T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding

Objective Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. Approach and Results We used wild-type mice and Selplg(-/-) and Fut7(-/-) mice defective in P-selectin binding and lymphopenic Rag2(-/-) mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4(+) memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. Conclusions The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke

IL-23 (Interleukin-23)-producing conventional dendritic cells control the detrimental IL-17 (Interleukin-17) response in stroke

Background and Purpose—Inflammatory mechanisms can exacerbate ischemic tissue damage and worsen clinical outcome in patients with stroke. Both αβ and γδ T cells are established mediators of tissue damage in stroke, and the role of dendritic cells (DCs) in inducing the early events of T cell activation and differentiation in stroke is not well understood. Methods—In a murine model of experimental stroke, we defined the immune phenotype of infiltrating DC subsets based on flow cytometry of surface markers, the expression of ontogenetic markers, and cytokine levels. We used conditional DC depletion, bone marrow chimeric mice, and IL-23 (interleukin-23) receptor-deficient mice to further explore the functional role of DCs. Results—We show that the ischemic brain was rapidly infiltrated by IRF4+/CD172a+ conventional type 2 DCs and that conventional type 2 DCs were the most abundant subset in comparison with all other DC subsets. Twenty-four hours after ischemia onset, conventional type 2 DCs became the major source of IL-23, promoting neutrophil infiltration by induction of IL-17 (interleukin-17) in γδ T cells. Functionally, the depletion of CD11c+ cells or the genetic disruption of the IL-23 signaling abrogated both IL-17 production in γδ T cells and neutrophil infiltration. Interruption of the IL-23/ IL-17 cascade decreased infarct size and improved neurological outcome after stroke. Conclusions—Our results suggest a central role for interferon regulatory factor 4-positive IL-23–producing conventional DCs in the IL-17–dependent secondary tissue damage in stroke

CNS-border associated macrophages respond to acute ischemic stroke attracting granulocytes and promoting vascular leakage

The central nervous system (CNS) contains several types of immune cells located in specific anatomic compartments. Macrophages reside at the CNS borders surrounding the brain vessels, in leptomeningeal spaces and the choroid plexus, where they interact with the vasculature and play immunological surveillance and scavenging functions. We investigated the phenotypic changes and role of these macrophages in response to acute ischemic stroke. Given that CD163 expression is a hallmark of perivascular and meningeal macrophages in the rat and human brain, we isolated CD163+ brain macrophages by fluorescence activated cell sorting. We obtained CD163+ cells from control rats and 16 h following transient middle cerebral artery occlusion, after verifying that infiltration of CD163+ peripheral myeloid cells is negligible at this acute time point. Transcriptome analysis of the sorted CD163+ cells identified ischemia-induced upregulation of the hypoxia inducible factor-1 pathway and induction of genes encoding for extracellular matrix components and leukocyte chemoattractants, amongst others. Using a cell depletion strategy, we found that CNS border-associated macrophages participate in granulocyte recruitment, promote the expression of vascular endothelial growth factor (VEGF), increase the permeability of pial and cortical blood vessels, and contribute to neurological dysfunction in the acute phase of ischemia/reperfusion. We detected VEGF expression surrounding blood vessels and in some CD163+ perivascular macrophages in the brain tissue of ischemic stroke patients deceased one day after stroke onset. These findings show ischemia-induced reprogramming of the gene expression profile of CD163+ macrophages that has a rapid impact on leukocyte chemotaxis and blood-brain barrier integrity, and promotes neurological impairment in the acute phase of stroke

Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota

Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection.Funded by Fundació la Marató de TV3 (ref. 201723-30-31-32) to Drs Planas, Urra, and Sancho; the Ministerio de Ciencia e Innovación (MICINN)/AEI/10.13039/501100011033 and European Regional Development Fund (ERDF) A way of making Europe by the European Union (PID2020-113202RB-I00 to Dr Planas); CSIC Interdisciplinary Thematic Platform Plataforma Temática Interdisciplinar (PTI)+Neuro-Aging of the Consejo Superior de Investigaciones Científicas (Dr Planas) funded M. Gallizioli. The work of Instituto de Productos Lácteos (IPLA)-Consejo Superior de Investigaciones Científicas (CSIC) group was partly financed by grant AYUD/2021/ 50981 from Principality of Asturias. The work of Institut de Química Avançada de Catalunya (IQAC)-CSIC was financed by Ministerio de Economía y Competitividad (AGL2017-83599-R) and MICINN (PID2020-117009RB-I00). Dr Díaz-Marugan was funded by La Caixa Foundation (ID 100010434; code LCF/BQ/DE16/11570021). Dr Arboleya received a postdoctoral Juan de la Cierva contract (MICINN, Ref. IJCI-2017-32156). Work in Dr Sancho laboratory was funded by Centro de Investigaciones Cardiovasculares (CNIC), European Union’s Horizon 2020 research and innovation program under grant agreement ERC-2016-Consolidator Grant 725091, and MICINN (PID2019-108157RB/AEI/10.13039/501100011033).Peer reviewe

Identity and functions of dendritic cell subsets in ischaemia-induced neuroinflammation

[eng] Cerebral ischaemia induces several inflammatory processes in the brain. Among them, the infiltration of immune cells is a hallmark of the pathology. Dendritic cells (DCs) are usually present in low numbers in the meninges and the choroid plexus, but rarely in the parenchyma. Upon ischaemia, the number of DCs increases, and the cells infiltrate the brain tissue, where they carry out different functions. In an experimental murine model of stroke, we set out to investigate the infiltration of several subsets of DCs to the brain and their functional role. Early after stroke, we show a rapid and significant influx of DCs, especially of conventional type 2 DCs (cDC2), which are the most abundant subset at all time points analysed. Twenty- four hours after stroke, these cells were the major source of IL-23, which was able to stimulate its receptor on γδ T cells, inducing their production of IL-17. In turn, IL-17 is responsible for the stimulation of the production of Cxcl1 by astrocytes, ultimately leading to the infiltration of neutrophils to the ischaemic brain and to the exacerbation of the tissue damage. We demonstrate that the interruption of the IL- 23/IL-17 axis decreases the infarct size and improves the neurological outcome of stroke in mice, suggesting that cDC2 may play a detrimental role in the early phase of the immune response to stroke. The analysis of the infiltration of DCs to the brain in inflammatory conditions has historically been difficult for the absence of univocal markers and for the similarity of their phenotype with other brain cells, especially microglia. The knowledge about the origin, phenotype and functions of brain DCs is therefore underdeveloped. One of the most commonly used markers for the study of DCs is CD11c, which is also expressed by a subset of microglia. The population of CD11c+ cells present in the brain increases after stroke, and we show that CD11c+ cells include proliferating microglia and infiltrating DCs. Despite their similarities, we demonstrate by RNA- Seq analysis that these two cell types exhibit a differential transcriptional profile, with interesting peculiarities in pattern recognition receptor and chemokine receptor expression. DCs extracted from the ischaemic brain outclass microglia in antigen presentation capacity, indicating a functional specialisation. We show that microglia are responsible for the production of chemokines that attract DCs to the brain, especially conventional type 1 DCs (cDC1). This specific subpopulation of DCs appears to have beneficial functions, reducing the infarct size and improving the functional outcome of ischaemic stroke. Altogether, the studies presented in this thesis shed light on the features discriminating DCs from microglia and uncover previously unknown roles of diverse subpopulations of infiltrating DCs in the outcome of ischaemic stroke

Identity and functions of dendritic cell subsets in ischaemia-induced neuroinflammation

