolated immature cDC1, but not isolated cDC2. However, both human and mouse cDC2 could induce IDO1 and acquire tolerogenic function when co-cultured with mature cDC1 through the action of cDC1-derived l-kynurenine. Accordingly, cDC1-specific inactivation of IDO1 in vivo exacerbated disease in experimental autoimmune encephalomyelitis. This study identifies a previously unrecognized metabolic communication in which IDO1-expressing cDC1 cells extend their immunoregulatory capacity to the cDC2 subset through their production of tryptophan metabolite l-kynurenine. This metabolic axis represents a potential therapeutic target in treating autoimmune demyelinating diseases.

Induction of a colitogenic phenotype in Th1-like cells depends on interleukin-23 receptor signaling.

Interleukin-23 receptor plays a critical role in inducing inflammation and autoimmunity. Here, we report that Th1-like cells differentiated in vitro with IL-12 + IL-21 showed similar IL-23R expression to that of pathogenic Th17 cells using eGFP reporter mice. Fate mapping established that these cells did not transition through a Th17 cell state prior to becoming Th1-like cells, and we observed their emergence in vivo in the T cell adoptive transfer colitis model. Using IL-23R-deficient Th1-like cells, we demonstrated that IL-23R was required for the development of a highly colitogenic phenotype. Single-cell RNA sequencing analysis of intestinal T cells identified IL-23R-dependent genes in Th1-like cells that differed from those expressed in Th17 cells. The perturbation of one of these regulators (CD160) in Th1-like cells inhibited the induction of colitis. We thus uncouple IL-23R as a purely Th17 cell-specific factor and implicate IL-23R signaling as a pathogenic driver in Th1-like cells inducing tissue inflammation.

Neurological Manifestations of COVID-19 Feature T Cell Exhaustion and Dedifferentiated Monocytes in Cerebrospinal Fluid.

Patients suffering from Coronavirus disease 2019 (COVID-19) can develop neurological sequelae, such as headache and neuroinflammatory or cerebrovascular disease. These conditions-termed here as Neuro-COVID-are more frequent in patients with severe COVID-19. To understand the etiology of these neurological sequelae, we utilized single-cell sequencing and examined the immune cell profiles from the cerebrospinal fluid (CSF) of Neuro-COVID patients compared with patients with non-inflammatory and autoimmune neurological diseases or with viral encephalitis. The CSF of Neuro-COVID patients exhibited an expansion of dedifferentiated monocytes and of exhausted CD4+ T cells. Neuro-COVID CSF leukocytes featured an enriched interferon signature; however, this was less pronounced than in viral encephalitis. Repertoire analysis revealed broad clonal T cell expansion and curtailed interferon response in severe compared with mild Neuro-COVID patients. Collectively, our findings document the CSF immune compartment in Neuro-COVID patients and suggest compromised antiviral responses in this setting.

CD5L/AIM Regulates Lipid Biosynthesis and Restrains Th17 Cell Pathogenicity.

Th17 cells play a critical role in host defense against extracellular pathogens and tissue homeostasis but can induce autoimmunity. The mechanisms implicated in balancing "pathogenic" and "non-pathogenic" Th17 cell states remain largely unknown. We used single-cell RNA-seq to identify CD5L/AIM as a regulator expressed in non-pathogenic, but not in pathogenic Th17 cells. Although CD5L does not affect Th17 differentiation, it is a functional switch that regulates the pathogenicity of Th17 cells. Loss of CD5L converts non-pathogenic Th17 cells into pathogenic cells that induce autoimmunity. CD5L mediates this effect by modulating the intracellular lipidome, altering fatty acid composition and restricting cholesterol biosynthesis and, thus, ligand availability for Rorγt, the master transcription factor of Th17 cells. Our study identifies CD5L as a critical regulator of the Th17 cell functional state and highlights the importance of lipid metabolism in balancing immune protection and disease induced by T cells.

Fas Promotes T Helper 17 Cell Differentiation and Inhibits T Helper 1 Cell Development by Binding and Sequestering Transcription Factor STAT1.

