Microglia-derived Macrophages Research Articles

Role of NLRP3 Inflammasomes in Monocyte and Microglial Recruitments in Choroidal Neovascularization.

Although the pathogenesis of choroidal neovascularization (CNV) is largely unknown in age-related macular degeneration (AMD), inflammasomes may contribute to CNV development and progression. To understand the role NLRP3 inflammasomes in CNV, we used Ccr2RFPCx3cr1GFP dual-reporter mice and immunostaining techniques to confirm localization of NLRP3 inflammasomes in the laser-induced CNV (LCNV) lesions. Confocal microscopy was used to image and quantify LCNV volumes. MCC950 was used as NLRP3 inhibitor. ELISA and quantitative RT-PCR were used to confirm the activation of NLRP3 by monitoring the expression of IL-1β protein and mRNA in choroidal tissues from LCNV mice. In addition, NLRP3 (-/-) LCNV mice were used to investigate whether NLRP3 inflammasomes contribute to the development of LCNV lesions. We observed that red fluorescent protein (RFP)-positive monocyte-derived macrophages and GFP-positive microglia-derived macrophages, in addition to other cell types, were localized in LCNV lesions at day 7 post-laser injury. In addition, NLRP3 inflammasomes are associated with LCNV lesions. Inhibition of NLRP3 inflammasomes, using MCC950, caused an increased Ccr2RFP-positive macrophages, Cx3cr1GFP-positive microglia, and other cells, resulting in an increase in total lesion size. NLRP3 (-/-) LCNV mice showed significantly increased lesion size compared with age-matched controls. Inhibition of NLRP3 resulted in decreased IL-1β mRNA and protein expression in the choroidal tissues, suggesting that increased lesion size may not be directly related to IL-1β.

Tumor and immune cell types interact to produce heterogeneous phenotypes of pediatric high-grade glioma.

Pediatric high-grade gliomas (PHGG) are aggressive brain tumors with 5-year survival rates ranging from <2% to 20% depending upon subtype. PHGG presents differently from patient to patient and is intratumorally heterogeneous, posing challenges in designing therapies. We hypothesized that heterogeneity occurs because PHGG comprises multiple distinct tumor and immune cell types in varying proportions, each of which may influence tumor characteristics. We obtained 19 PHGG samples from our institution's pediatric brain tumor bank. We constructed a comprehensive transcriptomic dataset at the single-cell level using single-cell RNA-Seq (scRNA-Seq), identified known glial and immune cell types, and performed differential gene expression and gene set enrichment analysis. We conducted multi-channel immunofluorescence (IF) staining to confirm the transcriptomic results. Our PHGG samples included 3 principal predicted tumor cell types: astrocytes, oligodendrocyte progenitors (OPCs), and mesenchymal-like cells (Mes). These cell types differed in their gene expression profiles, pathway enrichment, and mesenchymal character. We identified a macrophage population enriched in mesenchymal and inflammatory gene expression as a possible source of mesenchymal tumor characteristics. We found evidence of T-cell exhaustion and suppression. PHGG comprises multiple distinct proliferating tumor cell types. Microglia-derived macrophages may drive mesenchymal gene expression in PHGG. The predicted Mes tumor cell population likely derives from OPCs. The variable tumor cell populations rely on different oncogenic pathways and are thus likely to vary in their responses to therapy.

Insights into the co-evolution of glioblastoma and associated macrophages

Glioblastoma (GBM) is one of the most immunosuppressive and heterogeneous tumors with limited treatment options. Most studies relied on treatment-experienced patient samples to elucidate the origins of tumor heterogeneity, introducing bias into the analysis. The analysis of samples from multifocal GBM patients, in which independent lesions arise from the same progenitor and undergo parallel evolution, enables the study of the natural evolution of GBM while removing the effect of therapy on the emergence of heterogeneity. This enables the identification of critical events in the evolution of GBM and the unbiased study of subtype progression, diversity, and invasive potential. The tumor microenvironment of GBM undergoes significant changes throughout tumor progression. Recent studies have highlighted the switch from an abundance of resident microglia-derived macrophages in earlier stages to the prevalence of blood-derived macrophages in later stages of GBM. There is conclusive evidence that these alterations cannot be viewed in isolation and that the tumor microenvironment co-evolves with tumor cells during cancer progression. Together with an increasingly hypoxic environment, this culminates in highly immunosuppressive conditions, resulting in a feedback loop further reinforcing evolutionary changes in the tumor. A new study now provides a unique look at the natural evolution of GBM, identifies critical events in its development, and has the potential to help improve the diagnosis and therapy of this deadly disease.

