Post-ischemic Inflammation Research Articles

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

Inflammatory Ly6ChiCCR2+ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2+ monocytes and CCR2+ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2+ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6Chi monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6ChiCCR2+ monocytes, including a subgroup of Arginase-1+ cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2+/+ bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2+ monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2+ monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke.

Glucosamine-mediated immunomodulation after stroke is sexually dimorphic

Growing evidence suggests that galectin-3 (Gal-3) is instrumental in orchestrating innate immune response and microglia activation following different brain pathologies. However, its role remains controversial. We recently showed that a readily available natural product glucosamine may act as a strong modulator of Gal-3. Glucosamine is a naturally occurring sugar and a precursor in the synthesis of glycosylated proteins. It is often used as a supplement to treat symptoms of various inflammatory conditions. Our recent work suggests that by increasing the synthesis and availability of Gal-3 ligands and/or by regulating its expression levels, glucosamine may significantly modulate Gal-3 signaling. Because evidence suggests that Gal-3 might be differentially regulated after ischemic injury in the brains of female mice, here we examined and compared the immunomodulatory potential of glucosamine in male and female stroke. The mice were subjected to transient middle cerebral artery occlusion (MCAO), followed by different reperfusion periods. The short-term 5 days treatment with glucosamine (150 ​mg/kg i.p.) was initiated 2 ​hrs after stroke. To visualize the effects of glucosamine treatment on post-stroke inflammation, we took advantage of a transgenic mouse model bearing the dual reporter system luciferase/GFP under transcriptional control of a murine TLR2 promoter (TLR2-luc-GFP) allowing in vivo bioluminescence imaging of innate immune response and microglial activation. We report that after stroke, both, male and female mice strongly up-regulate the TLR2 bioluminescence signals from activated microglia, however, the observed in vivo immunomodulatory effects of glucosamine after stroke were sex-dependent. Analysis of cytokine profiles at protein level, in glucosamine-treated male mice 72hsr after stroke, revealed down regulation of pro-inflammatory cytokines, an increase in levels of anti-inflammatory cytokines including IL-4, IL13 and colony stimulating factors MCFC and GM-CSF and a significant decrease in the size of ischemic lesion in male mice. Conversely, in female mice glucosamine markedly increases the pro-inflammatory signaling and exacerbates ischemic injury. Analysis of the downstream signaling target of glucosamine/Gal-3 revealed that glucosamine administration restored PPAR-γ activity in male but not in female mice 3 days following MCAO. Together, our results suggest that glucosamine acts as a fine tuner of post-ischemic inflammation in a sex dependent-manner and may have therapeutic potential after stroke in males. Based on our results propose that targeting immune system after stroke may require adapted sex-specific therapeutic approaches.

Inhibition of Endothelial PHD2 Suppresses Post-Ischemic Kidney Inflammation through Hypoxia-Inducible Factor-1.

Prolyl-4-hydroxylase domain-containing proteins 1-3 (PHD1 to PHD3) regulate the activity of the hypoxia-inducible factors (HIFs) HIF-1 and HIF-2, transcription factors that are key regulators of hypoxic vascular responses. We previously reported that deficiency of endothelial HIF-2 exacerbated renal ischemia-reperfusion injury, whereas inactivation of endothelial PHD2, the main oxygen sensor, provided renoprotection. Nevertheless, the molecular mechanisms by which endothelial PHD2 dictates AKI outcomes remain undefined. To investigate the function of the endothelial PHD2/HIF axis in ischemic AKI, we examined the effects of endothelial-specific ablation of PHD2 in a mouse model of renal ischemia-reperfusion injury. We also interrogated the contribution of each HIF isoform by concurrent endothelial deletion of both PHD2 and HIF-1 or both PHD2 and HIF-2. Endothelial deletion of Phd2 preserved kidney function and limited transition to CKD. Mechanistically, we found that endothelial Phd2 ablation protected against renal ischemia-reperfusion injury by suppressing the expression of proinflammatory genes and recruitment of inflammatory cells in a manner that was dependent on HIF-1 but not HIF-2. Persistence of renoprotective responses after acute inducible endothelial-specific loss of Phd2 in adult mice ruled out a requirement for PHD2 signaling in hematopoietic cells. Although Phd2 inhibition was not sufficient to induce detectable HIF activity in the kidney endothelium, in vitro experiments implicated a humoral factor in the anti-inflammatory effects generated by endothelial PHD2/HIF-1 signaling. Our findings suggest that activation of endothelial HIF-1 signaling through PHD2 inhibition may offer a novel therapeutic approach against ischemic AKI.