The death receptor Fas removes activated lymphocytes through apoptosis. Previous transcriptional profiling predicted that Fas positively regulates interleukin-17 (IL-17)-producing T helper 17 (Th17) cells. Here, we demonstrate that Fas promoted the generation and stability of Th17 cells and prevented their differentiation into Th1 cells. Mice with T-cell- and Th17-cell-specific deletion of Fas were protected from induced autoimmunity, and Th17 cell differentiation and stability were impaired. Fas-deficient Th17 cells instead developed a Th1-cell-like transcriptional profile, which a new algorithm predicted to depend on STAT1. Experimentally, Fas indeed bound and sequestered STAT1, and Fas deficiency enhanced IL-6-induced STAT1 activation and nuclear translocation, whereas deficiency of STAT1 reversed the transcriptional changes induced by Fas deficiency. Thus, our computational and experimental approach identified Fas as a regulator of the Th17-to-Th1 cell balance by controlling the availability of opposing STAT1 and STAT3 to have a direct impact on autoimmunity.

Enhanced pathogenicity of Th17 cells due to natalizumab treatment: Implications for MS disease rebound.

After natalizumab (NAT) cessation, some multiple sclerosis (MS) patients experience a severe disease rebound. The rebound pathophysiology is still unclear; however, it has been linked to interleukin-17-producing T-helper (Th17) cells. We demonstrate that during NAT treatment, MCAM+CCR6+Th17 cells gradually acquire a pathogenic profile, including proinflammatory cytokine production, pathogenic transcriptional signatures, brain endothelial barrier impairment, and oligodendrocyte damage via induction of apoptotic pathways. This is accompanied by an increase in Th17 cell frequencies in the cerebrospinal fluid of NAT-treated patients. Notably, Th17 cells derived from NAT-treated patients, who later developed a disease rebound upon treatment cessation, displayed a distinct transcriptional pathogenicity profile associated with altered migratory properties. Accordingly, increased brain infiltration of patient Th17 cells was illustrated in a humanized mouse model and brain histology from a rebound patient. Therefore, peripheral blood-accumulated MCAM+CCR6+Th17 cells might be involved in rebound pathophysiology, and monitoring of changes in Th17 cell pathogenicity in patients before/during NAT treatment cessation might enable rebound risk assessment in the future.

Deterministic programming of human pluripotent stem cells into microglia facilitates studying their role in health and disease.

Microglia, the resident immune cells of the central nervous system (CNS), are derived from yolk-sac macrophages that populate the developing CNS during early embryonic development. Once established, the microglia population is self-maintained throughout life by local proliferation. As a scalable source of microglia-like cells (MGLs), we here present a forward programming protocol for their generation from human pluripotent stem cells (hPSCs). The transient overexpression of PU.1 and C/EBPß in hPSCs led to a homogenous population of mature microglia within 16 d. MGLs met microglia characteristics on a morphological, transcriptional, and functional level. MGLs facilitated the investigation of a human tauopathy model in cortical neuron-microglia cocultures, revealing a secondary dystrophic microglia phenotype. Single-cell RNA sequencing of microglia integrated into hPSC-derived cortical brain organoids demonstrated a shift of microglia signatures toward a more-developmental in vivo-like phenotype, inducing intercellular interactions promoting neurogenesis and arborization. Taken together, our microglia forward programming platform represents a tool for both reductionist studies in monocultures and complex coculture systems, including 3D brain organoids for the study of cellular interactions in healthy or diseased environments.

Association of symptom severity and cerebrospinal fluid alterations in recent onset psychosis in schizophrenia-spectrum disorders - An individual patient data meta-analysis.

