Chronic reactive gliosis following regulatory T cell depletion during acute MCMV encephalitis

Long‐term, persistent central nervous system inflammation is commonly seen following brain infection. Using a murine model of viral encephalitis (murine cytomegalovirus, MCMV) we have previously shown that post‐encephalitic brains are maintained in an inflammatory state consisting of glial cell reactivity, retention of brain‐infiltrating tissue‐resident memory CD8⁺ T‐cells, and long‐term persistence of antibody‐producing cells of the B‐lineage. Here, we report that this neuroinflammation occurs concomitantly with accumulation and retention of immunosuppressive regulatory T‐cells (Tregs), and is exacerbated following their ablation. However, the extent to which these Tregs function to control neuroimmune activation following MCMV encephalitis is unknown. In this study, we used Foxp3‐diphtheria toxin receptor‐GFP (Foxp3‐DTR‐GFP) transgenic mice, which upon administration of low‐dose diphtheria toxin (DTx) results in the specific depletion of Tregs, to investigate their function. We found treatment with DTx during the acute phase of viral brain infection (0–4 dpi) resulted in depletion of Tregs from the brain, exacerbation of encephalitis (i.e., increased presence of CD4⁺ and CD8⁺ T‐cells), and chronic reactive phenotypes of resident glial cells (i.e., elevated MHC Class II as well as PD‐L1 levels, sustained microgliosis, and increased glial fibrillary acidic protein (GFAP) expression on astrocytes) versus untreated, infected animals. This chronic proinflammatory environment was associated with reduced cognitive performance in spatial learning and memory tasks (Barnes Maze) by convalescent animals. These data demonstrate that chronic glial cell activation, unremitting post‐encephalitic neuroinflammation, and its associated long‐term neurological sequelae in response to viral brain infection are modulated by the immunoregulatory properties of Tregs. GLIA 2015;63:1982–1996     

Expression of allograft inflammatory factor-1 and haeme oxygenase-1 in brains of rats infected with the neurotropic Borna disease virus

Experimental infection of Lewis rats with Borna disease virus (BDV) causes an immune-mediated nonpurulent meningoencephalitis. Viral persistence in the central nervous system is accompanied by mononuclear infiltrates, activated monocytic/microglial cells and reactive astrocytes. The immune-mediated process was further characterized by expression analysis of allograft inflammatory factor-1 (AIF-1), a novel marker of monocyte/microglial activation and of glial fibrillary acid protein (GFAP) between day 3 and day 50 post infection (p.i.). Potential neuroprotective effects of these cells were studied by the induction of haeme oxygenase-1 (HO-1), a defensive molecule against oxidative stress in various brain insults. In BDV-infected rat brains, mononuclear infiltrates and AIF-1 expression increased up to day 28 p.i. During early time points p.i., AIF-1 expression was mainly found in inflammatory lesions and adjacent brain parenchyma. Already 24 days p.i., a widespread upregulation of AIF-1 was observed which declined only moderately beyond day 28 p.i. HO-1 induction was maximal between days 18 and 28 p.i. Increased amounts of GFAP-positive astrocytes were present beyond 24 days p.i. Viral antigen expression increased simultaneously to the inflammatory reaction and persisted up to 50 days p.i. Widespread upregulation of AIF-1 indicates an early, long-lasting microglial activation, which might be involved in the immunesurveillance of the immune-mediated inflammatory events. The early peak of HO-1 most likely represents a neuroprotective, anti-inflammatory response by invading monocytes, microglial cells and astrocytes during the formation of encephalitic lesions and acute viral replication.     

Reactive nitrogen species contribute to blood-labyrinth barrier disruption in suppurative labyrinthitis complicating experimental pneumococcal meningitis in the rat

Sensorineural hearing damage is a frequent complication of bacterial meningitis, affecting as many as 30% of survivors of pneumococcal meningitis. There is a substantial body of evidence that oxidants, such as reactive nitrogen species (RNS), are central mediators of brain damage in experimental bacterial meningitis. In the present study, we investigated whether RNS also contribute to the pathophysiology of suppurative labyrinthitis in our well-established rat model of pneumococcal meningitis. In all infected rats, but not in uninfected controls, we observed suppurative labyrinthitis. Cochlear inflammation was accompanied by severe blood-labyrinth barrier (BLB) disruption as evidenced by increased Evans Blue extravasation. Furthermore, increased cochlear expression of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) was detected by immunohistochemistry. Colocalization of iNOS and tyrosine nitration (a marker of RNS attack) indicated that nitric oxide (NO) produced by iNOS contributes to oxidative cochlear damage through the action of RNS. To determine the pathophysiological role of RNS in BLB disruption, rats were treated with peroxynitrite scavengers (MnTBAP and uric acid, UA). Six h after adjunctive treatment with 300 mg/kg i.p. UA or 15 mg/kg i.p. MnTBAP+100 mg/kg i.p. ceftriaxone, BLB disruption was significantly reduced compared with that in infected animals treated only with ceftriaxone. Therefore, we conclude that RNS are involved in the breaching of the BLB during meningogenic pneumococcal labyrinthitis.     

Oxidative stress is associated with region-specific neuronal death during thiamine deficiency.

Thiamine deficiency (TD) is a model of chronic impairment of oxidative metabolism and selective neuronal loss. TD leads to region-specific neuronal death and elevation of inducible nitric oxide synthase (iNOS) in macrophages/microglia in mouse brain. Identification of the initial site of neuronal death in the submedial thalamic nucleus allowed us to test the role of iNOS and oxidative stress in TD-induced neuronal death. The pattern of neuronal loss, which begins after 9 days of TD, overlapped with induction of the oxidative stress marker heme oxygenase-1 (HO-1) in microglia. Neuronal death and microglial HO-1 induction spread to engulf the whole thalamus after 11 days of TD. As in past studies, reactive iron and ferritin accumulated in microglia beginning on day 10. The lipid peroxidation product, 4-hydroxynonenal (HNE) accumulated in the remaining thalamic neurons only after 11 days of TD. These responses were not likely mediated by iNOS because HO-1 induction and HNE accumulation were comparable in iNOS knockout mice and wild-type controls. These results show that region and cell specific oxidative stress is associated with selective neurodegeneration during TD. Thus, TD is a useful model to help elucidate neuron-microglial interaction in neurodegenerative diseases associated with oxidative stress.     

Relapsing herpes simplex encephalitis following antiviral therapy

Although treatment for herpes simplex virus (HSV) encephalitis with antiviral agents has improved survival, occasional patients experience unexplained clinical exacerbations. This report presents evidence that some relapses may occur from recurrent viral encephalitis. An adult male developed the classic symptoms of HSV encephalitis. The cerebrospinal fluid (CSF), electroencephalogram, and isotope brain scan suggested a localized encephalitis involving the left temporal lobe. The pateint was treated for 10 days with high doses of cytosine arabinoside instead of the currently recommended adenine arabinoside. Progression of encephalitis stopped, and clinical recovery occurred. The HSV antibody titer increased eightfold. Fifty-four days after the initial encephalitis, the patient relapsed with a subacute progressive encephalitis involving the same brain area. The CSF demonstrated oligoclonal bands, elevated immunoglobulin G levels (100 mg/dl), and a high HSV antibody titer (1:8,192 by indirect hemagglutination test). From a left temporal lobe biopsy taken 74 days after onset of the initial encephalitis, herpes simplex virus type 1 was isolated. Without renewed antiviral drug therapy, the patient slowly recovered.     

Induction of HO-1 and NOS in doppel-expressing mice devoid of PrP: implications for doppel function

Ectopic expression of the doppel (Dpl) protein, a homologue of the prion protein (PrP), was recently associated with cerebellar Purkinje cell degeneration observed in two aging prion protein knock-out (Prnp(0/0)) mouse lines. We investigated the possible role of Dpl in oxidative metabolism. Two Prnp(0/0) mouse lines of similar genetic background were studied. One line expresses Dpl in the brain and displays Dpl-associated cerebellar abnormalities. The other has no elevated expression of Dpl and no cerebellar abnormalities. We observed a correlation between Dpl expression and the induction of both heme oxygenase 1 (HO-1) and nitric oxide synthase systems (nNOS and iNOS). These responses are suggestive of increased oxidative stress in the brains of the Dpl-expressing Prnp(0/0) mice. No induction was observed with Hsp-60, indicating a specific response by the HO/NOS system. We proposed that Dpl expression exacerbates oxidative damage that is antagonistic to the protective function of wild-type PrP.     

Aspirin inhibits stress-induced increase in plasma glutamate, brain oxidative damage and ATP fall in rats

The precise mechanisms by which stress induces brain damage are still being elucidated. The high-output, inducible isoform of nitric oxide (NO) synthase (iNOS) is expressed in rat brain after immobilisation stress and its inhibition protects against cell damage in this condition. We have hereby explored some mechanisms involved in iNOS expression and studied the effects of aspirin, a NSAID with neuroprotective actions, in this model. Acute (6 h) stress exposure in rats caused brain expression of iNOS, an increase in plasma glutamate and brain TNF-alpha, induction of oxidative indicators in brain and a fall in brain ATP levels. Prior administration of aspirin (10 mg/kg i.p.) inhibited all these effects caused by stress, suggesting possible therapeutic implications of this drug in this condition.     

Proinflammatory mediators released by activated microglia induces neuronal death in Japanese encephalitis

While a number of studies have documented the importance of microglia in central nervous system (CNS) response to injury, infection and disease, little is known regarding its role in viral encephalitis. We therefore, exploited an experimental model of Japanese Encephalitis, to better understand the role played by microglia in Japanese Encephalitis Virus (JEV) infection. Lectin staining performed to assess microglial activation indicated a robust increase in reactive microglia following infection. A difference in the topographic distribution of activated, resting, and phagocytic microglia was also observed. The levels of various proinflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (Cox-2), IL-6, IL-1beta, TNF-alpha, and MCP-1 that have been implicated in microglial response to an activational state was significantly elevated following infection. These cytokines exhibited region selective expression in the brains of infected animals, with the highest expression observed in the hippocampus. Moreover, the expression of neuronal specific nuclear protein NeuN was markedly downregulated during progressive infection indicating neuronal loss. In vitro studies further confirmed that microglial activation and subsequent release of various proinflammatory mediators induces neuronal death following JEV infection. Although initiation of immune responses by microglial cells is an important protective mechanism in the CNS, unrestrained inflammatory responses may result in irreparable brain damage. Our findings suggest that the increased microglial activation following JEV infection influences the outcome of viral pathogenesis. It is likely that the increased microglial activation triggers bystander damage, as the animals eventually succumb to infection.     

N-acetylcysteine attenuates early induction of heme oxygenase-1 following traumatic brain injury

Traumatic brain injury (TBI) results in a cascade of events that includes the production of reactive oxygen species. Heme oxygenase-1 (HO-1) is induced in glial cells following head trauma, suggestive of oxidative stress. We have studied the temporal and spatial effects of the antioxidant N-acetylcysteine (NAC) on HO-1 levels following lateral fluid-percussion injury by immunoblotting and immunohistochemistry. In the injured cerebral cortex, maximal HO-1 induction was seen 6 h post-TBI and was maintained for up to 24 h following the insult, while the ipsilateral hippocampus and thalamus showed marked induction at 24 h postinjury. In all three brain regions, little or no HO-1 immunoreactivity was observed on the contralateral side. Astrocytes exhibited positive immunoreactivity for HO-1 in the injured cerebral cortex, hippocampus, and thalamus, while some neurons and microglia were also immunoreactive in the injured cortex. The administration of NAC 5 min following TBI resulted in a marked reduction in this widespread induction of HO-1, concomitant with a decrease in the volume of injury in all three brain regions. Together, these findings indicate that HO-1 induction is related to both oxidative and injury characteristics of the affected tissue, suggesting that protein expression of this gene is a credible marker of oxidative damage in this model of TBI.     

A case study of the cognitive and behavioral deficits of temporal lobe damage in herpes simplex encephalitis

Herpes simplex viral encephalitis is a fairly common nonepidemic encephalitis which produces severe neurological sequelae in survivors. Most viral infections of the central nervous system produce diffuse damage, but the herpes simplex virus demonstrates a predilection for localization in the temporal and orbitofrontal regions of the brain. This case study illustrates the highly significant language difficulties, marked memory deficits, and propensity for physical aggression following temporal lobe damage brought about by herpes encephalitis, and presents the usefulness of a new diagnostic measure in delineating such a variable cognitive pattern.     

诱生型血红素氧合酶mRNA、诱生型一氧化氮合酶mRNA在脑缺血中的表达

目的观察诱生型血红素氧合酶(HO-1)mRNA、诱生型一氧化氮合酶(iNOS)mRNA在局灶性脑缺血中的表达及其不同作用。方法采用逆转录酶多聚酶链反应(RT-PCR)方法,测定HO-1mRNA、iNOSmRNA在局灶性缺血脑组织中不同时间点的表达变化。结果iNOSmRNA的表达在缺血后2 h出现,24 h达最高峰,以后逐渐下降。HO-1mRNA表达在缺血后2 h即出现,缺血后12 h达最高峰。结论脑缺血的病理生理过程中存在着一氧化氮(NO)及一氧化碳(CO)两种信使系统之间的相互作用。HO-1mRNA及iNOSmRNA的表达上调并具有时相性。缺血后期HO-1mRNA仍然维持在一定的水平,可能具有对抗后期iNOSmRNA增高所产生的NO毒性作用。     

Neurotransmitter synthesizing enzymes in experimental viral encephalitis

Stereotactic intracerebral inoculation of a non-neuroadapted strain of herpes simplex virus type 1 into the left neostriatum of Sprague-Dawley rats induced clinical acute encephalitis within 3 to 5 days postinoculation, with microscopic evidence of inflammation in brain parenchyma, but with no gross areas of tissue destruction. Viral presence in brain was unequivocally confirmed by tissue culture, immunofluorescence and electron microscopy. Levels of activity of neurotransmitter synthesizing enzymes tyrosine hydroxylase (TH), glutamate decarboxylase (GAD), and choline acetyltransferase (ChAT) in the substantia nigra, caudate-putamen and frontal cortex of acutely encephalitic animals were not significantly different from those of PBS-inoculated controls; neither were there significant differences between the inoculated and non-inoculated sides of the individual animals. Our results show that locally injected herpes simplex virus may spread in brain causing neurological symptoms and death without major local structural changes or loss of neurotransmitter synthesizing enzymes. The degree and distribution of cell dysfunction and cell loss in viral encephalitis basically determine any alterations of enzyme activities specific to the involved cell population. The literature on neurotransmitter enzymes and experimental viral encephalitis is reviewed.     

Oxidative tissue injury in multiple sclerosis is only partly reflected in experimental disease models

Recent data suggest that oxidative injury may play an important role in demyelination and neurodegeneration in multiple sclerosis (MS). We compared the extent of oxidative injury in MS lesions with that in experimental models driven by different inflammatory mechanisms. It was only in a model of coronavirus-induced demyelinating encephalomyelitis that we detected an accumulation of oxidised phospholipids, which was comparable in extent to that in MS. In both, MS and coronavirus-induced encephalomyelitis, this was associated with massive microglial and macrophage activation, accompanied by the expression of the NADPH oxidase subunit p22phox but only sparse expression of inducible nitric oxide synthase (iNOS). Acute and chronic CD4⁺ T cell-mediated experimental autoimmune encephalomyelitis lesions showed transient expression of p22phox and iNOS associated with inflammation. Macrophages in chronic lesions of antibody-mediated demyelinating encephalomyelitis showed lysosomal activity but very little p22phox or iNOS expressions. Active inflammatory demyelinating lesions induced by CD8⁺ T cells or by innate immunity showed macrophage and microglial activation together with the expression of p22phox, but low or absent iNOS reactivity. We corroborated the differences between acute CD4⁺ T cell-mediated experimental autoimmune encephalomyelitis and acute MS lesions via gene expression studies. Furthermore, age-dependent iron accumulation and lesion-associated iron liberation, as occurring in the human brain, were only minor in rodent brains. Our study shows that oxidative injury and its triggering mechanisms diverge in different models of rodent central nervous system inflammation. The amplification of oxidative injury, which has been suggested in MS, is only reflected to a limited degree in the studied rodent models.     

14-3-3 protein in CSF: an early predictor of SIV CNS disease

In neurons, 14-3-3 proteins regulate diverse processes, including signal transduction, neurotransmitter production, and apoptosis by binding to target proteins, but the role 14-3-3 proteins play in the pathogenesis of central nervous system (CNS) disease remains unclear. To examine the relationship between presence of 14-3-3 protein in cerebrospinal fluid (CSF) and encephalitis in the SIV/macaque model of HIV CNS disease, CSF levels of 14-3-3 protein were measured by quantitative immunoblotting throughout infection in 6 SIV-infected pigtailed macaques. Beginning during asymptomatic infection and continuing until death, CSF levels of 14-3-3 were elevated in 4 of 6 SIV-infected animals. Animals with 14-3-3 protein in CSF had the highest viral loads in the CSF after acute infection and the highest levels of both viral RNA and protein in brain (p < 0.001). In contrast, the presence of 14-3-3 protein in CSF was not associated with CNS microglial/macrophage activation measured by quantitative immunohistochemical staining for CD68 (p = 0.13). CSF levels of 14-3-3 protein may be a valuable marker of early neuronal damage, CNS viral replication, and CNS disease progression in HIV-infected individuals.     

莪术提取物对单纯疱疹病毒性脑炎模型小鼠脑血流动力学及氧化应激的作用

目的 探讨莪术提取物对单纯疱疹病毒性脑炎(Herpes simplex encephalitis, HSE)模型小鼠脑血流动力学及氧化应激的影响及其可能作用机制。方法 选取48只C57BL/6雄性小鼠脑部注射Ⅰ型单纯疱疹病毒建立HSE小鼠模型,造模成功小鼠随机分为模型组、莪术提取物低、高剂量(50、100 mg/kg)组、西药组(150 mg/kg阿昔洛韦)各12只,另设对照组12只;实验期间记录小鼠一般情况和存活天数并计算生命延长率;酶联免疫吸附法(Enzyme linked immunosorbent assay, ELISA)检测小鼠血清丙二醛(Malondialdehyde, MDA)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-Px)、超氧化物岐化酶(Superoxide dismutase, SOD)水平;激光散斑成像系统检测小鼠脑血流变化;苏木精-伊红染色法(Hematoxylin eosin staining, HE)染色观察小鼠脑组织病理学变化;实时荧光定量聚合酶链反应法(Real time fluorescent quantitati...     

Infiltration of immune T cells in the brain of mice with herpes simplex virus-induced encephalitis

Herpes simplex virus (HSV) infection of mice can induce viral encephalitis. Using two-fluorochrome immunofluorescence, our present study shows that though there is extensive myelin loss and necrosis in the brain stem of mice with HSV encephalitis, only some oligodendrocytes, astrocytes and microglial cells are infected. T cells that express CD4 or CD8 and a large number of CD4+, F4/80+ macrophages are present in perivascular infiltrates close to and in contact with HSV-infected cells in areas of massive myelin loss. These findings suggest that the resultant infiltration of immune cells into the brain during HSV-1 infection may cause as much damage as the virus itself.     

抑制血红素加氧酶-1对三氧化二砷抗胶质瘤的增敏作用

目的 探讨抑制血红素加氧酶-1(HO-1)是否可以增强三氧化二砷(ATO)对胶质瘤细胞的氧化损伤作用.方法 分别用HO-1激动剂钴原卟啉IX(CoPPIX)和HO-1抑制剂锌原卟啉IX(ZnPPIX)预处理细胞后,用ATO处理胶质瘤细胞系U251MG,通过检测细胞死亡、线粒体膜电位的下降、SubG1等评价ATO对胶质瘤细胞的损伤作用,并使用流式细胞技术检测ATO诱导的活性氧蓄积,用Western blot分析HO-1的诱导、P38和JNK的磷酸化情况.结果 ATO可以诱导U251MG细胞出现明显的细胞死亡、线粒体膜电位(MMP)下降、活性氧蓄积以及HO-1蛋白表达,这些指标均可被活性氧抑制剂L-N-乙酰半胱氨酸(LANC)抑制.HO-1诱导剂CoPPIX可以明显抑制As2O3诱导的细胞死亡和活性氧生成,而HO-1抑制剂ZnPPIX则可以显著增强上述作用.结论 抑制胶质瘤细胞HO-1的表达能显著增强ATO的抗胶质瘤作用,考虑到HO-1在胶质瘤内高表达,ATO联合HO-1抑制剂可能是一种有效治疗胶质瘤的新方法.     

The production of focal herpes encephalitis in mice by stereotaxic inoculation of virus. Anatomical and behavioral effects

A low virulence strain of herpes simplex type 1 was microinjected into the hippocampus of BALB/c mice. Intense replication of virus at the inoculum site was followed by spread of viral antigen to the afferent connections of the hippocampus. Surviving animals showed focal damage of limbic structures and specific behavioral abnormalities generally consistent with hippocampal damage. This procedure thus produces an animal model which more closely resembles human herpes encephalitis than those previously reported.     

Herpes simplex virus type 1 infection induces oxidative stress and the release of bioactive lipid peroxidation by-products in mouse P19N neural cell cultures

To determine whether herpes simplex virus type 1 (HSV-1) infection causes oxidative stress and lipid peroxidation in cultured neural cells, mouse P19 embryonal carcinoma cells were differentiated into cells with neural phenotypes (P19N cells) by retinoic acid and were then infected with HSV-1. Cellular levels of reactive oxygen species (ROS) and the release of lipid peroxidation by-products into the tissue culture medium were then measured by the generation of fluorescent markers hydroxyphenyl fluorescein and a stable chromophore produced by lipid peroxidation products, malondialdehyde (MDA) and hydroxyalkenals (4-HAEs; predominantly 4-hydroxy-2-nonenal [HNE]), respectively. HSV-1 infection increased ROS levels in neural cells as early as 1 h post infection (p.i.) and ROS levels remained elevated at 24 h p.i. This viral effect required viral entry and replication as heat- and ultraviolet light-inactivated HSV-1 were ineffective. HSV-1 infection also was associated with increased levels of MDA/HAE in the culture medium at 2 and 4 h p.i., but MDA/HAE levels were not different from those detected in mock infected control cultures at 1, 6, and 24 h p.i. HSV-1 replication in P19N cells was inhibited by the antioxidant compound ebselen and high concentrations of HNE added to the cultures, but was increased by low concentrations of HNE. These findings indicate that HSV-1 infection of neural cells causes oxidative stress that is required for efficient viral replication. Furthermore, these observations raise the possibility that soluble, bioactive lipid peroxidation by-products generated in infected neural cells may be important regulators of HSV-1 pathogenesis in the nervous system.