RESUMEN
The IL-23 pathway is genetically linked to autoimmune disease in humans and is required for pathogenic Th17 cell function in mice. However, because IL-23R-expressing mature Th17 cells are rare and poorly defined in mice at steady-state, little is known about IL-23 signaling. In this study, we show that the endogenous CCR6(+) memory T cell compartment present in peripheral lymphoid organs of unmanipulated mice expresses Il23r ex vivo, displays marked proinflammatory responses to IL-23 stimulation in vitro, and is capable of transferring experimental autoimmune encephalomyelitis. The prolyl-tRNA synthetase inhibitor halofuginone blocks IL-23-induced Stat3 phosphorylation and IL-23-dependent proinflammatory cytokine expression in endogenous CCR6(+) Th17 cells via activation of the amino acid starvation response (AAR) pathway. In vivo, halofuginone shows therapeutic efficacy in experimental autoimmune encephalomyelitis, reducing both established disease progression and local Th17 cell effector function within the CNS. Mechanistically, AAR activation impairs Stat3 responses downstream of multiple cytokine receptors via selective, posttranscriptional suppression of Stat3 protein levels. Thus, our study reveals latent pathogenic functions of endogenous Th17 cells that are regulated by both IL-23 and AAR pathways and identifies a novel regulatory pathway targeting Stat3 that may underlie selective immune regulation by the AAR.
Asunto(s)
Aminoácidos/deficiencia , Encefalomielitis Autoinmune Experimental/inmunología , Activación de Linfocitos , Factor de Transcripción STAT3/inmunología , Células Th17/inmunología , Animales , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/patología , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-23/genética , Interleucina-23/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Fosforilación/genética , Fosforilación/inmunología , Piperidinas , Quinazolinonas , Receptores CCR6/genética , Receptores CCR6/inmunología , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Células Th17/patologíaRESUMEN
The orphan nuclear receptor, retinoic acid receptor-related orphan nuclear receptor γt (RORγt), is required for the development and pathogenic function of interleukin-17A-secreting CD4(+) T helper type 17 (Th17) cells. Whereas small molecule RORγt antagonists impair Th17 cell development and attenuate autoimmune inflammation in vivo, the broader effects of these inhibitors on RORγt-dependent gene expression in vivo has yet to be characterized. We show that the RORγt inverse agonist TMP778 acts potently and selectively to block mouse Th17 cell differentiation in vitro and to impair Th17 cell development in vivo upon immunization with the myelin antigen MOG35-55 plus complete Freund's adjuvant. Importantly, we show that TMP778 acts in vivo to repress the expression of more than 150 genes, most of which fall outside the canonical Th17 transcriptional signature and are linked to a variety of inflammatory pathologies in humans. Interestingly, more than 30 genes are related with SMAD3, a transcription factor involved in the Th17 cell differentiation. These results reveal novel disease-associated genes regulated by RORγt during inflammation in vivo, and provide an early read on potential disease indications and safety concerns associated with pharmacological targeting of RORγt.
Asunto(s)
Diferenciación Celular/inmunología , Expresión Génica/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/inmunología , Células Th17/inmunología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Células Cultivadas , Femenino , Adyuvante de Freund/inmunología , Expresión Génica/efectos de los fármacos , Perfilación de la Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Inmunización/métodos , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/agonistas , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fragmentos de Péptidos/inmunología , Células Th17/efectos de los fármacos , Células Th17/metabolismoRESUMEN
The proinflammatory cytokine interleukin-1beta (IL-1beta) plays a significant role in leukocyte recruitment to the CNS. Although acute effects of IL-1beta signaling in the mouse brain have been well described, studies elucidating the downstream effects of sustained upregulation have been lacking. Using the recently described IL-1beta(XAT) transgenic mouse model, we triggered sustained unilateral hippocampal overexpression of IL-1beta. Transgene induction led to blood-brain barrier leakage, induction of MCP-1 (monocyte chemoattractant protein 1) (CCL2), ICAM-1 (intercellular adhesion molecule 1), and dramatic infiltration of CD45-positive leukocytes comprised of neutrophils, T-cells, macrophages, and dendritic cells. Despite prolonged cellular infiltration of the hippocampus, there was no evidence of neuronal degeneration. Surprisingly, neutrophils were observed in the hippocampal parenchyma as late as 1 year after transgene induction. Their presence was coincident with upregulation of the potent neutrophil chemotactic chemokines KC (keratinocyte-derived chemokine) (CXCL1) and MIP-2 (macrophage inflammatory protein 2) (CXCL2). Knock-out of their sole receptor CXCR2 abrogated neutrophil infiltration but failed to reduce leakage of the blood-brain barrier.
