Yu-Ping Peng

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.

Effect of endogenous catecholamines in lymphocytes on lymphocyte function.

Our previous work has showed that lymphocytes can synthesize catecholamines (CAs). However, role and mechanism of the endogenous CAs in lymphocytes in modulation of immune function are less known. In the present study, we used alpha-methyl-p-tyrosine (alpha-MT), an inhibitor of tyrosine hydroxylase (TH), and pargyline, an inhibitor of monoamine oxydase, to block the synthesis and degradation of CAs in lymphocytes and then observed changes of lymphocyte proliferation induced by concanavalin A (Con A). Phentolamine and propranolol, antagonists respectively to alpha- and beta-adrenoreceptors, were employed to investigate the receptor mechanism. We found that TH mRNA in the Con A-activated lymphocytes was 2.4 times higher in relative density than that in the resting lymphocytes. Similarly, the intracellular and supernatant CAs, including DA, NE and E, of the Con A-stimulated lymphocytes were significantly raised relative to those of the resting cells. alpha-MT (10(-11), 10(-10) and 10(-9) M) facilitated the Con A-induced lymphocyte proliferation, but pargyline (10(-11), 10(-10) and 10(-9) M) attenuated the cell proliferation. Meanwhile, alpha-MT and pargyline respectively led to decrease and increase in the intracellular and supernatant CAs (DA, NE and E) of the Con A-stimulated lymphocytes. Propranolol completely blocked, but phentolamine partly reversed, the suppressive effect of pargyline on the Con A-induced lymphocyte proliferation. Content of cAMP was remarkably increased in the lymphocytes treated with pargyline alone, but it dropped to control level after these cells were treated with propranolol plus pargyline. These results on the one hand further demonstrate the ability of lymphocytes to synthesize CAs and the enhancive ability of the activated lymphocytes to synthesize CAs, and on the other hand reveal an important role of the endogenous CAs in regulation of function of lymphocytes themselves. Besides, our present findings suggest that CAs synthesized by lymphocytes can secrete out of the lymphocytes via paracrine or autocrine pathway and affect lymphocyte function by beta-adrenoreceptor and cAMP mediating mechanism. Thus, it can be implied that CAs in lymphocytes are also involved in the cross-talk in the neuro-endocrine-immune networks.

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.

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.

Adrenoreceptor-coupled signal-transduction mechanisms mediating lymphocyte apoptosis induced by endogenous catecholamines.

Our previous work has shown that lymphocytes synthesize catecholamines (CAs) and the endogenous CAs accelerate apoptosis of concanavalin A (Con A)-activated lymphocyte. Here, we explored the involvement of adrenoreceptors (ARs) and signal molecules coupled to the ARs in the endogenous CA-mediated modulation of lymphocyte apoptosis. Pargyline, an inhibitor of CA degradation, up-regulated the expression of cleaved caspase-3 protein and increased the number of apoptotic cells. Antagonists of alpha(1)-ARs and beta(2)-ARs, not antagonists of alpha(2)-ARs or beta(1)-ARs, blocked these effects of pargyline. The facilitating effects of pargyline on lymphocyte apoptosis were mimicked by activators of adenylate cyclase and PKC, but reversed by inhibitors of PKA, PLC and PKC. Pargyline-stimulated CREB activation and Smac/DIABLO expression were prevented by the inhibitors of PKA, PLC and PKC. These results imply that endogenous CA-induced lymphocyte apoptosis is mediated by cAMP-PKA- and PLC-PKC-linked CREB-Smac/DIABLO pathways coupled with alpha(1)-ARs and beta(2)-ARs.

Lymphocyte-Derived Catecholamines Induce a Shift of Th1/Th2 Balance toward Th2 Polarization

Aims: Our previous work has shown that lymphocytes synthesize and secrete catecholamines (CAs), which regulate lymphocyte proliferation and apoptosis. In the present study, we explored the effect of the lymphocyte-derived CAs on differentiation and function of T helper (Th) cells. Methods: Lymphocytes were separated from the mesenteric lymph nodes of mice and stimulated by concanavalin A (Con A). These cells were treated with alpha-methyl-p- tyrosine (α-MT), an inhibitor of tyrosine hydroxylase (TH) that is a rate-limiting enzyme for synthesis of CAs, and pargyline, an inhibitor of monoamine oxidase that degrades CAs. Results: Treatment of Con A-stimulated lymphocytes with α-MT (10–6m) reduced CAs both in the cultured lymphocytes and in the culture supernatants. Simultaneously, α-MT upregulated expression of mRNAs and proteins of T-box expressed in T cells (T-bet) and interferon-γ (IFN-γ) but downregulated expression of mRNAs and proteins of GATA binding protein 3 (GATA-3) and interleukin-4 (IL-4) in Con A-activated lymphocytes. In contrast, pargyline (10–6m) increased intracellular and supernatant CA contents in Con A-activated lymphocytes. Meanwhile, the treatment with pargyline downregulated expression of T-bet and IFN-γ but upregulated expression of GATA-3 and IL-4 in these lymphocytes. Conclusion: CAs synthesized and secreted by lymphocytes regulate differentiation and function of Th cells, with an effect facilitating the shift of Th1/Th2 balance toward Th2 polarization.