Profiling of microglia nodules in multiple sclerosis reveals propensity for lesion formation.

Microglia nodules (HLA-DR+ cell clusters) are associated with brain pathology. In this post-mortem study, we investigated whether they represent the first stage of multiple sclerosis (MS) lesion formation. We show that microglia nodules are associated with more severe MS pathology. Compared to microglia nodules in stroke, those in MS show enhanced expression of genes previously found upregulated in MS lesions. Furthermore, genes associated with lipid metabolism, presence of T and B cells, production of immunoglobulins and cytokines, activation of the complement cascade, and metabolic stress are upregulated in microglia nodules in MS. Compared to stroke, they more frequently phagocytose oxidized phospholipids and possess a more tubular mitochondrial network. Strikingly, in MS, some microglia nodules encapsulate partially demyelinated axons. Taken together, we propose that activation of microglia nodules in MS by cytokines and immunoglobulins, together with phagocytosis of oxidized phospholipids, may lead to a microglia phenotype prone to MS lesion formation.

Microglia Transcriptional Profiling in Major Depressive Disorder Shows Inhibition of Cortical Gray Matter Microglia.

BACKGROUND: Microglia have been implicated in the pathophysiology of major depressive disorder (MDD), but information on biological mechanisms is limited. Therefore, we investigated the gene expression profile of microglial cells in relation to neuronal regulators of microglia activity in well-characterized MDD and control autopsy brains. METHODS: Pure, intact microglia were isolated at brain autopsy from occipital cortex gray matter (GM) and corpus callosum white matter of 13 donors with MDD and 10 age-matched control donors for RNA sequencing. Top differentially expressed genes were validated using immunohistochemistry staining. Because gene expression changes were only detected in GM microglia, neuronal regulators of microglia were investigated in cortical tissue and synaptosomes from the cortex by reverse transcriptase-quantitative polymerase chain reaction and Western blot. RESULTS: Transcriptome analysis revealed 92 genes differentially expressed in microglia isolated from GM, but none in microglia from white matter in donors with MDD, compared with control donors. Of these, 81 genes were less abundantly expressed in GM in MDD, including CD163, MKI67, SPP1, CD14, FCGR1A/C, and C1QA/B/C. Accordingly, pathways related to effector mechanisms, such as the complement system and phagocytosis, were differentially regulated in GM microglia in MDD. Immunohistochemistry staining revealed significantly lower expression of CD163 protein in MDD. Whole tissue analysis showed an increase in CD200 (p = .0009) and CD47 (p = .068) messenger RNA, and CD47 protein was significantly elevated (p = .0396) in synaptic fractions of MDD cases. CONCLUSIONS: Transcriptional profiling indicates an immune-suppressed microglial phenotype in MDD that is possibly caused by neuronal regulation.

Age-related changes in plasma biomarkers and their association with mortality in COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19)-induced mortality occurs predominantly in older patients. Several immunomodulating therapies seem less beneficial in these patients. The biological substrate behind these observations is unknown. The aim of this study was to obtain insight into the association between ageing, the host response and mortality in patients with COVID-19. METHODS: We determined 43 biomarkers reflective of alterations in four pathophysiological domains: endothelial cell and coagulation activation, inflammation and organ damage, and cytokine and chemokine release. We used mediation analysis to associate ageing-driven alterations in the host response with 30-day mortality. Biomarkers associated with both ageing and mortality were validated in an intensive care unit and external cohort. RESULTS: 464 general ward patients with COVID-19 were stratified according to age decades. Increasing age was an independent risk factor for 30-day mortality. Ageing was associated with alterations in each of the host response domains, characterised by greater activation of the endothelium and coagulation system and stronger elevation of inflammation and organ damage markers, which was independent of an increase in age-related comorbidities. Soluble tumour necrosis factor receptor 1, soluble triggering receptor expressed on myeloid cells 1 and soluble thrombomodulin showed the strongest correlation with ageing and explained part of the ageing-driven increase in 30-day mortality (proportion mediated: 13.0%, 12.9% and 12.6%, respectively). CONCLUSIONS: Ageing is associated with a strong and broad modification of the host response to COVID-19, and specific immune changes likely contribute to increased mortality in older patients. These results may provide insight into potential age-specific immunomodulatory targets in COVID-19.

The adhesion G protein-coupled receptor GPR56/ADGRG1 in cytotoxic lymphocytes.

