Search

Background/aims Neuroendocrine dysregulation has been associated with rheumatoid arthritis (RA). Tyrosine hydroxylase (TH), a rate-limiting enzyme for synthesis of neuroendocrine hormones such as epinephrine, is also expressed in T lymphocytes and regulates balance between helper T (Th) 17 cells and regulatory T (Treg) cells. Herein, we aimed to show that TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in collagen-induced arthritis (CIA), an animal model of RA, and these effects may be implemented by the mechanism of epinephrine action on α1-adrenoreceptor (α1-AR) in T cells. Methods CIA was prepared by intradermal injection of collagen type II in tail base of DBA1/J mice. On the 33rd day post-immunization, lentiviral vectors encoding TH or TH shRNA were injected into ankle joints of CIA mice. Limb inflammation of the mice was assessed beginning from day 21 until day 69 post-immunization by measurement of limb swelling, erythema and rigidity. Th17 and Treg differentiation and function in ankle joints were assessed on day 69 post-immunization by test of the expression of Th17 transcriptional factor ROR-γt and the levels of proinflammatory cytokines interleukin (IL)-17 and IL-22 as well as the expression of Treg transcriptional factor Foxp3 and the levels of antiinflammatory cytokines transforming growth factor (TGF)-β1 and IL-10. T cells were obtained from the spleen of mice that had been immunized with collagen type II 41 day earlier and treated with epinephrine or α1-AR agonist phenylephrine in vitro. Flow cytometry was used to analyze the percentages of CD25-IL-17+ cells and CD25+Foxp3+ cells in CD4+ T cells. Results TH gene overexpression in ankle joints of CIA mice reduced limb inflammation and Th17-related transcription factor expression and inflammatory cytokine production but increased Treg-related antiinflammatory cytokine production in the joints. In contrast, TH gene silence in ankle joints of CIA mice enhanced limb inflammation and Th17 cell activity but decreased Treg cell function in the joints. Epinephrine upregulated α1-AR expression in T cells derived from CIA mice. Both epinephrine and phenylephrine reduced CIA-induced Th17 transcription factor expression and inflammatory cytokine production but enhanced Treg antiinflammatory cytokine production in vitro. Conclusion Upregulating TH expression in joints alleviates joint inflammation and Th17/Treg imbalance in CIA at least partially by enhancing epinephrine action on α1-AR in T cells.

Neuroinflammation has been involved in pathogenesis of Parkinson's disease (PD), a chronic neurodegenerative disease characterized neuropathologically by progressive dopaminergic neuronal loss in the substantia nigra (SN). We recently have shown that helper T (Th)17 cells facilitate dopaminergic neuronal loss in vitro. Herein, we demonstrated that interleukin (IL)-17A, a proinflammatory cytokine produced mainly by Th17 cells, contributed to PD pathogenesis depending on microglia. Mouse and rat models for PD were prepared by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or striatal injection of 1-methyl-4-phenylpyridinium (MPP+), respectively. Both in MPTP-treated mice and MPP+-treated rats, blood-brain barrier (BBB) was disrupted and IL-17A level increased in the SN but not in cortex. Effector T (Teff) cells that were adoptively transferred via tail veins infiltrated into the brain of PD mice but not into that of normal mice. The Teff cell transfer aggravated nigrostriatal dopaminergic neurodegeneration, microglial activation and motor impairment. Contrarily, IL-17A deficiency alleviated BBB disruption, dopaminergic neurodegeneration, microglial activation and motor impairment. Anti-IL-17A-neutralizing antibody that was injected into lateral cerebral ventricle in PD rats ameliorated the manifestations mentioned above. IL-17A activated microglia but did not directly affect dopaminergic neuronal survival in vitro. IL-17A exacerbated dopaminergic neuronal loss only in the presence of microglia, and silencing IL-17A receptor gene in microglia abolished the IL-17A effect. IL-17A-treated microglial medium that contained higher concentration of tumor necrosis factor (TNF)-α facilitated dopaminergic neuronal death. Further, TNF-α-neutralizing antibody attenuated MPP+-induced neurotoxicity. The findings suggest that IL-17A accelerates neurodegeneration in PD depending on microglial activation and at least partly TNF-α release.

Parkinson's disease (PD) is a chronic neurodegenerative disease. Recently, neuroinflammation driven by CD4

Alzheimer's disease (AD), the most common chronic neurodegenerative disease, is pathologically characterized by the formation of neurofibrillary tangles because of hyperphosphorylation of tau protein and extracellular deposits of amyloid-β (Aβ) protein termed senile plaques. Recent studies indicate that neuronal apoptosis caused by chronic neuroinflammation is one of the important pathogenesis of AD. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. However, it is poorly known whether the anti-inflammatory property of TGF-β1 is involved in a neuroprotection in AD. Here, an AD cell model of hippocampal neurons induced by Aβ1-42 was used to show an anti-inflammatory and neuroprotective effect of TGF-β1 through its receptor transforming growth factor-β receptor type I (TβR-I). As expected, Aβ1-42-induced an upregulation in neuronal expression of amyloid precursor protein (APP), tumor necrosis factor-α, cyclooxygenase-2, Bax, cleaved caspase-3, and cleaved caspase-9, and a downregulation in the expression of Bcl-2, as well as an increase in the number of NeuN/terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) double-positive cells. TGF-β1 pretreatment reduced the Aβ1-42-induced effects of upregulating APP, tumor necrosis factor-α, Bax, cleaved caspase-3 and cleaved caspase-9, and downregulating Bcl-2, in addition to increasing NeuNTUNEL cell number. TβR-I expression in hippocampal neurons was downregulated by Aβ1-42 exposure, but upregulated by TGF-β1 pretreatment. Silencing of the TβR-I gene in the neurons abolished the anti-inflammatory and antiapoptotic effects of TGF-β1 in the Aβ1-42-induced AD cell model. These findings suggest that TGF-β1 protects neurons against Aβ1-42-induced neuronal inflammation and apoptosis by activation of TβR-I.