Current status and perspectives in the development of therapeutic agents targeting post-ischemic inflammation in the acute stage of stroke

We have developed a partial peptide of RANKL (receptor activator of NF-κB ligand) that suppresses TLR (toll-like receptor)-related inflammation via RANKL/RANK (receptor activator of nuclear factor-κB) signals in the acute phase of ischemic stroke. This peptide has been found to be a therapeutic agent for ischemic stroke that can be used in combination with tPA in a mouse model. Based on the findings, we are working on translational research to aim for clinical application of this peptide through collaboration with pharmacy companies. However, the problem is that the need for development of medication in the acute stage of ischemic stroke is currently low in pharmaceutical companies due to the failure of many investigational drugs in the past. To overcome the problem, we are examining the effects of this peptide in other diseases included in the company's priority areas and explaining the environmental changes in the clinical trials due to the development of endovascular treatment in the acute stage of ischemic stroke.

Analysis on transcriptionomic characteristics of Naoxintong Capsules in prevention of post-ischemic inflammation based on RNA-Seq technology

In this research, high-throughput sequencing was used to investigate the mechanism of Naoxintong Capsules(NXTC) in prevention of post-ischemic inflammation. First, microglia BV-2 inflammatory model was induced by 1.0 μg·mL~(-1) LPS to investigate the effect of intestinal absorption solution of NXTC(NXTCIA) at different concentrations(62.5, 31.25, 15.63, 7.81 μg·mL~(-1)) on LPS-induced BV-2 inflammatory factors in microglia. Then, an RNA-Seq high-throughput sequencing method was performed to identify the differentially expressed mRNAs in microglia BV-2 after pre-treatment with NXTC. GO and KEGG enrichment analysis was used to screen the potential biological processes and related signaling pathways of NXTC in inhibiting inflammation. The results showed that four NXTCIA concentrations could significantly inhibit the release of LPS-induced inflammatory mediators in BV-2 in a dose-dependent manner. Furthermore, high-throughput sequencing results showed that 392 mRNA transcripts were reversed following pre-treatment with NXTC. GO enrichment analysis showed that the transcripts reversed by NXTC were mainly involved in Toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway. Taken together, our findings showed that NXTC treatment could provide protective effects against inflammatory response and the mechanism might be related to the regulation of Toll-like receptor signaling pathway, chemokine signaling pathway, and TNF signaling pathway.

Exposure to Cigarette Smoke Augments Post-ischemic Brain Injury and Inflammation via Mobilization of Neutrophils and Monocytes.

Cigarette smoke is a major preventable risk factor of ischemic stroke. Cigarette smoke induces a significant increase in circulating leukocytes. However, it remains unclear to what extent and by what mechanisms smoke priming influences stroke severity. Here we report that exposure to cigarette smoke exacerbated ischemic brain injury in mice subjected to transient middle cerebral artery occlusion (MCAO). The augmentation of neurodeficits and brain infarction was accompanied by increased production of pro-inflammatory factors and brain infiltration of neutrophils and monocytes. Prior to brain ischemia, exposure to cigarette smoke induced mobilization of peripheral neutrophils, and monocytes. Furthermore, the detrimental effects of smoke priming on ischemic brain injury were abolished either by pharmacological inhibition of the recruitment of neutrophils and monocytes or by blockade of the NLRP3 inflammasome, an effector protein of neutrophils and monocytes. Our findings suggest that cigarette smoke-induced mobilization of peripheral neutrophils and monocytes augments ischemic brain injury.

Inhibition of inflammatory cells delays retinal degeneration in experimental retinal vein occlusion in mice.

