Microglia, immune cells of the central nervous system (CNS), are important for tissue development and maintenance and are implicated in CNS disease, but we lack understanding of human fetal microglia development. Single-cell gene expression and bulk chromatin profiles of microglia at 9 to 18 gestational weeks (GWs) of human fetal development were generated. Microglia were heterogeneous at all studied GWs. Microglia start to mature during this developmental period and increasingly resemble adult microglia with CNS-surveilling properties. Chromatin accessibility increases during development with associated transcriptional networks reflective of adult microglia. Thus, during early fetal development, microglia progress toward a more mature, immune-sensing competent phenotype, and this might render the developing human CNS vulnerable to environmental perturbations during early pregnancy.

V‐type immunoglobulin domain‐containing suppressor of T‐cell activation (VISTA) is a negative checkpoint regulator (NCR) involved in inhibition of T cell‐mediated immunity. Expression changes of other NCRs (PD‐1, PD‐L1/L2, CTLA‐4) during inflammation of the central nervous system (CNS) were previously demonstrated, but VISTA expression in the CNS has not yet been explored. Here, we report that in the human and mouse CNS, VISTA is most abundantly expressed by microglia, and to lower levels by endothelial cells. Upon TLR stimulation, VISTA expression was reduced in primary neonatal mouse and adult rhesus macaque microglia in vitro. In mice, microglial VISTA expression was reduced after lipopolysaccharide (LPS) injection, during experimental autoimmune encephalomyelitis (EAE), and in the accelerated aging Ercc1 Δ/− mouse model. After LPS injection, decreased VISTA expression in mouse microglia was accompanied by decreased acetylation of lysine residue 27 in histone 3 in both its promoter and enhancer region. ATAC‐sequencing indicated a potential regulation of VISTA expression by Pu.1 and Mafb, two transcription factors crucial for microglia function. Finally, our data suggested that VISTA expression was decreased in microglia in multiple sclerosis lesion tissue, whereas it was increased in Alzheimer's disease patients. This study is the first to demonstrate that in the CNS, VISTA is expressed by microglia, and that VISTA is differentially expressed in CNS pathologies.

The aim was to investigate whether microRNA (miRNA) expression is modulated by inhaled corticosteroid (ICS) treatmentWe performed genome-wide miRNA analysis on bronchial biopsies of 69 moderate/severe chronic obstructive pulmonary disease (COPD) patients at baseline and after 6- and 30-month treatment with the ICS fluticasone propionate or placebo. The effect of ICS on miRNA expression was validated in differentiated primary bronchial epithelial cultures, and functional studies were conducted in BEAS-2B cells. MiRNAs affected by ICS and their predicted targets were compared to an independent miRNA dataset of bronchial brushings from COPD patients and healthy controls.Treatment with ICS for both 6 and 30 months significantly altered the expression of four miRNAs, including miR-320d, which was increased during ICS treatment compared with placebo. The ICS-induced increase of miR-320d was confirmed in primary airway epithelial cells. MiR-320d negatively correlated targets were enriched for pro-inflammatory genes and were increased in the bronchial brushes of patients with lower lung function in the independent dataset. Overexpression of miR-320d in BEAS-2B cells dampened cigarette smoke extract-induced pro-inflammatory activity via inhibition of nuclear factor-κB.Collectively, we identified miR-320d as a novel mediator of ICS, regulating the pro-inflammatory response of the airway epithelium.

Astrocytes fulfil many functions in the central nervous system (CNS), including contribution to the blood brain barrier, synapse formation, and trophic support. In addition, they can mount an inflammatory response and are heterogeneous in morphology and function. To extensively characterize astrocyte subtypes, we FACS‐isolated and gene expression profiled distinct astrocyte subtypes from three central nervous system regions; forebrain, hindbrain and spinal cord. Astrocyte subpopulations were separated based on GLAST/SLC1A3 and ACSA‐2/ATP1B2 cell surface expression. The local brain environment proved key in establishing different transcriptional programs in astrocyte subtypes. Transcriptional differences between subtypes were also apparent in experimental autoimmune encephalomyelitis (EAE) mice, where these astrocyte subtypes showed distinct responses. While gene expression signatures associated with blood–brain barrier maintenance were lost, signatures involved in neuroinflammation and neurotoxicity were increased in spinal cord astrocytes, especially during acute disease stages. In chronic stages of EAE, this reactive astrocyte signature was slightly decreased, while obtaining a more proliferative profile, which might be relevant for glia scar formation and tissue regeneration. Morphological heterogeneity of astrocytes previously indicated the presence of astrocyte subtypes, and here we show diversity based on transcriptome variation associated with brain regions and differential responsiveness to a neuroinflammatory insult (EAE).

