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     

A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1^H46R); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1^H46R transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.     

Brain-derived neurotrophic factor facilitates glutamate and inhibits GABA release from hippocampal synaptosomes through different mechanisms

Brain-derived neurotrophic factor (BDNF) has an acute excitatory effect on rat hippocampal synaptic transmission. To compare the action of BDNF upon the release of excitatory and inhibitory neurotransmitters in the hippocampus, we studied the effect of acutely applied BDNF on the K+-evoked glutamate and on the K+-evoked gamma-aminobutyric acid (GABA) release from rat hippocampal nerve terminals (synaptosomes). The acute application of BDNF (30-100 ng/ml) enhanced the K+-evoked [3H]glutamate release. This effect involved tyrosine-kinase B (TrkB) receptor phosphorylation and Ca2+ entry into synaptosomes through voltage-sensitive calcium channels, since it was abolished by K252a (200 nM), which prevents TrkB-mediated phosphorylation, and by CdCl2 (0.2 mM), a blocker of voltage-sensitive calcium channels. In contrast, BDNF (3-100 ng/ml) inhibited K+-evoked [3H]GABA release from hippocampal synaptosomes. This action was also mediated by phosphorylation of the TrkB receptor, but was independent of Ca2+ entry into synaptosomes through voltage-sensitive calcium channels. Blockade of transport of GABA with SKF 89976a (20 microM) prevented the inhibitory action of BDNF upon GABA release, indicating that BDNF influences the activity of GABA transporters. It is concluded that BDNF influences in an opposite way, through distinct mechanisms, the release of glutamate and the release of GABA from hippocampal synaptosomes.     

Enriched environment decreases microglia and brain macrophages inflammatory phenotypes through adiponectin-dependent mechanisms: Relevance to depressive-like behavior

Regulation of neuroinflammation by glial cells plays a major role in the pathophysiology of major depression. While astrocyte involvement has been well described, the role of microglia is still elusive. Recently, we have shown that Adiponectin (ApN) plays a crucial role in the anxiolytic/antidepressant neurogenesis-independent effects of enriched environment (EE) in mice; however its mechanisms of action within the brain remain unknown. Here, we show that in a murine model of depression induced by chronic corticosterone administration, the hippocampus and the hypothalamus display increased levels of inflammatory cytokines mRNA, which is reversed by EE housing. By combining flow cytometry, cell sorting and q-PCR, we show that microglia from depressive-like mice adopt a pro-inflammatory phenotype characterized by higher expression levels of IL-1β, IL-6, TNF-α and IκB-α mRNAs. EE housing blocks pro-inflammatory cytokine gene induction and promotes arginase 1 mRNA expression in brain-sorted microglia, indicating that EE favors an anti-inflammatory activation state. We show that microglia and brain-macrophages from corticosterone-treated mice adopt differential expression profiles for CCR2, MHC class II and IL-4recα surface markers depending on whether the mice are kept in standard environment or EE. Interestingly, the effects of EE were abolished when cells are isolated from ApN knock-out mouse brains. When injected intra-cerebroventricularly, ApN, whose level is specifically increased in cerebrospinal fluid of depressive mice raised in EE, rescues microglia phenotype, reduces pro-inflammatory cytokine production by microglia and blocks depressive-like behavior in corticosterone-treated mice. Our data suggest that EE-induced ApN increase within the brain regulates microglia and brain macrophages phenotype and activation state, thus reducing neuroinflammation and depressive-like behaviors in mice.     

Interleukin-18 from neurons and microglia mediates depressive behaviors in mice with post-stroke depression

