Dopamine D2 receptor on CD4+ T cells is protective against inflammatory responses and signs in a mouse model of rheumatoid arthritis.

BACKGROUND: Dopamine is a neurotransmitter and has been found to regulate lymphocytes by acting on dopamine receptors (DRs). CD4+ T cells express all the five subtypes of DRs, D1R to D5R. Although CD4+ T cells have been involved in pathogenesis of rheumatoid arthritis (RA), roles of DRs expressed on these cells in RA are poorly understood. This study determined whether D2R expressed on CD4+ T cells regulates inflammatory responses and signs in collagen type II (CII)-induced arthritis (CIA), a mouse model of RA. METHODS: DBA/1 mice and C57BL/6 mice with global D1r or D2r deficiency (D1r-/- or D2r-/-) or CD4+ T cell-specific D2r deletion (D2rfl/fl/CD4Cre) were used to prepare CIA model by intradermal injection of CII. D2R agonist sumanirole was intraperitoneally administered in CIA mice. CD4+ T cells obtained from CIA mice were exposed to sumanirole or/and D2R antagonist L-741,626 in vitro. Arthritic symptoms were assessed by clinical arthritis scores. Flow cytometric assay measured frequencies of CD4+ T cell subsets (Th1, Th2, Th17 and Treg cells). Expression of specific transcription factors for the CD4+ T cell subsets was tested by Western blot. Cytokine production was estimated by quantitative PCR and ELISA. RESULTS: CIA mice manifested a bias of CD4+ T cells towards Th1 and Th17 cells. D2r-/- CIA mice showed a stronger bias towards Th1 and Th17 phenotypes than CIA mice, while D1r-/- CIA mice did not show the changes. CD4+ T cell-specific D2r deletion exacerbated both the polarization towards Th1 and Th17 cells and the symptoms of arthritis. Sumanirole administration in CIA mice ameliorated the bias of CD4+ T cells towards Th1 and Th17 phenotypes as well as arthritic symptoms. Sumanirole treatment of in vitro CD4+ T cells obtained from CIA mice promoted the shift to Treg cells, and the effect of sumanirole was blocked by L-741,626. CONCLUSIONS: D2R expressed on CD4+ T cells is protective against imbalance between pro-inflammatory and anti-inflammatory T cells and arthritic symptoms in CIA.

Activation of ß2-adrenergic Receptor Alleviates Collagen-induced Arthritis by Ameliorating Th17/Treg Imbalance.

Background: Recent research in our laboratory shows that CD4+ T cells express the ß2 adrenergic receptor (ß2-AR), and the sympathetic neurotransmitter norepinephrine regulates the function of T cells via ß2-AR signaling. However, the immunoregulatory effect of ß2-AR and its related mechanisms on rheumatoid arthritis is unknown. Objective: To explore the effects of ß2-AR in collagen-induced arthritis (CIA) on the imbalance of T helper (Th) 17/ regulatory T (Treg) cells. Methods: In DBA1/J mice, collagen type II was injected intradermally at the tail base to prepare the CIA model. The specific ß2-AR agonist, terbutaline (TBL), was administered intraperitoneally beginning on day 31 and continuing until day 47 after primary vaccination, twice a day. Magnetic beads were used to sort CD3+ T cells subsets from spleen tissues. Results: In vivo, ß2-AR agonist TBL alleviated arthritis symptoms in the CIA mice including histopathology of the ankle joints, four limbs' arthritis score, the thickness of ankle joints, and rear paws. After TBL treatment, in the ankle joints, the levels of proinflammatory factors (IL-17/22) notably decreased and the levels of immunosuppressive factors (IL-10/TGF-ß) significantly increased. In vitro, ROR-γt protein expression, Th17 cell number, mRNA expression and the releasing of IL-17/22 from CD3+ T cells reduced following TBL administration. Moreover, TBL enhanced the anti-inflammatory responses of Treg cells. Conclusion: These results suggest that ß2-AR activation exerts anti-inflammatory effects through the amelioration of Th17/Treg imbalance in the CIA disease.

Interleukin 17A deficiency alleviates neuroinflammation and cognitive impairment in an experimental model of diabetic encephalopathy.

