Introduction: Blood-derived macrophages become activated and contribute to a toxic environment in the brain after intracerebral hemorrhage (ICH). However, after three days, these macrophages become reparative and essential to tissue repair and recovery. The mechanisms by which this happens are poorly understood, but both myelin and cholesterol have been implicated in suppressing the macrophage response to different inflammatory signals. We have previously demonstrated through RNA sequencing that myelin suppresses responses to a broad range of diverse stimuli, while cholesterol only suppresses the response to lipopolysaccharides (LPS). The LXR agonist T091317 also suppressed responses, but not to the extent of myelin. We hypothesized that myelin would suppress cytokine production in the macrophage response to S100A9 and LPS. Methods and Results: Murine bone marrow-derived macrophages were pretreated with myelin for 18 hours before stimulation with S100A9 or LPS. Cytokine concentrations of TNF, IL-6, CCL2, and IL-10 were measured by cytometric bead array and analyzed by flow cytometry. Myelin seemed to have no significant effect on TNF production (Fig 1A). Myelin limited IL-6 production in response to S100A9 but not to LPS, but the LXR antagonist GSK2033 was unable to reverse this suppression (Fig 1B). Myelin increased CCL2 production in response to both stimulations in an LXR-dependent way (Fig 1C). IL-10 production was decreased in the myelin-treated conditions, and GSK2033 was unable to reverse this (Fig 1D). Conclusions: The measurement of protein responses may be more informative than transcriptional responses and it can help validate the RNA seq. Myelin suppressed some cytokine production, but not to the extent of what was shown in the RNA seq. This suggests that myelin may play a role in the suppressive response of macrophages to ICH, but other components are involved as well.

BACKGROUND:Fingolimod is an immunomodulatory drug that has shown promising effects in stroke treatment, including improvements in neurofunctional recovery and a reduction in infarct size. Fingolimod modulates the sphingosine-1-phosphate receptors, which leads to the internalization of sphingosine-1-phosphate receptors on T and B lymphocytes, thereby preventing their egress from secondary lymphoid organs. Here, we report a secondary analysis from the Stroke Preclinical Assessment Network trial. We assessed the effects of fingolimod versus vehicle on stroke outcomes to better evaluate its therapeutic potential. METHODS:The animal population (n=409) comprised male and female animals treated with fingolimod or vehicle. We used 4 clinically relevant models: young healthy mice (10-12 weeks-old), aging mice (16±1 month-old), obesity induced-hyperglycemic mice fed with a high-fat diet for 12 weeks (16 weeks-old), and spontaneously hypertensive rats (16±1 weeks-old). Stroke was induced by the middle cerebral artery occlusion for 1 hour, followed by reperfusion. Animals received a total of 6 intraperitoneal injections of 0.5 mg/kg twice daily of fingolimod or vehicle. Functional outcomes in the corner test and foot-faults test were measured at days 7 and 28. Lesion size and brain morphometry were evaluated at days 2 and 30 by magnetic resonance imaging. RESULTS:Overall, fingolimod did not improve morphological and functional outcomes. However, fingolimod effects varied depending on sex or the comorbidity model. Fingolimod promoted a better outcome in the corner test in aging females. In contrast, it favored a worse outcome in obesity-induced hyperglycemic mice at day 7. Despite having no effect on survival rates or lesion size, fingolimod attenuated the midline retraction at day 30 in aging males, consistent with less atrophy. CONCLUSIONS:Although fingolimod did not significantly benefit the overall primary functional outcome, its effects varied with sex and comorbidity models, underscoring how the therapeutic potential of a particular drug can differ in a heterogeneous population.