Association of glioma CD44 expression with glial dynamics in the tumour microenvironment and patient prognosis

Because of the heterogeneity of lower-grade gliomas (LGGs), patients show various survival outcomes that are not reliably predicted by histological classification. The tumour microenvironment (TME) contributes to the initiation and progression of brain LGGs. Identifying potential prognostic markers based on the immune and stromal components in the TME will provide new insights into the dynamic modulation of these two components of the TME in LGGs. We applied ESTIMATE to calculate the ratio of immune and stromal components from The Cancer Genome Atlas database. After combined differential gene expression analysis, protein–protein interaction network construction and survival analysis, CD44 was screened as an independent prognostic factor and subsequently validated utilizing data from the Chinese Glioma Genome Atlas database. To decipher the association of glioma cell CD44 expression with stromal cells in the TME and tumour progression, RT–qPCR, cell viability and wound healing assays were employed to determine whether astrocytes enhance glioma cell viability and migration by upregulating CD44 expression. Surprisingly, M1 macrophages were identified as positively correlated with CD44 expression by CIBERSORT analysis. CD44+ glioma cells were further suggested to interact with microglia-derived macrophages (M1 phenotype) via osteopontin signalling on the basis of single-cell sequencing data. Overall, we found that astrocytes could elevate the CD44 expression level of glioma cells, enhancing the recruitment of M1 macrophages that may promote glioma stemness via osteopontin-CD44 signalling. Thus, glioma CD44 expression might coordinate with glial activities in the TME and serve as a potential therapeutic target and prognostic marker for LGGs.

Sodium Tanshinone IIA Sulfonate Protects Against Cerebral Ischemia–reperfusion Injury by Inhibiting Autophagy and Inflammation

Sodium tanshinone IIA sulfonate (STS) can protect against brain damage induced by stroke. However, the neural protection mechanism of STS remains unclear. We investigated whether STS performs its protective function by suppressing autophagy and inflammatory activity during brain injury. We established a transient middle cerebral artery occlusion and reperfusion (MCAO/R) model by blocking the left middle cerebral artery with a thread inserted through the internal carotid artery for 1 h, followed by reperfusion for 48 h either with or without STS and the autophagy inhibitor 3-methyladenine (3-MA). Neuroprotective effects were determined by evaluating infarction, brain edema, and neurological deficits. The numbers of microglia-derived macrophages, monocyte-derived microglia, T cells, and B cells in the brains were measured, based on the surface marker analyses of CD45, CD11b, B220, CD3, and CD4 using fluorescence-assisted cell sorting. STS (10, 20, 40 mg/kg) was able to significantly reduce infarct volumes, improve neurological deficits, and reduce brain water contents. STS treatment reduced neuroinflammation, as assessed by the infiltration of macrophages and neutrophils, corresponding with reduced numbers of macrophages, T cells, and B cells in ischemia/reperfusion (I/R) brains. In addition, STS treatment also attenuated the upregulation of autophagy associated proteins, such as LC3-II, Beclin-1 and Sirt 6, which was induced by MCAO. These results demonstrated that STS can provide remarkable protection against ischemic stroke, possibly via the inhibition of autophagy and inflammatory activity.

