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.

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.

Dimerization of the plant molybdenum insertase Cnx1E is required for synthesis of the molybdenum cofactor.

The molybdenum cofactor (Moco) is a redox active prosthetic group, essentially required for numerous enzyme-catalyzed two electron transfer reactions. Moco is synthesized by an evolutionarily old and highly conserved multistep pathway. In the last step of Moco biosynthesis, the molybdenum center is inserted into the final Moco precursor adenylated molybdopterin (MPT-AMP). This unique and yet poorly characterized maturation reaction finally yields physiologically active Moco. In the model plant Arabidopsis, the two domain enzyme, Cnx1, is required for Moco formation. Recently, a genetic screen identified novel Arabidopsis cnx1 mutant plant lines each harboring a single amino acid exchange in the N-terminal Cnx1E domain. Biochemical characterization of the respective recombinant Cnx1E variants revealed two different amino acid exchanges (S197F and G175D) that impair Cnx1E dimerization, thus linking Cnx1E oligomerization to Cnx1 functionality. Analysis of the Cnx1E structure identified Cnx1E active site-bound molybdate and magnesium ions, which allowed to fine-map the Cnx1E MPT-AMP-binding site.

Novel pathomechanisms in inflammatory neuropathies.

Inflammatory neuropathies are rare autoimmune-mediated disorders affecting the peripheral nervous system. Considerable progress has recently been made in understanding pathomechanisms of these disorders which will be essential for developing novel diagnostic and therapeutic strategies in the future. Here, we summarize our current understanding of antigenic targets and the relevance of new immunological concepts for inflammatory neuropathies. In addition, we provide an overview of available animal models of acute and chronic variants and how new diagnostic tools such as magnetic resonance imaging and novel therapeutic candidates will benefit patients with inflammatory neuropathies in the future. This review thus illustrates the gap between pre-clinical and clinical findings and aims to outline future directions of development.

Stroke induces disease-specific myeloid cells in the brain parenchyma and pia.

Inflammation triggers secondary brain damage after stroke. The meninges and other CNS border compartments serve as invasion sites for leukocyte influx into the brain thus promoting tissue damage after stroke. However, the post-ischemic immune response of border compartments compared to brain parenchyma remains poorly characterized. Here, we deeply characterize tissue-resident leukocytes in meninges and brain parenchyma and discover that leukocytes respond differently to stroke depending on their site of residence. We thereby discover a unique phenotype of myeloid cells exclusive to the brain after stroke. These stroke-associated myeloid cells partially resemble neurodegenerative disease-associated microglia. They are mainly of resident microglial origin, partially conserved in humans and exhibit a lipid-phagocytosing phenotype. Blocking markers specific for these cells partially ameliorates stroke outcome thus providing a potential therapeutic target. The injury-response of myeloid cells in the CNS is thus compartmentalized, adjusted to the type of injury and may represent a therapeutic target.

Intraocular dendritic cells characterize HLA-B27-associated acute anterior uveitis.

Uveitis describes a heterogeneous group of inflammatory eye diseases characterized by infiltration of leukocytes into the uveal tissues. Uveitis associated with the HLA haplotype B27 (HLA-B27) is a common subtype of uveitis and a prototypical ocular immune-mediated disease. Local immune mechanisms driving human uveitis are poorly characterized mainly due to the limited available biomaterial and subsequent technical limitations. Here, we provide the first high-resolution characterization of intraocular leukocytes in HLA-B27-positive (n = 4) and -negative (n = 2) anterior uveitis and an infectious endophthalmitis control (n = 1) by combining single-cell RNA-sequencing with flow cytometry and protein analysis. Ocular cell infiltrates consisted primarily of lymphocytes in both subtypes of uveitis and of myeloid cells in infectious endophthalmitis. HLA-B27-positive uveitis exclusively featured a plasmacytoid and classical dendritic cell (cDC) infiltrate. Moreover, cDCs were central in predicted local cell-cell communication. This suggests a unique pattern of ocular leukocyte infiltration in HLA-B27-positive uveitis with relevance to DCs.

