RESUMO
Following peripheral nerve injury, extracellular ATP-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.
RESUMO
Following peripheral nerve injury, extracellular adenosine 5'-triphosphate (ATP)-mediated purinergic signaling is crucial for spinal cord microglia activation and neuropathic pain. However, the mechanisms of ATP release remain poorly understood. Here, we show that volume-regulated anion channel (VRAC) is an ATP-releasing channel and is activated by inflammatory mediator sphingosine-1-phosphate (S1P) in microglia. Mice with microglia-specific deletion of Swell1 (also known as Lrrc8a), a VRAC essential subunit, had reduced peripheral nerve injury-induced increase in extracellular ATP in spinal cord. The mutant mice also exhibited decreased spinal microgliosis, dorsal horn neuronal hyperactivity, and both evoked and spontaneous neuropathic pain-like behaviors. We further performed high-throughput screens and identified an FDA-approved drug dicumarol as a novel and potent VRAC inhibitor. Intrathecal administration of dicumarol alleviated nerve injury-induced mechanical allodynia in mice. Our findings suggest that ATP-releasing VRAC in microglia is a key spinal cord determinant of neuropathic pain and a potential therapeutic target for this debilitating disease.
Assuntos
Neuralgia , Traumatismos dos Nervos Periféricos , Camundongos , Animais , Microglia , Dicumarol/uso terapêutico , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Medula Espinal , Trifosfato de Adenosina/farmacologia , Proteínas de MembranaRESUMO
T helper 17 (Th17) cells develop in response to T cell receptor signals (TCR) in the presence of specific environments, and produce the inflammatory cytokine IL17A. These cells have been implicated in a number of inflammatory diseases and represent a potential target for ameliorating such diseases. The kinase ITK, a critical regulator of TCR signals, has been shown to be required for the development of Th17 cells. However, we show here that lung inflammation induced by Saccharopolyspora rectivirgula (SR) induced Hypersensitivity pneumonitis (SR-HP) results in a neutrophil independent, and ITK independent Th17 responses, although ITK signals are required for γδ T cell production of IL17A. Transcriptomic analysis of resultant ITK independent Th17 cells suggest that the SR-HP-induced extrinsic inflammatory signals may override intrinsic T cell signals downstream of ITK to rescue Th17 responses in the absence of ITK. These findings suggest that the ability to pharmaceutically target ITK to suppress Th17 responses may be dependent on the type of inflammation.
Assuntos
Alveolite Alérgica Extrínseca , Pneumonia , Proteínas Tirosina Quinases , Células Th17 , Alveolite Alérgica Extrínseca/enzimologia , Alveolite Alérgica Extrínseca/imunologia , Alveolite Alérgica Extrínseca/metabolismo , Citocinas/metabolismo , Humanos , Inflamação/metabolismo , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/imunologia , Pneumonia/metabolismo , Proteínas Tirosina Quinases/imunologia , Células Th17/enzimologia , Células Th17/imunologia , Células Th17/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Type 1 regulatory T (Tr1) cells differentiate in response to signals engaging the T cell receptor (TCR), express high levels of the immunosuppressive cytokine IL-10, but not Foxp3, and can suppress inflammation and promote immune tolerance. Here we show that ITK, an important modulator of TCR signalling, is required for the TCR-induced development of Tr1 cells in various organs, and in the mucosal system during parasitic and viral infections. ITK kinase activity is required for mouse and human Tr1 cell differentiation. Tr1 cell development and suppressive function of Itk deficient cells can be restored by the expression of the transcription factor interferon regulatory factor 4 (IRF4). Downstream of ITK, Ras activity is responsible for Tr1 cell induction, as expression of constitutively active HRas rescues IRF4 expression and Tr1 cell differentiation in Itk-/- cells. We conclude that TCR/ITK signalling through the Ras/IRF4 pathway is required for functional development of Tr1 cells.
Assuntos
Fatores Reguladores de Interferon/metabolismo , Proteínas Tirosina Quinases/metabolismo , Linfócitos T Reguladores/fisiologia , Proteínas ras/metabolismo , Animais , Diferenciação Celular , Humanos , Fatores Reguladores de Interferon/genética , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Orthomyxoviridae/imunologia , Fator 1 de Ligação ao Domínio I Regulador Positivo/metabolismo , Proteínas Tirosina Quinases/genética , Transdução de Sinais , Infecções por Strongylida/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Proteínas ras/genéticaRESUMO
Th1, Th2, Th9 and Th17 cells are conventional CD4+ effector T cells identified as secretors of prototypical cytokines IFNγ, IL4, IL9, and IL-17A respectively. Recently, populations of natural Th17 and Th1 cells (nTh17 and nTh1) with innate-like phenotype have been identified in the thymus that are distinct from conventional Th17 and Th1 cells. The absence of the Tec family kinase Interleukin-2 inducible T cell kinase (Itk) results in T cell immunodeficiency in mice and humans. Here we show that Itk negatively regulates the development of nTh1 cells that express IFNγ in a Tbet independent manner, and whose expansion can be enhanced by IL4. Furthermore, we show that robust induction of IL4 responses during Trichinella spiralis infection enhance the presence of nTh1 cells. We conclude T cell receptor signaling via Itk controls the development of natural Th1 cells, which are expanded by the presence of IL4.