RESUMO
The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.
Assuntos
Axônios/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Transtornos dos Movimentos , Proteínas Proto-Oncogênicas/deficiência , Receptores Proteína Tirosina Quinases/deficiência , Remielinização/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/ultraestrutura , Cuprizona/toxicidade , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalite/induzido quimicamente , Encefalite/patologia , Regulação da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores da Monoaminoxidase/toxicidade , Transtornos dos Movimentos/etiologia , Transtornos dos Movimentos/genética , Transtornos dos Movimentos/patologia , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/patologia , Bainha de Mielina/ultraestrutura , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Propriocepção/efeitos dos fármacos , Propriocepção/genética , Proteínas Proto-Oncogênicas/genética , Desempenho Psicomotor/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/genética , Reflexo de Endireitamento/efeitos dos fármacos , Reflexo de Endireitamento/genética , Remielinização/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor Tirosina Quinase AxlRESUMO
Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.
Assuntos
Axônios/efeitos dos fármacos , Doenças Desmielinizantes/tratamento farmacológico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/administração & dosagem , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Interferon beta/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Axônios/patologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Mediadores da Inflamação/metabolismo , Infusões Intraventriculares , Injeções Subcutâneas , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Interferon beta/administração & dosagem , Masculino , Camundongos , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Oligodendroglia/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Medula Espinal/imunologiaRESUMO
We have previously described reduced myelination and corresponding myelin basic protein (MBP) expression in the central nervous system of Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) deficient motheaten (me/me) mice compared with normal littermate controls. Deficiency in myelin and MBP expression in both brains and spinal cords of motheaten mice correlated with reduced MBP mRNA expression levels in vivo and in purified oligodendrocytes in vitro. Therefore, SHP-1 activity seems to be a critical regulator of oligodendrocyte gene expression and function. Consistent with this role, this study demonstrates that oligodendrocytes of motheaten mice and SHP-1-depleted N20.1 cells produce higher levels of reactive oxygen species (ROS) and exhibit corresponding markers of increased oxidative stress. In agreement with these findings, we demonstrate that increased production of ROS coincides with ROS-induced signaling pathways known to affect myelin gene expression in oligodendrocytes. Antioxidant treatment of SHP-1-deficient oligodendrocytes reversed the pathological changes in these cells, with increased myelin protein gene expression and decreased expression of nuclear factor (erythroid-2)-related factor 2 (Nrf2) responsive gene, heme oxygenase-1 (HO-1). Furthermore, we demonstrate that SHP-1 is expressed in human white matter oligodendrocytes, and there is a subset of multiple sclerosis subjects that demonstrate a deficiency of SHP-1 in normal-appearing white matter. These studies reveal critical pathways controlled by SHP-1 in oligodendrocytes that relate to susceptibility of SHP-1-deficient mice to both developmental defects in myelination and to inflammatory demyelinating diseases.