BACKGROUND:The inflammatory response within the central nervous system is a key driver of secondary brain injury after aneurysmal subarachnoid hemorrhage (aSAH). Less is known about the impact that inflammation has on complications like persistent post-hemorrhagic hydrocephalus. To explore the association between inflammation, disease severity, and permanent shunt placement, we characterized the early cytokine profiles of the blood and cerebrospinal fluid (CSF) of patients with aSAH. METHODS:Biological samples were collected from aSAH patients admitted to a single-center Neurosciences Intensive Care Unit between 2014 and 2024. Control CSF samples were collected from patients undergoing permanent shunt placement for normal pressure hydrocephalus. A multiplex bead-based immunoassay was used to analyze a panel of cytokines in plasma and CSF samples. Clinical variables, including demographics, disease severity, and permanent shunt placement were collected. RESULTS:Plasma and/or CSF samples were collected from 83 patients (58 aSAH patients, 25 controls). In aSAH patients, CC motif chemokine ligand-2 (CCL2), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF) were all elevated in CSF compared to plasma (p < 0.05 for all comparisons) and in the CSF of aSAH patients as compared to controls (p < 0.001 for all comparisons). However, only G-CSF and VEGF were associated with clinical severity at presentation when considering Hunt and Hess score as a dichotomized variable (p = 0.026 and p = 0.043, respectively). In multivariable models adjusted for age, sex, and modified Fisher Scale score, early CSF concentrations of IL-6 and IL-8 were associated with increased need for permanent shunt placement (p = 0.030 and p = 0.040, respectively). CONCLUSIONS:Within 72 hours of aSAH, proinflammatory cytokines can be detected at higher concentrations in CSF than in plasma, and at higher concentrations in aSAH patients compared to controls. Early concentrations of certain pro-inflammatory cytokines are associated with increased likelihood of persistent post-hemorrhagic shunt dependent hydrocephalus, independent of initial disease severity. These data support preclinical models of CNS inflammation after aSAH and suggest that early innate inflammation contributes to hydrocephalus.

Introduction: In intracerebral hemorrhage (ICH), blood-derived macrophages initially contribute to a neurotoxic inflammatory response. Understanding the endogenous signals that control this response could help limit these effects. Uptake of myelin debris limits macrophage inflammatory cytokine production in response to LPS, possibly due to the cholesterol present in myelin. However, the pathways by which myelin suppresses macrophage responses and the broader impacts of myelin on macrophage phenotype are poorly understood. Methods and Results: RNA sequencing was used to examine impacts of myelin in diverse conditions. BMDMs were pretreated with myelin, cholesterol or T0901317, an agonist of the cholesterol-response transcription factor LXR. The cells were stimulated with Type I cytokines, Type II cytokines, and different TLR agonists to mimic the diverse stimuli encountered in the inflamed brain (Fig 1A). Myelin suppressed responses to all stimulations, as did T0901317. Cholesterol suppressed the response to LPS and had little effect on other stimulations, including the ICH-relevant alarmin S100A9 (Fig 2). Gene Ontology Enrichment showed that many of the inflammatory gene pathways suppressed by myelin were unique for each stimulation although lipid metabolism pathways were shared across stimulations. Conclusions: Myelin broadly limits macrophage responses to diverse stimulations, partially though not completely through cholesterol and activation of LXR. Cholesterol does not suppress the response to S100A9, suggesting that other components of myelin or activation of phagocytic receptors contribute to suppression of the macrophage response during ICH.

Meningeal lymphatic vessels (MLVs) promote tissue clearance and immune surveillance in the central nervous system (CNS). Vascular endothelial growth factor-C (VEGF-C) regulates MLV development and maintenance and has therapeutic potential for treating neurological disorders. Herein, we investigated the effects of VEGF-C overexpression on brain fluid drainage and ischemic stroke outcomes in mice. Intracerebrospinal administration of an adeno-associated virus expressing mouse full-length VEGF-C (AAV-mVEGF-C) increased CSF drainage to the deep cervical lymph nodes (dCLNs) by enhancing lymphatic growth and upregulated neuroprotective signaling pathways identified by single nuclei RNA sequencing of brain cells. In a mouse model of ischemic stroke, AAV-mVEGF-C pretreatment reduced stroke injury and ameliorated motor performances in the subacute stage, associated with mitigated microglia-mediated inflammation and increased BDNF signaling in brain cells. Neuroprotective effects of VEGF-C were lost upon cauterization of the dCLN afferent lymphatics and not mimicked by acute post-stroke VEGF-C injection. We conclude that VEGF-C prophylaxis promotes multiple vascular, immune, and neural responses that culminate in a protection against neurological damage in acute ischemic stroke.