Negative checkpoint regulators (NCR) are intensely pursued as targets to modulate the immune response in cancer and autoimmunity. A large variety of NCR is expressed by central nervous system (CNS)-resident cell types and is associated with CNS homeostasis, interactions with peripheral immunity and CNS inflammation and disease. Immunotherapy blocking NCR affects the CNS as patients can develop neurological issues including encephalitis and multiple sclerosis (MS). How these treatments affect the CNS is incompletely understood, since expression and function of NCR in the CNS are only beginning to be unravelled. V-type immunoglobulin-like suppressor of T cell activation (VISTA) is an NCR that is expressed primarily in the haematopoietic system by myeloid and T cells. VISTA regulates T cell quiescence and activation and has a variety of functions in myeloid cells including efferocytosis, cytokine response and chemotaxis. In the CNS, VISTA is predominantly expressed by microglia and macrophages of the CNS. In this review, we summarize the role of NCR in the CNS during health and disease. We highlight expression of VISTA across cell types and CNS diseases and discuss the function of VISTA in microglia and during CNS ageing, inflammation and neurodegeneration. Understanding the role of VISTA and other NCR in the CNS is important considering the adverse effects of immunotherapy on the CNS, and in view of their therapeutic potential in CNS disease.

Background An innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo. Methods Two experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array. Results Microglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia. Conclusions Our results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.

Tomatidine, a natural steroidal alkaloid from unripe green tomatoes has been shown to exhibit many health benefits. We recently provided in vitro evidence that tomatidine reduces the infectivity of Dengue virus (DENV) and Chikungunya virus (CHIKV), two medically important arthropod-borne human infections for which no treatment options are available. We observed a potent antiviral effect with EC50 values of 0.82 μM for DENV-2 and 1.3 μM for CHIKV-LR. In this study, we investigated how tomatidine controls CHIKV infectivity. Using mass spectrometry, we identified that tomatidine induces the expression of p62, CD98, metallothionein and thioredoxin-related transmembrane protein 2 in Huh7 cells. The hits p62 and CD98 were validated, yet subsequent analysis revealed that they are not responsible for the observed antiviral effect. In parallel, we sought to identify at which step of the virus replication cycle tomatidine controls virus infectivity. A strong antiviral effect was seen when in vitro transcribed CHIKV RNA was transfected into Huh7 cells treated with tomatidine, thereby excluding a role for tomatidine during CHIKV cell entry. Subsequent determination of the number of intracellular viral RNA copies and viral protein expression levels during natural infection revealed that tomatidine reduces the RNA copy number and viral protein expression levels in infected cells. Once cells are infected, tomatidine is not able to interfere with active RNA replication yet it can reduce viral protein expression. Collectively, the results delineate that tomatidine controls viral protein expression to exert its antiviral activity. Lastly, sequential passaging of CHIKV in presence of tomatidine did not lead to viral resistance. Collectively, these results further emphasize the potential of tomatidine as an antiviral treatment towards CHIKV infection.

BackgroundAn innate immune memory response can manifest in two ways: immune training and immune tolerance, which refers to an enhanced or suppressed immune response to a second challenge, respectively. Exposing monocytes to moderate-to-high amounts of bacterial lipopolysaccharide (LPS) induces immune tolerance, whereas fungal β-glucan (BG) induces immune training. In microglia, it has been shown that different LPS inocula in vivo can induce either immune training or tolerance. Few studies focused on impact of BG on microglia and were only performed in vitro. The aim of the current study was to determine whether BG activates and induces immune memory in microglia upon peripheral administration in vivo.MethodsTwo experimental designs were used. In the acute design, mice received an intraperitoneal (i.p.) injection with PBS, 1 mg/kg LPS or 20 mg/kg BG and were terminated after 3 h, 1 or 2 days. In the preconditioning design, animals were first challenged i.p. with PBS, 1 mg/kg LPS or 20 mg/kg BG. After 2, 7 or 14 days, mice received a second injection with PBS or 1 mg/kg LPS and were sacrificed 3 h later. Microglia were isolated by fluorescence-activated cell sorting, and cytokine gene expression levels were determined. In addition, a self-developed program was used to analyze microglia morphological changes. Cytokine concentrations in serum were determined by a cytokine array.ResultsMicroglia exhibited a classical inflammatory response to LPS, showing significant upregulation of Tnf, Il6, Il1β, Ccl2, Ccl3 and Csf1 expression, three h after injection, and obvious morphological changes 1 and 2 days after injection. With an interval of 2 days between two challenges, both BG and LPS induced immune training in microglia. The training effect of LPS changed into immune tolerance after a 7-day interval between 2 LPS challenges. Preconditioning with BG and LPS resulted in increased morphological changes in microglia in response to a systemic LPS challenge compared to naïve microglia.ConclusionsOur results demonstrate that preconditioning with BG and LPS both induced immune training of microglia at two days after the first challenge. However, with an interval of 7 days between the first and second challenge, LPS-preconditioning resulted in immune tolerance in microglia.