Role of dihydrotestosterone in post-stroke peripheral immunosuppression after cerebral ischemia

Stroke is a sexually dimorphic disease with male gender considered a disadvantage in terms of risk and disease outcome. In intact males, stroke induces peripheral immunosuppression, characterized by decreased splenocyte numbers and proliferation and altered percentages of viable T, B, and CD11b⁺ cells. To investigate whether the potent androgen and known immunomodulator, dihydrotestosterone (DHT), exacerbates post-stroke immunosuppression in castrated male mice after focal stroke, we evaluated the effect of middle cerebral artery occlusion (MCAO) on peripheral and central nervous system (CNS) immune responses in castrated mice with or without controlled levels of DHT. MCAO reduced spleen cell numbers in both groups, but altered T cell and B cell percentages in remaining splenocytes and concomitantly increased the percentage of CD11b⁺ blood cells solely in DHT-replaced animals at 24 h. Furthermore, DHT-replacement reduced splenocyte proliferation which was accompanied by an increased percentage of immunosuppressive regulatory T cells relative to castrates 96 h post-MCAO. In brain, the percentages of immune cell populations in the ischemic hemisphere relative to the non-ischemic hemisphere were similar between castrated and DHT-replaced mice after MCAO. These data suggest DHT modulates peripheral immunosuppression after MCAO but with relatively little effect on early immune response of the recovering CNS.     

Inhibition of CXCL12 signaling attenuates the postischemic immune response and improves functional recovery after stroke

After stroke, brain inflammation in the ischemic hemisphere hampers brain tissue reorganization and functional recovery. Housing rats in an enriched environment (EE) dramatically improves recovery of lost neurologic functions after experimental stroke. We show here that rats housed in EE after stroke induced by permanent occlusion of the middle cerebral artery (pMCAO), showed attenuated levels of proinflammatory cytokines in the ischemic core and the surrounding peri-infarct area, including a significant reduction in the stroke-induced chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor-1 (CXCL12). To mimic beneficial effects of EE, we studied the impact of inhibiting CXCL12 action on functional recovery after transient MCAO (tMCAO). Rats treated with the specific CXCL12 receptor antagonist 1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclo-tetradecan (AMD3100) showed improved recovery compared with saline-treated rats after tMCAO, without a concomitant reduction in infarct size. This was accompanied by a reduction of infiltrating immune cells in the ischemic hemisphere, particularly cluster of differentiation 3-positive (CD3⁺) and CD3⁺/CD4⁺ T cells. Spleen atrophy and delayed death of splenocytes, induced by tMCAO, was prevented by AMD3100 treatment. We conclude that immoderate excessive activation of the CXCL12 pathway after stroke contributes to depression of neurologic function after stroke and that CXCR4 antagonism is beneficial for the recovery after stroke.     

Cortical response to levodopa in Parkinson's disease patients with dyskinesias

Levodopa‐induced dyskinesias are a common and disabling side effect of dopaminergic therapy in Parkinson's disease, but their neural mechanisms in vivo are still poorly understood. Besides striatal pathology, the importance of cortical dysfunction has been increasingly recognized. The supplementary motor area in particular, may have a relevant role in dyskinesias onset given its involvement in endogenously generated actions. The aim of the present study was to investigate the levodopa‐related cortical excitability changes along with the emergence of levodopa‐induced peak‐of‐dose dyskinesias in subjects with Parkinson's disease. Thirteen patients without dyskinesias and ten with dyskinesias received 200/50 mg fast‐acting oral levodopa/benserazide following overnight withdrawal (12 hr) from their dopaminergic medication. We targeted transcranial magnetic stimulation to the supplementary motor area, ipsilateral to the most dopamine‐depleted striatum defined with single‐photon emission computed tomography with [¹²³I]N‐ω‐fluoropropyl‐2β‐carbomethoxy‐3β‐(4‐iodophenyl)nortropane, and recorded transcranial magnetic stimulation‐evoked potentials with high‐density electroencephalography before and at 30, 60, and 180 min after levodopa/benserazide intake. Clinical improvement from levodopa/benserazide paralleled the increase in cortical excitability in both groups. Subjects with dyskinesias showed higher fluctuation of cortical excitability in comparison to non‐dyskinetic patients, possibly reflecting dyskinetic movements. Together with endogenous brain oscillation, levodopa‐related dynamics of brain state could influence the therapeutic response of neuromodulatory interventions.     

CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke

CD4⁺ T lymphocytes are key mediators of tissue damage after ischemic stroke. However, their infiltration kinetics and interactions with other immune cells in the delayed phase of ischemia remain elusive. We hypothesized that CD4⁺ T cells facilitate delayed autoreactive B cell responses in the brain, which have been previously linked to post-stroke cognitive impairment (PSCI). Therefore, we treated myelin oligodendrocyte glycoprotein T cell receptor transgenic 2D2 mice of both sexes with anti-CD4 antibody following 60-minute middle cerebral artery occlusion and assessed lymphocyte infiltration for up to 72 days. Anti-CD4-treatment eliminated CD4⁺ T cells from the circulation and ischemic brain for 28 days and inhibited B cell infiltration into the brain, particularly in animals with large infarcts. Absence of CD4⁺ T cells did not influence infarct maturation or survival. Once the CD4⁺ population recovered in the periphery, both CD4⁺ T and B lymphocytes entered the infarct site forming follicle-like structures. Additionally, we provide further evidence for PSCI that could be attenuated by CD4 depletion. Our findings demonstrate that CD4⁺ T cells are essential in delayed B cell infiltration into the ischemic brain after stroke. Importantly, lymphocyte infiltration after stroke is a long-lasting process. As CD4 depletion improved cognitive functions in an experimental set-up, these findings set the stage to elaborate more specific immune modulating therapies in treating PSCI.     

T‐cells and macrophages peak weeks after experimental stroke: Spatial and temporal characteristics

The activities of the central and peripheral immune systems impact neurological outcome after ischemic stroke. However, studies investigating the temporal profile of leukocyte infiltration, especially T‐cell recruitment, are sparse. Our aim was to investigate leukocyte infiltration at different time points after experimental stroke in mice. Permanent middle cerebral artery occlusion was performed on 11 weeks old C57BL/6J mice, allowed to survive for 1, 3, 8, 14 or 28 days. In addition to infarct size measurements, detailed immunohistochemical analyses of T‐cell and macrophage influx were performed. A recently introduced F‐19 MR probe (V‐sense), designed to track macrophages, was furthermore tested. Fourteen and 28 days after permanent middle cerebral artery occlusion a significant increase in CD3⁺ T‐cells was found within the ipsilateral hemisphere compared to controls, especially within the infarct core and the corpus callosum. The number of CD68⁺ cells within the infarct core was significantly increased at days 8, 14 and 28. This temporal pattern was also seen in MRI. After experimental stroke within the infarcted cortex we found a delayed (day 14) infiltration of T‐cells and macrophages. Furthermore, our data show that T‐cells are present in higher numbers in the corpus callosum compared to the rest of the brain (except from the infarct core where they were highest).     

Therapeutic potential of experimental autoimmune encephalomyelitis by Fasudil,a Rho kinase inhibitor

The migration of aberrant inflammatory cells into the central nervous system plays an important role in the pathogenesis of demyelinating diseases potentially through the Rho/Rho‐kinase (Rock) pathway, but direct evidence from human and animal models remains inadequate. Here we further confirm that Fasudil, a selective Rock inhibitor, has therapeutic potential in a mouse model of myelin oligodendrocyte glycoprotein (MOG)‐induced experimental autoimmune encephalomyelitis (EAE). The results show that Fasudil decreased the development of EAE in C57BL/6 mice. Immunohistochemistry disclosed that expression of Rock‐II in the perivascular spaces and vascular endothelial cells of spleens, spinal cords, and brains was elevated in EAE and was inhibited in the Fasudil‐treated group. T‐cell proliferation specific to MOG_35–55 was markedly reduced, together with a significant down‐regulation of interleukin (IL)‐17, IL‐6, and MCP‐1. In contrast, secretion of IL‐4 was increased, and IL‐10 was slightly elevated. There were no differences in the percentages of CD4⁺CD25⁺, CD8⁺CD28^?, and CD8⁺CD122⁺ in mononuclear cells. Histological staining disclosed a marked decrease of inflammatory cells in spinal cord and brain of Fasudil‐treated mice. These results, together with previous studies showing the inhibitory effect of Fasudil on T‐cell migration, might expand its clinical application as a new therapy for multiple sclerosis by decreasing cell migration and regulating immune balance. © 2010 Wiley‐Liss, Inc.     

