The Formyl Peptide Receptor 2 Regulates Microglial Phenotype Through Immunometabolism: Implications for Alzheimer’s Disease

Microglia are key players in the pathology of Alzheimer’s disease (AD), driving chronic inflammation, oxidative stress, and the altered metabolism seen in the brains of patients. With clinical trials continuing to fail, new approaches towards drug development are critical. Strategies to reduce microglial activation may therefore be a viable therapeutic approach to tackling AD. Formyl peptide receptor 2 (Fpr2), which drives peripheral inflammatory resolution, is expressed in microglia. However, its functional role in neuroinflammation is unclear. This thesis provides evidence to support the peripheral findings of Fpr2 stimulation, wherein it may also hold promise for exploitation as a therapeutic for neurodegenerative disorders, including AD. We also highlight novel findings surrounding the modulation of both oxidative stress and microglial metabolism associated with Fpr2 activation. Under inflammatory conditions, we report that selective agonists for Fpr2 modulate the microglial inflammatory response, actively shifting from a pro-inflammatory to a pro-resolving phenotype, emphasised by the reduction of pro-inflammatory cytokines and concomitant increases in both pro-resolving cytokines and phagocytosis. Metabolic shifting away from glycolysis was also observed for pro-resolving microglia. Moreover, we describe for the first time that Fpr2 completely reverses reactive oxygen species (ROS) production from the mitochondria and NADPH oxidase enzymes following an inflammatory stimulus. We also highlight that the toxic oligomeric amyloid (oAβ) facilitates microglial ROS production and subsequent metabolic changes without triggering an inflammatory response. oAβ facilitated NADPH oxidase activation, which in turn resulted in the activation of glucose 6-phosphate dehydrogenase (G6PD), the rate limiting step for the pentose phosphate pathway. This metabolic pathway is responsible for producing NADPH, which in turn NADPH oxidases exploit for further ROS production. These changes resulted in noticeable reductions in both microglial glycolysis and oxidative phosphorylation. We present data underlining that Fpr2/3 stimulation reverses oAβ-induced ROS production, with a resultant reduction in G6PD activity and the return of homeostatic glycolysis. These oAβ-induced microglial changes triggered the apoptosis of SH-SY5Y cells in co-culture with BV-2 microglia. However, supporting our interest in Fpr2/3 for therapeutic approaches to neurodegenerative diseases, post-treatment with a select agonist for the receptor successfully prevented apoptosis of these neuronal like SH-SY5Y cells. This original data unveils novel functions of Fpr2/3 in the central nervous system (CNS), supplementing the well-established pro-resolving functions the receptor facilitates within the periphery. The combination of pro-resolving, anti-oxidative, immunometabolic and anti-apoptotic functions of Fpr2/3 support the exploitation of this receptor for therapeutic research into multiple different CNS disorders, including AD

Hyalocyte origin, structure, and imaging

Hyalocytes have been recognized as resident tissue macrophages of the vitreous body since the mid-19th century. Despite this, knowledge about their origin, turnover, and dynamics is limited. Historically, initial studies on the origin of hyalocytes used light and electron microscopies. Modern investigations across species including rodents and humans will be described. Novel imaging is now available to study human hyalocytes in vivo. The shared ontogeny with retinal microglia and their eventual interdependence as well as differences will be discussed. Owing to a common origin as myeloid cells, hyalocytes and retinal microglia have similarities, but hyalocytes appear to be distinct as resident macrophages of the vitreous body.</p

Mechanisms of leukocyte migration across the blood–retina barrier

Immune-mediated inflammation in the retina is regulated by a combination of anatomical, physiological and immuno-regulatory mechanisms, referred to as the blood–retina barrier (BRB). The BRB is thought to be part of the specialised ocular microenvironment that confers protection or “immune privilege” by deviating or suppressing destructive inflammation. The barrier between the blood circulation and the retina is maintained at two separate anatomical sites. These are the endothelial cells of the inner retinal vasculature and the retinal pigment epithelial cells on Bruch’s membrane between the fenestrated choroidal vessels and the outer retina. The structure and regulation of the tight junctions forming the physical barrier are described. For leukocyte migration across the BRB to occur, changes are needed in both the leukocytes themselves and the cells forming the barrier. We review how the blood–retina barrier is compromised in various inflammatory diseases and discuss the mechanisms controlling leukocyte subset migration into the retina in uveoretinitis in more detail. In particular, we examine the relative roles of selectins and integrins in leukocyte interactions with the vascular endothelium and the pivotal role of chemokines in selective recruitment of leukocyte subsets, triggering adhesion, diapedesis and migration of inflammatory cells into the retinal tissue

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body

BackgroundThe eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive.ResultsIn this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes.ConclusionOur study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface

Microglial Corpse Clearance: Lessons From Macrophages

From development to aging and disease, the brain parenchyma is under the constant threat of debris accumulation, in the form of dead cells and protein aggregates. To prevent garbage buildup, the brain is equipped with efficient phagocytes: the microglia. Microglia are similar, but not identical to other tissue macrophages, and in this review, we will first summarize the differences in the origin, lineage and population maintenance of microglia and macrophages. Then, we will discuss several principles that govern macrophage phagocytosis of apoptotic cells (efferocytosis), including the existence of redundant recognition mechanisms ("find-me" and "eat-me") that lead to a tight coupling between apoptosis and phagocytosis. We will then describe that resulting from engulfment and degradation of apoptotic cargo, phagocytes undergo an epigenetic, transcriptional and metabolic rewiring that leads to trained immunity, and discuss its relevance for microglia and brain function. In summary, we will show that neuroimmunologists can learn many lessons from the well-developed field of macrophage phagocytosis biology.This work was supported by grants from the Spanish Ministry of Science, Innovation and Universities with FEDER funds (BFU2015-66689 and RYC-2013-12817), a Basque Government Department of Education project (PI_2016_1_0011), and a Fundacion Tatiana Perez de Guzman el Bueno project (P-048-FTPGB 2018) to AS. In addition, MM-R is recipient of a predoctoral fellowship from the University of the Basque Country UPV/EHU. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No funding is available for open access publications from our institution or other sources