Introduction: In intracerebral hemorrhage, blood-derived macrophages contribute to a neurotoxic inflammatory response. The endogenous signals that are able to suppress this response are poorly understood. Macrophages likely phagocytose myelin in the CNS during intracerebral hemorrhage and uptake of myelin limits macrophage cytokine production in response to LPS. The components of myelin responsible for its suppressive effects are not well understood, although cholesterol is a likely candidate. Additionally, the broader effects of myelin on macrophage phenotype have not been investigated. Methods and Results: Murine bone marrow-derived macrophages were treated with cholesterol before stimulation with S100A9 or LPS. The inflammatory response was measured by production of TNF, IL-6, CCL2, and IL-10 by ELISA. Cholesterol limited inflammatory cytokine production in response to LPS but not to S100A9. The LXR antagonist GSK2033 partially blocked the suppressive effects of cholesterol but activation of LXR with T0901317 was not sufficient to suppress cytokine production. An RNA-Seq screen was performed to examine broader effects of myelin and cholesterol uptake. BMDMs were pretreated with cholesterol, myelin, or the LXR agonist T0901317 before stimulation with S100A9, LPS, TNF, IFNg, IL-4, IL-10, TGFb, Poly(I:C), or Pam3CSK4. Myelin suppressed responses to all of these stimulations, as did T0901317 but to a lesser extent. Cholesterol suppressed the macrophage response to LPS but not the other stimulations. Conclusions: Myelin broadly limits macrophage responses to pro-inflammatory signals. Cholesterol alone limits the response to LPS, partially through activation of LXR, but LXR-independent mechanisms appear to be involved as well. Cholesterol does not limit the response to other stimulations, suggesting that other components of myelin or activation of phagocytic receptors contribute to suppression of the macrophage response.

Background:The inflammatory response within the central nervous system is a key driver of secondary brain injury after hemorrhagic stroke, both in patients with intracerebral hemorrhage (ICH) and aneurysmal subarachnoid hemorrhage (aSAH). In this study, we aimed to characterize inflammatory molecules in the blood and cerebrospinal fluid (CSF) of patients within 72 hours of hemorrhage to understand how such molecules vary across disease types and disease severity. Methods:Biological samples were collected from patients admitted to a single-center Neurosciences Intensive Care Unit with a diagnosis of ICH or aSAH between 2014 and 2022. Control CSF samples were collected from patients undergoing CSF diversion for normal pressure hydrocephalus. A panel of immune molecules in the plasma and CSF samples was analyzed using Cytometric Bead Array assays. Clinical variables, including demographics, disease severity, and intensive care unit length of stay were collected. Results:Plasma and/or CSF samples were collected from 260 patients (188 ICH patients, 54 aSAH patients, 18 controls). C-C motif chemokine ligand-2 (CCL2), interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF), interleukin-8 (IL-8), and vascular endothelial growth factor (VEGF), were detectable in the CSF within the first 3 days after hemorrhage, and all were elevated compared to plasma. Compared with controls, CCL2, IL-6, IL-8, G-CSF, and VEGF were elevated in the CSF of both ICH and aSAH patients (p<0.01 for all comparisons). VEGF was increased in ICH patients compared to aSAH patients (p<0.01). CCL2, G-CSF, and VEGF in the CSF were associated with more severe disease in aSAH patients only. Conclusions:Within 3 days of hemorrhagic stroke, proinflammatory molecules can be detected in the CSF at higher concentrations than in the plasma. Early concentrations of some pro-inflammatory molecules may be associated with markers of disease severity.