Selective knockout of astrocytic Na+/H+ exchanger isoform 1 reduces astrogliosis,BBB damage,infarction, and improves neurological function after ischemic stroke

Stimulation of Na⁺/H⁺ exchanger isoform 1 (NHE1) in astrocytes causes ionic dysregulation under ischemic conditions. In this study, we created a Nhe1^flox/flox (Nhe1^f/f) mouse line with exon 5 of Nhe1 flanked with two loxP sites and selective ablation of Nhe1 in astrocytes was achieved by crossing Nhe1^f/f mice with Gfap‐Cre^ERT2 Cre‐recombinase mice. Gfap‐Cre^ERT2+/?;Nhe1^f/f mice at postnatal day 60–90 were treated with either corn oil or tamoxifen (Tam, 75 mg/kg/day, i.p.) for 5 days. After 30 days post‐injection, mice underwent transient middle cerebral artery occlusion (tMCAO) to induce ischemic stroke. Compared with the oil‐vehicle group (control), Tam‐treated Gfap‐Cre^ERT2+/?;Nhe1^f/f (Nhe1 KO) mice developed significantly smaller ischemic infarction, less edema, and less neurological function deficits at 1–5 days after tMCAO. Immunocytochemical analysis revealed less astrocytic proliferation, less cellular hypertrophy, and less peri‐lesion gliosis in Nhe1 KO mouse brains. Selective deletion of Nhe1 in astrocytes also reduced cerebral microvessel damage and blood–brain barrier (BBB) injury in ischemic brains. The BBB microvessels of the control brains show swollen endothelial cells, opened tight junctions, increased expression of proinflammatory protease MMP‐9, and significant loss of tight junction protein occludin. In contrast, the Nhe1 KO mice exhibited reduced BBB breakdown and normal tight junction structure, with increased expression of occludin and reduced MMP‐9. Most importantly, deletion of astrocytic Nhe1 gene significantly increased regional cerebral blood flow in the ischemic hemisphere at 24 hr post‐MCAO. Taken together, our study provides the first line of evidence for a causative role of astrocytic NHE1 protein in reactive astrogliosis and ischemic neurovascular damage.     

CXCR4+CD45− BMMNC subpopulation is superior to unfractionated BMMNCs for protection after ischemic stroke in mice

Cell-based therapy is considered to be a promising therapeutic strategy for stroke treatment. Although unfractionated bone marrow mononuclear cells (BMMNCs) have been tried in both preclinical and clinical trials, the effective subpopulations need to be identified. In this study, we used fluorescence-activated cell sorting to harvest the CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ BMMNC subpopulations from transgenic mice that express enhanced green fluorescent protein. We then allogeneically grafted unfractionated BMMNCs or a subpopulation into mice subjected to transient middle cerebral artery occlusion (tMCAO) and compared the effects on stroke outcomes. We found that CXCR4⁺CD45⁻ BMMNCs, but not CXCR4⁺CD45⁺ BMMNCs, more effectively reduced infarction volume and neurologic deficits than did unfractionated BMMNCs. Brain tissue from the ischemic hemisphere of mice treated with CXCR4⁺CD45⁻ BMMNCs had higher levels of vascular endothelial growth factor and lower levels of TNF-α than did tissue from mice treated with unfractionated BMMNCs. In contrast, CXCR4⁺CD45⁺ BMMNCs showed an increase in TNF-α. Additionally, CXCR4⁺CD45⁺ and CXCR4⁺CD45⁻ populations exhibited more robust migration into the lesion areas and were better able to express cell-specific markers of different linages than were the unfractionated BMMNCs. Endothelial and astrocyte cell markers did not colocalize with eGFP⁺ cells in the brains of tMCAO mice that received CXCR4⁺CD45⁺ BMMNCs. In vitro, the CXCR4⁺CD45⁻ BMMNCs expressed significantly more Oct-4 and Nanog mRNA than did the unfractionated BMMNCs. However, we did not detect gene expression of these two pluripotent markers in CXCR4⁺CD45⁺ BMMNCs. Taken together, our study shows for the first time that the CXCR4⁺CD45⁻ BMMNC subpopulation is superior to unfractionated BMMNCs in ameliorating cerebral damage in a mouse model of tMCAO and could represent a new therapeutic approach for stroke treatment.     