Assuntos
Sistema Nervoso Central/patologia , Regulação da Expressão Gênica/genética , Esclerose Múltipla/patologia , Oligodendroglia/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Glutationa/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/genética , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , NF-kappa B/metabolismo , Carbonilação Proteica/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genéticaRESUMO
AKT3, a member of the serine/threonine kinase AKT family, is involved in a variety of biologic processes. AKT3 is expressed in immune cells and is the major AKT isoform in the CNS representing 30% of the total AKT expressed in spinal cord, and 50% in the brain. Myelin-oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) is a mouse model in which lymphocytes and monocytes enter the CNS, resulting in inflammation, demyelination, and axonal injury. We hypothesized that during EAE, deletion of AKT3 would negatively affect the CNS of AKT3(-/-) mice, making them more susceptible to CNS damage. During acute EAE, AKT3(-/-)mice were more severely affected than wild type (WT) mice. Evaluation of spinal cords showed that during acute and chronic disease, AKT3(-/-) spinal cords had more demyelination compared with WT spinal cords. Quantitative RT-PCR determined higher levels of IL-2, IL-17, and IFN-γ mRNA in spinal cords from AKT3(-/-) mice than WT. Experiments using bone marrow chimeras demonstrated that AKT3(-/-) mice receiving AKT3-deficient bone marrow cells had elevated clinical scores relative to control WT mice reconstituted with WT cells, indicating that altered function of both CNS cells and bone marrow-derived immune cells contributed to the phenotype. Immunohistochemical analysis revealed decreased numbers of Foxp3(+) regulatory T cells in the spinal cord of AKT3(-/-) mice compared with WT mice, whereas in vitro suppression assays showed that AKT3-deficient Th cells were less susceptible to regulatory T cell-mediated suppression than their WT counterparts. These results indicate that AKT3 signaling contributes to the protection of mice against EAE.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Mediadores da Inflamação/fisiologia , Glicoproteína Mielina-Oligodendrócito/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/fisiologia , Transdução de Sinais/imunologia , Doença Aguda , Animais , Doença Crônica , Encefalomielite Autoimune Experimental/induzido quimicamente , Predisposição Genética para Doença , Mediadores da Inflamação/antagonistas & inibidores , Região Lombossacral , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/antagonistas & inibidores , Glicoproteína Mielina-Oligodendrócito/fisiologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/fisiologia , Proteínas Proto-Oncogênicas c-akt/deficiência , Proteínas Proto-Oncogênicas c-akt/genética , Índice de Gravidade de Doença , Transdução de Sinais/genética , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
BACKGROUND AND OBJECTIVES: Tolebrutinib is a covalent BTK inhibitor designed and selected for potency and CNS exposure to optimize impact on BTK-dependent signaling in CNS-resident cells. We applied a translational approach to evaluate three BTK inhibitors in Phase 3 clinical development in MS with respect to their relative potency to block BTK-dependent signaling and exposure in the CNS METHODS: We used in vitro kinase and cellular activation assays, alongside pharmacokinetic sampling of cerebrospinal fluid (CSF) in the non-human primate cynomolgus to estimate the ability of these candidates (evobrutinib, fenebrutinib, and tolebrutinib) to block BTK-dependent signaling inside the CNS. RESULTS: In vitro kinase assays demonstrated that tolebrutinib reacted with BTK 65-times faster than evobrutinib, while fenebrutinib, a classical reversible antagonist with a Ki value of 4.7 nM and slow off-rate (1.54 x 10-5 s-1), also had an association rate 1760-fold slower (0.00245 µM-1 * s-1). Estimates of cellular potency were largely consistent with the in vitro kinase assays, with an estimated IC50 of 0.7 nM for tolebrutinib against 33.5 nM for evobrutinib and 2.9 nM for fenebrutinib. We then observed that evobrutinib, fenebrutinib, and tolebrutinib achieved similar levels of exposure in non-human primate CSF after oral doses of 10 mg/kg. However, tolebrutinib CSF exposure (4.8 ng/mL) (kp,uu CSF=0.40) exceeded the IC90 (the estimated concentration inhibiting 90% of kinase activity) value, while evobrutinib (3.2 ng/mL) (kp,uu CSF=0.13) and fenebrutinib (12.9 ng/mL) (kp,uu CSF=0.15) failed to reach the estimated IC90 values. CONCLUSIONS: Tolebrutinib was the only candidate of the three that attained relevant CSF exposure in non-human primates.
