Role of microglia in stroke.

Microglia play key roles in the post-ischemic inflammatory response and damaged tissue removal reacting rapidly to the disturbances caused by ischemia and working to restore the lost homeostasis. However, the modified environment, encompassing ionic imbalances, disruption of crucial neuron-microglia interactions, spreading depolarization, and generation of danger signals from necrotic neurons, induce morphological and phenotypic shifts in microglia. This leads them to adopt a proinflammatory profile and heighten their phagocytic activity. From day three post-ischemia, macrophages infiltrate the necrotic core while microglia amass at the periphery. Further, inflammation prompts a metabolic shift favoring glycolysis, the pentose-phosphate shunt, and lipid synthesis. These shifts, combined with phagocytic lipid intake, drive lipid droplet biogenesis, fuel anabolism, and enable microglia proliferation. Proliferating microglia release trophic factors contributing to protection and repair. However, some microglia accumulate lipids persistently and transform into dysfunctional and potentially harmful foam cells. Studies also showed microglia that either display impaired apoptotic cell clearance, or eliminate synapses, viable neurons, or endothelial cells. Yet, it will be essential to elucidate the viability of engulfed cells, the features of the local environment, the extent of tissue damage, and the temporal sequence. Ischemia provides a rich variety of region- and injury-dependent stimuli for microglia, evolving with time and generating distinct microglia phenotypes including those exhibiting proinflammatory or dysfunctional traits and others showing pro-repair features. Accurate profiling of microglia phenotypes, alongside with a more precise understanding of the associated post-ischemic tissue conditions, is a necessary step to serve as the potential foundation for focused interventions in human stroke.

Endothelial cells and macrophages as allies in the healthy and diseased brain.

Diseases of the central nervous system (CNS) are often associated with vascular disturbances or inflammation and frequently both. Consequently, endothelial cells and macrophages are key cellular players that mediate pathology in many CNS diseases. Macrophages in the brain consist of the CNS-associated macrophages (CAMs) [also referred to as border-associated macrophages (BAMs)] and microglia, both of which are close neighbours or even form direct contacts with endothelial cells in microvessels. Recent progress has revealed that different macrophage populations in the CNS and a subset of brain endothelial cells are derived from the same erythromyeloid progenitor cells. Macrophages and endothelial cells share several common features in their life cycle-from invasion into the CNS early during embryonic development and proliferation in the CNS, to their demise. In adults, microglia and CAMs have been implicated in regulating the patency and diameter of vessels, blood flow, the tightness of the blood-brain barrier, the removal of vascular calcification, and the life-time of brain endothelial cells. Conversely, CNS endothelial cells may affect the polarization and activation state of myeloid populations. The molecular mechanisms governing the pas de deux of brain macrophages and endothelial cells are beginning to be deciphered and will be reviewed here.

Lack of the Histone Deacetylase SIRT1 Leads to Protection against Endoplasmic Reticulum Stress through the Upregulation of Heat Shock Proteins.

Histone deacetylase SIRT1 represses gene expression through the deacetylation of histones and transcription factors and is involved in the protective cell response to stress and aging. However, upon endoplasmic reticulum (ER) stress, SIRT1 impairs the IRE1α branch of the unfolded protein response (UPR) through the inhibition of the transcriptional activity of XBP-1 and SIRT1 deficiency is beneficial under these conditions. We hypothesized that SIRT1 deficiency may unlock the blockade of transcription factors unrelated to the UPR promoting the synthesis of chaperones and improving the stability of immature proteins or triggering the clearance of unfolded proteins. SIRT1+/+ and SIRT1-/- fibroblasts were exposed to the ER stress inducer tunicamycin and cell survival and expression of heat shock proteins were analyzed 24 h after the treatment. We observed that SIRT1 loss significantly reduced cell sensitivity to ER stress and showed that SIRT1-/- but not SIRT1+/+ cells constitutively expressed high levels of phospho-STAT3 and heat shock proteins. Hsp70 silencing in SIRT1-/- cells abolished the resistance to ER stress. Furthermore, accumulation of ubiquitinated proteins was lower in SIRT1-/- than in SIRT1+/+ cells. Our data showed that SIRT1 deficiency enabled chaperones upregulation and boosted the proteasome activity, two processes that are beneficial for coping with ER stress.

