Microglia play critical roles in brain development, homeostasis, and neurological disorders. Here, we report that human microglial-like cells (iMGLs) can be differentiated from iPSCs to study their ...function in neurological diseases, like Alzheimer’s disease (AD). We find that iMGLs develop in vitro similarly to microglia in vivo, and whole-transcriptome analysis demonstrates that they are highly similar to cultured adult and fetal human microglia. Functional assessment of iMGLs reveals that they secrete cytokines in response to inflammatory stimuli, migrate and undergo calcium transients, and robustly phagocytose CNS substrates. iMGLs were used to examine the effects of Aβ fibrils and brain-derived tau oligomers on AD-related gene expression and to interrogate mechanisms involved in synaptic pruning. Furthermore, iMGLs transplanted into transgenic mice and human brain organoids resemble microglia in vivo. Together, these findings demonstrate that iMGLs can be used to study microglial function, providing important new insight into human neurological disease.
•Fully defined and efficient generation of human microglial-like cells from iPSCs•Whole-transcriptome and functional validation of iPSC-derived microglia (iMGLs)•Novel in vitro and in vivo applications for studying neurological diseases•iMGLs can be used to interrogate AD gene function
Abud et al. describe a fully defined protocol for the generation of human iPSC-derived microglia-like cells (iMGLs). Whole-transcriptome and novel functional analyses were used to validate microglial identity. iMGLs provide a platform for studying microglial function in health and disease.
Despite the development of highly effective treatments for relapsing-remitting multiple sclerosis (MS), limited progress has been made in addressing primary progressive or secondary progressive MS, ...both of which lead to loss of oligodendrocytes and neurons and axons, and to irreversible accumulation of disability. Neuroinflammation is central to all forms of MS. The current effective therapies for relapsing-remitting MS target the peripheral immune system; these treatments, however, have repeatedly failed in progressive MS. Greater understanding of inflammation driven by CNS-resident cells - including astrocytes and microglia - is, therefore, required to identify novel potential therapeutic opportunities. Advances in imaging, biomarker analysis and genomics suggest that microglia and astrocytes have central roles in the progressive disease process. In this Review, we provide an overview of the involvement of astrocytes and microglia at major sites of pathology in progressive MS. We discuss current and future therapeutic approaches to directly target glial cells, either to inhibit pathogenic functions or to restore homeostatic functions lost during the course of the disease. We also discuss how bidirectional communication between astrocytes and microglia needs to be considered, as therapeutic targeting of one is likely to alter the functions of the other.
Alzheimer's disease (AD) and Parkinson's disease (PD) are characterized by brain accumulation of aggregated amyloid-beta (Aβ) and alpha-synuclein (αSYN), respectively. In order to develop effective ...therapies, it is crucial to understand how the Aβ/αSYN aggregates can be cleared. Compelling data indicate that neuroinflammatory cells, including astrocytes and microglia, play a central role in the pathogenesis of AD and PD. However, how the interplay between the two cell types affects their clearing capacity and consequently the disease progression remains unclear.
The aim of the present study was to investigate in which way glial crosstalk influences αSYN and Aβ pathology, focusing on accumulation and degradation. For this purpose, human-induced pluripotent cell (hiPSC)-derived astrocytes and microglia were exposed to sonicated fibrils of αSYN or Aβ and analyzed over time. The capacity of the two cell types to clear extracellular and intracellular protein aggregates when either cultured separately or in co-culture was studied using immunocytochemistry and ELISA. Moreover, the capacity of cells to interact with and process protein aggregates was tracked using time-lapse microscopy and a customized "close-culture" chamber, in which the apical surfaces of astrocyte and microglia monocultures were separated by a <1 mm space.
Our data show that intracellular deposits of αSYN and Aβ are significantly reduced in co-cultures of astrocytes and microglia, compared to monocultures of either cell type. Analysis of conditioned medium and imaging data from the "close-culture" chamber experiments indicate that astrocytes secrete a high proportion of their internalized protein aggregates, while microglia do not. Moreover, co-cultured astrocytes and microglia are in constant contact with each other via tunneling nanotubes and other membrane structures. Notably, our live cell imaging data demonstrate that microglia, when attached to the cell membrane of an astrocyte, can attract and clear intracellular protein deposits from the astrocyte.
Taken together, our data demonstrate the importance of astrocyte and microglia interactions in Aβ/αSYN clearance, highlighting the relevance of glial cellular crosstalk in the progression of AD- and PD-related brain pathology.