Delayed diapedesis of CD8 T cells contributes to long-term pathology after ischemic stroke in male mice

ObjectiveStroke is a debilitating disorder with significant annual mortality and morbidity rates worldwide. Immune cells are recruited to the injured brain within hours after stroke onset and can exhibit either protective or detrimental effects on recovery. However, immune cells, including CD8 T cells, persist in the injured brain for weeks, suggesting a longer-term role for the adaptive immune system during functional recovery. The aim of this study was to determine if the delayed secondary diapedesis of CD8 T cells into the ischemic brain negatively impacts functional recovery after transient ischemic stroke in male mice.ResultsMice exhibited an increased number of leukocytes in the ipsilesional hemispheres at 14 days (3-fold; p < 0.001) and 30 days (2.2-fold; p = 0.02) after transient middle cerebral artery occlusion (tMCAo) compared to 8 days post-tMCAo, at which time acute neuroinflammation predominantly resolves. Moreover, mice with higher ipsilesional CD8 T cells at 30 days (R² = 0.52, p < 0.01) exhibited worse functional recovery. To confirm a detrimental role of chronic CD8 T cell diapedesis on recovery, peripheral CD8 T cells were depleted beginning 10 days post-tMCAo. Delayed CD8 T cell depletion improved motor recovery on the rotarod (F_(1,28) = 4.264; p = 0.048) compared to isotype control-treated mice. CD8 T cell-depleted mice also exhibited 2-fold (p < 0.001) reduced leukocyte infiltration at 30 days post-tMCAo. Specifically, macrophage, neutrophil, and CD4 T cell numbers were reduced in the ipsilesional hemisphere of the CD8 T cell-depleted mice independent of inflammatory status of the post-stroke CNS (e.g. microglial phenotype and cytokine production). RNAseq identified a unique profile for brain infiltrating CD8 T cells at 30 days post-tMCAo, with 46 genes differentially expressed relative to CD8 T cells at 3 days post-tMCAo.ConclusionOur data reveal a role for CD8 T cells in the chronic phase post-stroke that can be therapeutically targeted. We demonstrate long-term CD8 T cell recruitment into the ipsilesional hemisphere that affects both immune cell numbers present in the injured brain and functional recovery through one month after stroke onset.     

The NLRP3 inflammasome drives inflammation in ischemia/reperfusion injury after transient middle cerebral artery occlusion in mice

PurposeCerebral ischemia induces a profound neuro-inflammatory response, but the underlying molecular mechanisms are poorly understood. Inflammasomes (NLRP1, NLRP3, NLRC4, AIM2) are intracellular multi-protein complexes which can induce sets of pro-inflammatory cyto- and chemokines, and thereby guide inflammation. We, here, assessed the functional role of NLRP3 in ischemia/reperfusion (I/R) injury in a mouse model of transient cerebral ischemia.MethodsIschemic stroke was induced in C57Bl/6 mice by 60 min transient middle cerebral artery occlusion (tMCAO) and 3, 7 or 23 h of reperfusion, a paradigm of I/R injury. The expression patterns of inflammasomes in the ischemic hemispheres were evaluated by semiquantitative real-time PCR and Western Blot analysis accompanied by protein localization using immunocytochemistry. Finally, animals were treated with the inflammasome inhibitors Sulforaphane, Genipin, MCC950 or vehicle, directly before or upon recanalization after tMCAO. Stroke outcome was assessed, including infarct size and functional deficits, local inflammatory response, neuronal survival as well as blood–brain barrier function on day 1 after tMCAO.ResultsAfter tMCAO the relative gene expression levels of NLRP3 increased 20-30x within 1 day in the ischemic hemisphere which translated into an increased expression of NLRP3 in neurons. Accordingly, the gene expression levels of the NLRP3-modulator, Bruton’s Tyrosine Kinase (BTK), and the NLRP3-inducible cytokine IL-1β significantly rose. Lesser or non-significant changes were seen for the other inflammasomes. Application of inflammasome inhibitors covering all inflammasomes or specifically NLRP3 significantly reduced infarct volumes when given before or after tMCAO and was accompanied by clear evidence for reduced activation of caspase 1. This stroke attenuating effect coincided with less immune cell infiltration in the ischemic hemisphere and preservation of the blood–brain barrier integrity.ConclusionsOur data show that induction of the NLRP3 inflammasome in neurons drives neuroinflammation in acute ischemic stroke. Early blockade of NLRP3 protects from I/R injury by mitigating inflammation and stabilizing the blood–brain barrier.     