The role of microglia in retinal inflammation is still ambiguous. Branch retinal vein occlusion initiates an inflammatory response whereby resident microglia cells are activated. They trigger infiltration of neutrophils that exacerbate blood–retina barrier damage, regulate postischemic inflammation and irreversible loss of neuroretina. Suppression of microglia‐mediated inflammation might bear potential for mitigating functional impairment after retinal vein occlusion (RVO). To test this hypothesis, we depleted microglia by PLX5622 (a selective tyrosine kinase inhibitor that targets the colony‐stimulating factor‐1 receptor) in fractalkine receptor reporter mice (Cx3cr1 gfp/+) subjected to various regimens of PLX5622 treatment and experimental RVO. Effectiveness of microglia suppression and retinal outcomes including retinal thickness as well as ganglion cell survival were compared to a control group of mice with experimental vein occlusion only. PLX5622 caused dramatic suppression of microglia. Despite vein occlusion, reappearance of green fluorescent protein positive cells was strongly impeded with continuous PLX5622 treatment and significantly delayed after its cessation. In depleted mice, retinal proinflammatory cytokine signaling was diminished and retinal ganglion cell survival improved by almost 50% compared to nondepleted animals 3 weeks after vein occlusion. Optical coherence tomography suggested delayed retinal degeneration in depleted mice. In summary, findings indicate that suppression of cells bearing the colony‐stimulating factor‐1 receptor, mainly microglia and monocytes, mitigates ischemic damage and salvages retinal ganglion cells. Blood–retina barrier breakdown seems central in the disease mechanism, and complex interactions between different cell types composing the blood–retina barrier as well as sustained hypoxia might explain why the protective effect was only partial.

Dual Roles of Reactive Oxygen Species in Exacerbated Proinflammatory Effects of Caveolin‐2 Deficiency in Ischemia/Reperfusion and the Anti‐inflammatory Effects of Ethanol Preconditioning

Caveolin‐2 (Cav‐2), a member of caveolin protein family, is ubiquitously co‐expressed ubiquitously with caveolin‐1 (Cav‐1) in vascular endothelial cells. Cav‐2 interacts with Cav‐1 to form a hetero‐oligomeric complex within caveolae. In contrast with the extensively studied Cav‐1 including the functional role of Cav‐l expressed in ECs in ischemic injury, much less is known regarding the effects of Cav‐2. We hypothesized that (i) genetic deletion of Cav‐2 (CAV‐2 KO) would promote proinflammatory effects in the absence of I/R in wild‐type (WT) mice; (ii) postischemic inflammation will be exacerbated in CAV‐2 KO mice compared to WT animals; (iii) the proinflammatory effects induced by I/R are due to generation of reactive oxygen species (ROS) in CAV‐2 KO mice; and (iv) ingestion of ethanol at low to moderate levels (EPC) 24 hours prior to ischemia (45 min) followed by reperfusion (60 min) (I/R) will limit postischemic inflammation in CAV‐2 KO mice by a ROS‐dependent mechanism. Intravital fluorescence microscopy was used to visualize leukocyte rolling (LR) and adhesion (LA) in single postcapillary venules of the small intes­tine. Prior to I/R, male CAV‐2 KO mice were treated with EPC, MnTBAP (cell‐permeable SOD mimetic) 1 hour prior to I/R, or MnTBAP 20 minutes prior to EPC, respectively, with postcapillary venular LR and LA quantified during the first hour of reperfusion. Our data shown that LR and LA were markedly elevated in male CAV‐2 KO mice under baseline condition relative to that noted in WT animals. I/R‐induced LR and LA were increased to a greater extent in CAV‐2 KO mice compared to WT animals. These postischemic increases in LR and LA were abolished by treatment with EPC or by exposure to MnTBAP 1 hour prior to I/R. MnTBAP pretreatment 20 minutes prior to EPC also abolished the protective effects of EPC. These results indicate that genetic deletion of CAV‐2 produces proinflammatory effects under baseline conditions and exacerbates these effects after I/R by a ROS‐dependent mechanism. Antecedent EPC prevents the proinflammatory responses to I/R in CAV‐2 KO mice. This protective effect of EPC was initiated by ROS.Support or Funding InformationNIH: R01‐AA022108, R01‐HL081860; DOD: W81XWH‐15‐1‐0624.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pomalidomide Reduces Ischemic Brain Injury in Rodents