Assuntos
Tirosina Quinase da Agamaglobulinemia , Macaca fascicularis , Esclerose Múltipla , Inibidores de Proteínas Quinases , Tirosina Quinase da Agamaglobulinemia/antagonistas & inibidores , Tirosina Quinase da Agamaglobulinemia/metabolismo , Animais , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/farmacocinética , Esclerose Múltipla/tratamento farmacológico , Humanos , Sistema Nervoso Central/efeitos dos fármacos , Pirazinas/farmacologia , Pirazinas/farmacocinética , Piperazinas , Piperidinas , Piridonas , PirimidinasRESUMO
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Here we document for the first time that the cytokine IL-33 is upregulated in both the periphery and the CNS of MS patients. Plasma IL-33 was elevated in MS patients compared to normal subjects and a three-month treatment of MS patients with interferon ß-1a resulted in a significant decrease of IL-33 levels. Similarly, stimulated cultured lymphocytes and macrophages from MS patients had elevated IL-33 levels compared to normal subjects. In parallel, the transcription factor NF-κB that mediates IL-33 transcription was also elevated in leukocytes of MS patients. IL-33 was elevated in normal-appearing white matter and plaque areas from MS brains and astrocytes were identified as an important source of IL-33 expression in the CNS. In summary, IL-33 levels are elevated in the periphery and CNS of MS patients, implicating IL-33 in the pathogenesis of MS.
Assuntos
Sistema Nervoso Central/imunologia , Interleucinas/imunologia , Linfócitos/imunologia , Esclerose Múltipla/imunologia , Adulto , Células Cultivadas , Feminino , Humanos , Interleucina-33 , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/imunologia , Regulação para CimaRESUMO
The protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling and inflammatory gene expression, both in the immune system and in the central nervous system (CNS). Mice genetically lacking SHP-1 (me/me) display severe inflammatory demyelinating disease following inoculation with the Theiler's murine encephalomyelitis virus (TMEV) compared to infected wild-type mice. Therefore, it became essential to investigate the mechanisms of TMEV-induced inflammation in the CNS of SHP-1-deficient mice. Herein, we show that the expression of several genes relevant to inflammatory demyelination in the CNS of infected me/me mice is elevated compared to that in wild-type mice. Furthermore, SHP-1 deficiency led to an abundant and exclusive increase in the infiltration of high-level-CD45-expressing (CD45(hi)) CD11b(+) Ly-6C(hi) macrophages into the CNS of me/me mice, in concert with the development of paralysis. Histological analyses of spinal cords revealed the localization of these macrophages to extensive inflammatory demyelinating lesions in infected SHP-1-deficient mice. Sorted populations of CNS-infiltrating macrophages from infected me/me mice showed increased amounts of viral RNA and an enhanced inflammatory profile compared to wild-type macrophages. Importantly, the application of clodronate liposomes effectively depleted splenic and CNS-infiltrating macrophages and significantly delayed the onset of TMEV-induced paralysis. Furthermore, macrophage depletion resulted in lower viral loads and lower levels of inflammatory gene expression and demyelination in the spinal cords of me/me mice. Finally, me/me macrophages were more responsive than wild-type macrophages to chemoattractive stimuli secreted by me/me glial cells, indicating a mechanism for the increased numbers of infiltrating macrophages seen in the CNS of me/me mice. Taken together, these findings demonstrate that infiltrating macrophages in SHP-1-deficient mice play a crucial role in promoting viral replication by providing abundant viral targets and contribute to increased proinflammatory gene expression relevant to the effector mechanisms of macrophage-mediated demyelination.