Interleukin 17A (IL-17A) was previously shown to be a key pro-inflammatory factor in diabetes mellitus and associated complications. However, the role of IL-17A in diabetic encephalopathy remains poorly understood. In this study, we established a mouse model of diabetic encephalopathy that was deficient in IL-17A by crossing Il17a-/- mice with spontaneously diabetic Ins2Akita (Akita) mice. Blood glucose levels and body weights were monitored from 2-32 weeks of age. When mice were 32 weeks of age, behavioral tests were performed, including a novel object recognition test for assessing short-term memory and learning and a Morris water maze test for evaluating hippocampus-dependent spatial learning and memory. IL-17A levels in the serum, cerebrospinal fluid, and hippocampus were detected with enzyme-linked immunosorbent assays and real-time quantitative polymerase chain reaction. Moreover, proteins related to cognitive dysfunction (amyloid precursor protein, ß-amyloid cleavage enzyme 1, p-tau, and tau), apoptosis (caspase-3 and -9), inflammation (inducible nitric oxide synthase and cyclooxygenase 2), and occludin were detected by western blot assays. Pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1ß, and interferon-γ in serum and hippocampal tissues were measured by enzyme-linked immunosorbent assays. Microglial activation and hippocampal neuronal apoptosis were detected by immunofluorescent staining. Compared with that in wild-type mice, mice with diabetic encephalopathy had higher IL-17A levels in the serum, cerebrospinal fluid, and hippocampus; downregulation of occludin expression; lower cognitive ability; greater loss of hippocampal neurons; increased microglial activation; and higher expression of inflammatory factors in the serum and hippocampus. IL-17A knockout attenuated the abovementioned changes in mice with diabetic encephalopathy. These findings suggest that IL-17A participates in the pathological process of diabetic encephalopathy. Furthermore, IL-17A deficiency reduces diabetic encephalopathy-mediated neuroinflammation and cognitive defects. These results highlight a role for IL-17A as a mediator of diabetic encephalopathy and potential target for the treatment of cognitive impairment induced by diabetic encephalopathy.

Dopamine receptor D2 on CD4+ T cells is protective against neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a chronic neurodegenerative disease. Recently, neuroinflammation driven by CD4+ T cells has been involved in PD pathophysiology. Human and murine lymphocytes express all the five subtypes of dopamine receptors (DRs), DRD1 to DRD5. However, roles of DRs particularly DRD2 expressed on CD4+ T cells in PD remain elucidated. Global Drd1- or Drd2-knockout (Drd1-/- or Drd2-/-) mice or CD4+ T cell-specific Drd2-knockout (Drd2fl/fl/CD4Cre) mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD with the different mutants. On the 7th day following MPTP injection, mice were assessed for dopaminergic neurodegeneration, locomotor impairments, microglial activation, as well as CD4+ T-cell differentiation and function. Furthermore, in vitro CD4+ T cells were exposed to DRD2 agonist and antagonist and then differentiation and function of the cells were determined. MPTP induced dopaminergic neuronal loss in the nigrostriatal system, motor coordinative and behavioral impairments, microglial activation, and CD4+ T-cell polarization to pro-inflammatory T-helper (Th)1 and Th17 phenotypes. Importantly, either Drd2-/- or Drd2fl/fl/CD4Cre mice manifested more severe dopaminergic neurodegeneration, motor deficits, microglial activation, and CD4+ T-cell bias towards Th1 and Th17 phenotypes in response to MPTP, but Drd1-/- did not further alter MPTP intoxication. DRD2 agonist sumanirole inhibited shift of CD4+ T cells obtained from MPTP-intoxicated mice to Th1 and Th17 phenotypes and DRD2 antagonist L-741,626 reversed sumanirole effects. These findings suggest that DRD2 expressed on CD4+ T cells is protective against neuroinflammation and neurodegeneration in PD. Thus, developing a therapeutic strategy of stimulating DRD2 may be promising for mitigation of PD.

Intervention of Tyrosine Hydroxylase Expression Alters Joint Inflammation and Th17/Treg Imbalance in Collagen-Induced Arthritis.

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)-ß1 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.

TGF-ß1 Provides Neuroprotection via Inhibition of Microglial Activation in 3-Acetylpyridine-Induced Cerebellar Ataxia Model Rats.