Transforming growth factor (TGF)-β1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. Recently we have shown that TGF-β1 pretreatment in vitro protects against 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neuronal loss that characterizes in Parkinson’s disease (PD). Herein, we aimed to demonstrate that TGF-β1 administration in vivo after MPP+ toxicity has neuroprotection that is achieved by a mediation of microglia. A rat model of PD was prepared by injecting MPP+ unilaterally in the striatum. At 14 days after MPP+ injection, TGF-β1 was administrated in the right lateral cerebral ventricle. Primary ventral mesencephalic (VM) neurons and cerebral cortical microglia were treated by MPP+, respectively, and TGF-β1 was applied to neuronal or microglial cultures at 1 h after MPP+ treatment. As expected, MPP+ resulted in decrease in TGF-β1 production in the substantia nigra and in primary VM neurons and microglia. TGF-β1 intracerebroventricular administration alleviated MPP+-induced PD-like changes in pathology, motor coordination and behavior. Meanwhile, TGF-β1 ameliorated MPP+-induced microglial activation and inflammatory cytokine production in vivo. Interestingly, TGF-β1 treatment was not able to ameliorate MPP+-induced dopaminergic neuronal loss and caspase-3/9 activation in mono-neuron cultures, but TGF-β1 alleviated MPP+-induced microglial activation and inflammatory cytokine production in microglia-enriched cultures. This effect of TGF-β1 inhibiting microglial inflammatory response was blocked by Smad3 inhibitor SIS3. Importantly, neuronal exposure to supernatants of primary microglia that had been treated with TGF-β1 reduced dopaminergic neuronal loss and caspase-3/9 activation induced by MPP+-treated microglial supernatants. These findings establish that TGF-β1 exerts neuroprotective property in PD by inhibiting microglial inflammatory response via Smad3 signaling.

Background/Aims: Regulatory T (Treg) cells have been associated with neuroprotection by inhibiting microglial activation in animal models of Parkinson's disease (PD), a progressive neurodegenerative disease characterized by dopaminergic neuronal loss in the nigrostriatal system. Herein, we show that Treg cells directly protect dopaminergic neurons against 1-methyl-4-phenylpyridinium (MPP+) neurotoxicity via an interaction between the two transmembrane proteins CD47 and signal regulatory protein α (SIRPA). Methods: Primary ventral mesencephalic (VM) cells or VM neurons were pretreated with Treg cells before MPP+ treatment. Transwell co-culture of Treg cells and VM neurons was used to assess the effects of the Treg cytokines transforming growth factor (TGF)-β1 and interleukin (IL)-10 on dopaminergic neurons. Live cell imaging system detected a dynamic contact of Treg cells with VM neurons that were stained with CD47 and SIRPA, respectively. Dopaminergic neuronal loss, which was assessed by the number of tyrosine hydroxylase (TH)-immunoreactive cells, was examined after silencing CD47 in Treg cells or silencing SIRPA in VM neurons. Results: Treg cells prevented MPP+-induced dopaminergic neuronal loss and glial inflammatory responses. TGF-β1 and IL-10 secreted from Treg cells did not significantly prevent MPP+-induced dopaminergic neuronal loss in transwell co-culture of Treg cells and VM neurons. CD47 and SIRPA were expressed by Treg cells and VM neurons, respectively. CD47-labeled Treg cells dynamically contacted with SIRPA-labeled VM neurons. Silencing CD47 gene in Treg cells impaired the ability of Treg cells to protect dopaminergic neurons against MPP+ toxicity. Similarly, SIRPA knockdown in VM neurons reduced the ability of Treg cell neuroprotection. Rac1/Akt signaling pathway in VM neurons was activated by CD47-SIRPA interaction between Treg cells and the neurons. Inhibiting Rac1/Akt signaling in VM neurons compromised Treg cell neuroprotection. Conclusion: Treg cells protect dopaminergic neurons against MPP+ neurotoxicity by a cell-to-cell contact mechanism underlying CD47-SIRPA interaction and Rac1/Akt activation.

Regulatory T cells (Tregs), which secrete transforming growth factor (TGF)-β and interleukin (IL)-10, have essential role in anti-inflammatory and neurotrophic functions. Herein, we explore the neuroprotection of Tregs in Parkinson's disease (PD) by adoptive transfer of Tregs. Tregs, isolated by magnetic sorting, were activated in vitro and then were adoptively transferred to 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP)-treated mice. Neuroinflammation, dopaminergic neuronal loss and behavioral changes of PD mice were evaluated. Live cell imaging system detected a dynamic contact of Tregs with MN9D cells that were stained with CD45 and galectin-1, respectively. Tregs prevented MPTP-induced dopaminergic neuronal loss, behavioral changes, and attenuated the inflammatory reaction in the brain. When blockade the LFA-1 activity in Tregs or the ICAM-1 activity in endothelial cells, the percentage of Tregs in substantia nigra (SN) decreased. CD45 and galectin-1 were expressed by Tregs and MN9D cells, respectively. CD45-labeled Tregs dynamically contacted with galectin-1-labeled MN9D cells. Inhibiting CD45 in Tregs impaired the ability of Tregs to protect dopaminergic neurons against MPP+ toxicity. Similarly, galectin-1 knockdown in MN9D cells reduced the ability of Tregs neuroprotection. Adoptive transfer of Tregs protects dopaminergic neurons in PD mice by a cell-to-cell contact mechanism underlying CD45-galectin-1 interaction. Graphical Abstract.