Assuntos
Sistema Nervoso Central/imunologia , Inflamação/imunologia , Macrófagos/imunologia , Poliomielite/imunologia , Poliomielite/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Theilovirus/imunologia , Animais , Antígenos Ly/análise , Antígeno CD11b/análise , Ácido Clodrônico/farmacologia , Perfilação da Expressão Gênica , Fatores Imunológicos , Antígenos Comuns de Leucócito/análise , Procedimentos de Redução de Leucócitos , Macrófagos/química , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Medula Espinal/patologiaRESUMO
Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of proinflammatory cytokine signaling, TLR signaling, and inflammatory gene expression. Furthermore, mice genetically lacking SHP-1 (me/me) display a profound susceptibility to inflammatory CNS demyelination relative to wild-type mice. In particular, SHP-1 deficiency may act predominantly in inflammatory macrophages to increase CNS demyelination as SHP-1-deficient macrophages display coexpression of inflammatory effector molecules and increased demyelinating activity in me/me mice. Recently, we reported that PBMCs of multiple sclerosis (MS) patients have a deficiency in SHP-1 expression relative to normal control subjects indicating that SHP-1 deficiency may play a similar role in MS as to that seen in mice. Therefore, it became essential to examine the specific expression and function of SHP-1 in macrophages from MS patients. Herein, we document that macrophages of MS patients have deficient SHP-1 protein and mRNA expression relative to those of normal control subjects. To examine functional consequences of the lower SHP-1, the activation of STAT6, STAT1, and NF-kappaB was quantified and macrophages of MS patients showed increased activation of these transcription factors. In accordance with this observation, several STAT6-, STAT1-, and NF-kappaB-responsive genes that mediate inflammatory demyelination were increased in macrophages of MS patients following cytokine and TLR agonist stimulation. Supporting a direct role of SHP-1 deficiency in altered macrophage function, experimental depletion of SHP-1 in normal subject macrophages resulted in an increased STAT/NF-kappaB activation and increased inflammatory gene expression to levels seen in macrophages of MS patients. In conclusion, macrophages of MS patients display a deficiency of SHP-1 expression, heightened activation of STAT6, STAT1, and NF-kappaB and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in MS.
Assuntos
Macrófagos/enzimologia , Esclerose Múltipla Recidivante-Remitente/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Adulto , Sequência de Bases , Estudos de Casos e Controles , Citocinas/antagonistas & inibidores , Citocinas/genética , Citocinas/metabolismo , Primers do DNA/genética , Doenças Desmielinizantes/enzimologia , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Feminino , Expressão Gênica , Humanos , Técnicas In Vitro , Inflamação/enzimologia , Inflamação/genética , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/patologia , NF-kappa B/metabolismo , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT6/metabolismoRESUMO
IL-33 is a novel member of the IL-1 cytokine family and a potent inducer of type 2 immunity, as mast cells and Th2 CD4+ T cells respond to IL-33 with the induction of type 2 cytokines such as IL-13. IL-33 mRNA levels are extremely high in the CNS, and CNS glia possess both subunits of the IL-33R, yet whether IL-33 is produced by and affects CNS glia has not been studied. Here, we demonstrate that pathogen-associated molecular patterns (PAMPs) significantly increase IL-33 mRNA and protein expression in CNS glia. Interestingly, IL-33 was localized to the nucleus of astrocytes. Further, CNS glial and astrocyte-enriched cultures treated with a PAMP followed by an ATP pulse had significantly higher levels of supernatant IL-1beta and IL-33 than cultures receiving any single treatment (PAMP or ATP). Supernatants from PAMP + ATP-treated glia induced the secretion of IL-6, IL-13, and MCP-1 from the MC/9 mast cell line in a manner similar to exogenous recombinant IL-33. Further, IL-33 levels and activity were increased in the brains of mice infected with the neurotropic virus Theiler's murine encephalomyelitis virus. IL-33 also had direct effects on CNS glia, as IL-33 induced various innate immune effectors in CNS glia, and this induction was greatly amplified by IL-33-stimulated mast cells. In conclusion, these results implicate IL-33-producing astrocytes as a potentially critical regulator of innate immune responses in the CNS.
