Transcriptome analysis reveals the contribution of oligodendrocyte and radial glia-derived cues for maintenance of microglia identity.

Microglia are increasingly being recognized as druggable targets in neurodegenerative disorders, and good in vitro models are crucial to address cell biological questions. Major challenges are to recapitulate the complex microglial morphology and their in vivo transcriptome. We have therefore exposed primary microglia from adult rhesus macaques to a variety of different culture conditions including exposure to soluble factors as M-CSF, IL-34, and TGF-ß as well as serum replacement approaches, and compared their morphologies and transcriptomes to those of mature, homeostatic in vivo microglia. This enabled us to develop a new, partially serum-free, monoculture protocol, that yields high numbers of ramified cells. We also demonstrate that exposure of adult microglia to M-CSF or IL-34 induces similar transcriptomes, and that exposure to TGF-ß has much less pronounced effects than it does on rodent microglia. However, regardless of culture conditions, the transcriptomes of in vitro and in vivo microglia remained substantially different. Analysis of differentially expressed genes inspired us to perform 3D-spherical coculture experiments of microglia with oligodendrocytes and radial glia. In such spheres, microglia signature genes were strongly induced, even in the absence of neurons and astrocytes. These data reveal a novel role for oligodendrocyte and radial glia-derived cues in the maintenance of microglial identity, providing new anchor points to study microglia in health and disease.

Recombinant myelin oligodendrocyte glycoprotein quality modifies evolution of experimental autoimmune encephalitis in macaques.

Experimental autoimmune encephalitis (EAE) is a well-recognized model for the study of human acquired demyelinating diseases (ADD), a group of inflammatory disorders of the central nervous system (CNS) characterized by inflammation, myelin loss, and neurological impairment of variable severity. In rodents, EAE is typically induced by active immunization with a combination of myelin-derived antigen and a strong adjuvant as complete Freund's adjuvant (CFA), containing components of the mycobacterial wall, while myelin antigen alone or associated with other bacterial components, as lipopolysaccharides (LPS), often fails to induce EAE. In contrast to this, EAE can be efficiently induced in non-human primates by immunization with the recombinant human myelin oligodendrocyte glycoprotein (rhMOG), produced in Escherichia coli (E. coli), purified and formulated with incomplete Freund's adjuvant (IFA), which lacks bacterial elements. Here, we provide evidence indicating how trace amounts of bacterial contaminants within rhMOG may influence the course and severity of EAE in the cynomolgus macaque immunized with rhMOG/IFA. The residual amount of E. coli contaminants, as detected with mass spectrometry within rhMOG protein stocks, were found to significantly modulate the severity of clinical, radiological, and histologic hallmarks of EAE in macaques. Indeed, animals receiving the purest rhMOG showed milder disease severity, increased numbers of remissions, and reduced brain damage. Histologically, these animals presented a wider diversity of lesion types, including changes in normal-appearing white matter and prephagocytic lesions. Non-human primates EAE model with milder histologic lesions reflect more accurately ADD and permits to study of the pathogenesis of disease initiation and progression.

TLR-Induced IL-12 and CCL2 Production by Myeloid Cells Is Dependent on Adenosine A3 Receptor-Mediated Signaling.

TLR-induced signaling potently activates cells of the innate immune system and is subject to regulation at different levels. Inflammatory conditions are associated with increased levels of extracellular adenosine, which can modulate TLR-induced production of cytokines through adenosine receptor-mediated signaling. There are four adenosine receptor subtypes that induce different signaling cascades. In this study, we demonstrate a pivotal contribution of adenosine A3 receptor (A3R)-mediated signaling to the TLR4-induced expression of IL-12 in different types of human myeloid APC. In dendritic cells, IL-12 and CCL2 responses as evoked by TLR2, 3, 4, 5, and 8, as well as IL-12 responses evoked by whole pathogens, were all reduced when A3R-mediated signaling was blocked. As a result, concomitant production of IFN-γ and IL-17 by T cells was significantly inhibited. We further show that selective inhibition of A3R-mediated signaling reduced TLR-induced phosphorylation of the transcription factor STAT1 at tyrosine 701. Next-generation sequencing revealed that A3R-mediated signaling controls the expression of metallothioneins, known inhibitors of STAT1 phosphorylation. Together our results reveal a novel regulatory layer of innate immune responses, with a central role for metallothioneins and autocrine/paracrine signaling via A3Rs.

