Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide

[Background] Cyclooxygenase-2 (COX-2) is induced under inflammatory conditions, and prostaglandin E2 (PGE2) is one of the products of COX activity. PGE2 has pleiotropic actions depending on the activation of specific E-type prostanoid EP1-4 receptors. We investigated the involvement of PGE2 and EP receptors in glial activation in response to an inflammatory challenge induced by LPS.[Methods] Cultures of mouse microglia or astroglia cells were treated with LPS in the presence or absence of COX-2 inhibitors, and the production of PGE2 was measured by ELISA. Cells were treated with PGE2, and the effect on LPS-induced expression of TNF-α messenger RNA (mRNA) and protein was studied in the presence or absence of drug antagonists of the four EP receptors. EP receptor expression and the effects of EP2 and EP4 agonists and antagonists were studied at different time points after LPS.[Results] PGE2 production after LPS was COX-2-dependent. PGE2 reduced the glial production of TNF-α after LPS. Microglia expressed higher levels of EP4 and EP2 mRNA than astroglia. Activation of EP4 or EP2 receptors with selective drug agonists attenuated LPS-induced TNF-α in microglia. However, only antagonizing EP4 prevented the PGE2 effect demonstrating that EP4 was the main target of PGE2 in naïve microglia. Moreover, the relative expression of EP receptors changed during the course of classical microglial activation since EP4 expression was strongly depressed while EP2 increased 24 h after LPS and was detected in nuclear/peri-nuclear locations. EP2 regulated the expression of iNOS, NADPH oxidase-2, and vascular endothelial growth factor. NADPH oxidase-2 and iNOS activities require the oxidation of NADPH, and the pentose phosphate pathway is a main source of NADPH. LPS increased the mRNA expression of the rate-limiting enzyme of the pentose pathway glucose-6-phosphate dehydrogenase, and EP2 activity was involved in this effect.[Conclusions] These results show that while selective activation of EP4 or EP2 exerts anti-inflammatory actions, EP4 is the main target of PGE2 in naïve microglia. The level of EP receptor expression changes from naïve to primed microglia where the COX-2/PGE2/EP2 axis modulates important adaptive metabolic changes.This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) (SAF2014-56279R) and the European Community FP7 (InMiND project no. 278850). EBT had an FPU PhD fellowship of MINECO.Peer reviewe

Clemastine Induces an Impairment in Developmental Myelination

[EN] Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c(+) microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.This work was supported by Spanish Ministry of Education and Science (SAF 2016-75292- R); Spanish Ministry of Science andInnovation (PID 2019-109724RB-I00); Basque Government (PI-2016-1-0016); the University of the Basque Country (UPV/EHU);and Centro de Investigacion Biomedica en Red, EnfermedadesNeurodegenerativas (CIBERNED; grant CB06/05/0076). AP hasa predoctoral fellowship from the University of the Basque Country (UPV/EHU), AM has a predoctoral fellowship fromthe Spanish Ministry of Education and Science and AO-de-A hasa postdoctoral fellowship from the Basque Governmen

Neutrophil Gas6 expression is involved in neutrophil recognition by microglia for phagocytosis

Trabajo presentado en el 53rd Annual Scientific Meeting of the European Society for Clinical Investigation (ESCI), celebrado en Coimbra (Portugal), del 22 al 24 de mayo de 2019Background: Ischemic stroke induces a strong inflammatory response in the brain involving leukocyte infiltration. Microglia, the resident innate immune cells of the central nervous system, phagocytose infiltrating neutrophils1,2. However, the signals involved in neutrophil recognition are not well-known. The objective of this study was to investigate the involvement of the ¿eat-me¿ signal Gas6 in phagocytosis of neutrophils by microglia. Methods: We used wild type (wt) mice, Gas6-/- mice, and DsRed reporter mice. We isolated microglia from the adult mouse brain and maintained the cells in culture for 7 days. We then exposed Gas6+/+ or Gas6-/- microglia to Gas6+/+ or Gas6-/- bone marrow-derived neutrophils, and studied the cells with a multiposition time-lapse microscopy for 10-14h. Cultures of Gas6+/+ and Gas6-/- cells were run in parallel in n=3 independent experiments with 2 replicates per genotype in each experiment. We quantified events by cell tracking with ImageJ. Results: Compared to Gas+/+ neutrophils, phagocytosis of Gas6-/- neutrophils by either Gas+/+ or Gas6-/- microglia was reduced (40%) regardless of microglia genotype (two-way ANOVA by neutrophil genotype and microglia genotype, neutrophil genotype p< 0.0001). We also observed signs of neutrophil NETosis, i.e. apparent restructuration of the nucleus, clear expulsion of the intracellular content, increase in cell size, and loss of fluorescence, likely due to enzymatic degradation. We estimated that approximately 10% of neutrophils suffered NETosis. At this point cells looked empty, devoid of cellular organelles and lacked DNA. These structures remained in the culture but we observed that some of them (15%) were eventually engulfed by microglia. Conclusion: Microglia recognize and phagocyte neutrophils, including neutrophils that have undergo NETosis. Neutrophil Gas6 expression mediates neutrophil recognition by microglia for phagocytosis