To investigate the neuroprotective effects of transforming growth factor beta 1(TGF-β1) on the expression and secretion of cytokines induced by AβHippocampal neurons and microglia obtained from SD rat were co-cultured. TGF-β1 was applied on day 5 after the neurons and microglia co-cultures were incubated at the concentrations of 5 or 20 ng/ml, AβIn the hippocampal neuron-microglia co-cultures, AβTGF-β1 significantly inhibits the increase in inflammatory cytokines and the decrease in neurotrophic factor which are caused by Aβ

Tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines, is expressed in T lymphocytes. However, the role of T cell-expressed TH in rheumatoid arthritis (RA) is less clear. Herein, we aimed to show the contribution of TH expression by CD4+ T cells to alleviation of helper T (Th)17/regulatory T (Treg) imbalance in collagen-induced arthritis (CIA), a mouse model of RA. CIA was prepared by intradermal injection of collagen type II (CII) at tail base of DBA1/J mice. Expression of TH in the spleen and the ankle joints was measured by real-time polymerase chain reaction and Western blot analysis. Percentages of TH-expressing Th17 and Treg cells in splenic CD4+ T cells were determined by flow cytometry. Overexpression and knockdown of TH gene in CD4+ T cells were taken to evaluate effects of TH on Th17 and Treg cells in CIA. TH expression was upregulated in both the inflamed tissues (spleen and ankle joints) and the CD4+ T cells of CIA mice. In splenic CD4+ T cells, the cells expressing TH were increased during CIA. These cells that expressed more TH in CIA were mainly Th17 cells rather than Treg cells. TH gene overexpression in CD4+ T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion, whereas TH gene knockdown enhanced the Th17 cell activity. In contrast, TH gene overexpression increased Treg-related cytokine expression and secretion in CD4+ T cells of CIA mice, while TH gene knockdown decreased the Treg cell changes. Collectively, these findings show that CIA induces TH expression in CD4+ T cells, particularly in Th17 cells, and suggest that the increased TH expression during CIA represents an anti-inflammatory mechanism.

Neuroinflammation is principally linked to glial function and has been demonstrated to participate in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder characterized by beta-amyloid ccumulation and neurotransmission disruption. Previous findings suggest acetylcholine exerts anti-inflammatory and neuroprotective properties in several neurodegenerative disorders. However, the underlying mechanisms remain elusive. Here evaluation of the influence of acetylcholine on neuroinflammation and neurodegeneration in Alzheimer's disease is reported and further neuroprotective mechanisms are investigated. Investigation of microglia in lipopolysaccharide-induced hippocampal neuronal toxicity employed α7nAChR gene silencing and demonstrated that both the anti-inflammatory and neuroprotective effects of acetylcholine rely on α7nAChR pathways. As expected, in neuron-microglia co-cultures lipopolysaccharide induced an increase in expression of pro-inflammatory factors, including inducible nitric oxide synthase, interleukin-1α, and tumor necrosis factor-α, and decreased expression of neurotrophic factors such as insulin-like growth factor-1, and neuronal apoptosis. Acetylcholine protects against lipopolysaccharide-elicited neuronal injury by inhibiting the microglial inflammatory response and promoting microglial neurotrophic factor production via the action of α7nAChR on microglia. These findings establish that ACh activates α7nAChR in microglia, which in turn protects hippocampal neurons.

Microglia are the main immune cells in the central nervous system. In the present study, the mechanism for acetylcholine (ACh) inhibiting microglial inflammatory response was investigated. Primary culture of microglia was isolated from cerebral cortex of Sprague-Dawley (SD) rats. Lipopolysaccharide (LPS) was used to activate the microglia to induce inflammatory response, and then the microglia were treated with ACh for 24 h. Protein expressions of several inflammatory factors, insulin-like growth factor 1 (IGF-1) and α7 nicotinic acetylcholine receptor (α7nAChR) were detected by Western blot. Release of inflammatory factors and IGF-1 into media was detected by ELISA. After α7nAChR gene silence was achieved by lentivirus-transfection of α7nAChR-shRNA, the change of ACh effect was observed. The results showed that LPS induced microglial activation, up-regulated inducible nitric oxide synthase (iNOS) protein expression, increased the expressions and release of IL-1β and TNF-α, and decreased the expression and release of the neurotrophic factor, IGF-1. ACh could reverse these effects of LPS. Meanwhile, LPS reduced the protein expression of α7nAChR on the microglial cells, whereas ACh could reverse the effect. Silencing of α7nAChR gene in microglia abolished the ability of ACh to inhibit LPS-induced inflammatory responses. These results suggest that ACh exerts its protection against LPS-induced microglial inflammation via acting on α7nAChR on microglia, which may provide a novel target for the treatment of neuro-inflammatory diseases.

BACKGROUND Norepinephrine (NE), a neurotransmitter released from the sympathetic nerves, has been shown to be involved in rheumatoid arthritis (RA). However, its role in the sympathetic nervous system in RA is divergent. Herein, we demonstrate that the sympathetic neurotransmitter NE exerts an anti-inflammatory effect in collagen-induced arthritis (CIA), a mouse model of RA, by inhibiting Th17 cell differentiation and function via β2-adrenergic receptor (β2-AR) signaling. MATERIAL AND METHODS CIA was prepared by intradermal injection of collagen type II in the tail base of DBA1/J mice. On the 41st day post-immunization, the mice were used as CIA models. CD4+ T cells from the spleen were purified using magnetic cell sorting and activated with anti-CD3 anti-CD28 antibodies. Th17 cells were polarized from the CD4+ T cells using various antibodies and cytokines. RESULTS Co-expression of CD4 and β2-AR was observed in spleens of both intact and CIA mice. The β2-AR expression in the ankle and spleen was downregulated in CIA mice. CIA induced increases in production of interleukin (IL)-17 and IL-22, CD25-IL-17+ cell percentage, and ROR-γt expression in CD4+ T cells. Importantly, NE reduced the CIA-induced CD4+ T cell shift towards Th17 phenotype, and the β2-AR antagonist ICI118551 blocked the NE effect. Moreover, the β2-AR agonist terbutaline (Terb) inhibited CIA-induced CD4+ T cell proliferation and shift towards Th17 phenotype, and the protein kinase A (PKA) inhibitor H-89 abolished the agonist effect. Terb also reduced CIA-induced Th17 enhancement, and H-89 impaired the Terb effect. CONCLUSIONS NE inhibits Th17 cell differentiation and function in CIA condition by activation of β2-AR/PKA signaling.