Adverse immunostimulation caused by impurities: The dark side of biopharmaceuticals.

Drug safety is an important issue, especially in the experimental phases of development. Adverse immunostimulation (AI) is sometimes encountered following treatment with biopharmaceuticals, which can be life-threatening if it results in a severe systemic inflammatory reaction. Biopharmaceuticals that unexpectedly induce an inflammatory response still enter the clinic, even while meeting all regulatory requirements. Impurities (of microbial origin) in biopharmaceuticals are an often-overlooked cause of AI. This demonstrates that the current guidelines for quality control and safety pharmacology testing are not flawless. Here, based on two case examples, several shortcomings of the guidelines are discussed. The most important of these are the lack of sensitivity for impurities, lack of testing for pyrogens other than endotoxin, and the use of insensitive animal species and biomarkers in preclinical investigations. Moreover, testing for the immunotoxicity of biopharmaceuticals is explicitly not recommended by the international guidelines. Publication of cases of AI is pivotal, both to increase awareness and to facilitate scientific discussions on how to prevent AI in the future.

VISTA expression by microglia decreases during inflammation and is differentially regulated in CNS diseases.

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.

Fibronectin aggregates promote features of a classically and alternatively activated phenotype in macrophages.

BACKGROUND: Means to promote endogenous remyelination in multiple sclerosis (MS) benefit from insights into the role of inhibitory molecules that preclude remyelination. Fibronectin assembles into aggregates in MS, which impair oligodendrocyte differentiation and remyelination. Microglia and macrophages are required for complete remyelination and normally switch from a pro-inflammatory classical phenotype upon demyelination to a supportive alternative phenotype during remyelination. Here, we investigated the role of fibronectin aggregates in modulating microglia and macrophage behavior and phenotypes. METHODS: Bone marrow-derived macrophages and microglia from newborn rats were exposed to (a) plasma fibronectin coatings; (b) coatings of deoxycholate-insoluble fibronectin aggregates; (c) interferon-γ (IFNγ) treatment, as an inducer of the pro-inflammatory classically activated phenotype; (d) interleukin-4 (IL-4) treatment, to promote the pro-regenerative anti-inflammatory alternatively activated phenotype; or (e) left unstimulated on uncoated plastic. To examine the in vitro effects of the different stimulations on cell behavior and phenotype, proliferation, phagocytosis, morphology, and pro- and anti-inflammatory features were assessed. RESULTS: In line with a classically activated phenotype, exposure of microglia and macrophages to both plasma fibronectin and fibronectin aggregates induced an amoeboid morphology and stimulated phagocytosis by macrophages. Furthermore, as observed upon IFNγ treatment, coatings of aggregated, but not plasma fibronectin, promoted nitric oxide release by microglia and macrophages. Remarkably, fibronectin aggregates induced nitric oxide release in an integrin-independent manner. In addition, fibronectin aggregates, but not plasma fibronectin, increased the expression of arginase-1, similarly as observed upon treatment with IL-4. Proteomic analysis revealed that aggregates of fibronectin act as a scaffold for other proteins, including Hsp70 and thrombospondin-1, which may clarify the induction of both pro-inflammatory and anti-inflammatory features in macrophages cultured on fibronectin aggregate, but not plasma fibronectin coatings. CONCLUSIONS: Macrophages and microglia grown on aggregated fibronectin coatings adopt a distinct phenotype compared to plasma fibronectin coatings, showing pro-inflammatory and anti-inflammatory features. Therefore, the pathological fibronectin aggregates in MS lesions may impair remyelination by promoting and/or retaining several classically activated phenotypic features in microglia and macrophages.

Inflammasome-induced IL-1ß secretion in microglia is characterized by delayed kinetics and is only partially dependent on inflammatory caspases.

Inflammasomes are multiprotein complexes that link pathogen recognition and cellular stress to the processing of the proinflammatory cytokine interleukin-1ß (IL-1ß). Whereas inflammasome-mediated activation is heavily studied in hematopoietic macrophages and dendritic cells, much less is known about microglia, resident tissue macrophages of the brain that originate from a distinct progenitor. To directly compare inflammasome-mediated activation in different types of macrophages, we isolated primary microglia and hematopoietic macrophages from adult, healthy rhesus macaques. We analyzed the expression profile of NOD (nucleotide-binding oligomerization domain)-like receptors, adaptor proteins, and caspases and characterized inflammasome activation and regulation in detail. We here demonstrate that primary microglia can respond to the same innate stimuli as hematopoietic macrophages. However, microglial responses are more persistent due to lack of negative regulation on pro-IL-1ß expression. In addition, we show that while caspase 1, 4, and 5 activation is pivotal for inflammasome-induced IL-1ß secretion by hematopoietic macrophages, microglial secretion of IL-1ß is only partially dependent on these inflammatory caspases. These results identify key cell type-specific differences that may aid the development of strategies to modulate innate immune responses in the brain.