Silencing the lncRNA Maclpil in pro-inflammatory macrophages attenuates acute experimental ischemic stroke via LCP1 in mice

Long noncoding RNAs (lncRNA) expression profiles change in the ischemic brain after stroke, but their roles in specific cell types after stroke have not been studied. We tested the hypothesis that lncRNA modulates brain injury by altering macrophage functions. Using RNA deep sequencing, we identified 73 lncRNAs that were differentially expressed in monocyte-derived macrophages (MoDMs) and microglia-derived macrophages (MiDMs) isolated in the ischemic brain three days after stroke. Among these, the lncRNA, GM15628, is highly expressed in pro-inflammatory MoDMs but not in MiDMs, and are functionally related to its neighbor gene, lymphocyte cytosolic protein 1 (LCP1), which plays a role in maintaining cell shape and cell migration. We termed this lncRNA as Macrophage contained LCP1 related pro-inflammatory lncRNA, Maclpil. Using cultured macrophages polarized by LPS, M(LPS), we found that downregulation of Maclpil in M(LPS) decreased pro-inflammatory gene expression while promoting anti-inflammatory gene expression. Maclpil inhibition also reduced the migration and phagocytosis ability of MoDMs by inhibiting LCP1. Furthermore, adoptive transfer of Maclpil silenced M(LPS), reduced ischemic brain infarction, improved behavioral performance and attenuated penetration of MoDMs in the ischemic hemisphere. We conclude that by blocking macrophage, Maclpil protects against acute ischemic stroke by inhibiting neuroinflammation.

Abstract WP117: High-dimensional Analysis of Inflammatory Cells in Ischemic Stroke Mice

Background: Inflammatory leukocytes in the ischemic brain modulate brain injury, but their functions are not fully understood. The purpose of this study is to establish a novel technique of mass cytometry (CyTOF) to identify the functions and roles of inflammatory cells in ischemic stroke mice, an alternative method to flow cytometry, without fluorescence cross contaminations. Methods: Leukocytes were isolated from the spleen, bone marrow, and ischemic brain of ischemic stroke mice. These cell samples were simultaneously stained with 16 metal-labeled immune cell phenotype markers, and then detected by CyTOF. Data were analyzed by Cytobank program. Results: By using the manual gating strategy, the results showed that stroke resulted in significant increases of microglia-derived macrophages (MiMΦs), monocyte-derived macrophages (MoMΦs), natural killer (NK), plasmacytoid dendritic cells (pDCs), conventional DCs (cDCs), B cells, CD4 + T, CD8 + T cells, γδ T, and regulatory T (Treg) cells ( p &lt;0.05). For the first time, we discovered that the number of Eff/mem CD4, and Eff/mem CD8 T cells was significantly increased in the ischemic brain ( p &lt;0.05), suggesting that these effector/memory T cells are important for brain injury. In addition, SAPDE analysis showed that individual leukocytes in the ischemic brain on day 3 after stroke had higher protein expressions of CD45, CD11b, CD11c, B220, CD3, CD4, CD8, CD44, and NK1.1 than those from sham brains, suggesting that these proteins may be actively involved in brain injury. As expected, the number of leukocytes was significantly reduced ( p &lt;0.05) in the spleen, an indicator of immunosuppression after stroke. Nevertheless, in the bone marrow, Treg were increased at day 1, neutrophils and CD8 T cells increased at day 3 after ischemia ( p &lt;0.05), while the number of the remaining subtypes was reduced. Conclusions: The novel technique of CyTOF is a powerful tool to quantify leukocyte cell types and protein expressions in individual cells. Our results suggest that effector/memory T cells are involved in brain injury, and upregulation of cell surface receptors in individual leukocytes may be important for their effects on brain injury.

Role of microglia in inflammatory demyelination lesion in the central nervous system

In the lesion of experimental autoimmune encephalomyelitis (EAE), two different kinds of macrophages are activated: microglia derived macrophages (MiDM) and monocytes derived macrophages (MDM). These macrophages elicit different functions in the development of demyelination lesions in EAE. MDMs are infiltrated in the acute phase of EAE, and initiate demyelination at the nodes of Ranvier. On the other hands, MiDM always activated at the peak and the chronic stage of the disease. These macrophages express totally different cytokines at different time course of the disease: MDM are activated in the early stage of the disease and express genes that are mainly related to inflammation, while MiDM are activated at the peak and recovery stage and express genes that are related to homeostasis. Our findings provide the idea to develop new therapeutic strategy not only for demyelinating disease but also other neurological diseases with neuro-inflammation.