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

BACKGROUND: 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. METHODS: Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. RESULTS: Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. CONCLUSIONS: Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Ductular reaction-associated neutrophils promote biliary epithelium proliferation in chronic liver disease.

BACKGROUND & AIMS: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response. METHODS: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion. RESULTS: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion. CONCLUSIONS: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response. IMPACT AND IMPLICATIONS: Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease.

Transcriptomics and translatomics identify a robust inflammatory gene signature in brain endothelial cells after ischemic stroke.

Vascular endothelial function is challenged during cerebral ischemia and reperfusion. The endothelial responses are involved in inflammatory leukocyte attraction, adhesion and infiltration, blood-brain barrier leakage, and angiogenesis. This study investigated gene expression changes in brain endothelial cells after acute ischemic stroke using transcriptomics and translatomics. We isolated brain endothelial mRNA by: (i) translating ribosome affinity purification, enabling immunoprecipitation of brain endothelial ribosome-attached mRNA for translatome sequencing and (ii) isolating CD31+ endothelial cells by fluorescence-activating cell sorting for classical transcriptomic analysis. Both techniques revealed similar pathways regulated by ischemia but they showed specific differences in some transcripts derived from non-endothelial cells. We defined a gene set characterizing the endothelial response to acute stroke (24h) by selecting the differentially expressed genes common to both techniques, thus corresponding with the translatome and minimizing non-endothelial mRNA contamination. Enriched pathways were related to inflammation and immunoregulation, angiogenesis, extracellular matrix, oxidative stress, and lipid trafficking and storage. We validated, by flow cytometry and immunofluorescence, the protein expression of several genes encoding cell surface proteins. The inflammatory response was associated with the endothelial upregulation of genes related to lipid storage functions and we identified lipid droplet biogenesis in the endothelial cells after ischemia. The study reports a robust translatomic signature of brain endothelial cells after acute stroke and identifies enrichment in novel pathways involved in membrane signaling and lipid storage. Altogether these results highlight the endothelial contribution to the inflammatory response, and identify novel molecules that could be targets to improve vascular function after ischemic stroke.

Adjunct Thrombolysis Enhances Brain Reperfusion following Successful Thrombectomy.

OBJECTIVE: This study was undertaken to investigate whether adjunct alteplase improves brain reperfusion following successful thrombectomy. METHODS: This single-center, randomized, double-blind, placebo-controlled study included 36 patients (mean [standard deviation] = 70.8 [13.5] years old, 18 [50%] women) with large vessel occlusion undergoing thrombectomy resulting in near-normal (expanded Thrombolysis in Cerebral Infarction [eTICI] b50/67/2c, n = 23, 64%) or normal angiographic reperfusion (eTICI 3, n = 13, 36%). Seventeen patients were randomized to intra-arterial alteplase (0.225mg/kg), and 19 received placebo. At 48 hours, patients had brain perfusion/diffusion-weighted magnetic resonance imaging (MRI) and MRI-spectroscopy. The primary outcome was the difference in the proportion of patients with areas of hypoperfusion on MRI. Secondary outcomes were the infarct expansion ratio (final to initial infarction volume), and the N-acetylaspartate (NAA) peak relative to total creatine as a marker of neuronal integrity. RESULTS: The prevalence of hypoperfusion was 24% with intra-arterial alteplase, and 58% with placebo (adjusted odds ratio = 0.20, 95% confidence interval [CI] = 0.04-0.91, p = 0.03). Among 14 patients with final eTICI 3 scores, hypoperfusion was found in 1 of 7 (14%) in the alteplase group and 3 of 7 (43%) in the placebo group. Abnormal brain perfusion was associated with worse functional outcome at day 90. Alteplase significantly reduced the infarct expansion ratio compared with placebo (median [interquartile range (IQR)] = 0.7 [0.5-1.2] vs 3.2 [1.8-5.7], p = 0.01) and resulted in higher NAA peaks (median [IQR] = 1.13 [0.91-1.36] vs 1.00 [0.74-1.22], p < 0.0001). INTERPRETATION: There is a high prevalence of areas of hypoperfusion following thrombectomy despite successful reperfusion on angiography. Adjunct alteplase enhances brain reperfusion, which results in reduced expansion of the infarction and improved neuronal integrity. ANN NEUROL 2022;92:860-870.

