STING orchestrates the neuronal inflammatory stress response in multiple sclerosis.

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS.

B cells orchestrate tolerance to the neuromyelitis optica autoantigen AQP4.

Neuromyelitis optica is a paradigmatic autoimmune disease of the central nervous system, in which the water-channel protein AQP4 is the target antigen1. The immunopathology in neuromyelitis optica is largely driven by autoantibodies to AQP42. However, the T cell response that is required for the generation of these anti-AQP4 antibodies is not well understood. Here we show that B cells endogenously express AQP4 in response to activation with anti-CD40 and IL-21 and are able to present their endogenous AQP4 to T cells with an AQP4-specific T cell receptor (TCR). A population of thymic B cells emulates a CD40-stimulated B cell transcriptome, including AQP4 (in mice and humans), and efficiently purges the thymic TCR repertoire of AQP4-reactive clones. Genetic ablation of Aqp4 in B cells rescues AQP4-specific TCRs despite sufficient expression of AQP4 in medullary thymic epithelial cells, and B-cell-conditional AQP4-deficient mice are fully competent to raise AQP4-specific antibodies in productive germinal-centre responses. Thus, the negative selection of AQP4-specific thymocytes is dependent on the expression and presentation of AQP4 by thymic B cells. As AQP4 is expressed in B cells in a CD40-dependent (but not AIRE-dependent) manner, we propose that thymic B cells might tolerize against a group of germinal-centre-associated antigens, including disease-relevant autoantigens such as AQP4.

Swiss digital pathology recommendations: results from a Delphi process conducted by the Swiss Digital Pathology Consortium of the Swiss Society of Pathology.

Integration of digital pathology (DP) into clinical diagnostic workflows is increasingly receiving attention as new hardware and software become available. To facilitate the adoption of DP, the Swiss Digital Pathology Consortium (SDiPath) organized a Delphi process to produce a series of recommendations for DP integration within Swiss clinical environments. This process saw the creation of 4 working groups, focusing on the various components of a DP system (1) scanners, quality assurance and validation of scans, (2) integration of Whole Slide Image (WSI)-scanners and DP systems into the Pathology Laboratory Information System, (3) digital workflow-compliance with general quality guidelines, and (4) image analysis (IA)/artificial intelligence (AI), with topic experts for each recruited for discussion and statement generation. The work product of the Delphi process is 83 consensus statements presented here, forming the basis for "SDiPath Recommendations for Digital Pathology". They represent an up-to-date resource for national and international hospitals, researchers, device manufacturers, algorithm developers, and all supporting fields, with the intent of providing expectations and best practices to help ensure safe and efficient DP usage.

MicroRNA-92a-CPEB3 axis protects neurons against inflammatory neurodegeneration.

Neuroinflammation causes neuronal injury in multiple sclerosis (MS) and other neurological diseases. MicroRNAs (miRNAs) are important modulators of neuronal stress responses, but knowledge about their contribution to neuronal protection or damage during inflammation is limited. Here, we constructed a regulatory miRNA-mRNA network of inflamed motor neurons by leveraging cell type-specific miRNA and mRNA sequencing of mice undergoing experimental autoimmune encephalomyelitis (EAE). We found robust induction of miR-92a in inflamed spinal cord neurons and identified cytoplasmic polyadenylation element-binding protein 3 (Cpeb3) as a key target of miR-92a-mediated posttranscriptional silencing. We detected CPEB3 repression in inflamed neurons in murine EAE and human MS. Moreover, both miR-92a delivery and Cpeb3 deletion protected neuronal cultures against excitotoxicity. Supporting a detrimental effect of Cpeb3 in vivo, neuron-specific deletion in conditional Cpeb3 knockout animals led to reduced inflammation-induced clinical disability in EAE. Together, we identified a neuroprotective miR-92a-Cpeb3 axis in neuroinflammation that might serve as potential treatment target to limit inflammation-induced neuronal damage.

Targeting the TCA cycle can ameliorate widespread axonal energy deficiency in neuroinflammatory lesions.