Stroke is a leading cause of death and severe disability worldwide. After cerebral ischemia, inflammation plays a central role in the development of permanent neurological damage. Reactive oxygen species (ROS) are involved in the mechanism of post-ischemic inflammation. The activation of several inflammatory enzymes produces ROS, which subsequently suppress mitochondrial activity, leading to further tissue damage. Pomalidomide (POM) is a clinically available immunomodulatory and anti-inflammatory agent. Prior cellular studies demonstrate that POM can mitigate oxidative stress and lower levels of pro-inflammatory cytokines, particularly TNF-α, which plays a prominent role in ischemic stroke-induced brain damage and functional deficits. To evaluate the potential value of POM in cerebral ischemia, POM was initially administered to transgenic mice chronically over-expressing TNF-α surfactant protein (SP)-C promoter (SP-C/TNF-α mice) to assess whether systemically administered drug could lower systemic TNF-α level. POM significantly lowered serum levels of TNF-α and IL-5. Pharmacokinetic studies were then undertaken in mice to evaluate brain POM levels following systemic drug administration. POM possessed a brain/plasma concentration ratio of 0.71. Finally, rats were subjected to transient middle cerebral artery occlusion (MCAo) for 60 min, and subsequently treated with POM 30 min thereafter to evaluate action on cerebral ischemia. POM reduced the cerebral infarct volume in MCAo-challenged rats and improved motor activity, as evaluated by the elevated body swing test. POM’s neuroprotective actions on ischemic injury represent a potential therapeutic approach for ischemic brain damage and related disorders, and warrant further evaluation.

Коагуляционный гемостаз у крыс линии Вистар после кратковременной транзиторной ишемии головного мозга

Hemocoagulation properties in rats following a single short-term transient cerebral ischemia were studied for 21 days of the postischemic period using the coarulometric methods involving the automatic optical determination of fibrin clot formation time in the blood plasma. On the 3rd day we observed the lowered activity of the external, internal and general hemocoagulation pathways: the activated partial thromboplastin time (aPTT), prothrombin time (PT) and thrombin time (TT) were increased, indicating the hypocoagulation in the hemostatic system. These changes were accompanied by the increase of the blood fibrinogen levels, probably due to the post-ischemic inflammation. Subsequently, the fibrinogen levels were increased again on the 14th day of the post-ischemic period. That increase was accompanied by the shortening of the aPTT, indicating hypercoagulation following the internal blood clotting pathway mechanisms. At the same day, we observed the increased TT, indicating the acceleration of the fibrin clot formation in the final stage of blood coagulation. Hypercoagulative changes in the hemostasis system developing along the internal and external hemocoagulation pathways were evidenced by the shortening of the aPTT and PT in comparison with the values in the control rats on the 21st day of the post-ischemic period. Thus, the risk of secondary thrombosis persists for 2-3 weeks after a single short-term transient cerebral ischemia.

Development of a novel RANKL-based peptide, microglial healing peptide1-AcN (MHP1-AcN), for treatment of ischemic stroke

Although the regulation of post-ischemic inflammation is an important strategy to treat ischemic stroke, all clinical trials have failed to show its efficacy. To solve the problem, we previously developed a novel partial peptide of RANKL, microglial healing peptide 1 (MHP1), which could reduce ischemic injury by inhibiting Toll-like receptor (TLR) induced inflammation. However, optimization of the peptide was necessary to increase the stability and efficacies for clinical use. According to information gathered through HPLC/MS in serum, we have newly designed a series of modified MHP1 peptides and have found that N-terminal acetylation and C-terminal amidation in MHP1 (MHP1-AcN), can strengthen its anti-inflammatory effects and increase its stability with anti-osteoclastogenic effects. Anti-TLR activity was reported to be reduced in MHP1 when incubated at 37 °C for 24 hrs, but MHP1-AcN could keep the activity under the same condition. The therapeutic effect of MHP1-AcN was observed in transient ischemic stroke model at lower dose than MHP1. Importantly, MHP1-AcN did not affect thrombolytic effects of tissue plasminogen activator (tPA) and inhibited tPA-induced hemorrhagic transformation. These findings indicated that MHP1-AcN was stable and effective anti-TLR signal peptide and could be a promising agent for treating stroke patients receiving tPA and endovascular therapy.