Genetic Architecture of Ischaemic Strokes after COVID-19 Shows Similarities with Large Vessel Strokes.

We aimed to analyse whether patients with ischaemic stroke (IS) occurring within eight days after the onset of COVID-19 (IS-COV) are associated with a specific aetiology of IS. We used SUPERGNOVA to identify genome regions that correlate between the IS-COV cohort (73 IS-COV cases vs. 701 population controls) and different aetiological subtypes. Polygenic risk scores (PRSs) for each subtype were generated and tested in the IS-COV cohort using PRSice-2 and PLINK to find genetic associations. Both analyses used the IS-COV cohort and GWAS from MEGASTROKE (67,162 stroke patients vs. 454,450 population controls), GIGASTROKE (110,182 vs. 1,503,898), and the NINDS Stroke Genetics Network (16,851 vs. 32,473). Three genomic regions were associated (p-value < 0.05) with large artery atherosclerosis (LAA) and cardioembolic stroke (CES). We found four loci targeting the genes PITX2 (rs10033464, IS-COV beta = 0.04, p-value = 2.3 × 10-2, se = 0.02), previously associated with CES, HS6ST1 (rs4662630, IS-COV beta = -0.04, p-value = 1.3 × 10-3, se = 0.01), TMEM132E (rs12941838 IS-COV beta = 0.05, p-value = 3.6 × 10-4, se = 0.01), and RFFL (rs797989 IS-COV beta = 0.03, p-value = 1.0 × 10-2, se = 0.01). A statistically significant PRS was observed for LAA. Our results suggest that IS-COV cases are genetically similar to LAA and CES subtypes. Larger cohorts are needed to assess if the genetic factors in IS-COV cases are shared with the general population or specific to viral infection.

CD69 Plays a Beneficial Role in Ischemic Stroke by Dampening Endothelial Activation.

RATIONALE: CD69 is an immunomodulatory molecule induced during lymphocyte activation. Following stroke, T-lymphocytes upregulate CD69 but its function is unknown. OBJECTIVE: We investigated whether CD69 was involved in brain damage following an ischemic stroke. METHODS AND RESULTS: We used adult male mice on the C57BL/6 or BALB/c backgrounds, including wild-type mice and CD69-/- mice, and CD69+/+ and CD69-/- lymphocyte-deficient Rag2-/- mice, and generated chimeric mice. We induced ischemia by transient or permanent middle cerebral artery occlusion. We measured infarct volume, assessed neurological function, and studied CD69 expression, as well as platelet function, fibrin(ogen) deposition, and VWF (von Willebrand factor) expression in brain vessels and VWF content and activity in plasma, and performed the tail-vein bleeding test and the carotid artery ferric chloride-induced thrombosis model. We also performed primary glial cell cultures and sorted brain CD45-CD11b-CD31+ endothelial cells for mRNA expression studies. We blocked VWF by intravenous administration of anti-VWF antibodies. CD69-/- mice showed larger infarct volumes and worse neurological deficits than the wild-type mice after ischemia. This worsening effect was not attributable to lymphocytes or other hematopoietic cells. CD69 deficiency lowered the time to thrombosis in the carotid artery despite platelet function not being affected. Ischemia upregulated Cd69 mRNA expression in brain endothelial cells. CD69-deficiency increased fibrin(ogen) accumulation in the ischemic tissue, and plasma VWF content and activity, and VWF expression in brain vessels. Blocking VWF reduced infarct volume and reverted the detrimental effect of CD69-/- deficiency. CONCLUSIONS: CD69 deficiency promotes a prothrombotic phenotype characterized by increased VWF and worse brain damage after ischemic stroke. The results suggest that CD69 acts as a downregulator of endothelial activation.

Defining molecular identity and fates of CNS-border associated macrophages after ischemic stroke in rodents and humans.