Introduction Diabetic cardiomyopathy, a disorder of the heart muscle in diabetic patients, is one of the major causes of heart failure. The aim of present study was to investigate the therapeutic effect of hydrogen molecule on streptozotocin-induced diabetic cardiomyopathy in mice. Methods Diabetes was induced in adult male mice by consecutive peritoneal injection of streptozotocin (50 mg/kg/day) for 5 days. Then, they were treated with hydrogen water (1.3±0.2 mg/l) for 8 weeks (four groups, n= 83–88 in each group). Results Although treatment of diabetic mice with hydrogen water did not significantly affect blood glucose level, it significantly attenuated cardiac hypertrophy and reduced expression of atrial natriuretic factor and β-myosin heavy chain; it alleviated cardiac fibrosis and reduced expression of collagen I and III, transforming growth factor beta, alpha-smooth muscle actin, and osteopontin; it reduced cardiac caspase-3 activity and ratio of bax/bcl-2. Importantly, hydrogen water treatment improved cardiac function in streptozotocin-diabetic mice. Furthermore, it was found that hydrogen water treatment abated oxidative stress, suppressed inflammation, and attenuated endoplasmic reticulum stress in the hearts of streptozotocin-diabetic mice. In addition, hydrogen water treatment suppressed activation of Jun NH2-terminal kinase and p38 mitogen activated protein kinase signaling and nuclear factor κB signaling in the hearts of streptozotocin-diabetic mice. Conclusion Treatment with hydrogen molecule attenuated cardiac dysfunction in streptozotocin-induced diabetic mice, which was independent of glycemic control. Summary Treatment with hydrogen molecule attenuated cardiac dysfunction in streptozotocin-induced type 1 diabetic mice. Molecular hydrogen could thus be envisaged as a nutritional countermeasure for diabetic cardiomyopathy.

Cytosolic Ca(2+) overload induced by N-methyl-D-aspartate (NMDA) is one of the major causes for neuronal cell death during cerebral ischemic insult and neurodegenerative disorders. Previously, we have reported that the cytokine interleukin-6 (IL-6) reduces NMDA-induced cytosolic Ca(2+) overload by inhibiting both L-type voltage-gated calcium channel (L-VGCC) activity and intracellular Ca(2+) store release in cultured cerebellar granule neurons (CGNs). Here we aimed to show that NMDA-gated receptor channels (i.e., NMDA receptors, NMDARs) are an inhibitory target of IL-6 via a mediation of calcineurin (CaN) signaling. As expected, IL-6 decreased NMDAR-mediated cytosolic Ca(2+) overload and inward current in cultured CGNs. The NMDAR subunits, NR1, NR2A, NR2B and NR2C, were expressed in CGNs. Blocking either of NR2A, NR2B and NR2C with respective antagonist reduced NMDA-induced extracellular Ca(2+) influx and neuronal death. Importantly, the reduced percentages in extracellular Ca(2+) influx and neuronal death by either NR2B or NR2C antagonist were weaker in the presence of IL-6 than in the absence of IL-6, while the reduced percentage by NR2A antagonist was not significantly different between the presence and the absence of IL-6. AG490, an inhibitor of Janus kinase (JAK), abolished IL-6 protection against extracellular Ca(2+) influx, mitochondrial membrane depolarization, neuronal death, and CaN activity impairment induced by NMDA. The CaN inhibitor FK506 reduced these IL-6 neuroprotective properties. Collectively, these results suggest that IL-6 exerts neuroprotection by inhibiting activities of the NMDAR subunits NR2B and NR2C (but not NR2A) via the intermediation of JAK/CaN signaling.

Our previous work has shown that cerebellar interposed nucleus (IN) modulates immune function. Herein, we reveal mechanism underlying the immunomodulation. Treatment of bilateral cerebellar IN of rats with 3-mercaptopropionic acid (3-MP), a glutamic acid decarboxylase antagonist that reduces γ-aminobutyric acid (GABA) synthesis, enhanced cellular and humoral immune responses to bovine serum albumin, whereas injection of vigabatrin, a GABA-transaminase inhibitor that inhibits GABA degradation, in bilateral cerebellar IN attenuated the immune responses. The 3-MP or vigabatrin administrations in the cerebellar IN decreased or increased hypothalamic GABA content and lymphoid tissues' norepinephrine content, respectively, but did not alter adrenocortical or thyroid hormone levels in serum. In addition, a direct GABAergic projection from cerebellar IN to hypothalamus was found. These findings suggest that GABAergic neurons in cerebellar IN regulate immune system via hypothalamic and sympathetic pathways.

We previously have shown that cerebellar fastigial nucleus (FN) modulates immune function, but pathways or mechanisms underlying this immunomodulation require clarification. Herein, an anterograde and retrograde tracing of nerve tracts between the cerebellar FN and hypothalamus/thalamus was performed in rats. After demonstrating a direct cerebellar FN-hypothalamic/thalamic glutamatergic projection, 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of glutaminase that catalyzes glutamate synthesis, was injected bilaterally in the cerebellar FN and simultaneously, D,L-threo-β-hydroxyaspartic acid (THA), an inhibitor of glutamate transporters on cell membrane, was bilaterally injected in the lateral hypothalamic area (LHA) or the ventrolateral (VL) thalamic nucleus. DON treatment in the FN alone decreased number of glutamatergic neurons that projected axons to the LHA and also diminished glutamate content in both the hypothalamus and the thalamus. These effects of DON were reduced by combined treatment with THA in the LHA or in the VL. Importantly, DON treatment in the FN alone attenuated percentage and cytotoxicity of natural killer (NK) cells and also lowered percentage and cytokine production of T lymphocytes. These DON-caused immune effects were reduced or abolished by combined treatment with THA in the LHA, but not in the VL. Simultaneously, DON treatment elevated level of norepinephrine (NE) in the spleen and mesenteric lymphoid nodes, and THA treatment in the LHA, rather than in the VL, antagonized the DON-caused NE elevation. These findings suggest that glutamatergic neurons in the cerebellar FN regulate innate and adaptive immune functions and the immunomodulation is conveyed by FN-hypothalamic glutamatergic projections and sympathetic nerves that innervate lymphoid tissues.