Human diseases may be modeled in animals to allow preclinical assessment of putative new clinical interventions. Recent, highly publicized failures of large clinical trials called into question the rigor, design, and value of preclinical assessment. We established the Stroke Preclinical Assessment Network (SPAN) to design and implement a randomized, controlled, blinded, multi-laboratory trial for the rigorous assessment of candidate stroke treatments combined with intravascular thrombectomy. Efficacy and futility boundaries in a multi-arm multi-stage statistical design aimed to exclude from further study highly effective or futile interventions after each of four sequential stages. Six independent research laboratories performed a standard focal cerebral ischemic insult in five animal models that included equal numbers of males and females: young mice, young rats, aging mice, mice with diet-induced obesity, and spontaneously hypertensive rats. The laboratories adhered to a common protocol and efficiently enrolled 2615 animals with full data completion and comprehensive animal tracking. SPAN successfully implemented treatment masking, randomization, prerandomization inclusion and exclusion criteria, and blinded assessment of outcomes. The SPAN design and infrastructure provide an effective approach that could be used in similar preclinical, multi-laboratory studies in other disease areas and should help improve reproducibility in translational science.

Abstract Introduction Monocyte-derived macrophages contribute to neurotoxic inflammation during hemorrhagic stroke. Cholesterol is an immunosuppressive molecule present at high levels in the brain, primarily as a component of myelin. Uptake of myelin and activation of the transcription factor LXR downstream of myelin and cholesterol uptake limits the macrophage response to LPS. However, the roles of these molecules in macrophage responses to the endogenous signals present during sterile inflammation are poorly understood. The present study seeks to determine what role myelin, cholesterol, and LXR activation play in modifying macrophage responses to diverse signals. Methods and Results Murine bone marrow-derived macrophages were treated with myelin, cholesterol, or the LXR agonist T0901317 18h before stimulation with S100A9, IFN-g, TNF-a, IL-4, IL-10, TGF-b, p(I:C), Pam3CSK4, or LPS. Production of TNF-a, IL-6, CCL2, and IL-10 were measured by multiplex ELISA. Transcriptome-wide effects were measured by RNA sequencing. Cholesterol limited inflammatory cytokine production in response to LPS but not to other stimuli. The LXR agonist T0901317 did not suppress cytokine production. Principal component analysis revealed that cholesterol had strong effects on the transcriptomic responses to LPS but not to the other stimulations. Myelin affected the transcriptomic response to all inflammatory stimulations. Conclusions Cholesterol limits the macrophage response to LPS, partially through activation of LXR, but does not broadly limit macrophage activation. Uptake of myelin has broader effects than cholesterol, suggesting that non-cholesterol components of myelin modulate the macrophage response. Supported by grants from NIH (R01 NS097728, T32 HL007950) and AHA (Postdoctoral Fellowship 830877)

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA, and sustained interferon (IFN) response all of which are recapitulated and required for pathology in the SARS-CoV-2 infected MISTRG6-hACE2 humanized mouse model of COVID-19 with a human immune system 1-20 . Blocking either viral replication with Remdesivir 21-23 or the downstream IFN stimulated cascade with anti-IFNAR2 in vivo in the chronic stages of disease attenuated the overactive immune-inflammatory response, especially inflammatory macrophages. Here, we show SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release IL-1 and IL-18 and undergo pyroptosis thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and its accompanying inflammatory response is necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Remarkably, this same blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 by production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

Introduction: In the acute phase of intracerebral hemorrhage (ICH), blood-derived macrophages contribute to an inflammatory response that increases neuronal death and worsens patient outcome. A better understanding of the endogenous signals that control this response would aid in development of therapeutics to suppress it. Uptake of free cholesterol limits the macrophage response to microbial ligands, in part through activation of the transcription factor LXR. During ICH, blood-derived macrophages in the hematoma are not exposed to microbes but are exposed to the endogenous inflammatory molecule S100A9 as well as cholesterol in the form of myelin and cell debris. Methods and Results: Murine bone marrow-derived macrophages were treated with cholesterol before stimulation with S100A9 or LPS, which are both TLR4 ligands. The inflammatory response was measured by production of the cytokines/chemokines TNF, IL-6, CCL2, and IL-10 by multiplex ELISA and/or qPCR at 3 and 6 hours after stimulation. Activation of LXR was measured by expression of the LXR target genes Abca1 and Abcg1 by qPCR. Cholesterol limited inflammatory cytokine production in response to LPS but not to S100A9. Treatment with the LXR agonist T0901317 activated Abca1 and Abcg1 more strongly than cholesterol but was not as effective at suppressing cytokine production. Treatment with the LXR antagonist GSK2033 suppressed cholesterol-mediated expression of Abca1 and Abcg1 but did not block all suppressive effects of cholesterol on cytokine production. Conclusions: Cholesterol limits the macrophage response to LPS. This is partially through activation of LXR, but LXR-independent mechanisms are involved as well. Cholesterol does not limit the initial inflammatory response to the ICH-relevant molecule S100A9, which may help explain the robust acute inflammatory response that develops during acute ICH in the presence of abundant cholesterol.