Tesis doctoral presentada para lograr el título de Doctorado en Biomedicina por la Universidad de Barcelona, Facultad de Medicina y Ciencias de la Salud.--2021-02-15.--Excelente Cum LaudeCerebral ischaemia induces several inflammatory processes in the brain. Among them, the infiltration of immune cells is a hallmark of the pathology. Dendritic cells (DCs) are usually present in low numbers in the meninges and the choroid plexus, but rarely in the parenchyma. Upon ischaemia, the number of DCs increases, and the cells infiltrate the brain tissue, where they carry out different functions. In an experimental murine model of stroke, we set out to investigate the infiltration of several subsets of DCs to the brain and their functional role. Early after stroke, we show a rapid and significant influx of DCs, especially of conventional type 2 DCs (cDC2), which are the most abundant subset at all time points analysed. Twenty- four hours after stroke, these cells were the major source of IL-23, which was able to stimulate its receptor on γδ T cells, inducing their production of IL-17. In turn, IL-17 is responsible for the stimulation of the production of Cxcl1 by astrocytes, ultimately leading to the infiltration of neutrophils to the ischaemic brain and to the exacerbation of the tissue damage. We demonstrate that the interruption of the IL- 23/IL-17 axis decreases the infarct size and improves the neurological outcome of stroke in mice, suggesting that cDC2 may play a detrimental role in the early phase of the immune response to stroke. The analysis of the infiltration of DCs to the brain in inflammatory conditions has historically been difficult for the absence of univocal markers and for the similarity of their phenotype with other brain cells, especially microglia. The knowledge about the origin, phenotype and functions of brain DCs is therefore underdeveloped. One of the most commonly used markers for the study of DCs is CD11c, which is also expressed by a subset of microglia. The population of CD11c+ cells present in the brain increases after stroke, and we show that CD11c+ cells include proliferating microglia and infiltrating DCs. Despite their similarities, we demonstrate by RNA- Seq analysis that these two cell types exhibit a differential transcriptional profile, with interesting peculiarities in pattern recognition receptor and chemokine receptor expression. DCs extracted from the ischaemic brain outclass microglia in antigen presentation capacity, indicating a functional specialisation. We show that microglia are responsible for the production of chemokines that attract DCs to the brain, especially conventional type 1 DCs (cDC1). This specific subpopulation of DCs appears to have beneficial functions, reducing the infarct size and improving the functional outcome of ischaemic stroke. Altogether, the studies presented in this thesis shed light on the features discriminating DCs from microglia and uncover previously unknown roles of diverse subpopulations of infiltrating DCs in the outcome of ischaemic stroke

Brain ischemia induces an INF-mediated response in microglia of mice and humans.

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Brain ischemia causes necrotic cell death, microglia reactivity and blood-brain barrier breakdown. Danger signals ¿ including nuclear proteins, nucleic acids and heat-shock proteins ¿ are released from injured cells and trigger immune responses by activating pattern recognition receptors (PRRs). Microglial cells are equipped with PRRs, can sense danger signals in the environment and induce inflammation. Our aim was to investigate the inflammatory response of microglia to ischemia in mice and humans. We induced cerebral ischemia in mice by 45-min middle cerebral artery occlusion followed by reperfusion. We compared the transcriptomic profile of microglia isolated from brain tissue of control and ischemic mice using fluorescence activated cell sorting followed by RNA-Seq. Enrichment analysis showed a strong anti-viral response induced by ischemia in microglia, highlighted by upregulation of type-I interferons (IFNs) including Ifnb and many IFN-stimulated genes (ISG). Accordingly, in whole brain tissue, ischemia increased Ifnb, Ifna7 and Ifna9 mRNA expression, as well as many ISG, including Dhx58, Cxcl10, Irf7, and Irg15. A time-course study in brain tissue samples obtained between 1h and 7 days post-ischemia showed ISG expression increase from about 16h post-ischemia, reaching a plateau at 4-7 days. We also detected enhanced expression of ISG in post-mortem human brain tissue of ischemic stroke patients. To find out the contribution of microglia to gene expression in whole brain tissue, we depleted microglia in mice with a CSF1R inhibitor (PLX5622). Ischemia-induced expression of Ifnb, Ifna7, or Ifna9 mRNA was not reduced after microglia depletion. This implies that cells other than microglia are the main source of IFNs in the injured brain. In contrast, we found that microglia depletion reduced cerebral ISG expression. The results show that type I IFNs generated after ischemia in cells other than microglia activate type I IFN receptors in microglia, inducing ISG expression and triggering a specific transcriptional program.Peer reviewe