Innate Immune Memory and Transcriptional Profiling of Microglia

Microglia are the local immune cells of the brain. They are important for brain development, neural network formation, and protect against infections and damage. Chronic activation of microglia, like in neurodegenerative conditions, leads to perturbed microglia functions and contributes to brain pathology. The aim of the research by Yang Heng, presented in this thesis, was to characterize how microglia are affected by peripheral inflammation, DNA damage-induced aging and post mortem intervals. Systemic administration of bacterial lipopolysaccharide or fungal β-glucan changed the inflammatory response of microglia to a following inflammatory challenge, a phenomenon known as innate immune memory. Depending on the interval between the challenges, either an enhanced or a reduced response was detected. Immune training of microglia was also observed in accelerated aging mice, that are deficient in DNA damage repair. Targeting this deficiency in DNA damage repair to microglia did not induce training, but resulted in microglia loss and replacement. Characterization of the gene expression profiles, or transcriptomes, of microglia has increased our understanding of their identity and function. Here, we demonstrate that the transcriptomes of microglia nuclei and cells are very similar, and that also includes nuclei from frozen brain tissue. This offers the opportunity to analyze large collections of already collected and frozen tissue samples. Brain tissue samples are collected after a post mortem delay, which had a surprisingly limited influence on microglia transcriptomes. These findings justify the use of the archived postmortem brain specimens to study microglia transcriptomes, under both physiological and pathological conditions

VEGFR3 and Notch signaling in angiogenesis

Blood and lymphatic vessels form extensive networks throughout the body, which function in order to deliver oxygen and nutrients to the tissues, to remove extravasated fluid and to absorb dietary lipids. The formation of new blood and lymphatic vessels (termed angiogenesis and lymphangiogenesis) is critical during embryonic development and in the adult, and is regulated by multiple signaling pathways. Vascular Endothelial Growth Factors (VEGFs) and their receptors (VEGFRs), as well as the Notch signaling system, are key governors of blood and lymphatic endothelial cell fate, and regulate angiogenesis and lymphangiogenesis in health and disease. Despite the numerous recent advances in the field of vascular biology, many steps in the complex processes of angiogenesis and lymphangiogenesis remain unclear. In this study we investigated the role of VEGFR3 signaling in blood endothelial cells, tip cell specification, as well as the interplay of the receptor with the VEGFR2 and the Notch signaling pathways during angiogenesis. VEGFR3 is a tyrosine kinase receptor that is mainly expressed in lymphatic endothelial cells in the adult. We observed VEGFR3 expression in sprouts that guide the blood vascular endothelium in angiogenic conditions. VEGFR3 blockade with a monoclonal antibody displayed synergistic properties with simultaneous VEGFR2 targeting in reducing angiogenesis and inhibiting tumor growth. Furthermore we found that Notch signaling suppresses VEGFR3 expression in endothelial cells, identifying VEGFR3 as a novel tip cell marker, which is normally repressed by Notch activation. In the next step we employed a combination of genetic and in vitro models to show that loss of VEGFR3 results in a hypervascular phenotype, accompanied by loss of Notch signaling. VEGFR3 could be stimulated by VEGFC and activate Notch in blood endothelial cells. Our results point towards a mechanism where VEGFC produced by macrophages at the vascular front acts via VEGFR3 to activate Notch and turn tip into stalk cells; thus promoting the formation of stable vascular loops. Furthermore we identified the transcription factor FOXC2 as the downstream target of the VEGFC/VEGFR3/Notch signaling cascade. These data reinforce the idea that VEGFR3 has two distinct signaling modalities, one ligand-dependent and one ligand-independent, and that different perturbations in VEGFR3 expression and function result in diverse vascular phenotypes. Subsequently we investigated the interplay of VEGFR2 and VEGFR3 in postnatal angiogenesis and lymphangiogenesis, using a genetic approach of conditional mutagenesis. Various combinations of genetic ablation of VEGFRs and pharmacological inhibition of Notch showed that VEGFR2 is irreplaceable during sprouting angiogenesis, also in endothelial cells with low Notch signaling, and that it acts upstream of VEGFR3 expression in angiogenic settings. On the other hand VEGFR3 suppressed VEGFR2 expression in a negative feedback loop. Finally we employed for the first time lymphatic endothelial specific deletion of VEGFRs in postnatal conditions and found no significant role for VEGFR2 in lymphatic vessel growth and remodeling, while VEGFR3 signaling was indispensable. Our results unravel previously unknown roles for VEGFR3 in sprouting angiogenesis and provide new insight into the signaling cross-talk of the receptor with other important regulators of blood vessel development. Increasing our understanding of the molecular mechanisms underlying this process is crucial in order to comprehend the pathophysiology of angiogenesis-related diseases, characterized by excessive or insufficient blood vessel growth, and promote the emergence of novel angiogenic therapies.Not availabl