Post-stroke depression (PSD) is a common and serious complication that is affecting one thirds of stroke patients which leaves them with a poor quality of life, high mortality rate, high recurrent rate, and slow recovery. Recent studies showed that serum interleukin-18 (IL-18) level is a biomarker for patients with PSD. However, the role of IL-18 in the pathology of PSD is still unclear. In this study, we demonstrated that the IL-18 level in the ischemic brain significantly increased in mice with depression-like behaviors that were caused by the combined use of chronic spatial restraint stress and middle cerebral artery occlusion. Interestingly, IL-18 expression was mainly found in neurons at early phase and in microglia at a later phase. Injection of the exogenous IL-18 into the amygdala, but not the hippocampus or the striatum caused severe depression-like behaviors. On the contrary, the blockage of endogenous IL-18 by IL-18 binding protein, a specific antagonist of IL-18, repressed depressive phenotypes in SIR mice. IL-18 KO mice exhibited the resistance to spatial restraint stress and cerebral ischemia injury. Finally, we found that IL-18 mediated depressive behaviors by the interaction of IL-18 receptor and NKCC1, a sodium-potassium chloride co-transporter that is related to GABAergic inhibition. Administration of NKCC1 antagonist bumetanide exerted a therapeutic effect on the in IL-18-induced depressive mice. In conclusion, we demonstrated that increased IL-18 in the brain causes depression-like behaviors by promoting the IL-18 receptor/NKCC1 signaling pathway. Targeting IL-18 and its downstream pathway is a promising strategy for the prevention and treatment of PSD.     

Functional recovery after spinal cord injury in mice through activation of microglia and dendritic cells after IL-12 administration

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.     

Encephalitogenic T lymphocytes develop from SJL/J hematopoietic cells transplanted into severe combined immimodeficient ( SCID) mice

Previously, we constructed chimeras by injecting hematopoietic cells from experimental autoimmune encephalomyelitis (EAE)-susceptible SJL (H-2^S) strain mice into severe combined immunodeficient (SCID) C.B-17scid /scid (H-2^d) mice. These SCID mouse-SJL mouse hematopoietic cell chimeras developed passive EAE following adoptive transfer of PLP S139–151-specific SJL T lymphocyte line cells, but were resistant to active EAE induced by primary immunization with PLP S139–151. In order to gain an understanding of the encephalitogenic potential of transplanted hematopoietic progenitors in SCID mouse-SJL mouse chimeras, we attempted to induce EAE in hematopoietic chimeras constructed with or without an additional SJL fetal thymus implant. Chimeras with the thymus implant were susceptible to passive and active EAE while chimeras without the thymus implant were susceptible to passive but not active EAE. Encephalitogenic, CD4⁺, TCR⁺ T lymphocytes were selected in vitro from PLP S139-151-immunized, thymus-implanted chimeras. These results showed that hematopoietic SJL progenitors developed into antigen-presenting accessory cells and immunocompetent encephalitogenic T lymphocytes following transplantation into SCID mice. The development of primary immune reactivity depended on a fetal thymus implant for expression in SCID mouse-SJL mouse chimeras.     

Minocycline prevents the depressive-like behavior through inhibiting the release of HMGB1 from microglia and neurons

BackgroundOur previous study reports the causal role of high mobility group box 1 (HMGB1) in the development of depression; and we find glycyrrhizic acid (GZA) can be a potential treatment for major depressive disorder (MDD) considering its inhibition of HMGB1 activity. This study aims to further explore the exact cell types that release HMGB1 in the hippocampus.MethodsWe detected the effects of microglia conditioned medium on primary astrocytes and neurons. The effects of minocycline on depressive-like behaviors were tested in BABLB/c mice after four weeks of chronic unpredictable mild stress (CUMS) exposure. Furthermore, the immunofluorescence (IF) assays, hematoxylin-eosin (HE) and TUNEL staining were used to observe hippocampal slices to evaluate the release of HMGB1. The cytoplasmic translocations of HMGB1 protein were assayed by western-blot.ResultsExposure to CUMS caused an active release of HMGB1 from microglia and neurons in the hippocampus. After minocycline administration for inhibiting the activation of microglia, both microglia and neurons reduced the release of HMGB1 and the protein level of central and peripheral HMGB1 recovered accordingly. Along with blocking the release of HMGB1, behavioral and cognitive deficits induced by CUMS were improved significantly by minocycline. In addition, the supernatant of primary microglia stimulated the secretion of HMGB1 in primary neurons, not in astrocytes, at 24 h after 4 h-LPS treatment.ConclusionAll the evidence supported our hypotheses that microglia and neurons are the main cell sources of HMGB1 release under CUMS condition, and that the release of HMGB1 by microglia may play an important role in the development of depressive-like behavior.     

Acetylcholine receptor-reactive T lymphocytes from healthy subjects and myasthenia gravis patients.