Inflammation in the central nervous system can impair the function of neuronal mitochondria and contributes to axon degeneration in the common neuroinflammatory disease multiple sclerosis (MS). Here we combine cell-type-specific mitochondrial proteomics with in vivo biosensor imaging to dissect how inflammation alters the molecular composition and functional capacity of neuronal mitochondria. We show that neuroinflammatory lesions in the mouse spinal cord cause widespread and persisting axonal ATP deficiency, which precedes mitochondrial oxidation and calcium overload. This axonal energy deficiency is associated with impaired electron transport chain function, but also an upstream imbalance of tricarboxylic acid (TCA) cycle enzymes, with several, including key rate-limiting, enzymes being depleted in neuronal mitochondria in experimental models and in MS lesions. Notably, viral overexpression of individual TCA enzymes can ameliorate the axonal energy deficits in neuroinflammatory lesions, suggesting that TCA cycle dysfunction in MS may be amendable to therapy.

Bridging the Gap: Immunotherapy in Progressive Multifocal Leukoencephalopathy: A New Hope?

Progressive multifocal leukoencephalopathy (PML) is a severe infection of the CNS occurring in immunocompromised individuals in which large demyelinating lesions are induced by polyomavirus JC (JCV). In the absence of effective antiviral treatment, control of the infection relies on restoring anti-JCV immunity. Thus, particularly in long-standing immunocompromising conditions such as organ transplantation, lymphoproliferative disorders, or idiopathic lymphopenia, new strategies to boost anti-JCV immune responses are needed. Here, we report the case of a patient developing PML in the context of kidney transplantation who received recombinant human interleukin 7 to foster immune responses against JCV. We give an overview of the immunologic mechanisms underlying the development of PML and immune restoration within the CNS after JCV infection. Immunotherapeutic strategies developed based on current understanding of the disease hold promise in managing patients with PML.

Antigen recognition detains CD8+ T cells at the blood-brain barrier and contributes to its breakdown.

Blood-brain barrier (BBB) breakdown and immune cell infiltration into the central nervous system (CNS) are early hallmarks of multiple sclerosis (MS). High numbers of CD8+ T cells are found in MS lesions, and antigen (Ag) presentation at the BBB has been proposed to promote CD8+ T cell entry into the CNS. Here, we show that brain endothelial cells process and cross-present Ag, leading to effector CD8+ T cell differentiation. Under physiological flow in vitro, endothelial Ag presentation prevented CD8+ T cell crawling and diapedesis resulting in brain endothelial cell apoptosis and BBB breakdown. Brain endothelial Ag presentation in vivo was limited due to Ag uptake by CNS-resident macrophages but still reduced motility of Ag-specific CD8+ T cells within CNS microvessels. MHC class I-restricted Ag presentation at the BBB during neuroinflammation thus prohibits CD8+ T cell entry into the CNS and triggers CD8+ T cell-mediated focal BBB breakdown.

Regulator of G-protein signaling 1 critically supports CD8+ TRM cell-mediated intestinal immunity.

Members of the Regulator of G-protein signaling (Rgs) family regulate the extent and timing of G protein signaling by increasing the GTPase activity of Gα protein subunits. The Rgs family member Rgs1 is one of the most up-regulated genes in tissue-resident memory (TRM) T cells when compared to their circulating T cell counterparts. Functionally, Rgs1 preferentially deactivates Gαq, and Gαi protein subunits and can therefore also attenuate chemokine receptor-mediated immune cell trafficking. The impact of Rgs1 expression on tissue-resident T cell generation, their maintenance, and the immunosurveillance of barrier tissues, however, is only incompletely understood. Here we report that Rgs1 expression is readily induced in naïve OT-I T cells in vivo following intestinal infection with Listeria monocytogenes-OVA. In bone marrow chimeras, Rgs1 -/- and Rgs1 +/+ T cells were generally present in comparable frequencies in distinct T cell subsets of the intestinal mucosa, mesenteric lymph nodes, and spleen. After intestinal infection with Listeria monocytogenes-OVA, however, OT-I Rgs1 +/+ T cells outnumbered the co-transferred OT-I Rgs1- /- T cells in the small intestinal mucosa already early after infection. The underrepresentation of the OT-I Rgs1 -/- T cells persisted to become even more pronounced during the memory phase (d30 post-infection). Remarkably, upon intestinal reinfection, mice with intestinal OT-I Rgs1 +/+ TRM cells were able to prevent the systemic dissemination of the pathogen more efficiently than those with OT-I Rgs1 -/- TRM cells. While the underlying mechanisms are not fully elucidated yet, these data thus identify Rgs1 as a critical regulator for the generation and maintenance of tissue-resident CD8+ T cells as a prerequisite for efficient local immunosurveillance in barrier tissues in case of reinfections with potential pathogens.

