Microglia Require CD4 T Cells to Complete the Fetal-to-Adult Transition
Emanuela PasciutoOliver T. BurtonCarlos P. RocaVasiliki LagouWenson D. RajanTom TheysRenzo MancusoRaúl Y. TitoLubna KouserZsuzsanna Callaerts‐VeghAlerie Guzman de la FuenteTeresa PrezzemoloLoriana MascaliAleksandra BrajicCarly E. WhyteLidia YshiiAnna Martínez‐MurianaMichelle NaughtonAndrew YoungAlena MoudráPierre LemaîtreSuresh PoovathingalJeroen RaesBart De StrooperDenise FitzgeraldJames DooleyAdrian Liston
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The brain is a site of relative immune privilege. Although CD4 T cells have been reported in the central nervous system, their presence in the healthy brain remains controversial, and their function remains largely unknown. We used a combination of imaging, single cell, and surgical approaches to identify a CD69+ CD4 T cell population in both the mouse and human brain, distinct from circulating CD4 T cells. The brain-resident population was derived through in situ differentiation from activated circulatory cells and was shaped by self-antigen and the peripheral microbiome. Single-cell sequencing revealed that in the absence of murine CD4 T cells, resident microglia remained suspended between the fetal and adult states. This maturation defect resulted in excess immature neuronal synapses and behavioral abnormalities. These results illuminate a role for CD4 T cells in brain development and a potential interconnected dynamic between the evolution of the immunological and neurological systems. VIDEO ABSTRACT.Keywords:
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Despite the significant role microglia play in the pathology of multiple sclerosis (MS), medications that act within the central nervous system (CNS) to inhibit microglia have not yet been identified as treatment options.We screened 1040 compounds with the aim of identifying inhibitors of microglia to reduce neuroinflammation.The NINDs collection of 1040 compounds, where most are therapeutic medications, was tested at 10 µM final concentration on lipopolysaccharide (LPS)-activated human microglia. An ELISA was run on the media to measure the level of TNF-α as an indicator of microglia activity. For compounds that reduce LPS-activated TNF-α levels by over 50%, considered as a potential inhibitor of interest, toxicity tests were conducted to exclude non-specific cytotoxicity. Promising compounds were subjected to further analyses, including toxicity to other CNS cell types, and multiplex assays.Of 1040 compounds tested, 123 reduced TNF-α levels of LPS-activated microglia by over 50%. However, most of these were cytotoxic to microglia at the concentration tested while 54 were assessed to be non-toxic. Of the latter, spironolactone was selected for further analyses. Spironolactone reduced TNF-α levels of activated microglia by 50-60% at 10 µM, and this concentration did not kill microglia, neurons or astrocytes. In multiplex assays, spironolactone reduced several molecules in activated microglia. Finally, during the screening, we identified 9 compounds that elevated further the TNF-α levels in LPS-activated microglia.Many of the non-toxic compounds identified in this screen as inhibitors of microglia, including spironolactone, may be explored as viable therapeutic options in MS.
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Immune privilege is a term applied to organs that have a unique relationship with the immune response. These sites prohibit the spread of inflammation, since even minor episodes can threaten organ integrity and function. Once thought to be a passive process relying on physical barriers, immune privilege is now viewed as an active process, which uses multiple mechanisms to maintain organ function. The prototypic organ of immune privilege has been the eye, where the spread of inflammation can threaten vision. Nearly 10 years ago, we discussed the finding that Fas ligand (FasL) was constitutively expressed in the eye and played a major role in immune privilege by inducing apoptosis in inflammatory cells that enter the eye. In this review, we reexamine the original evidence for the role of FasL in immune privilege, update progress on some of the concepts, and discuss some of the issues that remain unresolved.
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Although anatomical barriers and soluble mediators have been implicated in immune privilege, it appears that the apoptotic cell death of Fas+ cells by tissue-associated CD95 ligand (Fas ligand, FasL) is an important component. One clinical example of the function of an immune privileged site is the success of human corneal transplants, where a very high percentage of transplants accept without tissue matching or immunosuppressive therapy. Since the mouse cornea expresses abundant Fas ligand and immune privilege has been implicated in the success of these transplants, we examined the role of FasL in corneal transplantation. Our results show that human corneas express functional FasL capable of killing Fas+ lymphoid cells in an in vitro culture system. Using a mouse model for corneal allograft transplantation, FasL+ orthografts were accepted at a rate of 45%, whereas FasL- grafts, or normal grafts transplanted to Fas- mice, were rejected 100% of the time. Histological analysis found that FasL+ grafts contained apoptotic mononuclear cells indicating the induction of apoptosis by the graft, while rejecting FasL- corneas contained numerous inflammatory cells without associated apoptosis. Taken together our results demonstrate that FasL expression on the cornea is a major factor in corneal allograft survival and, thus, we provide an explanation for one of the most successful tissue transplants performed in humans.