Sialylation of Campylobacter jejuni endotoxin promotes dendritic cell-mediated B cell responses through CD14-dependent production of IFN-ß and TNF-α.

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis and often precedes development of Guillain-Barré syndrome (GBS), a life-threatening paralytic disease. The incorporation of the carbohydrate sialic acid into C. jejuni lipooligosaccharides (LOS) is associated with increased severity of gastroenteritis and with induction of GBS; however, the underlying mechanisms remain completely unknown. In this study, we demonstrate that sialic acids in C. jejuni endotoxin enhance the rapid production of IFN-ß and TNF-α by human dendritic cells (DCs). Using neutralizing Abs and receptors it was shown that these DC-derived cytokines promote the proliferation of human mucosal B cells in a T cell-independent manner. The production of both IFN-ß and TNF-α by DCs in response to LOS requires CD14, and the amplified response of DCs to sialylated C. jejuni LOS is CD14 dependent. Together, these results indicate that sialylation of C. jejuni LOS increases DC activation and promotes subsequent B cell responses through CD14-driven production of IFN-ß and TNF-α. This enhanced DC/B cell response may explain the increased pathogenicity of sialylated C. jejuni and may be key to the initiation of B cell-mediated autoimmunity in GBS.

Statins amplify TLR-induced responses in microglia via inhibition of cholesterol biosynthesis.

Statins inhibit the endogenous intracellular mevalonate pathway and exposure to statins affects innate and adaptive immune responses. Different statins are currently under evaluation as (co)therapy in neuro-inflammatory diseases like multiple sclerosis. However, there are important discrepancies in the reported effects of statins on innate immune responses in different cell types. Studies to characterize such responses in clinically relevant primary cells are currently lacking. In this study, we investigated the effect of statins on Toll-like receptor (TLR)-induced responses of microglia, the resident macrophages of the central nervous system (CNS). Exposure of primary microglia from adult rhesus monkeys to different statins strongly amplified pro-inflammatory cytokine protein and mRNA levels in response to myeloid differentiation primary response gene 88-dependent TLR activation in particular. Rather than affecting nuclear facor-κB activation levels, statin exposure affected stress-activated protein/Jun-amino-terminal and p38 kinase signaling pathways. Mechanistic studies using specific pathway inhibitors and rescue experiments show that statin-induced inhibition of cholesterol biosynthesis, rather than inhibition of isoprenylation, was mainly responsible for the amplified TLR responses. Additionally, microglia were more sensitive to statin-mediated effects than bone marrow-derived macrophages of the same donor. This correlated to lower intrinsic microglial expression levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the enzyme targeted by statins. Amplification of TLR-induced responses in microglia by statin exposure might contribute to the generation of a more pro-inflammatory CNS microenvironment which can be of relevance for the pathogenesis of neuroinflammatory disorders.

The microtubule regulator stathmin is an endogenous protein agonist for TLR3.

TLR3 recognizes dsRNAs and is considered of key importance to antiviral host-defense responses. TLR3 also triggers neuroprotective responses in astrocytes and controls the growth of axons and neuronal progenitor cells, suggesting additional roles for TLR3-mediated signaling in the CNS. This prompted us to search for alternative, CNS-borne protein agonists for TLR3. A genome-scale functional screening of a transcript library from brain tumors revealed that the microtubule regulator stathmin is an activator of TLR3-dependent signaling in astrocytes, inducing the same set of neuroprotective factors as the known TLR3 agonist polyinosinic:polycytidylic acid. This activity of stathmin crucially depends on a long, negatively charged alpha helix in the protein. Colocalization of stathmin with TLR3 on astrocytes, microglia, and neurons in multiple sclerosis-affected human brain indicates that as an endogenous TLR3 agonist, stathmin may fulfill previously unsuspected regulatory roles during inflammation and repair in the adult CNS.