Assuntos
Encéfalo/metabolismo , Infecções por Cardiovirus/metabolismo , Interleucinas/genética , Neuroglia/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Encéfalo/imunologia , Encéfalo/virologia , Infecções por Cardiovirus/virologia , Núcleo Celular/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Técnicas Imunoenzimáticas , Interleucina-1beta , Interleucina-33 , Interleucinas/metabolismo , Mastócitos/metabolismo , Mastócitos/virologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Neuroglia/virologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Theilovirus/genética , Theilovirus/metabolismoRESUMO
Akt is a serine/threonine protein kinase that plays a major role in regulating multiple cellular processes. While the isoforms Akt1 and Akt2 are involved in apoptosis and insulin signaling, respectively, the role for Akt3 remains uncertain. Akt3 is predominantly expressed in the brain, and total deletion of Akt3 in mice results in a reduction in brain size and neurodegeneration following injury. Previously, we found that Akt3-/- mice have a significantly worse clinical course during myelin-oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE), an animal model in which autoreactive immune cells enter the CNS, resulting in inflammation, demyelination, and axonal injury. Spinal cords of Akt3-/- mice are severely demyelinated and have increased inflammation compared to WT, suggesting a neuroprotective role for Akt3 during EAE. To specifically address the role of Akt3 in neuroinflammation and maintaining neuronal integrity, we used several mouse strains with different manipulations to Akt3. During EAE, Akt3 Nmf350 mice (with enhanced Akt3 kinase activity) had lower clinical scores, a lag in disease onset, a delay in the influx of inflammatory cells into the CNS, and less axonal damage compared to WT mice. A significant increased efficiency of differentiation toward FOXP3 expressing iTregs was also observed in Akt3 Nmf350 mice relative to WT. Mice with a conditional deletion of Akt3 in CD4+ T-cells had an earlier onset of EAE symptoms, increased inflammation in the spinal cord and brain, and had fewer FOXP3+ cells and FOXP3 mRNA expression. No difference in EAE outcome was observed when Akt3 expression was deleted in neurons (Syn1-CKO). These results indicate that Akt3 signaling in T-cells and not neurons is necessary for maintaining CNS integrity during an inflammatory demyelinating disease.
Assuntos
Doenças Desmielinizantes/etiologia , Suscetibilidade a Doenças , Proteínas Proto-Oncogênicas c-akt/genética , Animais , Biomarcadores , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/etiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Imunofluorescência , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Medula Espinal/imunologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Recent studies in mice have demonstrated that the protein tyrosine phosphatase SHP-1 is a crucial negative regulator of cytokine signaling, inflammatory gene expression, and demyelination in central nervous system. The present study investigates a possible similar role for SHP-1 in the human disease multiple sclerosis (MS). The levels of SHP-1 protein and mRNA in PBMCs of MS patients were significantly lower compared to normal subjects. Moreover, promoter II transcripts, expressed from one of two known promoters, were selectively deficient in MS patients. To examine functional consequences of the lower SHP-1 in PBMCs of MS patients, we measured the intracellular levels of phosphorylated STAT6 (pSTAT6). As expected, MS patients had significantly higher levels of pSTAT6. Accordingly, siRNA to SHP-1 effectively increased the levels of pSTAT6 in PBMCs of controls to levels equal to MS patients. Additionally, transduction of PBMCs with a lentiviral vector expressing SHP-1 lowered pSTAT6 levels. Finally, multiple STAT6-responsive inflammatory genes were increased in PBMCs of MS patients relative to PBMCs of normal subjects. Thus, PBMCs of MS patients display a stable deficiency of SHP-1 expression, heightened STAT6 phosphorylation, and an enhanced state of activation relevant to the mechanisms of inflammatory demyelination.