Glial cells suppress postencephalitic CD8+ T lymphocytes through PD‐L1

Engagement of the programmed death (PD)?1 receptor on activated cells by its ligand (PD‐L1) is a mechanism for suppression of activated T‐lymphocytes. Microglia, the resident inflammatory cells of the brain, are important for pathogen detection and initiation of innate immunity, however, a novel role for these cells as immune regulators has also emerged. PD‐L1 on microglia has been shown to negatively regulate T‐cell activation in models of multiple sclerosis and acute viral encephalitis. In this study, we investigated the role of glial cell PD‐L1 in controlling encephalitogenic CD8⁺ T‐lymphocytes, which infiltrate the brain to manage viral infection, but remain to produce chronic neuroinflammation. Using a model of chronic neuroinflammation following murine cytomegalovirus (MCMV)‐induced encephalitis, we found that CD8⁺ T‐cells persisting within the brain expressed PD‐1. Conversely, activated microglia expressed PD‐L1. In vitro, primary murine microglia, which express low basal levels of PD‐L1, upregulated the co‐inhibitory ligand on IFN‐γ‐treatment. Blockade of the PD‐1: PD‐L1 pathway in microglial: CD8⁺ T‐cell co‐cultures increased T‐cell IFN‐γ and interleukin (IL)?2 production. We observed a similar phenomenon following blockade of this co‐inhibitory pathway in astrocyte: CD8⁺ T‐cell co‐cultures. Using ex vivo cultures of brain leukocytes, including microglia and CD8⁺ T‐cells, obtained from mice with MCMV‐induced chronic neuroinflammation, we found that neutralization of either PD‐1 or PD‐L1 increased IFN‐γ production from virus‐specific CD8⁺ T‐cells stimulated with MCMV IE1_168–176 peptide. These data demonstrate that microglia and astrocytes control antiviral T‐cell responses and suggest a therapeutic potential of PD1: PD‐L1 modulation to manage the deleterious consequences of uncontrolled neuroinflammation. GLIA 2014;62:1582–1594     

Lymphocyte infiltration of neocortex and hippocampus after a single brief seizure in mice

Various immune responses have been described in epileptic patients and animal models of epilepsy, but immune responses in brain after a single seizure are poorly understood. We studied immune responses in brain after a single brief generalized tonic–clonic seizure in mice. C57bl/6 mice, either unanesthetized or anesthetized (pentobarbital, ethyl chloride) received either electrical (15–30 mA, 100 Hz, 1 s) or sham stimulation (subcutaneous electrodes over frontal lobe, no current). Electrical stimulation of unanesthetized mice resulted in tonic–clonic convulsions with hind-limb extension (maximal seizure), tonic–clonic convulsions without hind-limb extension (submaximal seizure), or no seizure. In contrast, such stimulation of anesthetized mice did not result in seizure. Mice were killed at 1 h–7 days after seizure. Brains or regions dissected from brain (neocortex, hippocampus, midbrain, cerebellum) of each group were pooled, single cell suspensions prepared, and cells separated according to density. CD4⁺ (CD3⁺CD45^Hi) and CD8⁺ (CD3⁺CD45^Hi) T cell and CD45R⁺ (CD45^Hi) B cell numbers were determined by flow cytometry. At 24 h after a maximal seizure, CD4⁺ and CD8⁺ T cells and CD45R⁺ B cells appeared in brain, reaching peak numbers at 48 h, but were no longer detected at 7 days. CD4⁺ T cells and CD45R⁺ B cells were preferentially found in neocortex compared with hippocampus, whereas CD8⁺ T cells were preferentially found in hippocampus at 24 h after a maximal seizure. In contrast, virtually no lymphocytes were detected in brains of unstimulated or sham stimulated mice, unanesthetized stimulated mice after submaximal or no seizure, and anesthetized stimulated mice at 1 h–7 day. Neither Ly6-G+ neutrophils nor erythrocytes were detected in brains of any animals, nor was there any detectable increase of blood–brain barrier permeability by uptake of Evans Blue dye. The results indicate that lymphocyte entry into brain after a single brief seizure is due to a selective process of recruitment into cortical regions.     