Central nervous system (CNS)-border associated macrophages (BAMs) maintain their steady-state population during adulthood and are not replaced by circulating monocytes under physiological conditions. Their roles in CNS integrity and functions under pathological conditions remain largely unknown. Until recently, BAMs and microglia could not be unequivocally distinguished due to expression of common macrophage markers. We investigated the transcriptional profiles of immunosorted BAMs from rat sham-operated and ischemic brains using RNA sequencing. We found that BAMs express the distinct transcriptional signature than microglia and infiltrating macrophages. The enrichment of functional groups associated with the cell cycle in CD163+ cells isolated 3 days after the ischemic injury indicates the proliferative capacity of these cells. The increased number of CD163+ cells 3 days post-ischemia was corroborated by flow cytometry and detecting the increased number of CD163+ cells positive for a proliferation marker Ki67 at perivascular spaces. CD163+ cells in the ischemic brains up-regulated many inflammatory genes and parenchymal CD163+ cells expressed iNOS, which indicates acquisition of a pro-inflammatory phenotype. In mice, BAMs typically express CD206 and we found a subset of these cells expressing CD169. Chimeric mice generated by transplanting bone marrow of donor Cx3cr1gfpCCR2rfp mice to wild type hosts showed an increased number of CX3CR1+CD169+ perivascular macrophages 3 days post-ischemia. Furthermore, these cells accumulated in the brain parenchyma and we detected replacement of perivascular macrophages by peripheral monocytic cells in the sub-acute phase of stroke. In line with the animal results, post-mortem brain samples from human ischemic stroke cases showed time-dependent accumulation of CD163+ cells in the ischemic parenchyma. Our findings indicate a unique transcriptional signature of BAMs, their local proliferation and migration of inflammatory BAMs to the brain parenchyma after stroke in animal models and humans.

Leukocytes, Collateral Circulation, and Reperfusion in Ischemic Stroke Patients Treated With Mechanical Thrombectomy.

Background and Purpose- Peripheral immune cells are activated after stroke and may in turn influence the fate of ischemic brain tissue, thus exerting a dual role in ischemic stroke. We evaluated the contribution of neutrophil and lymphocyte counts to hemorrhagic complications and functional outcome in stroke patients treated with mechanical thrombectomy (MT) with varying degrees of collateral circulation and reperfusion. Methods- We retrospectively analyzed 433 consecutive ischemic stroke patients treated with MT. Neutrophil and lymphocyte counts and the neutrophil-to-lymphocyte ratio (NLR) were collected before MT and 1 day after symptom onset. Outcome measures included categories of hemorrhagic transformation, symptomatic intracerebral hemorrhage, 3-month functional dependence (modified Rankin Scale, 3-6), and mortality. Patients were categorized according to their baseline collateral status and the degree of reperfusion after MT. Results- Neutrophil counts and NLR increased, whereas lymphocyte counts decreased after MT (P<0.001), and changes in neutrophils and NLR at day 1 were significantly greater in patients with poor reperfusion. Neutrophil counts and NLR were significantly higher already at admission in patients with poor 3-month outcome. In adjusted analysis, the impact of neutrophilia on poor functional outcome was more substantial in patients with good collaterals achieving successful reperfusion (aOR, 3.09 per quartile; 95% CI, 1.95-4.90), whereas admission lymphopenia (aOR, 4.08 per decreasing quartile; 95% CI, 1.56-10.64) and higher NLR (aOR, 3.76 per quartile; 95% CI, 1.44-9.79) predicted subsequent symptomatic intracerebral hemorrhage in patients with poor collaterals and successful reperfusion. Conclusions- In patients treated with MT, neutrophil and lymphocyte counts are dynamic parameters associated with hemorrhagic complications and long-term outcome. The extent of collateral circulation and the success of brain reperfusion influence the strength of these associations and highlight the dual role of leukocytes in acute stroke.

Location of Neutrophils in Different Compartments of the Damaged Mouse Brain After Severe Ischemia/Reperfusion.