Assuntos
Expressão Gênica , Inflamação , Leucócitos Mononucleares/metabolismo , Esclerose Múltipla/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/deficiência , Arginase/análise , Estudos de Casos e Controles , Células Cultivadas , Vetores Genéticos , Humanos , Lentivirus/genética , Leucócitos Mononucleares/efeitos dos fármacos , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT6/análise , Fator de Transcrição STAT6/metabolismo , Estatística como Assunto , Fatores de TempoRESUMO
Tyro3, Axl, and Mertk, referred to as the TAM family of receptor tyrosine kinases, are instrumental in maintaining cell survival and homeostasis in mammals. TAM receptors interact with multiple signaling molecules to regulate cell migration, survival, phagocytosis and clearance of metabolic products and cell debris called efferocytosis. The TAMs also function as rheostats to reduce the expression of proinflammatory molecules and prevent autoimmunity. All three TAM receptors are activated in a concentration-dependent manner by the vitamin K-dependent growth arrest-specific protein 6 (Gas6). Gas6 and the TAMs are abundantly expressed in the nervous system. Gas6, secreted by neurons and endothelial cells, is the sole ligand for Axl. ProteinS1 (ProS1), another vitamin K-dependent protein functions mainly as an anti-coagulant, and independent of this function can activate Tyro3 and Mertk, but not Axl. This review will focus on the role of the TAM receptors and their ligands in the nervous system. We highlight studies that explore the function of TAM signaling in myelination, the visual cortex, neural cancers, and multiple sclerosis (MS) using Gas6-/- and TAM mutant mice models.
Assuntos
Sistema Nervoso/metabolismo , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , c-Mer Tirosina Quinase/fisiologia , Animais , Proteínas Sanguíneas/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Ligantes , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Proteína S , Transdução de Sinais , Vitamina K/fisiologia , Receptor Tirosina Quinase AxlRESUMO
In response to activation, CD4+ T cells upregulate autophagy. However, the functional consequences of that upregulation have not been fully elucidated. In this study, we identify autophagy as a tolerance-avoidance mechanism. Our data show that inhibition of autophagy during CD4+ T cell activation induces a long-lasting state of hypo-responsiveness that is accompanied by the expression of an anergic gene signature. Cells unable to induce autophagy after T cell receptor (TCR) engagement show inefficient mitochondrial respiration and decreased turnover of the protein tyrosine phosphatase PTPN1, which translates into defective TCR-mediated signaling. In vivo, inhibition of autophagy during antigen priming induces T cell anergy and decreases the severity of disease in an experimental autoimmune encephalomyelitis mouse model. Interestingly, CD4+ T cells isolated from the synovial fluid of juvenile idiopathic arthritis patients, while resistant to suboptimal stimulation-induced anergy, can be tolerized with autophagy inhibitors. We propose that autophagy constitutes a tolerance-avoidance mechanism, which determines CD4+ T cell fate.
Assuntos
Autofagia , Linfócitos T CD4-Positivos/imunologia , Anergia Clonal , Encefalomielite Autoimune Experimental/imunologia , Animais , Células Cultivadas , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
The molecular requirements for human myelination are incompletely defined, and further study is needed to fully understand the cellular mechanisms involved during development and in demyelinating diseases. We have established a human co-culture model to study myelination. Our earlier observations showed that addition of human γ-carboxylated growth-arrest-specific protein 6 (Gas6) to human oligodendrocyte progenitor cell (OPC) cultures enhanced their survival and maturation. Therefore, we explored the effect of Gas6 in co-cultures of enriched OPCs plated on axons of human fetal dorsal root ganglia explant. Gas6 significantly enhanced the number of myelin basic protein-positive (MBP+) oligodendrocytes with membranous processes parallel with and ensheathing axons relative to co-cultures maintained in defined medium only for 14 days. Gas6 did not increase the overall number of MBP+ oligodendrocytes/culture; however, it significantly increased the length of MBP+ oligodendrocyte processes in contact with and wrapping axons. Multiple oligodendrocytes were in contact with a single axon, and several processes from one oligodendrocyte made contact with one or multiple axons. Electron microscopy supported confocal Z-series microscopy demonstrating axonal ensheathment by MBP+ oligodendrocyte membranous processes in Gas6-treated co-cultures. Contacts between the axonal and oligodendrocyte membranes were evident and multiple wraps of oligodendrocyte membrane around the axon were visible supporting a model system in which to study events in human myelination and aspects of non-compact myelin formation.