The impact of transsphenoidal surgery on pituitary function in patients with non-functioning macroadenomas.

PURPOSE: Transsphenoidal surgery for non-functioning pituitary adenomas (NFPAs) can alter pituitary function. We assessed the rates of improvement and deterioration of pituitary function by axis and searched for predictive factors of these outcomes. METHODS: We reviewed consecutive medical files from patients having had transsphenoidal surgery for NFPA between 2004 and 2018. Pituitary functions and MRI imaging were analyzed prior and after surgery. The occurrence of recovery and new deficit were documented per axis. Prognostic factors of hormonal recovery and new deficits were searched. RESULTS: Among 137 patients analyzed, median tumor size of the NFPA was 24.8 mm and 58.4% of patients presented visual impairment. Before surgery, 91 patients (67%) had at least one abnormal pituitary axis (hypogonadism: 62.4%; hypothyroidism: 41%, adrenal insufficiency: 30.8%, growth hormone deficiency: 29.9%; increased prolactin: 50.8%). Following surgery, the recovery rate of pituitary deficiency of one axis or more was 46% and the rate of new pituitary deficiency was 10%. Rates of LH-FSH, TSH, ACTH and GH deficiency recovery were 35.7%, 30.4%, 15.4%, and 45.5% respectively. Rates of new LH-FSH, TSH, ACTH and GH deficiencies were 8.3%, 1.6%, 9.2% and 5.1% respectively. Altogether, 24.6% of patients had a global pituitary function improvement and only 7% had pituitary function worsening after surgery. Male patients and patients with hyperprolactinemia upon diagnosis were more likely to experience pituitary function recovery. No prognostic factors for the risk of new deficiencies were identified. CONCLUSION: In a real-life cohort of patients with NFPAs, recovery of hypopituitarism after surgery is more frequent than the occurrence of new deficiencies. Hence, hypopituitarism could be considered a relative indication for surgery in patients with NFPAs.

The alarmin interleukin-33 promotes the expansion and preserves the stemness of Tcf-1+ CD8+ T cells in chronic viral infection.

T cell factor 1 (Tcf-1) expressing CD8+ T cells exhibit stem-like self-renewing capacity, rendering them key for immune defense against chronic viral infection and cancer. Yet, the signals that promote the formation and maintenance of these stem-like CD8+ T cells (CD8+SL) remain poorly defined. Studying CD8+ T cell differentiation in mice with chronic viral infection, we identified the alarmin interleukin-33 (IL-33) as pivotal for the expansion and stem-like functioning of CD8+SL as well as for virus control. IL-33 receptor (ST2)-deficient CD8+ T cells exhibited biased end differentiation and premature loss of Tcf-1. ST2-deficient CD8+SL responses were restored by blockade of type I interferon signaling, suggesting that IL-33 balances IFN-I effects to control CD8+SL formation in chronic infection. IL-33 signals broadly augmented chromatin accessibility in CD8+SL and determined these cells' re-expansion potential. Our study identifies the IL-33-ST2 axis as an important CD8+SL-promoting pathway in the context of chronic viral infection.