The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia

Inflammation is considered as a major contributor to brain injury following cerebral ischemia. The therapeutic potential of both MLC601/MLC901, which are herbal extract preparations derived from Chinese Medicine, has been reported both in advanced stroke clinical trials and also in animal and cellular models. The aim of this study was to investigate the effects of MLC901 on the different steps of post-ischemic inflammation in focal ischemia in mice. In vivo injury was induced by 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. MLC901 was administered in post-treatment 90 min after the onset of ischemia and once a day during reperfusion. MLC901 treatment resulted in a reduction in infarct volume, a decrease of Blood Brain Barrier leakage and brain swelling, an improvement in neurological scores and a reduction of mortality rate at 24 hours after MCAO. These beneficial effects of MLC901 were accompanied by an inhibition of astrocytes and microglia/macrophage activation, a drastically decreased neutrophil invasion into the ischemic brain as well as by a negative regulation of pro-inflammatory mediator expression (cytokines, chemokines, matrix metalloproteinases). MLC901 significantly inhibited the expression of Prx6 as well as the transcriptional activity of NFκB and the activation of Toll-like receptor 4 (TLR4) signaling, an important pathway in the immune response in the ischemic brain. MLC901 effects on the neuroinflammation cascade induced by cerebral ischemia probably contribute, in a very significant way, in its potential therapeutic value.

Pomalidomide Ameliorates H₂O₂-Induced Oxidative Stress Injury and Cell Death in Rat Primary Cortical Neuronal Cultures by Inducing Anti-Oxidative and Anti-Apoptosis Effects.

Due to its high oxygen demand and abundance of peroxidation-susceptible lipid cells, the brain is particularly vulnerable to oxidative stress. Induced by a redox state imbalance involving either excessive generation of reactive oxygen species (ROS) or dysfunction of the antioxidant system, oxidative stress plays a central role in a common pathophysiology that underpins neuronal cell death in acute neurological disorders epitomized by stroke and chronic ones such as Alzheimer’s disease. After cerebral ischemia, for example, inflammation bears a key responsibility in the development of permanent neurological damage. ROS are involved in the mechanism of post-ischemic inflammation. The activation of several inflammatory enzymes produces ROS, which subsequently suppress mitochondrial activity, leading to further tissue damage. Pomalidomide (POM) is a clinically available immunomodulatory and anti-inflammatory agent. Using H2O2-treated rat primary cortical neuronal cultures, we found POM displayed neuroprotective effects against oxidative stress and cell death that associated with changes in the nuclear factor erythroid derived 2/superoxide dismutase 2/catalase signaling pathway. POM also suppressed nuclear factor kappa-light-chain-enhancer (NF-κB) levels and significantly mitigated cortical neuronal apoptosis by regulating Bax, Cytochrome c and Poly (ADP-ribose) polymerase. In summary, POM exerted neuroprotective effects via its anti-oxidative and anti-inflammatory actions against H2O2-induced injury. POM consequently represents a potential therapeutic agent against brain damage and related disorders and warrants further evaluation.

Pivotal role of innate myeloid cells in cerebral post-ischemic sterile inflammation.

Inflammatory responses play a multifaceted role in regulating both disability and recovery after ischemic brain injury. In the acute phase of ischemic stroke, resident microglia elicit rapid inflammatory responses by the ischemic milieu. After disruption of the blood-brain barrier, peripheral-derived neutrophils and mononuclear phagocytes infiltrate into the ischemic brain. These infiltrating myeloid cells are activated by the endogenous alarming molecules released from dying brain cells. Inflammation after ischemic stroke thus typically consists of sterile inflammation triggered by innate immunity, which exacerbates the pathologies of ischemic stroke and worsens neurological prognosis. Infiltrating immune cells sustain the post-ischemic inflammation for several days; after this period, however, these cells take on a repairing function, phagocytosing inflammatory mediators and cellular debris. This time-specific polarization of immune cells in the ischemic brain is a potential novel therapeutic target. In this review, we summarize the current understanding of the phase-dependent role of innate myeloid cells in ischemic stroke and discuss the cellular and molecular mechanisms of their inflammatory or repairing polarization from a therapeutic perspective.