Uveitis is a form of inflammation in the eye. It can occur in response to infection, or when the immune system mistakenly attacks the eye, in what is known as autoimmune uveitis. In approximately 80 percent of cases, the front part of the eye is affected. During an inflammatory episode, the liquid inside the front part of the eye fills with immune cells, but the nature of these cells remains unknown. This is because uveitis is rare, and doctors cannot routinely take samples from inside the eyes of affected individuals to diagnose the disease. This lack of samples makes research into this disease challenging. There are two main groups of immune cells that could be responsible for uveitis: myeloid cells and lymphoid cells. Myeloid cells form the first line of immune defense against infection by non-specifically attacking and removing pathogens . Lymphoid cells form the second line of immune defense, attacking specific pathogens. Lymphoid cells also have long-term memory, meaning they can 'remember' previous infections and fight them more effectively. Lymphoid cells receive instructions from a type of myeloid cell called a dendritic cell about what to attack. Dendritic cells relay their instructions to lymphoid cells using molecules called human leukocyte antigens (HLA). Autoimmune uveitis affecting the front part of the eye is common in individuals with an HLA type called HLA-B27, suggesting that communication between dendritic and lymphoid cells plays an important role in this type of inflammation. To make the most of limited patient samples, Kasper et al. used single cell techniques to examine the immune cells from the fluid inside the eye. Six samples came from people with autoimmune uveitis, and one from a person with an eye infection. The infection sample contained mainly myeloid cells that might attack bacteria responsible for the infection. In contrast, the autoimmune uveitis samples contained mainly lymphoid cells. Of these samples, four were from individuals with the gene that codes for the HLA-B27 molecule. These samples had a unique pattern of immune cells, with more dendritic cells than the samples from individuals that did not have this gene. This study included only a small number of individuals, but it shows that analysing single immune cells from the eye is possible in uveitis. This snapshot could help researchers understand the local immune response in the eye, and find an optimal treatment.

Single-cell profiling of CNS border compartment leukocytes reveals that B cells and their progenitors reside in non-diseased meninges.

The CNS is ensheathed by the meninges and cerebrospinal fluid, and recent findings suggest that these CNS-associated border tissues have complex immunological functions. Unlike myeloid lineage cells, lymphocytes in border compartments have yet to be thoroughly characterized. Based on single-cell transcriptomics, we here identified a highly location-specific composition and expression profile of tissue-resident leukocytes in CNS parenchyma, pia-enriched subdural meninges, dura mater, choroid plexus and cerebrospinal fluid. The dura layer of the meninges contained a large population of B cells under homeostatic conditions in mice and rats. Murine dura B cells exhibited slow turnover and long-term tissue residency, and they matured in experimental neuroinflammation. The dura also contained B lineage progenitors at the pro-B cell stage typically not found outside of bone marrow, without direct influx from the periphery or the skull bone marrow. This identified the dura as an unexpected site of B cell residence and potentially of development in both homeostasis and neuroinflammation.

Integrated single cell analysis of blood and cerebrospinal fluid leukocytes in multiple sclerosis.

Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) - an autoimmune disease of the CNS - increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell interaction.

Dysregulation of a specific immune-related network of genes biologically defines a subset of schizophrenia.

Currently, the clinical diagnosis of schizophrenia relies solely on self-reporting and clinical interview, and likely comprises heterogeneous biological subsets. Such subsets may be defined by an underlying biology leading to solid biomarkers. A transgenic rat model modestly overexpressing the full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1) protein (tgDISC1 rat) was generated that defines such a subset, inspired by our previous identification of insoluble DISC1 protein in post mortem brains from patients with chronic mental illness. Besides specific phenotypes such as DISC1 protein pathology, abnormal dopamine homeostasis, and changes in neuroanatomy and behavior, this animal model also shows subtle disturbances in overarching signaling pathways relevant for schizophrenia. In a reverse-translational approach, assuming that both the animal model and a patient subset share common disturbed signaling pathways, we identified differentially expressed transcripts from peripheral blood mononuclear cells of tgDISC1 rats that revealed an interconnected set of dysregulated genes, led by decreased expression of regulator of G-protein signaling 1 (RGS1), chemokine (C-C) ligand 4 (CCL4), and other immune-related transcripts enriched in T-cell and macrophage signaling and converging in one module after weighted gene correlation network analysis. Testing expression of this gene network in two independent cohorts of patients with schizophrenia versus healthy controls (n = 16/50 and n = 54/45) demonstrated similar expression changes. The two top markers RGS1 and CCL4 defined a subset of 27% of patients with 97% specificity. Thus, analogous aberrant signaling pathways can be identified by a blood test in an animal model and a corresponding schizophrenia patient subset, suggesting that in this animal model tailored pharmacotherapies for this patient subset could be achieved.