The design of chronic toxicology studies of monoclonal antibodies: implications for the reduction in use of non-human primates.

The changing environment of monoclonal antibody (mAb) development is impacting on the cost of drug development and the use of experimental animals, particularly non-human primates (NHPs). The drive to reduce these costs is huge and involves rethinking and improving nonclinical studies to make them more efficient and more predictive of man. While NHP use might be unavoidable in many cases because of the exquisite specificity and consequent species selectivity of mAbs, our increasing knowledge base can be used to improve drug development and maximise the output of experimental data. Data on GLP regulatory toxicology studies for 58mAbs were obtained from 10 companies across a wide range of therapeutic indications. These data have been used to investigate current practice and identify study designs that minimise NHP use. Our analysis shows that there is variation in the number of animals used for similar studies. This information has been used to develop practical guidance and make recommendations on the use of science-based rationale to design studies using fewer animals taking into account the current regulatory guidance. There are eight recommendations intended to highlight areas for consideration. They include guidance on the main group size, the inclusion of recovery groups and the number of dose groups used in short and long term chronic toxicology studies.

P2Y6 receptor-mediated signaling amplifies TLR-induced pro-inflammatory responses in microglia.

TLR-induced signaling initiates inflammatory responses in cells of the innate immune system. These responses are amongst others characterized by the secretion of high levels of pro-inflammatory cytokines, which are tightly regulated and adapted to the microenvironment. Purinergic receptors are powerful modulators of TLR-induced responses, and we here characterized the effects of P2Y6 receptor (P2RY6)-mediated signaling on TLR responses of rhesus macaque primary bone marrow-derived macrophages (BMDM) and microglia, using the selective P2RY6 antagonist MRS2578. We demonstrate that P2RY6-mediated signaling enhances the levels of TLR-induced pro-inflammatory cytokines in microglia in particular. TLR1, 2, 4, 5 and 8-induced responses were all enhanced in microglia, whereas such effects were much less pronounced in BMDM from the same donors. Transcriptome analysis revealed that the overall contribution of P2RY6-mediated signaling to TLR-induced responses in microglia leads to an amplification of pro-inflammatory responses. Detailed target gene analysis predicts that P2RY6-mediated signaling regulates the expression of these genes via modulation of the activity of transcription factors NFAT, IRF and NF-κB. Interestingly, we found that the expression levels of heat shock proteins were strongly induced by inhibition of P2RY6-mediated signaling, both under homeostatic conditions as well as after TLR engagement. Together, our results shed new lights on the specific pro-inflammatory contribution of P2RY6-mediated signaling in neuroinflammation, which might open novel avenues to control brain inflammatory responses.

The nearest neighbor nuclei method to objectify analysis of pertussis toxin-induced clustering.

The in vivo histamine sensitization test (HIST) has historically been performed to guarantee the safety of acellular per­tussis vaccine batches. Non-compliance of batches is primarily associated with the presence of low levels of pertussis toxin (PTx). Because of ethical, standardization and scientific reasons, a variety of alternative in vitro approaches have been studied to replace the lethal HIST. A broadly applied and partially accepted method is the CHO cell clustering test, which is based on the clustered growth pattern of CHO cells when exposed to minute amounts of PTx. One of the major hurdles for global application of the CHO clustering test is the manual assessment of the clusters, which is associated with suboptimal reproducibility of test outcomes and is time-consuming. Here, various parameters of CHO cell nuclei were evaluated in search for a reliable, objective read-out parameter. We demonstrate that the distance between each nucleus and its nearest neighbor (3N method) is the most suitable parameter to assess clustered cell growth. This method detects 2.8 mIU PTx/mL and thereby complies with the requirement set for the sensitivity of the CHO clustering test based on visual reading. In commercial acellular pertussis vaccines spiked with PTx, the method detects 45 mIU/mL PTx, which is substantially lower than the 181-725 mIU/mL PTx detected by visual interpretation. The 3N method thus allows objective and sensitive assessment of CHO clustering and thereby encourages broad and global implementation of the in vitro test as an alternative to the HIST.

Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

Lactoferrin Retargets Human Adenoviruses to TLR4 to Induce an Abortive NLRP3-Associated Pyroptotic Response in Human Phagocytes.