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease. Interleukin (IL)-10 is one of the most important and best anti-inflammatory cytokines. The objective of this report is to investigate whether IL-10 has any role in protecting ventral mesencephalic (VM) neurons in in vitro model of neuroinflammation. In this study, primary neuron-enriched culture was prepared from the VM tissues of E14 embryos of rats. The cells were pretreated with IL-10 (15 or 50 ng/mL) for 1 h followed by lipopolysaccharide (LPS, 50 ng/mL) application. We found LPS induced neuronal apoptosis and loss while pretreatment with IL-10 reduced neuronal damage after exposure of LPS toxicity. Furthermore, signal transduction pathways related to IL-10 in VM neurons were studied in inflammatory condition. We used both shRNA and pharmacologic inhibition to determine the role of the IL-10 receptor (IL-10R) and its downstream signaling pathways in LPS-induced VM neuronal toxicity. Silence of the IL-10R gene in VM neurons abolished IL-10 mediated protection and the properties of anti-inflammatory and anti-apoptosis. IL-10 also induced phosphorylation of signal transducer and activator of transcription (STAT) 3 in VM neurons. Pretreatment with the specific Janus kinase (JAK) inhibitor reduced STAT3 phosphorylation and blocked IL-10 mediated protection against LPS. These findings suggest that IL-10 provides neuroprotection by acting via IL-10R and its down-stream JAK-STAT3 signal pathways in VM neurons.

Objective: Recently, we have reported that lymphocyte-derived endogenous catecholamines (CAs) facilitate a shift in the T helper (Th)1/Th2 balance towards Th2. The purpose of this study was to explore the involvement of adrenoreceptors (ARs) in Th differentiation and function modulation by lymphocyte-derived CAs. Methods: Lymphocytes were separated from the mesenteric lymph nodes of mice, stimulated with concanavalin A (Con A) and treated with pargyline, an inhibitor of CA degradation. Results: Pargyline downregulated the expression of Th1-relative factors, T-bet, interferon (IFN)-γ and interleukin (IL)-2, but upregulated the expression of Th2-relative factors, GATA-3, IL-4 and IL-10. Pargyline reduced the percentage of IFN-γ-producing CD4+ cells and the CD4+IFN-γ+/CD4+IL-4+ cell ratio, although it did not alter the proportion of IL-4-producing CD4+ cells. In addition, the percentage of CD4+CD26+ T cells and the CD4+CD26+/CD4+CD30+ cell ratio were also reduced in the pargyline-treated group. Furthermore, Con A-activated T cells treated with pargyline produced a lower level of IFN-γ and a higher level of IL-4 than the control group. All these effects were blocked by the α1-AR antagonist corynanthine or the β2-AR antagonist ICI 118551, but not by the α2-AR antagonist yohimbine or β1-AR antagonist atenolol. Conclusions: These results imply that lymphocyte-derived CAs promote polarization of differentiation and function towards Th2 cells and that this effect is mediated by α1-AR and β2-AR.

Laggeric acid (1), a novel 4,5-seco-eudesmane sesquiterpene acid, along with 19 known compounds, was isolated from the MeOH extract of the whole plant of Laggera crispata. The structure of 1 was elucidated to be (4S,5S*,7R*,10R*)-4,5-seco-4,5-dihydroxy-11(13)-eudesmen-12-oic acid on the basis of spectroscopic analysis and a convenient Mosher's method.

The object of this study was to investigate the possible role of local voltage potentials (LVPs) in mapping the ventricular arrhythmias originating from right ventricular outflow (RVOT).Forty-seven patients with RVOT VAs (ventricular arrhythmias), referred for radiofrequency catheter ablation to our hospital, were analysed retrospectively for the prevalence, characteristics and electrophysiological evaluation of the LVPs recorded in successful and unsuccessful ablation sites.Radiofrequency ablation was successful immediately in all the 47 cases. Catheter ablation was performed at a mean of 8 +/- 6 sites per patient. There were 58 effective ablation sites, 5 cases with changing morphology of ventricular arrhythmias (VAs), and 318 invalid ablation sites. Activation times at effective ablation sites were slightly earlierthan those at invalid ablation sites (-28 +/- 8 ms vs-24 +/- 7 ms, P0.05). The LVPs appeared during VAs in 47 sites of the 58 effective ablation sites (81.0%), far more than the 22 sites of the 318 invalid ablation sites (6.9%) (P0.01). In two cases VAs recurred during follow-up. They received a second catheter ablation.Local ventricular potentials can be recorded in most patients with idiopathic VAs originating from the right outflow tract.The local potentials may facilitate successful radiofrequency ablation.