Persistent virus-specific and clonally expanded antibody-secreting cells respond to induced self-antigen in the CNS.

B cells contribute to the pathogenesis of both cellular- and humoral-mediated central nervous system (CNS) inflammatory diseases through a variety of mechanisms. In such conditions, B cells may enter the CNS parenchyma and contribute to local tissue destruction. It remains unexplored, however, how infection and autoimmunity drive transcriptional phenotypes, repertoire features, and antibody functionality. Here, we profiled B cells from the CNS of murine models of intracranial (i.c.) viral infections and autoimmunity. We identified a population of clonally expanded, antibody-secreting cells (ASCs) that had undergone class-switch recombination and extensive somatic hypermutation following i.c. infection with attenuated lymphocytic choriomeningitis virus (rLCMV). Recombinant expression and characterisation of these antibodies revealed specificity to viral antigens (LCMV glycoprotein GP), correlating with ASC persistence in the brain weeks after resolved infection. Furthermore, these virus-specific ASCs upregulated proliferation and expansion programs in response to the conditional and transient induction of the LCMV GP as a neo-self antigen by astrocytes. This class-switched, clonally expanded, and mutated population persisted and was even more pronounced when peripheral B cells were depleted prior to autoantigen induction in the CNS. In contrast, the most expanded B cell clones in mice with persistent expression of LCMV GP in the CNS did not exhibit neo-self antigen specificity, potentially a consequence of local tolerance induction. Finally, a comparable population of clonally expanded, class-switched, and proliferating ASCs was detected in the cerebrospinal fluid of relapsing multiple sclerosis (RMS) patients. Taken together, our findings support the existence of B cells that populate the CNS and are capable of responding to locally encountered autoantigens.

Endothelial cell-derived oxysterol ablation attenuates experimental autoimmune encephalomyelitis.

The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis (MS). However, the impact of endothelial-derived factors on CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream of Cholesterol-25-hydroxylase (Ch25h), promote neuroinflammation but their functions in the CNS are not well-understood. Using floxed-reporter Ch25h knock-in mice, we trace Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrate that Ch25h ablation specifically from ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h-deficient CNS ECs display altered lipid metabolism favoring polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) expansion, which suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h-deficiency combined with immature neutrophil mobilization into the blood circulation nearly completely protects mice from EAE. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by inhibiting the expansion of immunosuppressive myeloid cell populations.

Analysis of the T-cell repertoire and transcriptome identifies mechanisms of regulatory T-cell suppression of GVHD.

CD4+FOXP3+ regulatory T cells (Tregs) have demonstrated efficacy in the prevention and treatment of graft-versus-host disease (GVHD). Preclinical and clinical studies indicate that Tregs are able to protect from GVHD without interfering with the graft-versus-tumor (GVT) effect of hematopoietic cell transplantation (HCT), although the underlying molecular mechanisms are largely unknown. To elucidate Treg suppressive function during in vivo suppression of acute GVHD, we performed paired T-cell receptor (TCRα and ΤCRß genes) repertoire sequencing and RNA sequencing analysis on conventional T cells (Tcons) and Tregs before and after transplantation in a major histocompatibility complex -mismatched mouse model of HCT. We show that both Tregs and Tcons underwent clonal restriction, and Tregs did not interfere with the activation of alloreactive Tcon clones and the breadth of their TCR repertoire but markedly suppressed their expansion. Transcriptomic analysis revealed that Tregs predominantly affected the transcriptome of CD4 Tcons and, to a lesser extent, that of CD8 Tcons, thus modulating the transcription of genes encoding pro- and anti-inflammatory molecules as well as enzymes involved in metabolic processes, inducing a switch from glycolysis to oxidative phosphorylation. Finally, Tregs did not interfere with the induction of gene sets involved in the GVT effect. Our results shed light onto the mechanisms of acute GVHD suppression by Tregs and will support the clinical translation of this immunoregulatory approach.

Single-cell immune repertoire sequencing of B and T cells in murine models of infection and autoimmunity.