Opportunities to interrogate the immune responses in the injured tissue of living patients suffering from acute sterile injuries such as stroke and heart attack are limited. We leveraged a clinical trial of minimally invasive neurosurgery for patients with intracerebral hemorrhage (ICH), a severely disabling subtype of stroke, to investigate the dynamics of inflammation at the site of brain injury over time. Longitudinal transcriptional profiling of CD14(+) monocytes/macrophages and neutrophils from hematomas of patients with ICH revealed that the myeloid response to ICH within the hematoma is distinct from that in the blood and occurs in stages conserved across the patient cohort. Initially, hematoma myeloid cells expressed a robust anabolic proinflammatory profile characterized by activation of hypoxia-inducible factors (HIFs) and expression of genes encoding immune factors and glycolysis. Subsequently, inflammatory gene expression decreased over time, whereas anti-inflammatory circuits were maintained and phagocytic and antioxidative pathways up-regulated. During this transition to immune resolution, glycolysis gene expression and levels of the potent proresolution lipid mediator prostaglandin E-2 remained elevated in the hematoma, and unexpectedly, these elevations correlated with positive patient outcomes. Ex vivo activation of human macrophages by ICH-associated stimuli highlighted an important role for HIFs in production of both inflammatory and anti-inflammatory factors, including PGE(2), which, in turn, augmented VEGF production. Our findings define the time course of myeloid activation in the human brain after ICH, revealing a conserved progression of immune responses from proinflammatory to proresolution states in humans after brain injury and identifying transcriptional programs associated with neurological recovery.

Introduction: In response to intracerebral hemorrhage (ICH), monocytes are recruited to the brain parenchyma, where they differentiate into macrophages and contribute to a pathological inflammatory response. However, by day 3 after ICH, brain macrophages have adopted a more reparative phenotype and are important for clearance of apoptotic cells and recovery. The signals that control this inflammatory to reparative differentiation are incompletely understood, but cholesterol has been found to limit macrophage activation in multiple systems. The brain has the highest cholesterol content of any organ and we hypothesized that cholesterol uptake by macrophages limits inflammation and promotes the development of reparative macrophages following ICH. Methods and Results: Murine bone marrow-derived macrophages were stimulated with a cocktail of thrombin, S100A8, and IL-1b in order to mimic the Danger-Associated Molecular Patterns present in the brain after ICH (ICH-DAMP), LPS, or vehicle for 14-18 hours. Cytokine production was quantified by cytometric bead array and activation markers by flow cytometry. ICH-DAMP was found to upregulate CCL2, IL-6 and TNF, recapitulating the inflammatory phenotype seen in the first days after ICH. However, when cells were stimulated in the presence of cholesterol, production of CCL2, IL-6, and TNF were limited. Dectin-1 has inhibitory properties in some sterile injury models. ICH-DAMP was found to limit expression of dectin-1, and cholesterol reversed this inhibition. Exposure to exogenous cholesterol also upregulated the cholesterol transporter ABCA1, allowing cells to efflux excess cholesterol. The drug Valspodar was therefore used to block cholesterol efflux and was found to further limit ICH-DAMP-mediated upregulation of CCL2. Conclusion: These results suggest that the cholesterol in the brain may limit macrophage activation in response to the stimuli present during intracerebral hemorrhage.