Influence of low-dose alcohol consumption on post-ischemic inflammation: Role of cystathionine γ-lyase.

Low-dose alcohol consumption (LAC) has been shown to suppress post-ischemic inflammation and alleviate cerebral ischemia/reperfusion (I/R) injury. Cystathionine γ-Lyase (CSE) is one of the enzymes that endogenously produce hydrogen sulfide (H2S), which has an anti-inflammatory property at low concentration. We determined the potential role of CSE in the protective effect of LAC. Male C57BL/6J mice were divided into two groups, an ethanol group and a control group, and gavage fed with 0.7g/kg/day ethanol or volume-matched water once a day for 8 weeks. Transient focal cerebral ischemia was induced by unilateral middle cerebral artery occlusion (MCAO) for 90min. CSE inhibitors were intraperitoneally given 30min prior to the ischemia. Cerebral I/R injury, H2S production, adhesion molecules, IL-1 receptor accessory protein (IL-1RAcP), IL-1β, microglial activation, and neutrophil infiltration were evaluated at 24h of reperfusion. Eight-week ethanol feeding upregulated CSE in the cerebral cortex and reduced cerebral I/R injury. Moreover, ethanol increased post-ischemic H2S production and alleviated the post-ischemic inflammatory response (expression of adhesion molecules, IL-1RAcP, IL-1β, microglial activation, and neutrophil infiltration) in the peri-infarct cerebral cortex. Both inhibitors of CSE, DL-Propargylglycine (PAG) and β-cyano-L-alanine (BCA), abolished the protective effect of ethanol on cerebral I/R injury. In addition, PAG attenuated the inhibitory effect of ethanol on the post-ischemic inflammation. Thus, LAC may protect against cerebral I/R injury by suppressing post-ischemic inflammation via an upregulated CSE.

Endogenous Protection from Ischemic Brain Injury by Preconditioned Monocytes

Exposure to low-dose lipopolysaccharide (LPS) before cerebral ischemia is neuroprotective in stroke models, a phenomenon termed preconditioning (PC). Although it is well established that LPS-PC induces central and peripheral immune responses, the cellular mechanisms modulating ischemic injury remain unclear. Here, we investigated the role of immune cells in the brain protection afforded by PC and tested whether monocytes may be reprogrammed by ex vivo LPS exposure, thus modulating inflammatory injury after cerebral ischemia in male mice. We found that systemic injection of low-dose LPS induces a Ly6Chi monocyte response that protects the brain after transient middle cerebral artery occlusion (MCAO) in mice. Remarkably, adoptive transfer of monocytes isolated from preconditioned mice into naive mice 7 h after transient MCAO reduced brain injury. Gene expression and functional studies showed that IL-10, inducible nitric oxide synthase, and CCR2 in monocytes are essential for neuroprotection. This protective activity was elicited even if mouse or human monocytes were exposed ex vivo to LPS and then injected into male mice after stroke. Cell-tracking studies showed that protective monocytes are mobilized from the spleen and reach the brain and meninges, where they suppress postischemic inflammation and neutrophil influx into the brain parenchyma. Our findings unveil a previously unrecognized subpopulation of splenic monocytes capable of protecting the brain with an extended therapeutic window and provide the rationale for cell therapies based on the delivery of autologous or allogeneic protective monocytes in patients after ischemic stroke.SIGNIFICANCE STATEMENT Inflammation is a key component of the pathophysiology of the brain in stroke, a leading cause of death and disability with limited therapeutic options. Here, we investigate endogenous mechanisms of protection against cerebral ischemia. Using lipopolysaccharide (LPS) preconditioning (PC) as an approach to induce ischemic tolerance in mice, we found generation of neuroprotective monocytes within the spleen, from which they traffic to the brain and meninges, suppressing postischemic inflammation. Importantly, systemic LPS-PC can be mimicked by adoptive transfer of in vitro-preconditioned mouse or human monocytes at translational relevant time points after stroke. This model of neuroprotection may facilitate clinical efforts to increase the efficacy of BM mononuclear cell treatments in acute neurological diseases such as cerebral ischemia.