Our previous work has shown that the cerebellar fastigial nucleus (FN) is involved in modulation of lymphocyte function. Herein, we investigated effect of FN γ-aminobutyric acid (GABA)-ergic projections to the hypothalamus on lymphocytes to understand pathways and mechanisms underlying cerebellar immunomodulation. By injection of Texas red dextran amine (TRDA), an anterograde tracer, into FN, we found that the TRDA-labeled fibers from the FN traveled through the superior cerebellar peduncle (SCP), crossed in decussation of SCP (XSCP), entered the hypothalamus, and primarily terminated in the lateral hypothalamic area (LHA). Further, by injecting Fluoro-Ruby (FR), a retrograde tracer, in LHA, we observed that the FR-stained fibers retrogradely passed through XSCP and reached FN. Among these FR-positive neurons in the FN, there were GABA-immunoreactive cells. We then microinjected vigabatrin, which is an inhibitor of GABA-transaminase (GABA-T) that degrades GABA, bilaterally into FN. The vigabatrin treatment increased both number of GABA-immunoreactive neurons in FN-LHA projections and GABA content in the hypothalamus. Simultaneously, vigabatrin significantly reduced concanavalin A (Con A)-induced lymphocyte proliferation, anti-sheep red blood cell (SRBC) IgM antibody level, and natural killer (NK) cell number and cytotoxicity. In support of these findings, we inhibited GABA synthesis by using 3-mercaptopropionic acid (3-MP), which antagonizes glutamic acid decarboxylase (GAD). We found that the inhibition of GABA synthesis caused changes that were opposite to those when GABA was increased with vigabatrin. These findings show that the cerebellar FN has a direct GABAergic projection to the hypothalamus and that this projection actively participates in modulation of lymphocytes.

The epidermal growth factor receptor (EGFR) inhibitor erlotinib has been shown to induce complete remission of acute myeloid leukemia (AML) in two patients with concurrent lung cancer and raised attention for a role of EGFR in AML whereas a recent phase II clinical study with gefitinib in AML demonstrated a negative result on the outcome. However, from several studies, EGFR expression in AML is poorly defined and the role of EGFR in AML remains unclear. Herein, we report the results of EGFR expression in AML of large cohorts of adult and pediatric AML patients with the data of total protein and phosphorylation levels of EGFR. Our data conclude that there is the expression of EGFR at the protein level in a subset of AML, which was identified to be functionally active in ~15 % of AML patients. This suggests that future studies need to be conducted with a subset of AML patients characterized by high EGFR expression. Electronic supplementary material The online version of this article (doi:10.1186/s13045-016-0294-x) contains supplementary material, which is available to authorized users.

Tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines, is expressed in T lymphocytes. However, the role of T cell-expressed TH in rheumatoid arthritis (RA) is less clear. Herein, we aimed to show the contribution of TH expression by CD4+ T cells to alleviation of helper T (Th)17/regulatory T (Treg) imbalance in collagen-induced arthritis (CIA), a mouse model of RA. CIA was prepared by intradermal injection of collagen type II (CII) at tail base of DBA1/J mice. Expression of TH in the spleen and the ankle joints was measured by real-time polymerase chain reaction and Western blot analysis. Percentages of TH-expressing Th17 and Treg cells in splenic CD4+ T cells were determined by flow cytometry. Overexpression and knockdown of TH gene in CD4+ T cells were taken to evaluate effects of TH on Th17 and Treg cells in CIA. TH expression was upregulated in both the inflamed tissues (spleen and ankle joints) and the CD4+ T cells of CIA mice. In splenic CD4+ T cells, the cells expressing TH were increased during CIA. These cells that expressed more TH in CIA were mainly Th17 cells rather than Treg cells. TH gene overexpression in CD4+ T cells from CIA mice reduced Th17 cell percentage as well as Th17-related transcription factor and cytokine expression and secretion, whereas TH gene knockdown enhanced the Th17 cell activity. In contrast, TH gene overexpression increased Treg-related cytokine expression and secretion in CD4+ T cells of CIA mice, while TH gene knockdown decreased the Treg cell changes. Collectively, these findings show that CIA induces TH expression in CD4+ T cells, particularly in Th17 cells, and suggest that the increased TH expression during CIA represents an anti-inflammatory mechanism.

We have previously reported that dopamine D2-like receptors including D2, D3 and D4 receptors are more important in mediating modulation of T cells than dopamine D1-like receptors (D1 and D5 receptors). Here we aimed to clarify the role of D2-like receptors in regulation of differentiation and function of T lymphocyte subsets, including helper T (Th)1, Th2, Th17 and regulatory T (Treg) cells.Lymphocytes, separated from the mesenteric lymph nodes of mice, were stimulated with concanavalin A (Con A) and treated with the D2-like receptor agonist quinpirole or the antagonist haloperidol. Expression of lymphocyte cytokines and transcription factors and dopamine D2, D3 and D4 receptors were measured by real-time quantitative polymerase chain reaction and Western blot assay. Meanwhile, cAMP and phosphorylated cAMP-response element-binding (CREB) levels in the lymphocytes were examined by enzyme-linked immunosorbent assay and Western blot assay, respectively.Activation of D2-like receptors with the agonist quinpirole upregulated the expression of Th2- and Treg-specific transcription factors and cytokines in Con A-activated lymphocytes, but downregulated the expression of Th1- and Th17-specific transcription factors and cytokines. Simultaneously, quinpirole increased dopamine D3 and D4 receptor expression, but did not alter D2 receptor expression. However, quinpirole reduced both cAMP and phosphorylated CREB levels in Con A-activated lymphocytes. All these quinpirole effects were blocked by haloperidol, an antagonist of D2-like receptors.D2-like receptors, principally dopamine D3 and D4 receptors, promote differentiation and function of T lymphocytes towards anti-inflammatory T cell subsets by a negative link to cAMP-CREB pathway.