Cerebellar ataxias (CAs) consist of a heterogeneous group of neurodegenerative diseases hallmarked by motor deficits and deterioration of the cerebellum and its associated circuitries. Neuroinflammatory responses are present in CA brain, but how neuroinflammation may contribute to CA pathogenesis remain unresolved. Here, we investigate whether transforming growth factor (TGF)-ß1, which possesses anti-inflammatory and neuroprotective properties, can ameliorate the microglia-mediated neuroinflammation and thereby alleviate neurodegeneration in CA. In the current study, we administered TGF-ß1 via the intracerebroventricle (ICV) in CA model rats, by intraperitoneal injection of 3-acetylpyridine (3-AP), to reveal the neuroprotective role of TGF-ß1. The TGF-ß1 administration after 3-AP injection ameliorated motor impairments and reduced the calbindin-positive neuron loss and apoptosis in the brain stem and cerebellum. Meanwhile, 3-AP induced microglial activation and inflammatory responses in vivo, which were determined by morphological alteration and an increase in expression of CD11b, enhancement of percentage of CD40 + and CD86 + microglial cells, upregulation of pro-inflammatory mediators, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß, and a downregulation of neurotrophic factor, insulin-like growth factor (IGF)-1 in the brain stem and cerebellum. TGF-ß1 treatment significantly prevented all the changes caused by 3-AP. In addition, in vitro experiments, TGF-ß1 directly attenuated 3-AP-induced microglial activation and inflammatory responses in primary cultures. Purkinje cell exposure to supernatants of primary microglia that had been treated with TGF-ß1 reduced neuronal loss and apoptosis induced by 3-AP-treated microglial supernatants. Furthermore, the protective effect was similar to those treated with TNF-α-neutralizing antibody. These findings suggest that TGF-ß1 protects against neurodegeneration in 3-AP-induced CA rats via inhibiting microglial activation and at least partly TNF-α release.

Treg Cells Attenuate Neuroinflammation and Protect Neurons in a Mouse Model of Parkinson's Disease.

Regulatory T cells (Tregs), which secrete transforming growth factor (TGF)-ß 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.

IL-17A exacerbates neuroinflammation and neurodegeneration by activating microglia in rodent models of Parkinson's disease.

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.

Acetylcholine suppresses microglial inflammatory response via α7nAChR to protect hippocampal neurons.

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.

[Acetylcholine suppresses microglial inflammatory response in rats via acting on microglial α7nAChR].

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-1ß 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.

Norepinephrine Inhibits Th17 Cells via ß2-Adrenergic Receptor (ß2-AR) Signaling in a Mouse Model of Rheumatoid Arthritis.

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 ß2-adrenergic receptor (ß2-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 ß2-AR was observed in spleens of both intact and CIA mice. The ß2-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 ß2-AR antagonist ICI118551 blocked the NE effect. Moreover, the ß2-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 ß2-AR/PKA signaling.

TGF-ß1 protection against Aß1-42-induced hippocampal neuronal inflammation and apoptosis by TßR-I.

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-ß (Aß) 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)-ß1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. However, it is poorly known whether the anti-inflammatory property of TGF-ß1 is involved in a neuroprotection in AD. Here, an AD cell model of hippocampal neurons induced by Aß1-42 was used to show an anti-inflammatory and neuroprotective effect of TGF-ß1 through its receptor transforming growth factor-ß receptor type I (TßR-I). As expected, Aß1-42-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-ß1 pretreatment reduced the Aß1-42-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. TßR-I expression in hippocampal neurons was downregulated by Aß1-42 exposure, but upregulated by TGF-ß1 pretreatment. Silencing of the TßR-I gene in the neurons abolished the anti-inflammatory and antiapoptotic effects of TGF-ß1 in the Aß1-42-induced AD cell model. These findings suggest that TGF-ß1 protects neurons against Aß1-42-induced neuronal inflammation and apoptosis by activation of TßR-I.

[Effects of TGF-ß1 on the expression and secretion of cytokines induced by Aß1-42 in hippocampal neuron and microglia co-cultures].