GPR56/ADGRG1 is an adhesion G protein-coupled receptor connected to brain development, haematopoiesis, male fertility, and tumorigenesis. Nevertheless, expression of GPR56 is not restricted to developmental processes. Studies over the last years have demonstrated a marked presence of GPR56 in human cytotoxic NK and T cells. Expression of GPR56 in these cells is driven by the transcription factor HOBIT, corresponds with the production of cytolytic mediators and the presence of CX3 CR1 and CD57, indicates a state of terminal differentiation and cellular exhaustion, and disappears upon cellular activation. Functional studies indicate that GPR56 regulates cell migration and effector functions and thereby acts as an inhibitory immune checkpoint. We here discuss the current state of knowledge regarding GPR56 in cytotoxic lymphocytes.

Selective emergence of antibody-secreting cells in the multiple sclerosis brain.

BACKGROUND: Although distinct brain-homing B cells have been identified in multiple sclerosis (MS), it is unknown how these further evolve to contribute to local pathology. We explored B-cell maturation in the central nervous system (CNS) of MS patients and determined their association with immunoglobulin (Ig) production, T-cell presence, and lesion formation. METHODS: Ex vivo flow cytometry was performed on post-mortem blood, cerebrospinal fluid (CSF), meninges and white matter from 28 MS and 10 control brain donors to characterize B cells and antibody-secreting cells (ASCs). MS brain tissue sections were analysed with immunostainings and microarrays. IgG index and CSF oligoclonal bands were measured with nephelometry, isoelectric focusing, and immunoblotting. Blood-derived B cells were cocultured under T follicular helper-like conditions to evaluate their ASC-differentiating capacity in vitro. FINDINGS: ASC versus B-cell ratios were increased in post-mortem CNS compartments of MS but not control donors. Local presence of ASCs associated with a mature CD45low phenotype, focal MS lesional activity, lesional Ig gene expression, and CSF IgG levels as well as clonality. In vitro B-cell maturation into ASCs did not differ between MS and control donors. Notably, lesional CD4+ memory T cells positively correlated with ASC presence, reflected by local interplay with T cells. INTERPRETATION: These findings provide evidence that local B cells at least in late-stage MS preferentially mature into ASCs, which are largely responsible for intrathecal and local Ig production. This is especially seen in active MS white matter lesions and likely depends on the interaction with CD4+ memory T cells. FUNDING: Stichting MS Research (19-1057 MS; 20-490f MS), National MS Fonds (OZ2018-003).

Osteopontin associates with brain TRM-cell transcriptome and compartmentalization in donors with and without multiple sclerosis.

The human brain is populated by perivascular T cells with a tissue-resident memory T (TRM)-cell phenotype, which in multiple sclerosis (MS) associate with lesions. We investigated the transcriptional and functional profile of freshly isolated T cells from white and gray matter. RNA sequencing of CD8+ and CD4+ CD69+ T cells revealed TRM-cell signatures. Notably, gene expression hardly differed between lesional and normal-appearing white matter T cells in MS brains. Genes up-regulated in brain TRM cells were MS4A1 (CD20) and SPP1 (osteopontin, OPN). OPN is also abundantly expressed by microglia and has been shown to inhibit T cell activity. In line with their parenchymal localization and the increased presence of OPN in active MS lesions, we noticed a reduced production of inflammatory cytokines IL-2, TNF, and IFNγ by lesion-derived CD8+ and CD4+ T cells ex vivo. Our study reports traits of brain TRM cells and reveals their tight control in MS lesions.

Ultrastructural Axon-Myelin Unit Alterations in Multiple Sclerosis Correlate with Inflammation.

OBJECTIVE: Changes in the normal-appearing white matter (NAWM) in multiple sclerosis (MS) may contribute to disease progression. Here, we systematically quantified ultrastructural and subcellular characteristics of the axon-myelin unit in MS NAWM and determined how this correlates with low-grade inflammation. METHODS: Human brain tissue obtained with short postmortem delay and fixation at autopsy enables systematic quantification of ultrastructural characteristics. In this study, we performed high-resolution immunohis tochemistry and quantitative transmission electron microscopy to study inflammation and ultrastructural characteristics of the axon-myelin unit in MS NAWM (n = 8) and control white matter (WM) in the optic nerve. RESULTS: In the MS NAWM, there were more activated and phagocytic microglia cells (HLA+ P2RY12- and Iba1+ CD68+ ) and more T cells (CD3+ ) compared to control WM, mainly located in the perivascular space. In MS NAWM compared to control WM, there were, as expected, longer paranodes and juxtaparanodes and larger overlap between paranodes and juxtaparanodes. There was less compact myelin wrapping, a lower g-ratio, and a higher frequency of axonal mitochondria. Changes in myelin and axonal mitochondrial frequency correlated positively with the number of active and phagocytic microglia and lymphocytes in the optic nerve. INTERPRETATION: These data suggest that in MS NAWM myelin detachment and uncompact myelin wrapping occurs, potassium channels are unmasked at the nodes of Ranvier, and axonal energy demand is increased, or mitochondrial transport is stagnated, accompanied by increased presence of activated and phagocytic microglia and T cells. These subclinical alterations to the axon-myelin unit in MS NAWM may contribute to disease progression. ANN NEUROL 2023;93:856-870.