Peripheral blood lymphocytes from 23 of 114 (20%) myasthenia gravis (MG) patients showed positive T-cell proliferative responses to native acetylcholine receptor (AChR) purified from the electric fish Torpedo, compared with two of 25 (8%) healthy or other neurologic disease controls. Responsiveness appeared to fluctuate seasonally. Long-term T-cell lines and clones could be selected as readily from the two healthy responders as from the MG cases and showed similar culture behavior, CD4+ phenotype, and HLA class II restrictions. One clone from a control cross-reacted with recombinant human AChR alpha chain (r37-429A) and with the synthetic peptide 125-143(S-S) from its sequence. Both these human antigens stimulated primary proliferative responses at substantially higher frequencies (26 to 59%) than native xeno-AChR--in both patients and controls--demonstrating that truly autoreactive T cells are not inevitably deleted during normal T-cell development.     

Profile of the RNA in exosomes from astrocytes and microglia using deep sequencing:implications for neurodegeneration mechanisms

Glial cells play an important role in signal transduction,energy metabolism,extracellular ion homeostasis and neuroprotection of the central nervous system.However,few studies have explained the potential effects of exosomes from glial cells on central nervous system health and disease.In this study,the genes expressed in exosomes from astrocytes and microglia were identified by deep RNA sequencing.Kyoto Encyclopedia of Genes and Genomes analysis indicated that several pathways in these exosomes are responsible for promoting neurodegenerative diseases,including Alzheimer's disease,Parkinson's disease and Huntington's disease.Gene ontology analysis showed that extracellular exosome,mitochondrion and growth factor activity were enriched in exosomes from the unique astrocyte group,while extracellular exosome and mitochondrion were enriched in exosomes from the unique microglia group.Next,combined with the screening of hub genes,the protein-protein interaction network analysis showed that exosomes from astrocytes influence neurodegenerative diseases through metabolic balance and ubiquitin-dependent protein balance,whereas exosomes from microglia influence neurodegenerative diseases through immune inflammation and oxidative stress.Although there were differences in RNA expression between exosomes from astrocytes and microglia,the groups were related by the hub genes,ubiquitin B and heat shock protein family A(Hsp70) member 8.Ubiquitin B appeared to be involved in pleiotropic regulatory functions,including immune regulation,inflammation inhibition,protein catabolism,intracellular protein transport,exosomes and oxidative stress.The results revealed the clinical significance of exosomes from glia in neurodegenerative diseases.This study was approved by the Animal Ethics Committee of Nantong University,China(approval No.S20180102-152) on January 2,2018.     

The antinociceptive effect of trazodone in mice is mediated through both mu-opioid and serotonergic mechanisms

The antinociceptive effects of trazodone (a triazolopyridine derivative with antidepressant activity) and its interaction with various opioid, noradrenaline and serotonin receptor subtypes were evaluated. Mice were tested with a hotplate analgesia meter. Trazodone induced a dose-dependent antinociceptive effect following i.p. administration. The ED(50) for mice in the hotplate assay for trazodone was 24.8 mg/kg (9.8; 67.4; 95% CL). The effect of opioid, adrenergic and serotonergic receptor antagonists was examined as to their ability to block trazodone antinociception. Trazodone-induced antinociception was significantly inhibited by naloxone, beta-FNA and naloxonazine, but not by nor-BNI or naltrindole, implying involvement of mu1- and mu2-opioid mechanisms. When adrenergic and serotonergic antagonists were used, metergoline (p<0.05) but not phentolamine or yohimbine, decreased antinociception elicited by trazodone, implying a clear 5-HT mechanism of antinociception. When trazodone was administered together with various agonists of the opioid receptor subtypes, it significantly potentiated antinociception mediated by mu1- and mu2- opioid receptor subtypes. Summing up these results, we conclude that the antinociceptive effect of trazodone is mainly influenced by the mu1- +mu2-opioid receptor subtype combined with the serotonergic receptor. These results explain the diffuse clinical use of trazodone in the management of some pain syndromes, and in opioid- and alcohol-detoxification programs, but raise questions regarding a possible 'indirect' opioid-dependence induced by trazodone itself.     

The effects of MPTP on the activation of microglia/astrocytes and cytokine/chemokine levels in different mice strains

The effects of MPTP on two mouse strains with different MPTP sensitivities and immunological backgrounds were compared: MPTP-sensitive C57BL/6 mice (B6) with a propensity for Th1 and less MPTP-sensitive BALB/c mice (BALB) with a propensity for Th2. It was found that acute MPTP treatment induced behavioral dysfunction, activated microglia/astrocytes, and increased the levels of IL-10, IL-12(p40) IL-13, IFN-gamma, and MCP-1 in CSF in B6, but not in BALB. This suggests that variances in immunological backgrounds might be a major contributing factor in sensitivity differences to MPTP.     

The antinociceptive effect of mirtazapine in mice is mediated through serotonergic,noradrenergic and opioid mechanisms

The antinociceptive effects of the noradrenergic and specific serotonergic antidepressant (NaSSA) drug mirtazapine and its interaction with various opioid receptor subtypes were evaluated in mice with a hotplate analgesicmeter. Mirtazapine elicited an antinociceptive effect in a dose-dependent manner following doses from 1 to 7.5mg/kg. As the mirtazapine dose increased beyond 10mg/kg latencies returned to baseline, yielding a biphasic dose-response curve. The effect of opioid, adrenergic, and serotonergic receptor antagonists was examined as to their ability to block mirtazapine antinociception. Mirtazapine (at 10mg/kg)-induced antinociception was significantly inhibited by naloxone, nor-BNI, and naltrindole, but neither by beta-FNA nor by naloxonazine, implying the involvement of kappa(1)- and delta-opioid mechanisms. When adrenergic and serotonergic antagonists were used, both metergoline and yohimbine, decreased antinociception elicited by mirtazapine, implying a combined serotonergic and noradrenergic mechanism of antinociception. When mirtazapine was administered together with various agonists of the opioid receptor subtypes, it significantly potentiated antinociception mediated only by kappa(3)-opioid receptor subtypes. Summing up these results we conclude that the antinociceptive effect of mirtazapine is mainly influenced by the kappa(3)-opioid receptor subtype combined with both serotonergic and noradrenergic receptors. These results suggest a potential use of mirtazapine in the management of some pain syndromes, and raise questions regarding a possible indirect opioid-dependence induced by mirtazapine. However, further research is needed in order to establish both the exact clinical indications and the effective doses of mirtazapine when prescribed for pain.     

胶质瘤中LC3B与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的相关性及意义

目的研究不同级别胶质瘤组织中自噬相关蛋白LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞数量的相关性并探讨其意义。方法采用免疫组化、Western blot检测127例不同级别胶质瘤及40例瘤旁正常组织中LC3B的表达,免疫组化检测CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量,分析其相关性及意义。结果 (1)免疫组化及Western blot检测结果显示LC3B的表达在瘤旁正常组织、低级别胶质瘤及高级别胶质瘤中呈不同程度的升高,且组间比较差异具有显著性(P0.05)。(2)CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞在胶质瘤中的数量明显高于瘤旁正常组织(P0.05),且高级别胶质瘤明显高于低级别胶质瘤(P0.05)。(3)LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量在不同级别胶质瘤中均呈正相关,低级别胶质瘤中相关系数分别为0.466、0.599、0.537,高级别胶质瘤中相关系数分别为0.657、0.608、0.561。(4)胶质瘤中LC3B的表达及CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量均与肿瘤的大小有关(P0.05)。结论在胶质瘤组织中LC3B、CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞均呈高表达,且LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量呈正相关,LC3B可能是影响胶质瘤细胞免疫的因素之一。     

Uptake of fibrillar beta-amyloid by microglia isolated from MSR-A (type I and type II) knockout mice

To characterize the receptors involved in binding fibrillar amyloid A-beta (fA beta), we compared the uptake of fA beta in microglia from wildtype (MSR-A+/+) and MSR-A knockout (MSR-A-/-) mice. On average, there was a 60% reduction in the uptake of Cy3-fA beta in microglia from the MSR-A-/- mice. Cy3-fA beta uptake in the MSR-A-/- mice was still competable by scavenger receptor ligands, including acetylated low-density lipoprotein (Ac-LDL) and fucoidan. This indicates that uptake by MSR-B and/or other MSRs is also involved in the uptake of fA beta by microglia. However, the significant reduction in the uptake of fA beta in the MSR-A-/- microglia suggests that fA beta gets internalized mostly by MSR-As in microglia. Uptake of modified fA beta (ClqA beta) was similar in the MSR-A-/- microglia as in the wildtype indicating that the uptake of the opsonized fA beta is independent of MSR-A.