The emerging roles of microglia are currently being investigated in the healthy and diseased brain with a growing interest in their diverse functions. In recent years, it has been demonstrated that ...microglia are not only immunocentric, but also neurobiological and can impact neural development and the maintenance of neuronal cell function in both healthy and pathological contexts. In the disease context, there is widespread consensus that microglia are dynamic cells with a potential to contribute to both central nervous system damage and repair. Indeed, a number of studies have found that microenvironmental conditions can selectively modify unique microglia phenotypes and functions. One novel mechanism that has garnered interest involves the regulation of microglial function by microRNAs, which has therapeutic implications such as enhancing microglia-mediated suppression of brain injury and promoting repair following inflammatory injury. Furthermore, recently published articles have identified molecular signatures of myeloid cells, suggesting that microglia are a distinct cell population compared to other cells of myeloid lineage that access the central nervous system under pathological conditions. Thus, new opportunities exist to help distinguish microglia in the brain and permit the study of their unique functions in health and disease.
Infiltrating monocyte-derived macrophages (MDMs) and resident microglia dominate central nervous system (CNS) injury sites. Differential roles for these cell populations after injury are beginning to ...be uncovered. Here, we show evidence that MDMs and microglia directly communicate with one another and differentially modulate each other's functions. Importantly, microglia-mediated phagocytosis and inflammation are suppressed by infiltrating macrophages. In the context of spinal cord injury (SCI), preventing such communication increases microglial activation and worsens functional recovery. We suggest that macrophages entering the CNS provide a regulatory mechanism that controls acute and long-term microglia-mediated inflammation, which may drive damage in a variety of CNS conditions.
Sphingosine 1‐phosphate receptors (S1PR) are G protein‐coupled and compose a family with five subtypes, S1P1R–S1P5R. The drug Gilenya® (Novartis, Basel, Switzerland) (Fingolimod; FTY720) targets ...S1PRs and was the first oral therapy for patients with relapsing‐remitting multiple sclerosis (MS). The phosphorylated form of FTY720 (pFTY720) binds S1PRs causing initial agonism, then subsequent receptor internalization and functional antagonism. Internalization of S1P1R attenuates sphingosine 1‐phosphate (S1P)‐mediated egress of lymphocytes from lymph nodes, limiting aberrant immune function in MS. pFTY720 also exerts direct actions on neurons and glial cells which express S1PRs. In this study, we investigated the regulation of pro‐inflammatory chemokine release by S1PRs in enriched astrocytes and microglial cultures. Astrocytes and microglia were stimulated with lipopolysaccharide (LPS) and increases in C‐X‐C motif chemokine 5 (CXCL5), also known as LIX (lipopolysaccharide‐induced CXC chemokine) expression were quantified. Results showed that pFTY720 attenuated LPS‐induced CXCL5 (LIX) protein release from astrocytes, as did the S1P1R selective agonist, SEW2871. In addition, pFTY720 blocked messenger ribonucleic acid (mRNA) transcription of the chemokines, (i) CXCL5/LIX, (ii) C‐X‐C motif chemokine 10 (CXCL10) also known as interferon gamma‐induced protein 10 (IP10) and (iii) chemokine (C‐C motif) ligand 2 (CCL2) also known as monocyte chemoattractant protein 1 (MCP1). Interestingly, inhibition of sphingosine kinase attenuated LPS‐induced increases in mRNA levels of all three chemokines, suggesting that LPS‐TLR4 (Toll‐like receptor 4) signalling may enhance chemokine expression via S1P‐S1PR transactivation. Lastly, these observations were not limited to astrocytes since we also found that pFTY720 attenuated LPS‐induced release of CXCL5 from microglia. These data highlight a role for S1PR signalling in regulating the levels of chemokines in glial cells and support the notion that pFTY720 efficacy in multiple sclerosis may involve the direct modulation of astrocytes and microglia.
In addition to its immunomodulatory actions, the multiple sclerosis (MS) drug FTY720 binds to membrane sphingosine 1‐phosphate (S1P) receptors and regulates the release of inflammatory mediators from glial cells in the central nervous system. In this study, we found that lipopolysaccharide (LPS) induced the release of chemokines, in particular C‐X‐C motif chemokine 5 (CXCL5), from astrocytes and microglia and that phosphorylated FTY720 (pFTY720) blocks this process. Our data suggest that LPS induces transactivation of glial S1P receptors, in a sphingosine kinase‐dependent manner, resulting in chemokine synthesis and that pFTY720 interrupts this signalling cascade by causing intracellular accumulation of membrane S1P1R. Therefore, FTY720's therapeutic effects in MS may include modulation of neuroinflammation through direct regulation of S1PR signalling in astrocytes and microglia.