T-cell surveillance of the human brain in health and multiple sclerosis.

Circulating and tissue-resident T cells collaborate in the protection of tissues against harmful infections and malignant transformation but also can instigate autoimmune reactions. Similar roles for T cells in the brain have been less evident due to the compartmentized organization of the central nervous system (CNS). In recent years, beneficial as well as occasional, detrimental effects of T-cell-targeting drugs in people with early multiple sclerosis (MS) have increased interest in T cells patrolling the CNS. Next to studies focusing on T cells in the cerebrospinal fluid, phenotypic characteristics of T cells located in the perivascular space and the meninges as well as in the parenchyma in MS lesions have been reported. We here summarize the current knowledge about T cells infiltrating the healthy and MS brain and argue that understanding the dynamics of physiological CNS surveillance by T cells is likely to improve the understanding of pathological conditions, such as MS.

CDK7/12/13 inhibition targets an oscillating leukemia stem cell network and synergizes with venetoclax in acute myeloid leukemia.

The heterogeneous response of acute myeloid leukemia (AML) to current anti-leukemic therapies is only partially explained by mutational heterogeneity. We previously identified GPR56 as a surface marker associated with poor outcome across genetic groups, which characterizes two leukemia stem cell (LSC)-enriched compartments with different self-renewal capacities. How these compartments self-renew remained unclear. Here, we show that GPR56+ LSC compartments are promoted in a complex network involving epithelial-to-mesenchymal transition (EMT) regulators besides Rho, Wnt, and Hedgehog (Hh) signaling. Unexpectedly, Wnt pathway inhibition increased the more immature, slowly cycling GPR56+ CD34+ fraction and Hh/EMT gene expression, while Wnt activation caused opposite effects. Our data suggest that the crucial role of GPR56 lies in its ability to co-activate these opposing signals, thus ensuring the constant supply of both LSC subsets. We show that CDK7 inhibitors suppress both LSC-enriched subsets in vivo and synergize with the Bcl-2 inhibitor venetoclax. Our data establish reciprocal transition between LSC compartments as a novel concept underlying the poor outcome in GPR56high AML and propose combined CDK7 and Bcl-2 inhibition as LSC-directed therapy in this disease.

The Inhibitory Receptor GPR56 (Adgrg1) Is Specifically Expressed by Tissue-Resident Memory T Cells in Mice But Dispensable for Their Differentiation and Function In Vivo.

Tissue-resident memory T (TRM) cells with potent antiviral and antibacterial functions protect the epithelial and mucosal surfaces of our bodies against infection with pathogens. The strong proinflammatory activities of TRM cells suggest requirement for expression of inhibitory molecules to restrain these memory T cells under steady state conditions. We previously identified the adhesion G protein-coupled receptor GPR56 as an inhibitory receptor of human cytotoxic lymphocytes that regulates their cytotoxic effector functions. Here, we explored the expression pattern, expression regulation, and function of GPR56 on pathogen-specific CD8+ T cells using two infection models. We observed that GPR56 is expressed on TRM cells during acute infection and is upregulated by the TRM cell-inducing cytokine TGF-ß and the TRM cell-associated transcription factor Hobit. However, GPR56 appeared dispensable for CD8+ T-cell differentiation and function upon acute infection with LCMV as well as Listeria monocytogenes. Thus, TRM cells specifically acquire the inhibitory receptor GPR56, but the impact of this receptor on TRM cells after acute infection does not appear essential to regulate effector functions of TRM cells.

Phenotypic comparison of human alveolar macrophages before and after in vivo rhinovirus 16 challenge.

We used mass cytometry to extensively characterize bronchoalveolar lavage macrophages before and two days after in vivo rhinovirus 16 infection in a heterogeneous population of healthy and asthma/COPD subjects. Multivariate partial least squares discriminant analysis revealed distinct clusters of alveolar macrophages before versus after the virus, suggesting changes in overall phenotype.

Multiple Targets for Oxysterols in Their Regulation of the Immune System.