CD38 regulation in activated astrocytes: Implications for neuroinflammation and HIV‐1 brain infection

Reactive astrogliosis is a key pathological aspect of neuroinflammatory disorders including human immunodeficiency virus type 1 (HIV‐1)‐associated neurological disease. On the basis of previous data that showedastrocytes activated with interleukin (IL)‐1β induce neuronal injury, we analyzed global gene changes in IL‐1β‐activated human astrocytes by gene microarray. Among the up‐regulated genes, CD38, a 45‐kDa type II single chain transmembrane glycoprotein, was a top candidate, with a 17.24‐fold change that was validated by real‐time polymerase chain reaction. Key functions of CD38 include enzymatic activities and involvement in adhesion and cell signaling. Importantly, CD38⁺CD8⁺ T‐cell expression is a clinical correlate for progression of HIV‐1 infection and biological marker for immune activation. Thus, CD38 expression in HIV‐1 and/or IL‐1β‐stimulated human astrocytes and human brain tissues was analyzed. IL‐1β and HIV‐1 activation of astrocytes enhanced CD38 mRNA levels. Both CD38 immunoreactivity and adenosine 5′‐diphosphate (ADP)‐ribosyl cyclase activity were up‐regulated in IL‐1β‐activated astrocytes. CD38 knockdown using specific siRNAs significantly reduced astrocyte proinflammatory cytokine and chemokine production. However, CD38 mRNA levels were unchanged in IL‐1β knockdown conditions, suggesting that IL‐1β autocrine loop is not implicated in this process. Quantitative immunohistochemical analysis of HIV‐seropositive without encephalitis and HIV‐1 encephalitis brain tissues showed significant up‐regulation of CD38, which colocalized with glial fibrillary acidic protein–positive cells in areas of inflammation. These results suggest an important role of CD38 in the regulation of astrocyte dysfunction during the neuroinflammatory processes involved in neurodegenerative/neuroinflammatory disorders such as HIV‐1 encephalitis. © 2009 Wiley‐Liss, Inc.     

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia

We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45^+high cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8⁺ T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4⁺ T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.     

Glucocorticoids increase CD4CD25 cell percentage and Foxp3 expression in patients with multiple sclerosis

Objectives – To determine whether percentages of CD4⁺CD25^high T cells (a group of regulatory T cells, Treg) differ in patients with multiple sclerosis (MS) in relapse vs remission after glucocorticoid treatment and whether treatment for relapses changes Treg population and the expression of Foxp3, a key Treg‐associated molecule. Materials and methods – Peripheral blood mononuclear cells (PBMC) were obtained from 20 patients with MS during relapse, just before and 2 days after starting steroid treatment (i.v. methylprednisolone 1 g/day for 3 days) and then 6 weeks after treatment. CD4⁺CD25^hi cells were analysed by using flow cytometry. Cytokines were measured by using an ELISA and Foxp3, CD3 and CD25 expression by using quantitative real‐time PCR. Results – The percentage of CD4⁺CD25^hi cells, plasma IL‐10 and Foxp3/CD3 ratio increased 48 h after methylprednisolone initiation and returned to baseline values by 6 weeks post‐treatment. Conclusions – Results suggest that glucocorticoids increase Treg cell functional molecules and percentages. This may be a mechanism whereby steroids expedite recovery from MS relapses.     

Temporal kinetics of CD8+CD28+ and CD8+CD28− T lymphocytes in the injured rat spinal cord

This study aims to explore the temporal changes of cytotoxic CD8⁺CD28⁺ and regulatory CD8⁺ CD28⁻ T‐cell subsets in the lesion microenvironment after spinal cord injury (SCI) in rats, by combination of immunohistochemistry (IHC) and flow cytometry (FCM). In the sham‐opened spinal cord, few CD8⁺ T cells were found. After SCI, the CD8⁺ T cells were detected at one day post‐injury (dpi), then markedly increased and were significantly higher at 3, 7, and 14 dpi compared with one dpi (p < 0.01), the highest being seven dpi. In CD8⁺ T cells, more than 90% were CD28⁺, and there were only small part of CD28⁻ ( < 10%). After 14 days, the infiltrated CD8⁺ T cells were significantly decreased, and few could be found in good condition at 21 and 28 dpi. Annexin V and propidium iodide (PI) staining showed that the percentages of apoptotic/necrotic CD8⁺ cells at 14 dpi and 21 dpi were significantly higher than those of the other early time‐points (p < 0.01). These results indicate that CD8⁺ T cells could rapidly infiltrate into the injured spinal cords and survive two weeks, however, cytotoxic CD8⁺ T cells were dominant. Therefore, two weeks after injury might be the “time window” for treating SCI by prolonging survival times and increasing the fraction of CD8⁺ regulatory T‐cells. © 2016 Wiley Periodicals, Inc.     