Differential Detection and Distribution of Microglial and Hematogenous Macrophage Populations in the Injured Spinal Cord oflys-EGFP-kiTransgenic Mice

The acute inflammatory response that follows spinal cord injury (SCI) contributes to secondary injury that results in the expansion of the lesion and further loss of neurologic function. A cascade of receptor-mediated signaling events after SCI leads to activation of innate immune responses including the migration of microglia and active recruitment of circulating leukocytes. Because conventional techniques do not always distinguish macrophages derived from CNS-resident microglia from blood-derived monocytes, the role that each macrophage type performs cannot be assessed unambiguously in these processes. We demonstrate that, in the normal and spinal cord-injured lys-EGFP-ki transgenic mouse, enhanced green fluorescent protein (EGFP) is expressed only in mature hematopoietic granulomyelomonocytic cells and not in microglia. This allowed us to assess the temporal and spatial relationships between microglia-derived and hematogenous macrophages as well as neutrophils during a period of 6 weeks after clip compression SCI. Within the lesion, EGFP-positive monocyte-derived macrophages were found at the epicenter surrounded by EGFP-negative-activated microglia and microglia-derived macrophages. Neutrophils were not present when EGFP-positive monocyte-derived macrophages were depleted, indicating that neutrophil persistence in the lesion depended on the presence of these monocytes. Thus, these 2 distinct macrophage populations can be independently identified and tracked, thereby allowing their roles in acute and chronic stages of SCI-associated inflammation to be defined.

Cooperative Phagocytes: Resident Microglia and Bone Marrow Immigrants Remove Dead Photoreceptors in Retinal Lesions

Phagocytosis is essential for the removal of photoreceptor debris following retinal injury. We used two mouse models, mice injected with green fluorescent protein-labeled bone marrow cells or green fluorescent protein-labeled microglia, to study the origin and activation patterns of phagocytic cells after acute blue light-induced retinal lesions. We show that following injury, blood-borne macrophages enter the eye via the optic nerve and ciliary body and soon migrate into the injured retinal area. Resident microglia are also activated rapidly throughout the entire retina and adopt macrophage characteristics only in the injured region. Both blood-borne- and microglia-derived macrophages were involved in the phagocytosis of dead photoreceptors. No obvious breakdown of the blood-retinal barrier was observed. Ccl4, Ccl12, Tgfb1, Csf1, and Tnf were differentially expressed in both the isolated retina and the eyecup of wild-type mice. Debris-laden macrophages appeared to leave the retina into the general circulation, suggesting their potential to become antigen-presenting cells. These experiments provide evidence that both local and immigrant macrophages remove apoptotic photoreceptors and cell debris in the injured retina.

Cerebral hypoxia-ischemia and middle cerebral artery occlusion induce expression of the cannabinoid CB2 receptor in the brain

Until recently the cannabinoid CB2 receptor was believed to be absent from the central nervous system. In this study we have identified CB2 expressing cells that appear in the rat brain following stroke and hypoxic-ischemia. At 3 days following surgery CB2-positive macrophages, deriving from resident microglia and/or invading monocytes appear on the lesioned side of the brain. By day 7, a mixed population of CB2-positive cells is present. Microglia-derived macrophages are the key cells in the first stages of brain inflammation, and a pivotal step in the neurodegeneration that follows the acute stage of injury. Thus, CB2 may be important in the brain during injury, and in inflammatory neurodegenerative disorders. The presence of CB2-positive cells in the brain following stroke may provide a novel strategy for cannabinoid-mediated intervention into stroke induced neurodegeneration without the psychoactive effects of CB1 receptor stimulation.