T helper (Th)17 cells, a subset of CD4+ T lymphocytes, have strong pro-inflammatory property and appear to be essential in the pathogenesis of many inflammatory diseases. However, the involvement of Th17 cells in Parkinson’s disease (PD) that is characterized by a progressive degeneration of dopaminergic (DAergic) neurons in the nigrostriatal system is unclear. Here, we aimed to demonstrate that Th17 cells infiltrate into the brain parenchyma and induce neuroinflammation and DAergic neuronal death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)- or 1-methyl-4-phenylpyridinium (MPP+)-induced PD models. Blood–brain barrier (BBB) disruption in the substantia nigra (SN) was assessed by the signal of FITC-labeled albumin that was injected into blood circulation via the ascending aorta. Live cell imaging system was used to observe a direct contact of Th17 cells with neurons by staining these cells using the two adhesion molecules, leukocyte function-associated antigen (LFA)-1 and intercellular adhesion molecule (ICAM)-1, respectively. Th17 cells invaded into the SN where BBB was disrupted in MPTP-induced PD mice. Th17 cells exacerbated DAergic neuronal loss and pro-inflammatory/neurotrophic factor disorders in MPP+-treated ventral mesencephalic (VM) cell cultures. A direct contact of LFA-1-stained Th17 cells with ICAM-1-stained VM neurons was dynamically captured. Either blocking LFA-1 in Th17 cells or blocking ICAM-1 in VM neurons with neutralizing antibodies abolished Th17-induced DAergic neuronal death. These results establish that Th17 cells infiltrate into the brain parenchyma of PD mice through lesioned BBB and exert neurotoxic property by promoting glial activation and importantly by a direct damage to neurons depending on LFA-1/ICAM-1 interaction.

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca(2+) fluorescence intensity, cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca(2+) overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca(2+) overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.

Three new compounds, debilitriol (1), debilignanoside (2), and equisetumine (3), along with nine known compounds, were isolated from the whole plant of Equisetum debile. Their structures were elucidated by spectral and chemical methods.

Three new 3(4),9(10)-disecocycloartane peroxy triterpene lactones, named as pseudolarolides Q2, T1, and T2 (1–3, resp.), along with five known triterpenoids, were isolated from the seeds of Pseudolarix kaempferiGord. Their structures were elucidated to be (9R,10R,16R,23R,25R)-16,23-epoxy-9,10-epidioxy-3,4:9,10-disecocycloart-1(2)-ene-3,4:26,23-diolide (1), (1R,9R,10S,16R,23S,25R)-1,4:16,23-diepoxy-9,10-epidioxy-3,4;9,10-disecocycloartan-26(23)-olid-3-oic acid methyl ester (2), and (1R,9R,10S,16R,23R,25R)-1,4:16,23-diepoxy-9,10-epidioxy-3,4;9,10-disecocycloartan-26(23)-olid-3-oic acid methyl ester (3) on the basis of spectroscopic analysis.

The aim of present study was to investigate whether downregulation of dimethylarginine dimethylaminohydrolase (DDAH2) and endothelial nitric oxide synthase (eNOS) induced endothelial dysfunction in sinoaortic-denervated (SAD) rats. SAD rats exhibited significantly higher blood pressure (BP) variability and markedly lower baroreflex sensitivity. However, there was no significant difference in BP between SAD rats and sham-operated rats. In SAD rats, ultrastructural analysis revealed that endothelial cells were degenerated and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) study showed that apoptotic aortic endothelial cells increased. Circulating angiotensinII (AngII), asymmetric dimethylarginine (ADMA) and malondialdehyde (MDA) levels in SAD rats were similar to sham-operated rats, but aortic AngII and MDA levels locally increased. Endothelium-mediated relaxation of thoracic aorta isolated from SAD rats was impaired compared to sham-operated rats, whereas the sodium nitroprusside-induced relaxation was quite similar. Western blotting results showed that DDAH2 and eNOS expressions decreased significantly in the aortae of SAD rats. Treatment of primary cultured rat aortic endothelial cells with AngII (1 μM) resulted in a marked reduction of DDAH2 and eNOS expressions, and coadministration of losartan (1 μM), an AT 1 receptor antagonist, abolished the effect. In conclusion, downregulation of DDAH2 and eNOS induced endothelial dysfunction in SAD rats. DDAH2 and eNOS may be the potential targets for treatment of endothelial dysfunction.

Ten compounds, comprising a new C(8) normonoterpenoid glycoside, trachelinoside (1), structurally as 4-(2-O-beta-D-glucopyranosyl)-hydroxyethyl-5,5-dimethyldihydrofuran-2(3H)-one, together with nine known compounds, were isolated from the 85% ethanol extract of the vines and leaves of Trachelospermum jasminoides (Lindl.) Lem.

Background: The unfolded protein response (UPR) is a cellular stress response triggered by accumulation of misfolded proteins in the endoplasmatic reticulum. Bortezomib is a proteasome inhibitor that triggers the UPR. Our goal was to globally assess the expression and activation of cell stress proteins, including the UPR, and to determine if proteins in the UPR pathway are prognostic of clinical response or predictive of bortezomib resistance in pediatric acute myeloid leukemia (AML). Methods: We analyzed 5 UPR proteins (CAV1, EIF2S1, EIF2S1.pS51, ERN1, and GRP78) involved in the UPR by reverse phase protein arrays (RPPA) in ‘‘bulk'' (CD3-/19-) AML cells from 505 de novo pediatric AML patients who participated in the Children's Oncology Group AAML1031 phase 3 clinical trial. Progeny clustering was used to identify subgroups of patients (protein clusters) based on similar protein expression. Clusters were correlated with outcome, as well as patient characteristics. Results: Four UPR protein clusters (C1-4) were recognized based on relative protein expression levels. UPR clusters were correlated with cytogenetics (high risk underrepresented in C3+C4, P Conclusion: We analyzed UPR in pediatric AML and identified four protein clusters that were prognostic of OS and EFS. We were able to identify a subgroup of patients that benefited from the addition of bortezomib to ADE chemotherapy. We hypothesize that patients with low UPR activation are more susceptible to protein cell stress, and protein cell stress susceptibility is amplified by the addition of bortezomib with ADE. This suggests that certain subsets of pediatric AML patients benefit from the ADE+B therapy. The use of UPR screening could identify patients who would benefit from ADE+B therapy. Citation Format: Fieke W. Hoff, Yihua Qiu, Wendy Hu, Amina A. Qutub, Alon S. Gamis, Richard Aplenc, E Anders Kolb, Todd A. Alonzo, Eveline SJM de Bont, Steven M. Kornblau, Terzah Horton. Proteomic profiling of the unfolded protein response identifies patients benefiting from bortezomib in pediatric acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 451.