Neuroinflammation and blood-cerebrospinal fluid barrier (BCB) disruption could be key elements in schizophrenia-spectrum disorders(SSDs) etiology and symptom modulation. We present the largest two-stage individual patient data (IPD) meta-analysis, investigating the association of BCB disruption and cerebrospinal fluid (CSF) alterations with symptom severity in first-episode psychosis (FEP) and recent onset psychotic disorder (ROP) individuals, with a focus on sex-related differences. Data was collected from PubMed and EMBASE databases. FEP, ROP and high-risk syndromes for psychosis IPD were included if routine basic CSF-diagnostics were reported. Risk of bias of the included studies was evaluated. Random-effects meta-analyses and mixed-effects linear regression models were employed to assess the impact of BCB alterations on symptom severity. Published (6 studies) and unpublished IPD from n = 531 individuals was included in the analyses. CSF was altered in 38.8 % of individuals. No significant differences in symptom severity were found between individuals with and without CSF alterations (SMD = -0.17, 95 %CI -0.55-0.22, p = 0.341). However, males with elevated CSF/serum albumin ratios or any CSF alteration had significantly higher positive symptom scores than those without alterations (SMD = 0.34, 95 %CI 0.05-0.64, p = 0.037 and SMD = 0.29, 95 %CI 0.17-0.41p = 0.005, respectively). Mixed-effects and simple regression models showed no association (p > 0.1) between CSF parameters and symptomatic outcomes. No interaction between sex and CSF parameters was found (p > 0.1). BCB disruption appears highly prevalent in early psychosis and could be involved in positive symptoms severity in males, indicating potential difficult-to-treat states. This work highlights the need for considering BCB breakdownand sex-related differences in SSDs clinical trials and treatment strategies.

Bcl6 controls meningeal Th17-B cell interaction in murine neuroinflammation.

Ectopic lymphoid tissue containing B cells forms in the meninges at late stages of human multiple sclerosis (MS) and when neuroinflammation is induced by interleukin (IL)-17 producing T helper (Th17) cells in rodents. B cell differentiation and the subsequent release of class-switched immunoglobulins have been speculated to occur in the meninges, but the exact cellular composition and underlying mechanisms of meningeal-dominated inflammation remain unknown. Here, we performed in-depth characterization of meningeal versus parenchymal Th17-induced rodent neuroinflammation. The most pronounced cellular and transcriptional differences between these compartments was the localization of B cells exhibiting a follicular phenotype exclusively to the meninges. Correspondingly, meningeal but not parenchymal Th17 cells acquired a B cell-supporting phenotype and resided in close contact with B cells. This preferential B cell tropism for the meninges and the formation of meningeal ectopic lymphoid tissue was partially dependent on the expression of the transcription factor Bcl6 in Th17 cells that is required in other T cell lineages to induce isotype class switching in B cells. A function of Bcl6 in Th17 cells was only detected in vivo and was reflected by the induction of B cell-supporting cytokines, the appearance of follicular B cells in the meninges, and of immunoglobulin class switching in the cerebrospinal fluid. We thus identify the induction of a B cell-supporting meningeal microenvironment by Bcl6 in Th17 cells as a mechanism controlling compartment specificity in neuroinflammation.

Supporting the differential diagnosis of connective tissue diseases with neurological involvement by blood and cerebrospinal fluid flow cytometry.

OBJECTIVE: Neurological manifestations of autoimmune connective tissue diseases (CTD) are poorly understood and difficult to diagnose. We here aimed to address this shortcoming by studying immune cell compositions in CTD patients with and without neurological manifestation. METHODS: Using flow cytometry, we retrospectively investigated paired cerebrospinal fluid (CSF) and blood samples of 28 CTD patients without neurological manifestation, 38 CTD patients with neurological manifestation (N-CTD), 38 non-inflammatory controls, and 38 multiple sclerosis (MS) patients, a paradigmatic primary neuroinflammatory disease. RESULTS: We detected an expansion of plasma cells in the blood of both N-CTD and CTD compared to non-inflammatory controls and MS. Blood plasma cells alone distinguished the clinically similar entities N-CTD and MS with high discriminatory performance (AUC: 0.81). Classical blood monocytes indicated higher disease activity in systemic lupus erythematosus (SLE) patients. Surprisingly, immune cells in the CSF did not differ significantly between N-CTD and CTD, while CD4+ T cells and the CD4+/CD8+ ratio were elevated in the blood of N-CTD compared to CTD. Several B cell-associated parameters partially overlapped in the CSF in MS and N-CTD. We built a machine learning model that distinguished N-CTD from MS with high discriminatory power using either blood or CSF. CONCLUSION: We here find that blood flow cytometry alone surprisingly suffices to distinguish CTD with neurological manifestations from clinically similar entities, suggesting that a rapid blood test could support clinicians in the differential diagnosis of N-CTD.

