-/-bone marrow chimeras (BMCs) at days 7 and 15 after ICH. Right: Quantification of residual hematoma volume in the WT and Ccr2 -/-BMCs. n = 11 at day 7; n = 9 at day 15. *P < 0.05 by Student's t test. (B) Cylinder test and apomorphine turning test from WT and Ccr2 -/-BMCs at day 15 after ICH. n = 6/group for cylinder test; n = 8/ group for apomorphine turning test. *P < 0.05 by Student's t test. (C) Cylinder test, neurological deficit score, and corner test in control-and anti-CCR2 antibody-treated mice at days 1 and 3 after collagenase ICH. n = 7/group. *P < 0.05 by 1-way repeated-measures ANOVA and Bonferroni's post hoc test. (D) Top: Representative coronal sections show hematoma from control-and anti-CCR2 antibody-treated WT mice after blood injection ICH at 7 days. Bottom: Quantification of hematoma volume, n = 3/ group. *P < 0.05 versus control by Student's t test. (E) Top: Representative coronal sections show hematoma in the isotype control-and anti-CCR2 antibody-treated mice from collagenase model at day 12. Bottom: Quantification of hematoma volume. n = 8/group. *P < 0.05 versus control by Student's t test. (F) The cylinder test, neurological deficit score, and corner test in isotype control-and anti-CCR2 antibody-treated ICH mice at days 3, 5, 7, 9, and 11 after collagenase ICH surgery. n = 8/group. *P < 0.05 versus isotype control group by 1-way repeated-measures ANOVA and Bonferroni's post hoc test. αCCR2, anti-CCR2 antibody.

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 E2 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 PGE2, 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.

Background and Purpose: Brain tissue-resident microglia and monocyte-derived macrophages (MDMs) are innate immune cells that contribute to the inflammatory response, phagocytosis of debris, and tissue repair after injury. We have previously reported that both microglia and MDMs transition from proinflammatory to reparative phenotypes over days after an intracerebral hemorrhage (ICH). However, their individual functional properties in the brain remain largely unknown. Here we characterized the differences between microglia and MDMs and further elucidate their distinct activation states and functional contributions to the pathophysiology and recovery after ICH. Methods: Autologous blood injection was used to model ICH in mice. Longitudinal transcriptomic analyses on isolated microglia and MDMs from mice at days 1, 3, 7 and 10 after ICH and naive controls identified core transcriptional programs that distinguish these cells. Imaging flow cytometry and in vivo phagocytosis assays were used to study phagocytic ability of microglia and MDMs. Antigen presentation was evaluated by ovalbumin-OTII CD4 T-cell proliferation assays with bone marrow–derived macrophages and primary microglia cultures. Results: MDMs had higher phagocytic activity and higher erythrophagocytosis in the ICH brain. Differential gene expression revealed distinct transcriptional signatures in the MDMs and microglia after ICH. MDMs had higher expression of MHCII (major histocompatibility complex class II) genes than microglia at all time points and greater ability to induce antigen-specific T-cell proliferation. Conclusions: The different ontogeny of microglia and MDMs lead to divergent responses and functions in the inflamed brain as these 2 cell populations differ in phagocytic functions and antigen-presenting capabilities in the brain after ICH.

ObjectiveIntracerebral hemorrhage carries a high mortality and survivors are frequently left with significant disability. Immunological mechanisms may play an important role in hemorrhage‐induced brain injury, however, research linking these mechanisms with clinical outcome remains limited. We aim to identify serum inflammatory mediators that are associated with outcome after intracerebral hemorrhage in order to translate data from experimental models to a patient cohort and identify potential targets worthy of reverse translation.MethodsA prospective cohort study at two comprehensive stroke centers enrolled patients with spontaneous intracerebral hemorrhage. Peripheral blood was collected at 6, 24, and 72 h from onset. Functional outcome was assessed at 90 days using the modified Rankin Scale (mRS). Serum inflammatory mediators were measured using multiplex ELISA. Multivariable modeling identified serum biomarkers independently associated with functional outcome at 90 days.Results115 patients completed the study. At 6 h after onset, patients with elevated CCL2 had worse mRS score at day 90 (OR 4.07, 95% CI 1.27–13.10, P = 0.02) after adjusting for age, gender, ICH volume, IVH, infratentorial location and NIHSS score. At 24 and 72 h after onset, elevation in CXCL10 was independently associated with worse 90 days mRS score (24 h: OR 8.08, 95% CI 2.69–24.30, P < 0.001; 72 h: OR 3.89, 95% CI 1.12–13.49, P = 0.03).InterpretationAcute and subacute elevations in specific immune factors are associated with poor outcome, highlighting potential pathways that may contribute to ongoing brain injury in patients with intracerebral hemorrhage.

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.