Background: Despite substantial increases in therapy intensity, the overall survival of pediatric acute myeloid leukemia (AML) is still guarded, with survival rates of approximately 60%. This indicates the need for new therapeutic strategies, as well as improved risk stratification. Chemotherapies target proteins rather than genetic events, yet little is known about the proteomic landscape in pediatric AML. This study provides a global assessment of pediatric AML protein expression and correlates protein expression with outcome. Methods: A reverse phase protein array (RPPA) probed with 298 validated antibodies was performed to determine protein expression in ‘‘bulk'' (CD3-/19-) AML cells from 505 diagnostic pediatric AML patients who participated in the Children's Oncology Group AAML1031 phase 3 clinical trial. Proteomic profiling was applied in the context of 31 protein functional groups (PFG) (e.g., cell cycle, apoptosis) to analyze their expression in relation to related proteins. Progeny clustering was performed to identify patients with correlated protein expression patterns within each PFG (protein cluster). Block clustering searched for protein clusters that recurrently co-occurred (protein constellation), and for subgroups of patients that expressed similar combinations of protein constellations (patient signatures). Signatures were correlated with patient and disease characteristics. Results: For each PFG, protein clusters (n=120) could be discerned that showed different protein expression states. From this we constructed 11 protein constellations and 10 patient signatures. Signatures were correlated with event-free survival (EFS) when we combined signatures into favorable (Sig. 4, 8), intermediate (Sig. 6, 7, 9) and unfavorable (Sig. 1-3, 5, 10) groups (p=0.01). Other significant clinical correlations between signatures included CEPBA (40% in Sig. 6, vs. 9% overall, p Conclusion: We studied the proteomic landscape in 505 pediatric AML patients, and identified 10 protein signatures based on 11 protein constellations. We identified signatures that did well with ADE therapy vs. signatures that did not. Recognition of deregulated proteins could help to select drugs that could potentially improve individualized therapies for the latter signatures. Citation Format: Fieke W. Hoff, Yihua Qiu, Wendy Hu, Amina A. Qutub, Alan S. Gamis, Richard Aplenc, E Anders Kolb, Todd A. Alonzo, Eveline SJM de Bont, Terzah M. Horton, Steven M. Kornblau. Proteomic landscape of de novo pediatric acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2699.

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.

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.

The aim of the present study was to examine the effect of the overexpression of tyrosine hydroxylase (TH), a rate-limiting enzyme for the synthesis of catecholamines (CAs), in lymphocytes on the differentiation and function of T helper (Th) cells. A recombinant TH overexpression plasmid (pEGFP-N1-TH) was constructed and transfected into mesenteric lymphocytes using nucleofection technology. These cells were stimulated with concanavalin A (Con A) for 48 h and then examined for TH expression and CA content, as well as for the percentage of Th1 and Th2 cells, cytokine concentrations and for the levels of signaling molecules. The lymphocytes overexpressing TH also expressed higher mRNA and protein levels of TH, and synthesized more CAs, including norepinephrine (NE), epinephrine (E) and dopamine (DA) than the mock-transfected control cells. TH gene overexpression in the lymphocytes reduced the percentage of interferon-γ (IFN-γ)-producing CD4+ cells and the ratio of CD4+IFN-γ+/CD4+IL-4+ cells, as well as the percentages of CD4+CD26+ and CD4+CD30+ cells and the ratio of CD4+CD26+/CD4+CD30+ cells. TH overexpression also reduced the secretion of IFN-γ and tumor necrosis factor (TNF) from lymphocytes. Moreover, NE inhibited the Con A-induced lymphocyte proliferation and decreased both cyclic adenosine monophosphate (cAMP) levels and p38 mitogen-activated protein kinase (MAPK) expression in the lymphocytes. Our findings thus indicate that TH gene overexpression promotes the polarization and differentiation of CD4+ cells towards Th2 cells, and this effect is mediated by the cAMP and p38 MAPK signaling pathways.

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.

Parkinson's disease (PD), a neurodegenerative disorder, has been reported to be associated with brain neuroinflammation in its pathogenesis. Herein, changes in peripheral immune system were determined to better understand PD pathogenesis and provide possible target for treatment of PD through improvement of immune disorder.1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was intraperitoneally injected into mice to prepare PD model. Expression levels of pro-inflammatory and anti-inflammatory cytokines and transcription factors of CD4+ T lymphocyte subsets in spleen and mesenteric lymph nodes and concentrations of the cytokines in serum were examined on day 7 after MPTP injection. Percentages of CD4+ T lymphocyte subsets were measured by flow cytometry.MPTP induced PD-like changes such as motor and behavioral deficits and nigrostriatal impairment. Expression levels of the pro-inflammatory cytokines including interferon (IFN)-γ, interleukin (IL)-2, IL-17 and IL-22, in spleen and mesenteric lymph nodes were upregulated and their concentrations in serum were elevated in PD progression. But, the concentrations of the anti-inflammatory cytokines including IL-4, IL-10 and transforming growth factor (TGF)-β were not altered in the two lymphoid tissues or serum of PD mice. In addition, expression of T-box in T cells (T-bet), the specific transcription factor of helper T (Th) 1 cells, was downregulated, but expression of transcription factor forkhead box p3 (Foxp3), the transcription factor of regulatory T (Treg) cells, was upregulated. In support of the results, the numbers of IFN-γ-producing CD4+ cells (Th1 cells) were reduced but CD4+CD25+ cells (Treg cells) were elevated in both the lymphoid tissues of PD mice.PD has a dysfunction of peripheral immune system. It manifests enhancement of proinflammatory response and CD4+ T cell differentiation bias towards Treg cells away from Th1 cells.