Cerebrospinal fluid proteomics indicates immune dysregulation and neuronal dysfunction in antibody associated autoimmune encephalitis.

Autoimmune Encephalitis (AE) spans a group of non-infectious inflammatory conditions of the central nervous system due to an imbalanced immune response. Aiming to elucidate the pathophysiological mechanisms of AE, we applied an unsupervised proteomic approach to analyze the cerebrospinal fluid (CSF) protein profile of AE patients with autoantibodies against N-methyl-d-aspartate receptor (NMDAR) (n = 9), leucine-rich glioma-inactivated protein 1 (LGI1) (n = 9), or glutamate decarboxylase 65 (GAD65) (n = 8) compared to 9 patients with relapsing-remitting multiple sclerosis as inflammatory controls, and 10 patients with somatic symptom disorder as non-inflammatory controls. We found a dysregulation of the complement system, a disbalance between pro-inflammatory and anti-inflammatory proteins on the one hand, and dysregulation of proteins involved in synaptic transmission, synaptogenesis, brain connectivity, and neurodegeneration on the other hand to a different extent in all AE subtypes compared to non-inflammatory controls. Furthermore, elevated levels of several proteases and reduction in protease inhibitors could be detected in all AE subtypes compared to non-inflammatory controls. Moreover, the different AE subtypes showed distinct protein profiles compared to each other and inflammatory controls which may facilitate future identification of disease-specific biomarkers. Overall, CSF proteomics provides insights into the complex pathophysiological mechanisms of AE, including immune dysregulation, neuronal dysfunction, neurodegeneration, and altered protease function.

Next-Generation Neuroimmunology: New Technologies to Understand Central Nervous System Autoimmunity.

Understanding neuroimmunological disorders is essential for developing new diagnostic and therapeutic strategies. Rodent models have provided valuable insights, but are sometimes equated with their human counterparts. Here, we summarize how novel technologies may enable an improved human-focused view of immune mechanisms. Recent studies have applied these new technologies to the brain parenchyma, its surrounding cerebrospinal fluid, and peripheral immune compartments. Therapeutic interventions have also facilitated translational understanding in a reverse way. However, with improved technology, access to patient samples remains a rate-limiting step in translational research. We anticipate that next-generation neuroimmunology is likely to integrate, in the immediate future, diverse technical tools for optimal diagnosis, prognosis, and treatment of neuroimmunological disorders.

Alemtuzumab-induced immune phenotype and repertoire changes: implications for secondary autoimmunity.

Alemtuzumab is a monoclonal antibody that causes rapid depletion of CD52-expressing immune cells. It has proven to be highly efficacious in active relapsing-remitting multiple sclerosis; however, the high risk of secondary autoimmune disorders has greatly complicated its use. Thus, deeper insight into the pathophysiology of secondary autoimmunity and potential biomarkers is urgently needed. The most critical time points in the decision-making process for alemtuzumab therapy are before or at Month 12, where the ability to identify secondary autoimmunity risk would be instrumental. Therefore, we investigated components of blood and CSF of up to 106 multiple sclerosis patients before and after alemtuzumab treatment focusing on those critical time points. Consistent with previous reports, deep flow cytometric immune-cell profiling (n = 30) demonstrated major effects on adaptive rather than innate immunity, which favoured regulatory immune cell subsets within the repopulation. The longitudinally studied CSF compartment (n = 18) mainly mirrored the immunological effects observed in the periphery. Alemtuzumab-induced changes including increased numbers of naïve CD4+ T cells and B cells as well as a clonal renewal of CD4+ T- and B-cell repertoires were partly reminiscent of haematopoietic stem cell transplantation; in contrast, thymopoiesis was reduced and clonal renewal of T-cell repertoires after alemtuzumab was incomplete. Stratification for secondary autoimmunity did not show clear immununological cellular or proteomic traits or signatures associated with secondary autoimmunity. However, a restricted T-cell repertoire with hyperexpanded T-cell clones at baseline, which persisted and demonstrated further expansion at Month 12 by homeostatic proliferation, identified patients developing secondary autoimmune disorders (n = 7 without secondary autoimmunity versus n = 5 with secondary autoimmunity). Those processes were followed by an expansion of memory B-cell clones irrespective of persistence, which we detected shortly after the diagnosis of secondary autoimmune disease. In conclusion, our data demonstrate that (i) peripheral immunological alterations following alemtuzumab are mirrored by longitudinal changes in the CSF; (ii) incomplete T-cell repertoire renewal and reduced thymopoiesis contribute to a proautoimmune state after alemtuzumab; (iii) proteomics and surface immunological phenotyping do not identify patients at risk for secondary autoimmune disorders; (iv) homeostatic proliferation with disparate dynamics of clonal T- and B-cell expansions are associated with secondary autoimmunity; and (v) hyperexpanded T-cell clones at baseline and Month 12 may be used as a biomarker for the risk of alemtuzumab-induced autoimmunity.