Assuntos
Axônios/ultraestrutura , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Básica da Mielina/metabolismo , Oligodendroglia/ultraestrutura , Axônios/metabolismo , Técnicas de Cocultura , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Gânglios Espinais/ultraestrutura , Humanos , Marcação In Situ das Extremidades Cortadas , Microscopia Confocal , Bainha de Mielina , Oligodendroglia/metabolismoRESUMO
Interferon-ß (IFN-ß) is a current effective treatment for multiple sclerosis (MS) and exerts its therapeutic effects by down-modulating the systemic immune response and cytokine signaling. In clinical practice there are several formulations of interferon including a low dose of IFN-ß 1a formulation of 30 µg IM once weekly (Avonex) and a high dose formulation of 44 µg SC three times weekly (Rebif). Recent studies suggest that Rebif is more efficacious compared to Avonex in preventing relapses and decreasing MRI activity in relapsing remitting MS (RRMS) patients. This study examines whether there are quantitative gene expression changes in interferon-treated RRMS patients that can explain the difference in efficacy and side effects between Rebif and Avonex. Herein, RRMS patients were treated for three months with IFN-ß 1a and the levels of plasma cytokines and gene expression in peripheral blood mononuclear cells were examined. Thirty-two normal subjects were compared to thirty-two RRMS patients, of which ten were treated with Rebif and ten with Avonex. Rebif and Avonex both significantly and equally suppressed plasma TNF-α and IL-6 levels. Rebif suppressed IL-13 significantly more than Avonex. Rebif also significantly suppressed the levels of the chemokines CCL17 and RANTES, the protease ADAM8, and COX-2 at a higher degree compared to Avonex. The STAT1-inducible genes IP-10 and caspase 1 were significantly increased with Rebif compared to Avonex. In conclusion, the higher dosed, more frequently administered IFN-ß 1a Rebif when compared to IFN-ß 1a Avonex has more potent immunomodulatory effects. These quantitative results might relate to efficacy and side-effect profile of the two IFN-ß 1a formulations and provide prospective practical clinical tools to monitor treatment and adjust dosage.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/fisiologia , Interferon beta/administração & dosagem , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Adjuvantes Imunológicos/administração & dosagem , Adulto , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Monitoramento de Medicamentos/métodos , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Interferon beta-1a , Interferon beta/uso terapêutico , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/genética , Esclerose Múltipla Recidivante-Remitente/imunologia , Prevenção Secundária , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
The protein tyrosine phosphatase SHP-1 is a critical regulator of cytokine signaling and inflammation. Mice homozygous for a null allele at the SHP-1 locus have a phenotype of severe inflammation and are hyper-responsive to the TLR4 ligand LPS. TLR4 stimulation in the CNS has been linked to both neuropathic pain and sickness behaviors. To determine if reduction in SHP-1 expression affects LPS-induced behaviors, responses of heterozygous SHP-1-deficient (me/+) and wild-type (+/+) mice to LPS were measured. Chronic (4-week) treatment with LPS induced avoidant behaviors indicative of fear/anxiety in me/+, but not +/+, mice. These behaviors were correlated with a LPS-induced type 2 cytokine, cytokine receptor, and immune effector arginase profile in the brains of me/+ mice not found in +/+ mice. Me/+ mice also had a constitutively greater level of TLR4 in the CNS than +/+ mice. Additionally, me/+ mice displayed constitutively increased thermal sensitivity compared to +/+ mice, measured by the tail-flick test. Moreover, me/+ glial cultures were more responsive to LPS than +/+ glia. Therefore, the reduced expression of SHP-1 in me/+ imparts haploinsufficiency with respect to the control of CNS TLR4 and pain signaling. Furthermore, type 2 cytokines become prevalent during chronic TLR4 hyperstimulation in the CNS and are associated positively with behaviors that are usually linked to type 1 pro-inflammatory cytokines. These findings question the notion that type 2 immunity is solely anti-inflammatory in the CNS and indicate that type 2 immunity induces/potentiates CNS inflammatory processes.