Antiserum against human glucose transporter 5 is highly specific for microglia among cells of the mononuclear phagocyte system

Human monocytes and a variety of tissue macrophages, including microglia, were studied immunohistochemically to determine the expression of a novel microglial marker, human glucose transporter 5 (hGLUT5), in these cells. The hGLUT5 was not expressed in most peripheral macrophages in the normal state, but weakly expressed in some foamy macrophages in atherosclerotic lesions. There was no hGLUT5 reactivity in blood monocytes. In the lesions of brain infarcts, foamy macrophages (predominantly monocyte-derived cells) in the ischemic core were mostly negative for hGLUT5, while activated and phagocytic microglia in the transitional zone were consistently positive. The present study indicated that unlike other microglial markers, hGLUT5 is rarely present in peripheral macrophages, and that hGLUT5 immunohistochemistry is useful in distinguishing microglia-derived macrophages from monocyte-derived macrophages in acute necrotic lesions.

Microglia-derived macrophages in early multiple sclerosis plaques

Microglia-derived macrophages in early multiple sclerosis plaques

In situ detection of transforming growth factor-β mRNA in experimental rat glioma and reactive glial cells

Malignant gliomas are associated with a state of systemic immunosuppression which appears to be partially mediated by transforming growth factor beta (TGF-β) secreted from glioma cells. In a recently described animal model of malignant glioma, massive activation of local microglial cells and formation of microglia-derived macrophages has been observed in the absence of detectable tumour regression. We have investigated the in situ expression of TGF-β in rat glioma as a possible cause of ineffective tumour destruction. Two weeks following unilateral injection of glioma cells, large tumours were observed in the affected hemisphere. In situ hybridization for TGF-β1 mRNA revealed an intense signal over the entire tumour area. In the peritumoural area, at sites of glial activation, a lower signal was obtained over cellular profiles containing nuclei typical for microglia, as well as other unidentified cellular profiles. No signal was obtained over the contralateral unaffected hemisphere. Northern blot analysis revealed a strong expression of TGF-β1 mRNA in tumour tissue, a lesser signal in the peritumoural reactive brain tissue and virtually no signal in normal tissue. Our data indicate that the experimental rat glioma has the potential to secrete TGF-β in vivo which might render the microglial infiltration ineffective. TGF-β expressed by activated microglial cells themselves might further inhibit their tumoricidal potential, thus contributing further to unrestrained tumour growth.

Immunophenotypic analysis of infiltrating leukocytes and microglia in an experimental rat glioma.

The appearance and cellular distribution of major histocompatibility complex (MHC), as well as lymphocytic and macrophage antigens has been studied in a fully developed experimental rat forebrain glioma. Activated microglial cells and microglia-derived macrophages expressing CR3 complement receptor molecules and MHC class II (Ia) antigen were found throughout the tumor, and with increased density along the tumor's periphery. MHC class I antigen expression was entirely absent from tumor cells, and found only occasionally on microglia. The expression of leukocyte common antigen, and CD4 and CD8 antigens was conspicuous throughout the tumor, and associated with lymphocytes, perivascular cells, and microglia. Cells expressing the ED2 macrophage epitope were almost exclusively of the perivascular type and revealed a distribution dissimilar to that of cells positive for Ia antigen. The ED2 epitope was found sporadically on ramified microglial cells. The results show that despite heavy infiltration with blood mononuclear and CNS microglial cells, the tumor showed no evidence of destruction caused by inflammatory cells. Possible mechanisms of tumor immunosuppressive activity preventing the full immunological activation of microglia and blood mononuclear cells are discussed.

Response of microglial cells to experimental rat glioma.

The response of indigenous CNS microglia to an experimentally induced glioma has been studied in rat brain using lectin histochemistry with the Griffonia simplicifolia B4-isolectin. The study was undertaken 2 weeks after tumor cell injection when tumor size was near maximal. Reactive microglial cells formed a dense band that surrounded most of the well-circumscribed tumor mass, and extended along the corpus callosum into the contralateral cerebral hemisphere. From the periphery inward, reactive microglia extended into the tumor tissue, where large numbers of them were found to be present as microglia-derived macrophages. The lectin stain, which also labels endothelial cells, revealed a highly vascularized tumor with ongoing neovascularization apparent as vascular sprouts. Moderate numbers of lectin-stained blood monocytes were localized primarily inside the vessel lumina. Our results show that microglial cells react to brain tumors; however, it remains to be determined whether the microglial response represents an active antitumor defense mechanism that could be manipulated during immunotherapeutic approaches.