Redefining the heterogeneity of peripheral nerve cells in health and autoimmunity.

Peripheral nerves contain axons and their enwrapping glia cells named Schwann cells (SCs) that are either myelinating (mySCs) or nonmyelinating (nmSCs). Our understanding of other cells in the peripheral nervous system (PNS) remains limited. Here, we provide an unbiased single cell transcriptomic characterization of the nondiseased rodent PNS. We identified and independently confirmed markers of previously underappreciated nmSCs and nerve-associated fibroblasts. We also found and characterized two distinct populations of nerve-resident homeostatic myeloid cells that transcriptionally differed from central nervous system microglia. In a model of chronic autoimmune neuritis, homeostatic myeloid cells were outnumbered by infiltrating lymphocytes which modulated the local cell-cell interactome and induced a specific transcriptional response in glia cells. This response was partially shared between the peripheral and central nervous system glia, indicating common immunological features across different parts of the nervous system. Our study thus identifies subtypes and cell-type markers of PNS cells and a partially conserved autoimmunity module induced in glia cells.

Integrated single-cell transcriptomics of cerebrospinal fluid cells in treatment-naïve multiple sclerosis.

Multiple sclerosis (MS) is a chronic and often disabling autoimmune disease of the central nervous system (CNS). Cerebrospinal fluid (CSF) surrounds and protects the CNS. Analysis of CSF can aid the diagnosis of CNS diseases, help to identify the prognosis, and underlying mechanisms of diseases. Several recent studies have leveraged single-cell RNA-sequencing (scRNA-seq) to identify MS-associated changes in CSF cells that are considerably more altered than blood cells in MS. However, not all alterations were replicated across all studies. We therefore integrated multiple available scRNA-seq datasets of CSF cells from MS patients with early relapsing-remitting (RRMS) disease. We provide a searchable and interactive resource of this integrated analysis ( https://CSFinMS.bxgenomics.com ) facilitating diverse visualization and analysis methods without requiring computational skills. In the present joint analysis, we replicated the known expansion of B lineage and the recently described expansion of natural killer (NK) cells and some cytotoxic T cells and decrease of monocytes in the CSF in MS. The previous observation of the abundance of Th1-like Th17 effector memory cells in the CSF was not replicated. Expanded CSF B lineage cells resembled class-switched plasmablasts/-cells (e.g., SDC1/CD138, MZB1) as expected. Our integrative analysis thus validates increased cell type diversity and B cell maturation in the CSF in MS and improves accessibility of available data.

Cerebrospinal fluid flow cytometry distinguishes psychosis spectrum disorders from differential diagnoses.