Differences in the post-stroke innate immune response between young and old

Aging is associated to progressive changes impairing fundamental cellular and tissue functions, and the relationships amongst them through the vascular and immune systems. Aging factors are key to understanding the pathophysiology of stroke since they increase its risk and worsen its functional outcome. Most currently recognised hallmarks of aging are also involved in the cerebral responses to stroke. Notably, age-associated chronic low-grade inflammation is related to innate immune responses highlighted by induction of type-I interferon. The interferon program is prominent in microglia where it interrelates cell damage, danger signals, and phagocytosis with immunometabolic disturbances and inflammation. Microglia engulfment of damaged myelin and cell debris may overwhelm the cellular capacity for waste removal inducing intracellular lipid accumulation. Acute inflammation and interferon-stimulated gene expression are also typical features of acute stroke, where danger signal recognition by microglia trigger immunometabolic alterations underscored by lipid droplet biogenesis. Aging reduces the capacity to control these responses causing increased and persistent inflammation, metabolic dysregulation, and impaired cellular waste disposal. In turn, chronic peripheral inflammation during aging induces immunosenescence further worsening stroke-induced immunodepression, thus increasing the risk of post-stroke infection. Aging also alters gut microbiota composition inducing dysbiosis. These changes are enhanced by age-related diseases, such as atherosclerosis and type-II diabetes, that further promote vascular aging, predispose to stroke, and exacerbate brain inflammation after stroke. Current advances in aging research suggest that some age-associated alterations may be reversed. Future work will unravel whether such evolving anti-aging research may enable designing strategies to improve stroke outcome in the elderly.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. Study supported by grant PID2020-113202RB-I00 funded by Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI), Gobierno de España/ 10.13039/501100011033 and “European Regional Development Fund (ERDF). A way of making Europe”. The Interdisciplinary Thematic Platform PTI-NEURO-AGING+ of the Spanish National Research Council (CSIC) provided support for this work, and funded MG. MAR had a predoctoral fellowship (PRE2018-085737) funded by MCIN/AEI/ 10.13039/501100011033 and by “European Social Fund (ESF) Investing in your future”. DB acknowledges the funding provided by the Fundação para a Ciência e a Tecnologia (FCT-Portugal-PTDC/MED-IMU/0870/2020)

Repopulated microglial cells after depletion in old mice maintain the aging features but are protective in brain ischemia

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 202

Border-associated macrophages influence the endothelial response to brain ischemia

Trabajo presentado en el 19th National Meeting of the Spanish Society of Neuroscience, celebrado en Lleida (España), del 3 al 5 de noviembre de 2021Brain perivascular macrophages are resident in the perivascular space of arterioles and venules between the vasculature and the glia limitans and they are surrounded by basal lamina. Together with meningeal and choroid plexus macrophages they remain for long in the brain since early stages of development and display low or negligible exchange with the periphery during the lifespan under steady state conditions. Thus, brain macrophages reside at the edges of the brain parenchyma in strategic locations for communication with the periphery. To emphasize such features these cells have been termed border-associated macrophages. In this study we hypothesized that BAMs interact with the vasculature and affect the response of vascular endothelial cells after brain ischemia. In order to investigate the role of BAMs we took advantage of the fact that subsets of these macrophages express CD169 (Siglec1) under steady-state conditions. We obtained CD169-DTR mice, which express the diphtheria toxin receptor under the promoter of CD169. Administration of Diphtheria toxin (DTx) i.p. at days 1, 3 and 5, caused a strong reduction of BAMs, as assessed by immunofluorescence and cell counting. Treatment controls were CD169-DTR mice that received saline injection at the same time points. At day 8 after the first DTx administration, ischemia was induced by middle cerebral artery occlusion for 45 min followed by 24h reperfusion. At 24h, mice received an MRI scan and the brain was processed for fluorescence activating cell sorting in order to isolate CD31+ endothelial cells from the cerebral tissue. We then obtained endothelial mRNA to study gene expression. Ischemia induced upregulation of NOX-2 (Cybb) and Mcp-1 (Ccl2) mRNA in the vascular endothelium, according to previous results of our group. Our preliminary results in the BAM-depleted mice show an increased expression of CCL2 and some reduction of NOX-2 in the endothelium that we are currently validating.Supported by the ITN program of the EU ENTRAIN (H2020-MSCA-ITN-2018 - GA 813294), and MICINN (PID2020-113202RB-I00).Peer reviewe