Sphingosine-1-phosphate receptor (S1PR) modulators have entered clinical practice as immune-modulators for the treatment of multiple sclerosis (MS). Pharmacologic modulation of S1PR expression on ...lymphocytes inhibits these cells capacity to respond to the S1P gradient within regional lymph nodes (LNs) (and thymus) that promotes their exit into peripheral circulation. The resultant peripheral blood restricted lymphopenia is considered to underlie the capacity of S1PR modulators to reduce new inflammatory lesion formation in MS in the absence of global immune suppression. These modulators also regulate entry of selective lymphocyte populations and dendritic cells (DCs) into LNs and modulate sphingosine-1-phosphate (S1P) cell signaling networks that govern the generation of specific cell subsets within LNs. S1PR modulators that access the CNS can also have functional effects within this compartment since S1PRs are expressed by cells comprising the blood brain barrier (BBB) and by those within the parenchyma, including neurons, astrocytes, oligodendrocytes and microglia. Absence of S1P1 receptor (S1PR1) on astrocytes reduces disease severity in experimental autoimmune encephalomyelitis (EAE). Even under conditions that inhibit cell responses to the natural ligand, S1PR modulators can continue to induce active signaling responses; such responses may be relevant for promoting neuroprotection and augmenting tissue repair within the CNS.
Multifocal inflammatory lesions featuring destruction of lipid-rich myelin are pathologic hallmarks of multiple sclerosis. Lesion activity is assessed by the extent and composition of myelin uptake ...by myeloid cells present in such lesions. In the inflamed CNS, myeloid cells are comprised of brain-resident microglia, an endogenous cell population, and monocyte-derived macrophages, which infiltrate from the systemic compartment. Using microglia isolated from the adult human brain, we demonstrate that myelin phagocytosis is dependent on the polarization state of the cells. Myelin ingestion is significantly enhanced in cells exposed to TGF-β compared with resting basal conditions and markedly reduced in classically activated polarized cells. Transcriptional analysis indicated that TGF-β-treated microglia closely resembled M0 cells. The tyrosine kinase phagocytic receptor MerTK was one of the most upregulated among a select number of differentially expressed genes in TGF-β-treated microglia. In contrast, MerTK and its known ligands, growth arrest-specific 6 and Protein S, were downregulated in classically activated cells. MerTK expression and myelin phagocytosis were higher in CNS-derived microglia than observed in monocyte-derived macrophages, both basally and under all tested polarization conditions. Specific MerTK inhibitors reduced myelin phagocytosis and the resultant anti-inflammatory biased cytokine responses for both cell types. Defining and modulating the mechanisms that regulate myelin phagocytosis has the potential to impact lesion and disease evolution in multiple sclerosis. Relevant effects would include enhancing myelin clearance, increasing anti-inflammatory molecule production by myeloid cells, and thereby permitting subsequent tissue repair.
Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unclear whether distinct ...microglia subpopulations or states contribute to different stages of pain development and maintenance. Using single-cell RNA-sequencing, we show that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in male as compared to female mice. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following peripheral nerve injury. Apolipoprotein E (Apoe) is the top upregulated gene in spinal cord microglia at chronic time points after peripheral nerve injury in mice. Furthermore, polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell RNA sequencing analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a link between ApoE and chronic pain in humans.
Cells of the adaptive and innate immune systems in the brain parenchyma and in the meningeal spaces contribute to physiologic functions and disease states in the central nervous system (CNS). Animal ...studies have demonstrated the involvement of immune constituents, along with major histocompatibility complex (MHC) molecules, in neural development and rare genetic disorders (e.g., colony stimulating factor 1 receptor CSF1R deficiency). Genome wide association studies suggest a comparable role of the immune system in humans. Although the CNS can be the target of primary autoimmune disorders, no current experimental model captures all of the features of the most common human disorder placed in this category, multiple sclerosis (MS). Such features include spontaneous onset, environmental contributions, and a recurrent/progressive disease course in a genetically predisposed host. Numerous therapeutic interventions related to antigen and cytokine specific therapies have demonstrated effectiveness in experimental autoimmune encephalomyelitis (EAE), the animal model used to define principles underlying immune‐mediated mechanisms in MS. Despite the similarities in the two diseases, most treatments used to ameliorate EAE have failed to translate to the human disease. As directly demonstrated in animal models and implicated by correlative studies in humans, adaptive and innate immune constituents within the systemic compartment and resident in the CNS contribute to the disease course of neurodegenerative and neurobehavioral disorders. The expanding knowledge of the molecular properties of glial cells provides increasing insights into species related variables. These variables affect glial bidirectional interactions with the immune system as well as their own production of “immune molecules” that mediate tissue injury and repair.
• Poor recapitulation of human neuroinflammatory disease by animal models;• Caused by species differences in the immune and glial systems• Resulting from >75 million years of evolutionary divergence, unique environmental niches, evolutionary pressures, and aggressive inbreeding.
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