Characterization of microglia diversity through live cell image analysis

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Microglia are very sensitive to changes in the environment and respond through morphological transformation, phagocytosis and metabolism adaptations. The characterization of microglia heterogeneity is still a challenge since microglia can adopt multiple morphologies and it is not easy to categorize them, particularly in vitro. Though it is well-known that morphology is usually related to function, we are still unable to interpret the meaning of a change in shape. In order to depict microglia behavior in healthy and pathological conditions, we developed image analysis programs to quantify neuronal death, microglia morphologies and phagocytosis. Primary mice neuron-glia cultures, in which microglia express the tdTomato protein, were exposed to excitotoxic or excitotoxic+inflammatory challenges and analyzed 8h later in time-lapse acquired in a confocal microscope. Neuronal death was assessed by SYTOX staining of nucleic debris and phagocytosis through the engulfment of green SYTOX positive particles in red microglia. We identified 7 morphologies (amoeboid, hypertrophic, fried-egg, bipolar and 3 “inflamed” morphologies) and found the morphometric features able to describe them. Through machine learning, we generated a classifier able to separate them and assign one of the 7 classes to microglia in sample images. In control or excitotoxicity-treated cultures, ameboid and hypertrophic morphologies were the most abundant and did not show changes in the distribution of the populations, or in phagocytosis. Conversely, excitotoxicity+inflammation decreased the amoeboid and hypertrophic populations, induced the appearance of inflamed morphologies and significantly increased the percentage of phagocytosing microglia. Our data suggest that in vitro accumulation of dead cells is not sufficient at least in our model to significantly modify microglia behavior at early time-points (up to 12h) and that inflammation is critical to promote phenotypical changes in microglia. The tools we generated can be useful to correlate microglia behavior with environmental changes and characterize the phenotype of disease-associated microglia.This project is funded by a MICINN grant (PID2020-113202RB-I00) and has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 654248Peer reviewe

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice

Inflammatory Ly6CCCR2 monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2 monocytes and CCR2 lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2 monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6C monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6CCCR2 monocytes, including a subgroup of Arginase-1 cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2 bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2 monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2 monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke

Additional file 3: Figure S3. of Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide

Time course of NOX2 protein expression after LPS in macrophages. (TIF 237 kb

Additional file 1: Figure S1. of Differential expression of E-type prostanoid receptors 2 and 4 in microglia stimulated with lipopolysaccharide

Illustration of microglia cultures. (TIF 473 kb

Characterization of microglia behaviour in healthy and pathological conditions with image analysis tools

Microglia are very sensitive to changes in the environment and respond through morphological, functional and metabolic adaptations. To depict the modifications microglia undergo under healthy and pathological conditions, we developed free access image analysis scripts to quantify microglia morphologies and phagocytosis. Neuron–glia cultures, in which microglia express the reporter tdTomato, were exposed to excitotoxicity or excitotoxicity + inflammation and analysed 8 h later. Neuronal death was assessed by SYTOX staining of nucleus debris and phagocytosis was measured through the engulfment of SYTOX+ particles in microglia. We identified seven morphologies: round, hypertrophic, fried egg, bipolar and three ‘inflamed’ morphologies. We generated a classifier able to separate them and assign one of the seven classes to each microglia in sample images. In control cultures, round and hypertrophic morphologies were predominant. Excitotoxicity had a limited effect on the composition of the populations. By contrast, excitotoxicity + inflammation promoted an enrichment in inflamed morphologies and increased the percentage of phagocytosing microglia. Our data suggest that inflammation is critical to promote phenotypical changes in microglia. We also validated our tools for the segmentation of microglia in brain slices and performed morphometry with the obtained mask. Our method is versatile and useful to correlate microglia sub-populations and behaviour with environmental changes

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice

Inflammatory Ly6CCCR2 monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2 monocytes and CCR2 lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2 monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6C monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6CCCR2 monocytes, including a subgroup of Arginase-1 cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2 bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2 monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2 monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke