Yi-Hua Qiu

Th17 Cell-Mediated Neuroinflammation Is Involved in Neurodegeneration of Aβ1-42-Induced Alzheimer’s Disease Model Rats

Neuroinflammation, especially innate immunocyte-mediated neuroinflammation, has been reported to participate in pathogenesis of Alzheimer’s disease (AD). However, the involvement of adaptive immune cells, such as CD4+ T lymphocytes, in pathogenesis of AD is not well clarified. Herein, we focus on T helper 17 (Th17) cells, a subpopulation of CD4+ T cells with high proinflammation, and show the implication of the cells in neurodegeneration of AD. Amyloid β1-42 (Aβ1-42) was bilaterally injected into hippocampus of rats to induce AD. On days 7 and 14 following the Aβ1-42 administration, escape latency of the rats in Morris water maze was increased, expression of amyloid precursor protein was upregulated, but expression of protein phosphatase 2A was downregulated in the hippocampus, and Nissl stain showed neuronal loss and gliosis in CA1 region. Infusion of FITC-linked albumin in blood circulation and combination with immunostaining of hippocampal sections for RORγ, a specific transcriptional factor of Th17 cells, demonstrated blood-brain barrier (BBB) disruption and Th17 cells’ infiltration into brain parenchyma of AD rats. Expression of Th17 proinflammatory cytokines, interleukin (IL)-17 and IL-22, was increased in the hippocampus, and concentrations of the two cytokines were elevated in both the cerebrospinal fluid and the serum in AD occurrence and development. Compared with intact or saline-treated control rats, AD animals indicated an upregulated expression of Fas and FasL in the hippocampus. Further, the immunofluorescent histochemistry on AD hippocampal sections with NeuN, RORγ, Fas and FasL displayed that Fas was principally expressed by neurons and FasL was predominantly expressed by Th17 cells, and that neuronal apoptosis shown by TUNEL and NeuN double-labeled cells increased. These results suggest that Th17 cells, which were infiltrated into AD brain parenchyma, participate in neuroinflammation and neurodegeneration of AD by release of proinflammatory cytokines and by direct action on neurons via Fas/FasL apoptotic pathway.

Interleukin-6 protects cultured cerebellar granule neurons against glutamate-induced neurotoxicity

Cytokine interleukin-6 (IL-6) has been showed to be an important mediator of neuroimmune responses. However, effects of IL-6 in the central nervous system (CNS) are quite complex and diverse, and mechanisms through which IL-6 influences neuronal functions are primarily unknown. In the present study, we explored protective effect of IL-6 that was chronically applied to cerebellar granule neurons (CGNs) in culture against neurodamage induced by glutamate and mechanisms involved in the neuroprotective effect of IL-6. The chronic IL-6 exposure significantly prevented the CGNs from the glutamate-induced attenuation of neuronal vitality. This neuroprotective effect of IL-6 depended on its concentrations. IL-6 at 2.5 ng/ml did not markedly improve the neuronal vitality, but IL-6 at 5 and 10 ng/ml notably improved the neuronal vitality. The glutamate-evoked neuronal apoptosis also was strikingly inhibited by the chronic IL-6 pretreatment. Intracellular Ca2+ in the CGNs lacking IL-6 pretreatment acutely rose as soon as these neurons were stimulated by glutamate and were maintained at higher levels during the whole 18-min period of glutamate attack. Although intracellular Ca2+ in the IL-6-pretreated CGNs also produced an acute and transient elevation in response to the glutamate insult, they quickly dropped and recovered to basal levels before the glutamate application. Anti-gp130 monoclonal antibody (mAb) blocked the suppressive effect of IL-6 on the glutamate-induced intracellular Ca2+ overload. These results reveal that IL-6 can protect neurons against glutamate-induced neurotoxicity, and suggest that the neuroprotective effect of IL-6 may be via gp130 signal transducing pathway to suppress the glutamate-evoked intracellular Ca2+ overload.

Neuroprotection of interleukin-6 against NMDA attack and its signal transduction by JAK and MAPK

Cytokine interleukin-6 (IL-6) has been well shown to be elevated in brain injury and diseases. However, the significance of IL-6 production in such neuropathologic states remains controversial, and the intracellular signal-transduction pathways involved in the brain IL-6 action are primarily unclear. We previously indicated that exogenous IL-6 protected neurons against glutamate and N-methyl-d-aspartate (NMDA) attacks and the effects of IL-6 was blocked by anti-gp130 antibody. Here, we provide further evidence for the IL-6 neuroprotection and show signal molecules transducing the IL-6 message. The cerebellar granule neurons from postnatal 8-day infant rats were exposed to IL-6 for 8 days, and also pretreated chronically with Janus kinase (JAK) inhibitor AG490 and mitogen-activated protein kinase (MAPK) inhibitor PD98059. NMDA stimulated the cultured neurons for 30 min to induce neuronal injury and death. Cell counting kit-8 assay and Western blot were employed to measure neuronal vitality and cleaved caspase-3 expression, respectively. The chronic IL-6 exposure prevented the suppression of the neuronal vitality and the enhancement of the cleaved caspase-3 level induced by NMDA. The neuroprotective effect of IL-6 depended on IL-6 concentration and neuronal damaged degree. IL-6-induced STAT3 phosphorylation was inhibited by AG490 but not by PD98059; and IL-6-induced ERK1/2 activation was blocked by PD98059 but not by AG490. Either AG490 or PD98059 blocked the IL-6 protection against the NMDA-elicited neuronal vitality decrease and caspase-3 activation increase. These findings suggest that IL-6 protects neurons from NMDA-induced excitoxicity and the IL-6 neuroprotection may be transduced by both JAK/STAT3 and RAS/MAPK pathways.

Protection of TGF-β1 against neuroinflammation and neurodegeneration in Aβ1-42-induced Alzheimer's disease model rats.

Neuroinflammation has been reported to be associated with Alzheimer’s disease (AD) pathogenesis. Neuroinflammation is generally considered as an outcome of glial activation; however, we recently demonstrated that T helper (Th)17 cells, a subpopulation of proinflammatory CD4+ T cells, are also involved in AD pathogenesis. Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, can be immunosuppressive, but its effects on lymphocyte-mediated neuroinflammation in AD pathogenesis have not been well addressed. In the current study we administered TGF-β1 via intracerebroventricle (ICV) and intranasal (IN) routes in AD model rats to investigate its antiinflammatory and neuroprotective effects. The AD rat model was prepared by bilateral hippocampal injection of amyloid-β (Aβ)1–42. TGF-β1 was administered via ICV one hour prior to Aβ1–42 injection or via both nares seven days after Aβ1–42 injection. ICV administration of TGF-β1 before Aβ1–42 injection remarkably ameliorated Aβ1–42-induced neurodegeneration and prevented Aβ1–42-induced increases in glia-derived proinflammatory mediators (TNF-α, IL-1β and iNOS), as well as T cell-derived proinflammatory cytokines (IFN-γ, IL-2, IL-17 and IL-22), in the hypothalamus, serum or cerebrospinal fluid (CSF) in a concentration-dependent manner. TGF-β1 pretreatment also prevented Aβ1–42-induced decreases in the neurotrophic factors, IGF-1, GDNF and BDNF, and in the antiinflammatory cytokine, IL-10. Similarly, IN administration of TGF-β1 after Aβ1–42 injection reduced neurodegeneration, elevation of proinflammatory mediators and cytokines, and reduction of neurotrophic and antiinflammatory factors, in the hypothalamus, serum or CSF. These findings suggest that TGF-β1 suppresses glial and T cell-mediated neuroinflammation and thereby alleviates AD-related neurodegeneration. The effectiveness of IN administered TGF-β1 in reducing Aβ1–42 neurotoxicity suggests a possible therapeutic approach in patients with AD.

Dopamine Receptors Modulate Cytotoxicity of Natural Killer Cells via cAMP-PKA-CREB Signaling Pathway

Dopamine (DA), a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK) cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R) with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R) with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB) level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA), prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC), counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The results may provide more targets of therapeutic strategy for neuroimmune diseases.

Effect of endogenous catecholamines on apoptosis of Con A-activated lymphocytes of rats

Our previous studies show that lymphocytes express tyrosine hydroxylase (TH) and synthesize catecholamines (CAs) including dopamine, epinephrine and norepinephrine, and that the lymphocytes-derived endogenous CAs affect function of lymphocytes via autocrine/paracrine pathways. Over recent years, induction of apoptosis has been suggested to be a possible mechanism underlying the endogenous CAs-mediated lymphocyte proliferation, differentiation and activation. However, direct effect of the lymphocytes-synthesized CAs on lymphocyte apoptosis is less known. In the present study, TH inhibitor alpha-methyl-p-tyrosine (alpha-MT) and monoamine oxydase inhibitor pargyline were employed to block the synthesis and degradation of CAs in lymphocytes activated by concanavalin A (Con A). Apoptotic cells and apoptosis-related genes and proteins, Bax, Bcl-2, Fas, Fas-Ligand (FasL) and caspase-3, were examined in the lymphocytes treated with alpha-MT or pargyline by means of Annexin V/propidium iodide (PI) staining, real-time PCR and Western blot analyses, respectively. The treatment with alpha-MT of 10(-6) M and 10(-5) M (not 10(-7) M) notably reduced intracellular and supernatant DA, E and NE of the Con A-activated lymphocytes in a dose-dependent manner, and correspondingly, the treatment induced a remarkable decrease of apoptotic lymphocytes but not necrotic cells. The expression of Bax, Fas, FasL and caspase-3 mRNAs and proteins was significantly inhibited in the Con A-activated lymphocytes after the cells were treated with alpha-MT of 10(-6) M and 10(-5) M; but the expression of Bcl-2 mRNA and protein was dramatically increased by the alpha-MT treatment. Contrarily, the treatment with pargyline of 10(-6) M and 10(-5) M (not 10(-7) M) evidently increased the intracellular and supernatant DA, E and NE contents of the Con A-activated lymphocytes in a dose-dependent manner, and meanwhile, it caused a striking increase of apoptotic lymphocytes but not necrotic cells. The expression of Bax, Fas, FasL and caspase-3 mRNAs and proteins in the Con A-stimulated lymphocytes was remarkably enhanced by the treatment with pargyline of 10(-6) M and 10(-5) M, but the expression of Bcl-2 mRNA and protein was notably attenuated by the pargyline treatment. These results imply that endogenous CAs synthesized and secreted by lymphocytes accelerate lymphocyte apoptosis by altering fine balance between the expression of antiapoptotic and proapoptotic markers at transcriptional and translational levels, and suggest that both the death receptor pathway and the mitochondrial pathway are involved in the endogenous CAs-induced apoptosis.

Effect of lesions of cerebellar fastigial nuclei on lymphocyte functions of rats.

The cerebellum, probably owing to its traditional concept limited to motor control, is less well studied in immunoregulation. To obtain more comprehension and knowledge on cerebellar functions, we investigated effect of cerebellar fastigial nucleus (FN), an output nucleus of the spinocerebellum, on lymphocyte functions, and explored central and peripheral pathways involved in the effect. Kainic acid (KA) was microinjected into bilateral FN of rats (0.4 microg KA in 0.4 microl saline for each side) to destroy neurons of the nuclei. On days 8, 16 and 32 following the FN lesions, methyl-thiazole-tetrazolium (MTT) assay and flow cytometry were used to measure proliferation of concanavalin A (Con A)-induced lymphocytes and cytotoxicity of natural killer (NK) cells against YAC-1 cells, respectively. Meanwhile, glutamate and monoamine neurotransmitters, including norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT), in the hypothalamus and the spleen were determined by means of high-performance liquid chromatography (HPLC) assay. Adrenocorticotropic hormone (ACTH) and cortisol in the plasma were also detected respectively by radioimmunoassay and chemiluminescent immunoassay after the FN lesions. We found that the Con A-induced lymphocyte proliferation and the NK cell cytotoxicity were both significantly enhanced on days 8, 16 and 32 following the effective lesions of the bilateral FN in comparison with those of matching control rats microinjected with saline in their FN. Contents of glutamate and NE, not DA and 5-HT, in the hypothalamus, and concentration of NE, not DA, in the spleen were all remarkably reduced on the 16th day following the FN lesions, when both the T lymphocyte proliferation and the NK cell cytotoxicity were dramatically increased. However, levels of ACTH and cortisol in the plasma had no notable differences between FN lesion rats and FN saline ones when the enhanced T and NK cell functions occurred. These findings reveal that the cerebellar FN participates in the modulation of lymphocyte functions and that the hypothalamus and sympathetic nerves innervating lymphoid organs are involved in this neuroimmunomodulation. Thus, a possible central and peripheral pathway for the spinocerebellum to regulate lymphocyte functions is suggested, i.e. cerebellum-hypothalamus-sympathetic nerves-lymphocytes, while the functional axis of hypothalamus-pituitary-adrenal gland may not contribute to mediation of the spinocerebellar immunomodulation.

TGF-β1 Protection against Aβ1–42-Induced Neuroinflammation and Neurodegeneration in Rats

Transforming growth factor (TGF)-β1, a cytokine that can be expressed in the brain, is a key regulator of the brain’s responses to injury and inflammation. Alzheimer’s disease (AD), the most common neurodegenerative disorder, involves inflammatory processes in the brain in addition to the hallmarks, amyloid-β (Aβ) plaques and neurofibrillary tangles. Recently, we have shown that T-helper (Th) 17 cells, a subpopulation of CD4+ T-cells with high proinflammation, also participate in the brain inflammatory process of AD. However, it is poorly known whether TGF-β1 ameliorates the lymphocyte-mediated neuroinflammation and, thereby, alleviates neurodegeneration in AD. Herein, we administered TGF-β1 via the intracerebroventricle (ICV) in AD model rats, by Aβ1–42 injection in both sides of the hippocampus, to show the neuroprotection of TGF-β1. The TGF-β1 administration after the Aβ1–42 injection ameliorated cognitive deficit and neuronal loss and apoptosis, reduced amyloid precursor protein (APP) expression, elevated protein phosphatase (PP)2A expression, attenuated glial activation and alleviated the imbalance of the pro-inflammatory/anti-inflammatory responses of T-lymphocytes, compared to the Aβ1–42 injection alone. These findings demonstrate that TGF-β1 provides protection against AD neurodegeneration and suggest that the TGF-β1 neuroprotection is implemented by the alleviation of glial and T-cell-mediated neuroinflammation.

Interleukin-10 Protection against Lipopolysaccharide-Induced Neuro-Inflammation and Neurotoxicity in Ventral Mesencephalic Cultures

Interleukin (IL)-10, an anti-inflammatory cytokine, is expressed in the brain and can inhibit microglial activation. Herein, we utilized lipopolysaccharide (LPS)-induced inflammatory Parkinson’s disease (PD) cell model to determine whether microglia and astrocytes are necessary targets for IL-10 neuroprotection. Primary ventral mesencephalic (VM) cultures with different composition of neurons, microglia and astrocytes were prepared. The cells were exposed to IL-10 (15, 50 or 150 ng/mL) 1 h prior to LPS (50 ng/mL) treatment. LPS induced dopaminergic and non-dopaminergic neuronal loss in VM cultures, VM neuron-enriched cultures, and neuron-microglia co-cultures, but not in neuron-astrocyte co-cultures. IL-10 reduced LPS-induced neuronal loss particularly in single VM neuron cultures. Pro-inflammatory mediators (TNF-α, IL-1β, inducible nitric oxide synthase and cyclooxygenase-2) were upregulated in both neuron-microglia and neuron-astrocyte co-cultures by LPS. In contrast, neurotrophic factors (brain-derived neurotrophic factor, insulin-like growth factor-1 or glial cell-derived neurotrophic factor) were downregulated in neuron-microglia co-cultures, but upregulated in neuron-astrocyte co-cultures by LPS. IL-10 reduced both the increase in production of the pro-inflammatory mediators and the decrease in production of the neurotrophic factors induced by LPS. These results suggest that astrocytes can balance LPS neurotoxicity by releasing more neurotrophic factors and that IL-10 exerts neuroprotective property by an extensive action including direct on neurons and indirect via inhibiting microglial activation.

Transforming growth factor-β1 acts via TβR-I on microglia to protect against MPP+-induced dopaminergic neuronal loss

Neuroinflammation is associated with pathogenesis of Parkinsons disease (PD), a neurodegenerative disorder characterized by a progressive loss of dopaminergic (DAergic) neurons within the substantia nigra. Transforming growth factor (TGF)-β1 exerts anti-inflammatory and neuroprotective properties. However, it is unclear if microglia are required for TGF-β1 neuroprotection in PD. Here we used both shRNA and pharmacologic inhibition to determine the role of microglial TGF-β receptor (TβR)-I and its downstream signaling pathways in 1-methyl-4-phenylpyridinium (MPP(+))-induced DAergic neuronal toxicity. As expected, MPP(+) reduced the number of tyrosine hydroxylase (TH)-immunoreactive cells in ventral mesencephalic cell cultures. We found that MPP(+) activated microglia as determined by an upregulation in expression of CD11b and inducible nitric oxide synthase (iNOS), an increase in expression and secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-1β, and a decrease in expression and secretion of the neurotrophic factor, insulin-like growth factor (IGF)-1. Pretreatment with TGF-β1 significantly inhibited all these changes caused by MPP(+). Expression of microglial TβR-I was upregulated by TGF-β1. Silencing of the TβR-I gene in microglia abolished both the neuroprotective and anti-inflammatory properties of TGF-β1. TGF-β1 increased microglial p38 MAPK and Akt phosphorylation, both of which were blocked by the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, respectively. Pretreatment of microglia with either SB203580 or LY294002 impaired the ability of TGF-β1 to inhibit MPP(+)-induced DAergic neuronal loss and microglial activation. These findings establish that TGF-β1 activates TβR-I and its downstream p38 MAPK and PI3K-Akt signaling pathways in microglia to protect against DAergic neuronal loss that characterizes in PD.