Ursolic Acid Ameliorates Inflammation in Cerebral Ischemia and Reperfusion Injury Possibly via High Mobility Group Box 1/Toll-Like Receptor 4/NFκB Pathway.

Toll-like receptors (TLRs) play key roles in cerebral ischemia and reperfusion injury by inducing the production of inflammatory mediators, such as interleukins (ILs) and tumor necrosis factor-alpha (TNF-α). According to recent studies, ursolic acid (UA) regulates TLR signaling and exhibits notable anti-inflammatory properties. In the present study, we explored the mechanism by which UA regulates inflammation in the rat middle cerebral artery occlusion and reperfusion (MCAO/R) model. The MCAO/R model was induced in male Sprague–Dawley rats (MCAO for 2 h, followed by reperfusion for 48 h). UA was administered intragastrically at 0.5, 24, and 47 h after reperfusion. The direct high mobility group box 1 (HMGB1) inhibitor glycyrrhizin (GL) was injected intravenously after 0.5 h of ischemia as a positive control. The degree of brain damage was estimated using the neurological deficit score, infarct volume, histopathological changes, and neuronal apoptosis. We assessed IL-1β, TNF-α, and IL-6 levels to evaluate post-ischemic inflammation. HMGB1 and TLR4 expression and phosphorylation of nuclear factor kappa-light-chain-enhancer of activated B cell (NFκB) were also examined to explore the underlying mechanism. UA (10 and 20 mg/kg) treatment significantly decreased the neurological deficit scores, infarct volume, apoptotic cells, and IL-1β, TNF-α, and IL-6 concentrations. The infarct area ratio was reduced by (33.07 ± 1.74), (27.05 ± 1.13), (27.49 ± 1.87), and (39.74 ± 2.14)% in the 10 and 20 mg/kg UA, GL, and control groups, respectively. Furthermore, UA (10 and 20 mg/kg) treatment significantly decreased HMGB1 release and the TLR4 level and inactivated NFκB signaling. Thus, the effects of intragastric administration of 20 mg/kg of UA and 10 mg/kg of GL were similar. We provide novel evidence that UA reduces inflammatory cytokine production to protect the brain from cerebral ischemia and reperfusion injury possibly through the HMGB1/TLR4/NFκB signaling pathway.

Abstract 259: S100A9 is a Master Regulator of Inflammation and Repair After Myocardial Infarction

Background: The innate immune response plays an important role in cardiac repair following an acute myocardial infarction. The pro-inflammatory alarmin S100A8/A9 is released in high amounts locally and systemically during acute coronary events. Here, we studied the effects of S100A9 blockade on the innate immune responses involved in post-ischemic myocardial inflammation and repair. Methods: We induced MI by permanent left coronary artery ligation in C57BL/6 mice, which were subsequently treated with the specific S100A8/A9 blocker ABR-238901 (30mg/kg) or with buffer for 21 days. Left ventricular function was assessed by echocardiography. Immune cell populations in the myocardium, blood, bone marrow and spleen were analysed by flow cytometry. Results: The treatment induced progressive deterioration of the left ventricular systolic function and increased left ventricular volumes in ABR-238901-treated mice compared with controls, suggesting defective repair and negative myocardial remodelling. After 21 days of treatment, infarction size was significantly higher in mice receiving S100A8/A9 blockade (16.1±5.4% vs. 9.9±3.4%, P=0.03). S100A9 blockade inhibited proliferation of hematopoietic stem cells in the bone marrow, and reduced neutrophil and monocyte trafficking from bone marrow and spleen to the circulation and myocardium. Neutrophil and monocyte counts increased in the bone marrow and spleen of mice receiving S100A8/A9 blockade, possibly due to impaired egression. The presence of reparatory CD11b + F4/80 + Ly6C low macrophages expressing the efferocytosis receptor MerTK was potently decreased in the myocardium by day 7 post-MI compared to controls (36 820 ± 2 538 vs. 72 371 ± 4 482 cells/heart, P=0.0001). The transcription factor Nur77 mediates phenotype switching from inflammatory Ly6C hi monocytes to reparatory Ly6C lo macrophages in MI. ABR-238901 lowered the expression of Nur77 in Ly6C hi/int monocytes in-vivo , total bone marrow derived monocytes in-vitro , and the activity of Nur77 in macrophages in-vitro . Conclusions: S100A8/A9 plays an important role in leukocyte trafficking after MI. Long-term S100A8/A9 blockade impairs efferocytosis and myocardial repair, leading to systolic dysfunction and detrimental remodelling.