Oxysterols, or cholesterol oxidation products, are naturally occurring lipids which regulate the physiology of cells, including those of the immune system. In contrast to effects that are mediated through nuclear receptors or by epigenetic mechanism, which take tens of minutes to occur, changes in the activities of cell-surface receptors caused by oxysterols can be extremely rapid, often taking place within subsecond timescales. Such cell-surface receptor effects of oxysterols allow for the regulation of fast cellular processes, such as motility, secretion and endocytosis. These cellular processes play critical roles in both the innate and adaptive immune systems. This review will survey the two broad classes of cell-surface receptors for oxysterols (G-protein coupled receptors (GPCRs) and ion channels), the mechanisms by which cholesterol oxidation products act on them, and their presence and functions in the different cell types of the immune system. Overall, this review will highlight the potential of oxysterols, synthetic derivatives and their receptors for physiological and therapeutic modulation of the immune system.

GPCRomics of Homeostatic and Disease-Associated Human Microglia.

G-protein-coupled receptors (GPCRs) are critical sensors affecting the state of eukaryotic cells. To get systematic insight into the GPCRome of microglia, we analyzed publicly available RNA-sequencing data of bulk and single cells obtained from human and mouse brains. We identified 17 rhodopsin and adhesion family GPCRs robustly expressed in microglia from human brains, including the homeostasis-associated genes CX3CR1, GPR34, GPR183, P2RY12, P2RY13, and ADGRG1. Expression of these microglial core genes was lost upon culture of isolated cells ex vivo but could be acquired by human induced pluripotent stem cell (iPSC)-derived microglial precursors transplanted into mouse brains. CXCR4 and PTGER4 were higher expressed in subcortical white matter compared to cortical grey matter microglia, and ADGRG1 was downregulated in microglia obtained from normal-appearing white and grey matter tissue of multiple sclerosis (MS) brains. Single-cell RNA sequencing of microglia from active lesions, obtained early during MS, revealed downregulation of homeostasis-associated GPCR genes and upregulation of CXCR4 expression in a small subset of MS-associated lesional microglia. Functional presence of low levels of CXCR4 on human microglia was confirmed using flow cytometry and transwell migration towards SDF-1. Microglia abundantly expressed the GPCR down-stream signaling mediator genes GNAI2 (αi2), GNAS (αs), and GNA13 (α13), the latter particularly in white matter. Drugs against several microglia GPCRs are available to target microglia in brain diseases. In conclusion, transcriptome profiling allowed us to identify expression of GPCRs that may contribute to brain (patho)physiology and have diagnostic and therapeutic potential in human microglia.

Clinical features and prognostic factors in Covid-19: A prospective cohort study.

BACKGROUND: Mortality rates are high among hospitalized patients with COVID-19, especially in those intubated on the ICU. Insight in pathways associated with unfavourable outcome may lead to new treatment strategies. METHODS: We performed a prospective cohort study of patients with COVID-19 admitted to general ward or ICU who underwent serial blood sampling. To provide insight in the pathways involved in disease progression, associations were estimated between outcome risk and serial measurements of 64 biomarkers in potential important pathways of COVID-19 infection (inflammation, tissue damage, complement system, coagulation and fibrinolysis) using joint models combining Cox regression and linear mixed-effects models. For patients admitted to the general ward, the primary outcome was admission to the ICU or mortality (unfavourable outcome). For patients admitted to the ICU, the primary outcome was 12-week mortality. FINDINGS: A total of 219 patients were included: 136 (62%) on the ward and 119 patients (54%) on the ICU; 36 patients (26%) were included in both cohorts because they were transferred from general ward to ICU. On the general ward, 54 of 136 patients (40%) had an unfavourable outcome and 31 (23%) patients died. On the ICU, 54 out of 119 patients (45%) died. Unfavourable outcome on the general ward was associated with changes in concentrations of IL-6, IL-8, IL-10, soluble Receptor for Advanced Glycation End Products (sRAGE), vascular cell adhesion molecule 1 (VCAM-1) and Pentraxin-3. Death on the ICU was associated with changes in IL-6, IL-8, IL-10, sRAGE, VCAM-1, Pentraxin-3, urokinase-type plasminogen activator receptor, IL-1-receptor antagonist, CD14, procalcitonin, tumor necrosis factor alfa, tissue factor, complement component 5a, Growth arrest-specific 6, angiopoietin 2, and lactoferrin. Pathway analysis showed that unfavourable outcome on the ward was mainly driven by chemotaxis and interleukin production, whereas death on ICU was associated with a variety of pathways including chemotaxis, cell-cell adhesion, innate host response mechanisms, including the complement system, viral life cycle regulation, angiogenesis, wound healing and response to corticosteroids. INTERPRETATION: Clinical deterioration in patients with severe COVID-19 involves multiple pathways, including chemotaxis and interleukin production, but also endothelial dysfunction, the complement system, and immunothrombosis. Prognostic markers showed considerable overlap between general ward and ICU patients, but we identified distinct differences between groups that should be considered in the development and timing of interventional therapies in COVID-19. FUNDING: Amsterdam UMC, Amsterdam UMC Corona Fund, and Dr. C.J. Vaillant Fonds.