Background and Purpose- Ischemia attracts neutrophils to the injured brain. However, neutrophil location and access to the damaged brain tissue is not yet entirely understood. We aimed to investigate neutrophil location in a mouse model of cerebral ischemia/reperfusion. Methods- Adult male C57BL/6 mice (n=52) received 45-minute intraluminal middle cerebral artery occlusion followed by 14, 24, 48, or 96 hours of reperfusion. Sham-operated mice (n=9) were subjected to the entire surgical procedure. We used wild-type mice and CatchupIVM mice expressing a red fluorescent protein in neutrophils. In addition, fluorescent neutrophils obtained from reporter DsRed (discosoma red fluorescent protein) mice were transferred intravenously to wild-type mice after ischemia. Mice received transcardial paraformaldehyde perfusion, the brain was cryoprotected, frozen, and cryostat sections were studied by immunofluorescence and confocal microscopy. Results- Ischemia induced a time-dependent increase in brain neutrophil numbers versus sham operation. We detected neutrophils in the leptomeninges, ventricles, capillary lumen, perivascular spaces, and parenchyma within the infarcted core. Most ischemic mice showed neutrophils in the leptomeninges and perivascular spaces, whereas the presence and number of neutrophils in the parenchyma was variable among ischemic mice. During the first 24 hours, only a few mice showed parenchymal neutrophils, but the frequency of mice showing neutrophils in the parenchyma and neutrophil numbers increased at 48 and 96 hours. We also detected signs of basement membrane disruption and hints of occasional neutrophil degranulation and formation of neutrophil extracellular traps. Conclusions- After ischemia/reperfusion, neutrophils accumulate in the leptomeninges and perivascular spaces, and eventually can reach the infarcted brain parenchyma.

Microglial cell loss after ischemic stroke favors brain neutrophil accumulation.

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood-brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia.

Murine iPSC-derived microglia and macrophage cell culture models recapitulate distinct phenotypical and functional properties of classical and alternative neuro-immune polarisation.

The establishment and validation of reliable induced pluripotent stem cell (iPSC)-derived in vitro models to study microglia and monocyte/macrophage immune function holds great potential for fundamental and translational neuro-immunology research. In this study, we first demonstrate that ramified CX3CR1+ iPSC-microglia (cultured within a neural environment) and round-shaped CX3CR1- iPSC-macrophages can easily be differentiated from newly established murine CX3CR1eGFP/+CCR2RFP/+ iPSC lines. Furthermore, we show that obtained murine iPSC-microglia and iPSC-macrophages are distinct cell populations, even though iPSC-macrophages may upregulate CX3CR1 expression when cultured within a neural environment. Next, we characterized the phenotypical and functional properties of murine iPSC-microglia and iPSC-macrophages following classical and alternative immune polarisation. While iPSC-macrophages could easily be triggered to adopt a classically-activated or alternatively-activated phenotype following, respectively, lipopolysaccharide + interferon γ or interleukin 13 (IL13) stimulation, iPSC-microglia and iPSC-macrophages cultured within a neural environment displayed a more moderate activation profile as characterised by the absence of MHCII expression upon classical immune polarisation and the absence of Ym1 expression upon alternative immune polarisation. Finally, extending our preceding in vivo studies, this striking phenotypical divergence was also observed for resident microglia and infiltrating monocytes within highly inflammatory cortical lesions in CX3CR1eGFP/+CCR2RFP/+ mice subjected to middle cerebral arterial occlusion (MCAO) stroke and following IL13-mediated therapeutic intervention thereon. In conclusion, our study demonstrates that the applied murine iPSC-microglia and iPSC-macrophage culture models are able to recapitulate in vivo microglia and monocyte/macrophage ontogeny and corresponding phenotypical/functional properties upon classical and alternative immune polarisation, and therefore represent a valuable in vitro platform to further study and modulate microglia and (infiltrating) monocyte immune responses under neuro-inflammatory conditions within a neural environment.

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 αß 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.

Adrenal hormones and circulating leukocyte subtypes in stroke patients treated with reperfusion therapy.