Psychotic disorders are common and disabling mental conditions. The relative importance of immune-related mechanisms in psychotic disorders remains subject of debate. Here, we present a large-scale retrospective study of blood and cerebrospinal fluid (CSF) immune cell profiles of psychosis spectrum patients. We performed basic CSF analysis and multi-dimensional flow cytometry of CSF and blood cells from 59 patients with primary psychotic disorders (F20, F22, F23, and F25) in comparison to inflammatory (49 RRMS and 16 NMDARE patients) and non-inflammatory controls (52 IIH patients). We replicated the known expansion of monocytes in the blood of psychosis spectrum patients, that we identified to preferentially affect classical monocytes. In the CSF, we found a relative shift from lymphocytes to monocytes, increased protein levels, and evidence of blood-brain barrier disruption in psychosis. In fact, these CSF features confidently distinguished autoimmune encephalitis from psychosis despite similar (initial) clinical features. We then constructed machine learning models incorporating blood and CSF parameters and demonstrated their superior ability to differentiate psychosis from non-inflammatory controls compared to individual parameters. Multi-dimensional and multi-compartment immune cell signatures can thus support the diagnosis of psychosis spectrum disorders with the potential to accelerate diagnosis and initiation of therapy.

Classification of neurological diseases using multi-dimensional CSF analysis.

Although CSF analysis routinely enables the diagnosis of neurological diseases, it is mainly used for the gross distinction between infectious, autoimmune inflammatory, and degenerative disorders of the CNS. To investigate, whether a multi-dimensional cellular blood and CSF characterization can support the diagnosis of clinically similar neurological diseases, we analysed 546 patients with autoimmune neuroinflammatory, degenerative, or vascular conditions in a cross-sectional retrospective study. By combining feature selection with dimensionality reduction and machine learning approaches we identified pan-disease parameters that were altered across all autoimmune neuroinflammatory CNS diseases and differentiated them from other neurological conditions and inter-autoimmunity classifiers that subdifferentiate variants of CNS-directed autoimmunity. Pan-disease as well as diseases-specific changes formed a continuum, reflecting clinical disease evolution. A validation cohort of 231 independent patients confirmed that combining multiple parameters into composite scores can assist the classification of neurological patients. Overall, we showed that the integrated analysis of blood and CSF parameters improves the differential diagnosis of neurological diseases, thereby facilitating early treatment decisions.

CNS Pericytes Modulate Local T Cell Infiltration in EAE.

Pericytes at the blood-brain barrier (BBB) are located between the tight endothelial cell layer of the blood vessels and astrocytic endfeet. They contribute to central nervous system (CNS) homeostasis by regulating BBB development and maintenance. Loss of pericytes results in increased numbers of infiltrating immune cells in the CNS in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS). However, little is known about their competence to modulate immune cell activation or function in CNS autoimmunity. To evaluate the capacity of pericytes to directly interact with T cells in an antigen-specific fashion and potentially (re)shape their function, we depleted major histocompatibility complex (MHC) class II from pericytes in a cell type-specific fashion and performed T cell-pericyte cocultures and EAE experiments. We found that pericytes present antigen in vitro to induce T cell activation and proliferation. In an adoptive transfer EAE experiment, pericyte-specific MHC II KO resulted in locally enhanced T cell infiltration in the CNS; even though, overall disease course of mice was not affected. Thus, pericytes may serve as non-professional antigen-presenting cells affecting states of T cell activation, thereby locally shaping lesion formation in CNS inflammation but without modulating disease severity.

Intravenous methadone causes acute toxic and delayed inflammatory encephalopathy with persistent neurocognitive impairments.

BACKGROUND: The mu-opioid agonist methadone is administered orally and used in opioid detoxification and in the treatment of moderate-to-severe pain. Acute oral methadone-use and -abuse have been associated with inflammatory and toxic central nervous system (CNS) damage in some cases and cognitive deficits can develop in long-term methadone users. In contrast, reports of intravenous methadone adverse effects are rare. CASE PRESENTATION: Here, we report a patient who developed acute bilateral hearing loss, ataxia and paraparesis subsequently to intravenous methadone-abuse. While the patient gradually recovered from these deficits, widespread magnetic resonance imaging changes progressed and delayed-onset encephalopathy with signs of cortical dysfunction persisted. This was associated with changes in the composition of monocyte and natural killer cell subsets in the cerebrospinal fluid. CONCLUSION: This case suggests a potential bi-phasic primary toxic and secondary inflammatory CNS damage induced by intravenous methadone.