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Microglia, the major inflammatory cells of the brain, play a pivotal role in the initiation and progression of Alzheimer's Disease (AD) by either phagozytosing amyloid‐β deposits or by releasing cytotoxic and pro‐inflammatory substances in response to activation by amyloid‐β aggregates, including amyloid‐β oligomers (AβO). We here propose microglial Kv1.3 channels as a novel target for curbing the harmful effects of Aβ‐induced microglia activation. Microglia isolated from the brains of adult 5xFAD mice expressed higher levels of Kv1.3 than microglia from age‐matched control mice. We further observed strong Kv1.3 immunoreactivities in microglia associated with amyloid plaques in brains of 5xFAD mice. Proof for the functional importance of Kv1.3 in microglia comes from our observations that the Kv1.3 blocker PAP‐1 inhibits AβO‐stimulated NO production as well as microglia‐mediated neurotoxicity in dissociated cultures and organotypic brain slices. A 6‐week course of daily PAP‐1 injections also reduced the degree of microglia activation in 5xFAD mice. In contrast, Kv1.3 blockade with PAP‐1 does not affect phagocytosis of Aβ aggregates by microglia. These observations suggest that Kv1.3 blockers might preferentially inhibit microglia mediated neuronal killing without affecting beneficial functions such as scavenging of debris. Supported by NIH and Alzheimer's Association
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Abstract Age-associated microglial dysfunction contributes to the accumulation of amyloid-β (Aβ) plaques in Alzheimer’s disease. Although several studies have shown age-related declines in the phagocytic capacity of myeloid cells, relatively few have examined phagocytosis of normally aged microglia. Furthermore, much of the existing data on aging microglial function have been generated in accelerated genetic models of Alzheimer’s disease. Here we found that naturally aged microglia phagocytosed less Aβ over time. To gain a better understanding of such dysfunction, we assessed differences in gene expression between young and old microglia that either did or did not phagocytose Aβ. Young microglia had both phagocytic and neuronal maintenance signatures indicative of normal microglial responses, whereas, old microglia, regardless of phagocytic status, exhibit signs of broad dysfunction reflective of underlying neurologic disease states. We also found downregulation of many phagocytic receptors on old microglia, including TREM2, an Aβ phagocytic receptor. TREM2 protein expression was diminished in old microglia and loss of TREM2 + microglia was correlated with impaired Aβ uptake, suggesting a mechanism for phagocytic dysfunction in old microglia. Combined, our work reveals that normally aged microglia have broad changes in gene expression, including defects in Aβ phagocytosis that likely underlies the progression to neurologic disease.
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The eye, like the brain and reproductive organs, possesses inherent immune privilege, and inflammation is self-regulated so as to preserve the organ functions. Studies over the past 30 years have provided insights of the multiple mechanisms of immune privilege. At present, three major lines of thought prevail regarding the molecular mechanisms of immune privilege in the eye: there are (1) anatomical, cellular, and molecular barriers in the eye; (2) eye-derived immunological tolerance, the so-called anterior chamber-associated immune deviation; and (3) immune suppressive intraocular microenvironment. In this review, the mechanisms of immune privilege that have been learned from ocular inflammation animal models, especially corneal transplantation, are described. Roles of new B7 family molecules on local immune regulation within the cornea are also introduced.
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Author(s): Rice, Rachel Anne | Advisor(s): Green, Kim N | Abstract: Microglia are the immune competent cells of the central nervous system (CNS). During development, microglia play critical roles in pruning synapses and refining neuronal connectivity. In the adult brain, microglia constantly survey the parenchyma for cellular damage or invading pathogens. Upon detection of such events, microglia become activated and shift to a phagocytic phenotype, secreting pro-inflammatory molecules and adopting an amoeboid morphology. As part of the resolution/repair process, microglia return to a surveillant state and produce anti-inflammatory molecules. Unfortunately, with severe insults, such as traumatic brain injury or chronic neurodegeneration, microglia remain activated and contribute to an inflammatory process that is never, or poorly, resolved. In this way, we hypothesize that microglia contribute deleteriously to functional outcomes.The goal of my dissertation is to determine the contributions of microglia to neuronal health and cognition in both the healthy and injured brain. The direct assessment of microglia-specific contributions is possible due to the discovery by our lab that microglia are dependent upon signaling through the colony-stimulating factor 1 receptor (CSF1R) for their survival. Treatment with a small-molecule CSF1R inhibitor eliminates g99% of microglia from the adult mouse brain. Critically, microglia fully repopulate the CNS upon withdrawal of the CSF1R inhibitor, effectively renewing this cellular compartment. Using a genetic model of inducible neuronal loss, I have determined that the elimination of microglia during a lesion is detrimental to cellular health, while the elimination of microglia following a lesion results in the reversal of many lesion-induced deficits. Importantly, this research suggests that the microglia-mediated immune response is beneficial during insult or injury, but deleterious after such an event. Moreover, repopulation of the brain with new microglia following neuronal lesioning largely resets the inflammatory milieu and confers functional benefits.Finally, long-term elimination of microglia was employed in order to determine if these cells shape the synaptic landscape in the healthy adult brain, as they do during development. Indeed, I found that microglial elimination in healthy adult mice results in brain-wide and robust increases in dendritic spine numbers and excitatory neuronal connectivity, indicating that microglia modulate synaptic function throughout the course of the lifetime.
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The expression of Fas ligand (FasL) in the eye is an important factor in the maintenance of immune privilege. Although FasL expression in donor corneas contributes to prolonged survival of orthotopic corneal allografts in solid organ transplantation, FasL gene-transfected tissues reportedly lead to graft destruction through neutrophil recruitment. Differences in the effects of FasL have been attributed to different roles of soluble FasL (sFasL) and membrane FasL (mFasL). This is based on the presumption that the signals through sFasL and mFasL differ, with one causing apoptosis and the other activating inflammation. It was recently reported that inflammation caused by FasL was inhibited at an immune-privileged site, and therefore the effects of FasL may depend on differences in the anatomic sites where FasL-expressing cells are located. In this article, we discuss the role of sFasL and mFasL in ocular immune privilege.
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