Ischemic stroke sets in motion a dialogue between the central nervous and the immune systems that includes the sympathetic/adrenal system. We investigated the course of immune cells and adrenocortical and adrenomedullary effectors in a cohort of 51 patients with acute stroke receiving reperfusion therapy (intravenous alteplase or mechanical thrombectomy) and its correlation with stroke outcomes and infarct growth. Cortisol increased rapidly and fleetingly after stroke, but 39% of patients who had larger infarctions on admission showed a positive delta cortisol at day 1. It was associated with enhanced infarct growth (p = 0.002) and poor outcome [OR (95% CI) 5.30 (1.30-21.69)], and correlated with less lymphocytes and T cells at follow up. Likewise, fewer circulating lymphocytes, T cells, and Tregs were associated with infarct growth. By contrast, metanephrines did not increase at clinical onset, and decreased over time. Higher levels of NMN correlated with more Treg and B cells. Eventually, complete reperfusion at the end of therapy headed the identification of more circulating Tregs at day 1. Then activation of cortical or medullar compartments of the adrenal gland result in specific signatures on leukocyte subpopulations. Manipulation of the adrenal gland hormone levels warrants further investigation.

Structural and functional brain alterations in a murine model of Angiotensin II-induced hypertension.

Hypertension is a main risk factor for the development of cerebral small vessel disease (cSVD) - a major contributor to stroke and the most common cause of vascular dementia. Despite the increasing socioeconomic importance arising from cSVD, currently only a few specific treatment strategies with proven efficacy are known. Fundamental to the lack of specific treatments is poor understanding of the disease pathogenesis and a lack of appropriate animal models resembling all symptoms of the human disease. However, chronic hypertensive rat models have been shown to bear similarities to most key features of cSVD. Despite a significantly larger toolbox available for genotypic and phenotypic modifications compared to rats, mouse models of hypertension are unusual when modeling cSVD and associated cognitive impairment experimentally. In the present study, we therefore characterized hypertension-mediated cerebrovascular alterations and accompanying structural and functional consequences by simultaneously treating adult wild-type mice (C57BL/6N) with Angiotensin II (AngII) and the nitric oxide synthases inhibitor L-NAME for 4 weeks. Hypertension associated to cerebral alterations reminiscent of early-onset cSVD and vascular cognitive impairment when combined with additional AngII bolus injections. Most importantly, preventing the elevation of blood pressure (BP) protected from the development of cSVD symptoms and associated cognitive decline. Our data strongly support the suitability of this particular mouse model of AngII-induced hypertension as an appropriate animal model for early-onset cSVD and hence, vascular cognitive impairment, pathologies commonly preceding vascular dementia.

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.

Neuroanatomical correlates of stroke-associated infection and stroke-induced immunodepression.

BACKGROUND: Infections represent the most frequent medical complications in stroke patients. Their main determinants are dysphagia and a transient state of immunodepression. We analyzed whether distinct anatomical brain regions were associated with the occurrence of stroke-associated infections or immunodepression. MATERIALS AND METHODS: In 106 patients with acute ischemic stroke, we evaluated the incidence of pneumonia, urinary tract infection, or other infections together with the characterization of biomarkers of immunodepression. Twenty control subjects served to provide reference values. Using voxel-based lesion-symptom mapping, the involvement of gray and white matter structures was correlated with clinical and laboratory findings in crude analyses and in volume adjusted models to rule out associations reflecting differences in the size of the infarction. RESULTS: Stroke-associated infection occurred in 22 (21%) patients and prevailed in patients with larger infarcts. Volume adjusted voxel-based lesion-symptom mapping revealed the involvement of the superior and middle temporal gyri, the orbitofrontal cortex, the superior longitudinal fasciculus and the inferior fronto-occipital fasciculus amongst infected patients. These associations were similar for pneumonia but not for urinary tract infections. Lymphopenia was associated with lesions of the superior and middle temporal gyri. Laterality did not influence stroke-associated infections or the presence of immunodepressive traits after volume control. The greatest overlap in the neuroanatomical correlates occurred between pneumonia and dysphagia. CONCLUSION: Infarct volume plays a relevant role in the occurrence of stroke-associated infections, but lesions in specific brain locations such as the superior and lateral temporal lobe and the orbitofrontal cortex are also associated with increased infectious risk, especially pneumonia.