Adaptive immune repertoires are composed by the ensemble of B and T-cell receptors within an individual, reflecting both past and current immune responses. Recent advances in single-cell sequencing enable recovery of the complete adaptive immune receptor sequences in addition to transcriptional information. Here, we recovered transcriptome and immune repertoire information for polyclonal T follicular helper cells following lymphocytic choriomeningitis virus (LCMV) infection, CD8+ T cells with binding specificity restricted to two distinct LCMV peptides, and B and T cells isolated from the nervous system in the context of experimental autoimmune encephalomyelitis. We could relate clonal expansion, germline gene usage, and clonal convergence to cell phenotypes spanning activation, memory, naive, antibody secretion, T-cell inflation, and regulation. Together, this dataset provides a resource for immunologists that can be integrated with future single-cell immune repertoire and transcriptome sequencing datasets.

Synergistic prostaglandin E synthesis by myeloid and endothelial cells promotes fetal hematopoietic stem cell expansion in vertebrates.

During development, hematopoietic stem cells (HSCs) are produced from the hemogenic endothelium and will expand in a transient hematopoietic niche. Prostaglandin E2 (PGE2) is essential during vertebrate development and HSC specification, but its precise source in the embryo remains elusive. Here, we show that in the zebrafish embryo, PGE2 synthesis genes are expressed by distinct stromal cell populations, myeloid (neutrophils, macrophages), and endothelial cells of the caudal hematopoietic tissue. Ablation of myeloid cells, which produce the PGE2 precursor prostaglandin H2 (PGH2), results in loss of HSCs in the caudal hematopoietic tissue, which could be rescued by exogeneous PGE2 or PGH2 supplementation. Endothelial cells contribute by expressing the PGH2 import transporter slco2b1 and ptges3, the enzyme converting PGH2 into PGE2. Of note, differential niche cell expression of PGE2 biosynthesis enzymes is also observed in the mouse fetal liver. Taken altogether, our data suggest that the triad composed of neutrophils, macrophages, and endothelial cells sequentially and synergistically contributes to blood stem cell expansion during vertebrate development.

G9a dictates neuronal vulnerability to inflammatory stress via transcriptional control of ferroptosis.

Neuroinflammation leads to neuronal stress responses that contribute to neuronal dysfunction and loss. However, treatments that stabilize neurons and prevent their destruction are still lacking. Here, we identify the histone methyltransferase G9a as a druggable epigenetic regulator of neuronal vulnerability to inflammation. In murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS), we found that the G9a-catalyzed repressive epigenetic mark H3K9me2 was robustly induced by neuroinflammation. G9a activity repressed anti-ferroptotic genes, diminished intracellular glutathione levels, and triggered the iron-dependent programmed cell death pathway ferroptosis. Conversely, pharmacological treatment of EAE mice with a G9a inhibitor restored anti-ferroptotic gene expression, reduced inflammation-induced neuronal loss, and improved clinical outcome. Similarly, neuronal anti-ferroptotic gene expression was reduced in MS brain tissue and was boosted by G9a inhibition in human neuronal cultures. This study identifies G9a as a critical transcriptional enhancer of neuronal ferroptosis and potential therapeutic target to counteract inflammation-induced neurodegeneration.

Neurodegenerative phagocytes mediate synaptic stripping in Neuro-HIV.

Glial cell activation is a hallmark of several neurodegenerative and neuroinflammatory diseases. During HIV infection, neuroinflammation is associated with cognitive impairment, even during sustained long-term suppressive antiretroviral therapy. However, the cellular subsets contributing to neuronal damage in the CNS during HIV infection remain unclear. Using post-mortem brain samples from eight HIV patients and eight non-neurological disease controls, we identify a subset of CNS phagocytes highly enriched in LGALS3, CTSB, GPNMB and HLA-DR, a signature identified in the context of ageing and neurodegeneration. In HIV patients, the presence of this phagocyte phenotype was associated with synaptic stripping, suggesting an involvement in the pathogenesis of HIV-associated neurocognitive disorder. Taken together, our findings elucidate some of the molecular signatures adopted by CNS phagocytes in HIV-positive patients and contribute to the understanding of how HIV might pave the way to other forms of cognitive decline in ageing HIV patient populations.