Despite decades of clinical and preclinical investigations, we still poorly grasp our innate immune response to human adenoviruses (HAdVs) and their vectors. In this study, we explored the impact of lactoferrin on three HAdV types that are being used as vectors for vaccines. Lactoferrin is a secreted globular glycoprotein that influences direct and indirect innate immune response against a range of pathogens following a breach in tissue homeostasis. The mechanism by which lactoferrin complexes increases HAdV uptake and induce maturation of human phagocytes is unknown. We show that lactoferrin redirects HAdV types from species B, C, and D to Toll-like receptor 4 (TLR4) cell surface complexes. TLR4-mediated internalization of the HAdV-lactoferrin complex induced an NLRP3-associated response that consisted of cytokine release and transient disruption of plasma membrane integrity, without causing cell death. These data impact our understanding of HAdV immunogenicity and may provide ways to increase the efficacy of HAdV-based vectors/vaccines.

Exposure of Microglia to Interleukin-4 Represses NF-κB-Dependent Transcription of Toll-Like Receptor-Induced Cytokines.

Interleukin (IL)-4 is a cytokine that affects both adaptive and innate immune responses. In the central nervous system, microglia express IL-4 receptors and it has been described that IL-4-exposed microglia acquire anti-inflammatory properties. We here demonstrate that IL-4 exposure induces changes in the cell surface protein expression profile of primary rhesus macaque microglia and enhances their potential to induce proliferation of T cells with a regulatory signature. Moreover, we show that Toll like receptor (TLR)-induced cytokine production is broadly impaired in IL-4-exposed microglia at the transcriptional level. IL-4 type 2 receptor-mediated signaling is shown to be crucial for the inhibition of microglial innate immune responses. TLR-induced nuclear translocalization of NF-κB appeared intact, and we found no evidence for epigenetic modulation of target genes. By contrast, nuclear extracts from IL-4-exposed microglia contained significantly less NF-κB capable of binding to its DNA consensus site. Further identification of the molecular mechanisms that underlie the inhibition of TLR-induced responses in IL-4-exposed microglia may aid the design of strategies that aim to modulate innate immune responses in the brain, for example in gliomas.

Activation of Toll-like receptors and dendritic cells by a broad range of bacterial molecules.

Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, their capacity to activate TLRs and to affect DC maturation, cytokine production and T cell polarizing capacity were determined. Different bacterial species differed in their potency to affect these parameters. In general, on the DC level differences were found in the maturation-inducing capacity of gram-negative and gram-positive bacteria. Remarkably, these differences did not result in differential polarization of the T cell response. With respect to TLRs, TLR4 activation by pathogens correlated with their ability to induce DC maturation, while for TLR2 and TLR5 such a correlation was absent. Taken together, this study provides insight into qualitative differences and general effects of pathogen-derived molecules on dendritic cells.

An Overview of in vitro Methods to Study Microglia.

Neuroinflammation is a common feature in neurodegenerative diseases and strategies to modulate neuroinflammatory processes are increasingly considered as therapeutic options. In such strategies, glia cells rather than neurons represent the cellular targets. Microglia, the resident macrophages of the central nervous system, are principal players in neuroinflammation and detailed cellular biological knowledge of this particular cell type is therefore of pivotal importance. The last decade has shed new light on the origin, characteristics and functions of microglia, underlining the need for specific in vitro methodology to study these cells in detail. In this review we provide a comprehensive overview of existing methodology such as cell lines, stem cell-derived microglia and primary dissociated cell cultures, as well as discuss recent developments. As there is no in vitro method available yet that recapitulates all hallmarks of adult homeostatic microglia, we also discuss the advantages and limitations of existing models across different species.

Modulation of the cytokine network in human adult astrocytes by human herpesvirus-6A.

Human herpesvirus-6A (HHV-6A) is a common pathogen whose role in CNS disorders including multiple sclerosis remains controversial. To understand how HHV-6A could influence inflammatory pathways in the CNS, we infected cultured human adult astrocytes and examined the expression of 268 cytokines, chemokines, growth factors and their receptors by gene profiling. HHV-6 infection alone had little effect on the astrocyte gene profile but strongly altered the astrocyte response to proinflammatory cytokines. Under those conditions astrocytes express higher levels of anti-inflammatory mediators including IL-10 and IL-11, chemotactic factors, growth factors and factors controlling type I interferon production. Our data suggest that HHV-6 itself does not evoke a pro-inflammatory response in astrocytes but rather triggers immune modulatory factors in the face of inflammation.