Abstract 374: Lack of Fibronectin Containing Extra Domain a Attenuates Acute Myocardial Ischemia/Reperfusion Injury in Hyperlipidemic Mice by Limiting Postischemic Thrombo-inflammation

Background: Reperfusion therapy is standard care for patients following acute myocardial infarction. Fibronectin-splicing variant containing extra domain A (Fn-EDA), which is an endogenous ligand for toll-like-receptor 4 (TLR4), is upregulated during vascular hypertension, myocardial injury, and atherosclerosis. An earlier study done in rabbit showed that Fn-EDA accumulates more rapidly (within 24 hrs) in the reperfused hearts. Very little is known about the mechanistic role of Fn-EDA in the pathophysiology of myocardial infarction (MI) in the comorbid condition of hyperlipidemia. Methods: Susceptibility to MI outcome was evaluated in hyperlipidemic Apoe -/- , Fn-EDA -/- Apoe -/- , TLR4 -/- Apoe -/- and Fn-EDA -/- TLR4 -/- Apoe -/- mice (male and female; 8-10 weeks old) by transient 1-hour ischemia/23 hours of reperfusion (I/R). Myocardial I/R injury outcome was evaluated by measuring infarct area, cardiac Troponin I (cTnI) levels in plasma, postischemic thrombo-inflammation (thrombi and neutrophil influx) and myocyte apoptosis. Results: Irrespective of gender, Fn-EDA -/- Apoe -/- mice (Apoe -/- mice expressing Fn deficient in EDA) exhibited smaller infarcts and decreased cTnI levels concomitant with reduced postischemic thrombosis, inflammation and myocyte apoptosis ( P <0.05 vs. Apoe -/ - mice). Genetic ablation of TLR4 attenuated myocardial I/R injury outcome in Apoe -/- mice ( P <0.05 vs. Apoe -/- mice), but did not further reduce in Fn-EDA -/- Apoe -/- mice, suggesting Fn-EDA modulates TLR4-dependent MI exacerbation. Bone marrow transplantation experiments revealed that nonhematopoietic cells-derived Fn-EDA exacerbates myocardial I/R injury through TLR4 expressed on the hematopoietic cell. Infusion of a specific inhibitor of Fn-EDA into Apoe -/- mice significantly reduced myocardial I/R injury. Conclusions: Hyperlipidemic mice deficient in Fn-EDA exhibit TLR4-dependent reduced myocardial I/R injury that was associated with decreased thrombo-inflammatory response. These findings suggest that targeting Fn-EDA combined with thrombolytic agents could be an effective therapeutic strategy to inhibit myocardial I/R injury in patients with hyperlipidemia.

Interleukin-1 receptor antagonist-mediated neuroprotection by umbilical cord-derived mesenchymal stromal cells following transplantation into a rodent stroke model

The human umbilical cord is a promising source of mesenchymal stromal cells (MSCs). Intravenous administration of human umbilical cord-derived MSCs (IV-hUMSCs) showed a favorable effect in a rodent stroke model by a paracrine mechanism. However, its underlying therapeutic mechanisms must be determined for clinical application. We investigated the therapeutic effects and mechanisms of our good manufacturing practice (GMP)-manufactured hUMSCs using various cell doses and delivery time points in a rodent model of stroke. IV-hUMSCs at a dose of 1 × 106 cells at 24 h after stroke improved functional deficits and reduced neuronal damage by attenuation of post-ischemic inflammation. Transcriptome and immunohistochemical analyses showed that interleukin-1 receptor antagonist (IL-1ra) was highly upregulated in ED-1-positive inflammatory cells in rats treated with IV-hUMSCs. Treatment with conditioned medium of hUMSCs increased the expression of IL-1ra in a macrophage cell line via activation of cAMP-response element-binding protein (CREB). These results strongly suggest that the attenuation of neuroinflammation mediated by endogenous IL-1ra is an important therapeutic mechanism of IV-hUMSCs for the treatment of stroke.