IFN-γ-dependent tumor-antigen cross-presentation by lymphatic endothelial cells promotes their killing by T cells and inhibits metastasis.

Tumor-associated lymphatic vessels promote metastasis and regulate antitumor immune responses. Here, we assessed the impact of cytotoxic T cells on the local lymphatic vasculature and concomitant tumor dissemination during an antitumor response. Interferon-γ (IFN-γ) released by effector T cells enhanced the expression of immunosuppressive markers by tumor-associated lymphatic endothelial cells (LECs). However, at higher effector T cell densities within the tumor, T cell-based immunotherapies induced LEC apoptosis and decreased tumor lymphatic vessel density. As a consequence, lymphatic flow was impaired, and lymph node metastasis was reduced. Mechanistically, T cell-mediated tumor cell death induced the release of tumor antigens and cross-presentation by tumor LECs, resulting in antigen-specific LEC killing by T cells. When LECs lacked the IFN-γ receptor expression, LEC killing was abrogated, indicating that IFN-γ is indispensable for reducing tumor-associated lymphatic vessel density and drainage. This study provides insight into how cytotoxic T cells modulate tumor lymphatic vessels and may help to improve immunotherapeutic protocols.

Tissue-resident CD8 T cells in central nervous system inflammatory diseases: present at the crime scene and guilty.

Tissue-resident memory T cells (TRM) represent a subset of antigen-experienced T cells that are constantly retained in a given tissue with limited trafficking through the circulation. These cells are characterized by expression of molecules enabling their tissue anchoring and downregulation of molecules promoting tissue egress. They reside at sites of previous antigen encounter and their number increases with age. TRM have been shown to provide rapid and efficient protection against tissue reinfection and TRM density correlates with efficient antitumor responses. Intriguingly, the density of CD8 TRM is increased in the central nervous system (CNS) of patients with neuroinflammatory diseases such as multiple sclerosis, or suffering from neurodegenerative diseases. In this review, we discuss current knowledge regarding the diversity of CNS-resident CD8 T cells and their role in CNS autoimmunity. Given their likely contribution to the protracted course of several inflammatory diseases of the CNS, their therapeutic targeting becomes an important challenge.

Towards a national strategy for digital pathology in Switzerland.

Precision medicine is entering a new era of digital diagnostics; the availability of integrated digital pathology (DP) and structured clinical datasets has the potential to become a key catalyst for biomedical research, education and business development. In Europe, national programs for sharing of this data will be crucial for the development, testing, and validation of machine learning-enabled tools supporting clinical decision-making. Here, the Swiss Digital Pathology Consortium (SDiPath) discusses the creation of a Swiss Digital Pathology Infrastructure (SDPI), which aims to develop a unified national DP network bringing together the Swiss Personalized Health Network (SPHN) with Swiss university hospitals and subsequent inclusion of cantonal and private institutions. This effort builds on existing developments for the national implementation of structured pathology reporting. Opening this national infrastructure and data to international researchers in a sequential rollout phase can enable the large-scale integration of health data and pooling of resources for research purposes and clinical trials. Therefore, the concept of a SDPI directly synergizes with the priorities of the European Commission communication on the digital transformation of healthcare on an international level, and with the aims of the Swiss State Secretariat for Economic Affairs (SECO) for advancing research and innovation in the digitalization domain. SDPI directly addresses the needs of existing national and international research programs in neoplastic and non-neoplastic diseases by providing unprecedented access to well-curated clinicopathological datasets for the development and implementation of novel integrative methods for analysis of clinical outcomes and treatment response. In conclusion, a SDPI would facilitate and strengthen inter-institutional collaboration in technology, clinical development, business and research at a national and international scale, promoting improved patient care via precision medicine.