OBJECTIVE: To investigate the neuroprotective effects of transforming growth factor beta 1(TGF-ß1) on the expression and secretion of cytokines induced by Aß1-42 in hippocampal neurons and microglial co-cultures. METHODS: Hippocampal neurons and microglia obtained from SD rat were co-cultured. TGF-ß1 was applied on day 5 after the neurons and microglia co-cultures were incubated at the concentrations of 5 or 20 ng/ml, Aß1-42 was added 1 h following TGF-ß1 application at a concentration of 5 µmol/L. They were incubated for 72 h and then assessed for further studies. Western blot analyses were employed to examine the expression of inducible nitric oxide synthase (iNOS); Real-time PCR and ELISA were used to detect the mRNA expression and secretion of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and insulin-like growth factor-1 (IGF-1). RESULTS: In the hippocampal neuron-microglia co-cultures, Aß1-42 induced upregulation of iNOS, TNF-α and IL-1ß, downregulation of IGF-1. TGF-ß1 pretreatment ameliorated the pro-inflammatory effects caused by Aß1-42. CONCLUSIONS: TGF-ß1 significantly inhibits the increase in inflammatory cytokines and the decrease in neurotrophic factor which are caused by Aß1-42-induced microglia activation.

Interleukin-10 inhibits neuroinflammation-mediated apoptosis of ventral mesencephalic neurons via JAK-STAT3 pathway.

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 50ng/mL) for 1h followed by lipopolysaccharide (LPS, 50ng/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.

TGF-ß1 Neuroprotection via Inhibition of Microglial Activation in a Rat Model of Parkinson's Disease.

Transforming growth factor (TGF)-ß1 is a pleiotropic cytokine with immunosuppressive and anti-inflammatory properties. Recently we have shown that TGF-ß1 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-ß1 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-ß1 was administrated in the right lateral cerebral ventricle. Primary ventral mesencephalic (VM) neurons and cerebral cortical microglia were treated by MPP+, respectively, and TGF-ß1 was applied to neuronal or microglial cultures at 1 h after MPP+ treatment. As expected, MPP+ resulted in decrease in TGF-ß1 production in the substantia nigra and in primary VM neurons and microglia. TGF-ß1 intracerebroventricular administration alleviated MPP+-induced PD-like changes in pathology, motor coordination and behavior. Meanwhile, TGF-ß1 ameliorated MPP+-induced microglial activation and inflammatory cytokine production in vivo. Interestingly, TGF-ß1 treatment was not able to ameliorate MPP+-induced dopaminergic neuronal loss and caspase-3/9 activation in mono-neuron cultures, but TGF-ß1 alleviated MPP+-induced microglial activation and inflammatory cytokine production in microglia-enriched cultures. This effect of TGF-ß1 inhibiting microglial inflammatory response was blocked by Smad3 inhibitor SIS3. Importantly, neuronal exposure to supernatants of primary microglia that had been treated with TGF-ß1 reduced dopaminergic neuronal loss and caspase-3/9 activation induced by MPP+-treated microglial supernatants. These findings establish that TGF-ß1 exerts neuroprotective property in PD by inhibiting microglial inflammatory response via Smad3 signaling.

Treg Cells Protect Dopaminergic Neurons against MPP+ Neurotoxicity via CD47-SIRPA Interaction.

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)-ß1 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-ß1 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.

Low expression of ASH2L protein correlates with a favorable outcome in acute myeloid leukemia.

ASH2L encodes a trithorax group protein that is a core component of all characterized mammalian histone H3K4 methyltransferase complexes, including mixed lineage leukemia (MLL) complexes. ASH2L protein levels in primary leukemia patient samples have not yet been defined. We analyzed ASH2L protein expression in 511 primary AML patient samples using reverse phase protein array (RPPA) technology. We discovered that ASH2L expression is significantly increased in a subset of patients carrying fms-related tyrosine kinase 3 (FLT3) mutations. Furthermore, we observed that low levels of ASH2L are associated with increased overall survival. We also compared ASH2L levels to the expression of 230 proteins previously analyzed on this array. ASH2L expression was inversely correlated with 32 proteins, mostly involved in cell adhesion and cell cycle inhibition, while a positive correlation was observed for 50 proteins, many of which promote cell proliferation. Together, these results indicate that a lower level of ASH2L protein is beneficial to AML patients.

Th17 Cells Induce Dopaminergic Neuronal Death via LFA-1/ICAM-1 Interaction in a Mouse Model of Parkinson's Disease.

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.

Epidermal growth factor receptor is expressed and active in a subset of acute myeloid leukemia.

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.

Expression of tyrosine hydroxylase in CD4+ T cells contributes to alleviation of Th17/Treg imbalance in collagen-induced arthritis.

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.