Asunto(s)
Barrera Hematoencefálica/fisiología , Hipocampo/metabolismo , Interleucina-1beta/metabolismo , Leucocitos/metabolismo , Neutrófilos/metabolismo , Regulación hacia Arriba/fisiología , Animales , Quimiocina CCL2/metabolismo , Quimiocina CXCL2 , Molécula 1 de Adhesión Intercelular/metabolismo , Interleucina-1beta/genética , Antígenos Comunes de Leucocito/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Monocinas/deficiencia , Monocinas/metabolismoRESUMEN
IL-17A-expressing CD4(+) T cells (Th17 cells) are generally regarded as key effectors of autoimmune inflammation. However, not all Th17 cells are pro-inflammatory. Pathogenic Th17 cells that induce autoimmunity in mice are distinguished from nonpathogenic Th17 cells by a unique transcriptional signature, including high Il23r expression, and these cells require Il23r for their inflammatory function. In contrast, defining features of human pro-inflammatory Th17 cells are unknown. We show that pro-inflammatory human Th17 cells are restricted to a subset of CCR6(+)CXCR3(hi)CCR4(lo)CCR10(-)CD161(+) cells that transiently express c-Kit and stably express P-glycoprotein (P-gp)/multi-drug resistance type 1 (MDR1). In contrast to MDR1(-) Th1 or Th17 cells, MDR1(+) Th17 cells produce both Th17 (IL-17A, IL-17F, and IL-22) and Th1 (IFN-γ) cytokines upon TCR stimulation and do not express IL-10 or other anti-inflammatory molecules. These cells also display a transcriptional signature akin to pathogenic mouse Th17 cells and show heightened functional responses to IL-23 stimulation. In vivo, MDR1(+) Th17 cells are enriched and activated in the gut of Crohn's disease patients. Furthermore, MDR1(+) Th17 cells are refractory to several glucocorticoids used to treat clinical autoimmune disease. Thus, MDR1(+) Th17 cells may be important mediators of chronic inflammation, particularly in clinical settings of steroid resistant inflammatory disease.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Crohn/metabolismo , Regulación de la Expresión Génica/inmunología , Células Th17/efectos de los fármacos , Células Th17/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Enfermedad de Crohn/inmunología , Citometría de Flujo , Glucocorticoides/farmacología , Humanos , Interferón gamma/metabolismo , Análisis por Micromatrices , Células Th17/inmunologíaRESUMEN
Human memory T cells (T(M) cells) that produce IL-17 or IL-22 are currently defined as Th17 or Th22 cells, respectively. These T cell lineages are almost exclusively CCR6(+) and are important mediators of chronic inflammation and autoimmunity. However, little is known about the mechanisms controlling IL-17/IL-22 expression in memory Th17/Th22 subsets. We show that common γ chain (γc)-using cytokines, namely IL-2, IL-7, and IL-15, potently induce Th17-signature cytokine expression (Il17a, Il17f, Il22, and Il26) in CCR6(+), but not CCR6(-), T(M) cells, even in CCR6(+) cells lacking IL-17 expression ex vivo. Inhibition of phosphoinositide 3-kinase (PI-3K) or Akt signaling selectively prevents Th17 cytokine induction by γc-cytokines, as does ectopic expression of the transcription factors FOXO1 or KLF2, which are repressed by PI-3K signaling. These results indicate that Th17 cytokines are tuned by PI-3K signaling in CCR6(+) T(M) cells, which may contribute to chronic or autoimmune inflammation. Furthermore, these findings suggest that ex vivo analysis of IL-17 expression may greatly underestimate the frequency and pathogenic potential of the human Th17 compartment.
Asunto(s)
Citocinas/inmunología , Memoria Inmunológica/fisiología , Fosfatidilinositol 3-Quinasas/inmunología , Receptores CCR6 , Transducción de Señal/inmunología , Células Th17/inmunología , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedad Crónica , Citocinas/genética , Humanos , Inflamación/genética , Inflamación/inmunología , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/inmunología , Transducción de Señal/genéticaRESUMEN
The interleukin (IL)-12p40 family of cytokines plays a critical role in the development of experimental autoimmune encephalomyelitis (EAE). However, the relative contributions of IL-12 and IL-23 to the pathogenic process remain to be elucidated. Here, we show that activation of uncommitted myelin-reactive T cells in the presence of either IL-12p70 or IL-23 confers encephalogenicity. Adoptive transfer of either IL-12p70- or IL-23-polarized T cells into naive syngeneic hosts resulted in an ascending paralysis that was clinically indistinguishable between the two groups. However, histological and reverse transcription-polymerase chain reaction analysis of central nervous system (CNS) tissues revealed distinct histopathological features and immune profiles. IL-12p70-driven disease was characterized by macrophage-rich infiltrates and prominent NOS2 up-regulation, whereas neutrophils and granulocyte-colony-stimulating factor (CSF) were prominent in IL-23-driven lesions. The monocyte-attracting chemokines CXCL9, 10, and 11 were preferentially expressed in the CNS of mice injected with IL-12p70-modulated T cells, whereas the neutrophil-attracting chemokines CXCL1 and CXCL2 were up-regulated in the CNS of mice given IL-23-modulated T cells. Treatment with anti-IL-17 or anti-granulocyte/macrophage-CSF inhibited EAE induced by transfer of IL-23-polarized, but not IL-12p70-polarized, cells. These findings indicate that autoimmunity can be mediated by distinct effector populations that use disparate immunological pathways to achieve a similar clinical outcome.