Interferon regulatory factor 4/5 signaling impacts on microglial activation after ischemic stroke in mice

Microglial activation is a key element in initiating and perpetuating inflammatory responses to stroke. Interferon regulatory factor 5 (IRF5) and IRF4 signaling have been found critical in mediating macrophage pro‐inflammatory (M1) and anti‐inflammatory (M2) phenotypes, respectively, in peripheral inflammation. We hypothesize that the IRF5/4 regulatory axis also mediates microglial activation after stroke. C57BL6 mice of 8–12 weeks were subject to a 90‐min middle cerebral artery occlusion, and the brains evaluated at 24 h, 3, 10 and 30 days after reperfusion. Flow cytometry was utilized to examine microglial activation and cytokine expression. RT‐PCR was performed for mRNA levels of IRF5/4 in sorted microglia. Microglial expression of IRF5/4 was examined by immunohistochemistry, and brain cytokine levels were determined by ELISA. Our results revealed that the IRF5 mRNA level in sorted microglia increased at 3 days of stroke; whereas IRF4 mRNA level exhibited biphasic increases, with a transient rise at 24 h and a peak at 10 days. The same pattern was seen in IRF5/4 protein colocalization with Iba‐1⁺ cells by IHC. Intracellular levels of TNF‐α and IL‐1β in microglia peaked at 3 days of stroke, and IL‐4⁺IL‐10⁺ double‐positive microglia significantly increased at day 10. Brain levels of these cytokines were consistent with microglial cytokine changes. Worse behavior test results were seen at 3 days vs. 10 days of stroke. We conclude that microglia phenotypes are dynamic to ischemic stroke, and IRF5/4 signaling may regulate microglial M1/M2 activation and impact on stroke outcomes.     

Endothelial nitric oxide synthase inhibition triggers inflammatory responses in the brain of male rats exposed to ischemia‐reperfusion injury

Nitric oxide (NO) derived from endothelial NO synthase (eNOS) plays a role in preserving and maintaining the brain's microcirculation, inhibiting platelet aggregation, leukocyte adhesion, and migration. Inhibition of eNOS activity results in exacerbation of neuronal injury after ischemia by triggering diverse cellular mechanisms, including inflammatory responses. To examine the relative contribution of eNOS in stroke‐induced neuroinflammation, we analyzed the effects of systemic treatment with l‐N‐(1‐iminoethyl)ornithine (L‐NIO), a relatively selective eNOS inhibitor, on the expression of MiR‐155‐5p, a key mediator of innate immunity regulation and endothelial dysfunction, in the cortex of male rats subjected to transient middle cerebral artery occlusion (tMCAo) followed by 24 hr of reperfusion. Inducible NO synthase (iNOS) and interleukin‐10 (IL‐10) mRNA expression were evaluated by real‐time polymerase chain reaction in cortical homogenates and in resident and infiltrating immune cells isolated from ischemic cortex. These latter cells were also analyzed for their expression of CD40, a marker of M1 polarization of microglia/macrophages.tMCAo produced a significant elevation of miR155‐5p and iNOS expression in the ischemic cortex as compared with sham surgery. eNOS inhibition by L‐NIO treatment further elevated the cortical expression of these inflammatory mediators, while not affecting IL‐10 mRNA levels. Interestingly, modulation of iNOS occurred in resident and infiltrating immune cells of the ischemic hemisphere. Accordingly, L‐NIO induced a significant increase in the percentage of CD40⁺ events in CD68⁺ microglia/macrophages of the ischemic cortex as compared with vehicle‐injected animals. These findings demonstrate that inflammatory responses may underlie the detrimental effects due to pharmacological inhibition of eNOS in cerebral ischemia.