Absence of B Cells in Brainstem and White Matter Lesions Associates With Less Severe Disease and Absence of Oligoclonal Bands in MS.

OBJECTIVE: To determine whether B-cell presence in brainstem and white matter (WM) lesions is associated with poorer pathological and clinical characteristics in advanced MS autopsy cases. METHODS: Autopsy tissue of 140 MS and 24 control cases and biopsy tissue of 24 patients with MS were examined for CD20+ B cells and CD138+ plasma cells. The presence of these cells was compared with pathological and clinical characteristics. In corresponding CSF and plasma, immunoglobulin (Ig) G ratio and oligoclonal band (OCB) patterns were determined. In a clinical cohort of 73 patients, the presence of OCBs was determined during follow-up and compared to status at diagnosis. RESULTS: In 34% of active and 71% of mixed active/inactive lesions, B cells were absent, which correlated with less pronounced meningeal B-cell infiltration (p < 0.0001). The absence of B cells and plasma cells in brainstem and WM lesions was associated with a longer disease duration (p = 0.001), less frequent secondary progressive MS compared with relapsing and primary progressive MS (p < 0.0001 and p = 0.046, respectively), a lower proportion of mixed active/inactive lesions (p = 0.01), and less often perivascular T-cell clustering (p < 0.0001). Moreover, a lower CSF IgG ratio (p = 0.006) and more frequent absence of OCBs (p < 0.0001) were noted. In a clinical cohort, numbers of patients without OCBs in CSF were increased at follow-up (27.4%). CONCLUSIONS: The absence of B cells is associated with a favorable clinical and pathological profile. This finding may reflect extremes of a continuum of genetic or environmental constitution, but also a regression of WM humoral immunopathology in the natural course of advanced MS.

White matter lesions in multiple sclerosis are enriched for CD20dim CD8+ tissue-resident memory T cells.

Brain CD8+ CD69+ tissue-resident memory T (TRM ) cells comprise a CD20dim subset, which is proportionally larger in CD103-negative TRM cells. In multiple sclerosis (MS) lesions, CD20dim TRM -cell proportions are increased. CD20-expression is associated with higher levels of CXCR6, Ki-67, and granzyme B, supporting CD20dim TRM cells as a relevant subset in MS.

IgG Immune Complexes Break Immune Tolerance of Human Microglia.

Microglia are phagocytic cells involved in homeostasis of the brain and are key players in the pathogenesis of multiple sclerosis (MS). A hallmark of MS diagnosis is the presence of IgG Abs, which appear as oligoclonal bands in the cerebrospinal fluid. In this study, we demonstrate that myelin obtained post mortem from 8 out of 11 MS brain donors is bound by IgG Abs. Importantly, we show that IgG immune complexes strongly potentiate activation of primary human microglia by breaking their tolerance for microbial stimuli, such as LPS and Poly I:C, resulting in increased production of key proinflammatory cytokines, such as TNF and IL-1ß. We identified FcγRI and FcγRIIa as the two main responsible IgG receptors for the breaking of immune tolerance of microglia. Combined, these data indicate that IgG immune complexes potentiate inflammation by human microglia, which may play an important role in MS-associated inflammation and the formation of demyelinating lesions.

Single-cell mass cytometry reveals complex myeloid cell composition in active lesions of progressive multiple sclerosis.

Myeloid cells contribute to inflammation and demyelination in the early stages of multiple sclerosis (MS), but it is still unclear to what extent these cells are involved in active lesion formation in progressive MS (PMS). Here, we have harnessed the power of single-cell mass cytometry (CyTOF) to compare myeloid cell phenotypes in active lesions of PMS donors with those in normal-appearing white matter from the same donors and control white matter from non-MS donors. CyTOF measurements of a total of 74 targeted proteins revealed a decreased abundance of homeostatic and TNFhi microglia, and an increase in highly phagocytic and activated microglia states in active lesions of PMS donors. Interestingly, in contrast to results obtained from studies of the inflammatory early disease stages of MS, infiltrating monocyte-derived macrophages were scarce in active lesions of PMS, suggesting fundamental differences of myeloid cell composition in advanced stages of PMS.