RESUMEN
Previous studies have identified a unique Treg population, which expresses the Th17 characteristic transcription factor RORγt. These RORγt+ Tregs possess enhanced immunosuppressive capacity, which endows them with great therapeutic potential. However, as a caveat, they are also capable of secreting pro-inflammatory IL-17A. Since the sum function of RORγt+ Tregs in glomerulonephritis (GN) remains unknown, we studied the effects of their absence. Purified CD4+ T cell populations, containing or lacking RORγt+ Tregs, were transferred into immunocompromised RAG1 knockout mice and the nephrotoxic nephritis model of GN was induced. Absence of RORγt+ Tregs significantly aggravated kidney injury, demonstrating overall kidney-protective properties. Analyses of immune responses showed that RORγt+ Tregs were broadly immunosuppressive with no preference for a particular type of T cell response. Further characterization revealed a distinct functional and transcriptional profile, including enhanced production of IL-10. Expression of the chemokine receptor CCR6 marked a particularly potent subset, whose absence significantly worsened GN. As an underlying mechanism, we found that chemokine CCL20 acting through receptor CCR6 signaling mediated expansion and activation of RORγt+ Tregs. Finally, we also detected an increase of CCR6+ Tregs in kidney biopsies, as well as enhanced secretion of chemokine CCL20 in 21 patients with anti-neutrophil cytoplasmic antibody associated GN compared to that of 31 healthy living donors, indicating clinical relevance. Thus, our data characterize RORγt+ Tregs as anti-inflammatory mediators of GN and identify them as promising target for Treg directed therapies.
Asunto(s)
Glomerulonefritis , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Ratones , Animales , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Linfocitos T Reguladores , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Riñón/patología , Ratones Noqueados , Células Th17 , Receptores CCR6/genética , Receptores CCR6/metabolismoRESUMEN
BACKGROUND: New therapies blocking the IL-6 receptor (IL-6R) have recently become available and are successfully being used to treat inflammatory diseases like arthritis. Whether IL-6 blockers may help patients with kidney inflammation currently remains unknown. METHODS: To learn more about the complex role of CD4+ T cell-intrinsic IL-6R signaling, we induced nephrotoxic nephritis, a mouse model for crescentic GN, in mice lacking T cell-specific IL-6Ra. We used adoptive transfer experiments and studies in reporter mice to analyze immune responses and Treg subpopulations. RESULTS: Lack of IL-6Ra signaling in mouse CD4+ T cells impaired the generation of proinflammatory Th17 cells, but surprisingly did not ameliorate the course of GN. In contrast, renal damage was significantly reduced by restricting IL-6Ra deficiency to T effector cells and excluding Tregs. Detailed studies of Tregs revealed unaltered IL-10 production despite IL-6Ra deficiency. However, in vivo and in vitro, IL-6Ra classic signaling induced RORγt+Foxp3+ double-positive Tregs (biTregs), which carry the trafficking receptor CCR6 and have potent immunoregulatory properties. Indeed, lack of IL-6Ra significantly reduced Treg in vitro suppressive capacity. Finally, adoptive transfer of T cells containing IL-6Ra-/- Tregs resulted in severe aggravation of GN in mice. CONCLUSIONS: Our data refine the old paradigm, that IL-6 enhances Th17 responses and suppresses Tregs. We here provide evidence that T cell-intrinsic IL-6Ra classic signaling indeed induces the generation of Th17 cells but at the same time highly immunosuppressive RORγt+ biTregs. These results advocate caution and indicate that IL-6-directed therapies for GN need to be cell-type specific.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Subunidad alfa del Receptor de Interleucina-6/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Animales , Línea Celular , Cruzamientos Genéticos , Femenino , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Inmunosupresores/uso terapéutico , Inflamación , Subunidad alfa del Receptor de Interleucina-6/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Necrosis , Receptores de Interleucina-6/genética , Células Th17/citologíaRESUMEN
Background Th17 cells are central pathogenic mediators of autoimmune disease, including many forms of GN. IL-10 receptor signaling (IL-10R) in regulatory T cells (Tregs) has been implicated in the downregulation of Th17 cells, but the underlying molecular mechanisms and functional relevance of this process remain unclear.Methods We generated mice with Treg-specific IL-10Ra deficiency and subjected these mice to nephrotoxic serum-induced nephritis as a model of crescentic GN. Immune responses and Treg phenotypes were extensively analyzed.Results Compared with controls, mice with IL-10Ra-/- Tregs showed a spontaneously overshooting Th17 immune response. This hyper-Th17 phenotype was further boosted during GN and associated with aggravated renal injury. Notably, abrogation of IL-10Ra signaling in Tregs increased dendritic cell activation and production of Th17-inducing cytokines. In contrast, Treg trafficking and expression of chemokine receptor CCR6 remained unaffected, indicating mechanisms of Th17 control, differing from those of previously identified CCR6+ Treg17 cells. Indeed, the capacity for direct in vitro suppression of Th17 responses by IL-10Ra-/- Tregs was significantly impaired. As underlying pathology, analyses conducted in vitro and in vivo using double-fluorescent reporter mice revealed strikingly decreased IL-10 production by IL-10Ra-/- Tregs. To assess, whether reduced IL-10 could explain the hyper Th17 phenotype, competitive cotransfer experiments were performed. Supporting our concept, IL-10Ra-/- T cells differentiated into Th17 cells at much higher frequencies than wild type T cells did during GN.Conclusions IL-10R engagement optimizes Treg-mediated suppression of Th17 immunity. We hypothesize a feed-forward loop, in which IL-10Ra signaling reinforces IL-10 secretion by Tregs which potently controls Th17 development via direct and indirect mechanisms. IL-10R thus may be a promising therapeutic target for the treatment of GN.
Asunto(s)
Glomerulonefritis/inmunología , Subunidad alfa del Receptor de Interleucina-10/metabolismo , Interleucina-10/metabolismo , Nefritis/metabolismo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Animales , Diferenciación Celular , Citocinas/metabolismo , Células Dendríticas/fisiología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Subunidad alfa del Receptor de Interleucina-10/genética , Masculino , Ratones , Ratones Noqueados , Nefritis/inmunología , Receptores CCR6/metabolismo , Células Th17/metabolismoRESUMEN
Th1 cells are central pathogenic mediators of crescentic GN (cGN). Mechanisms responsible for Th1 cell downregulation, however, remain widely unknown. Recently, it was proposed that activation of the Th1-characteristic transcription factor T-bet optimizes Foxp3+ regulatory T (Treg) cells to counteract Th1-type inflammation. Because very little is known about the role of T-bet+ Treg1 cells in inflammatory diseases, we studied the function of these cells in the nephrotoxic nephritis (NTN) model of cGN. The percentage of Treg1 cells progressively increased in kidneys of nephritic wild-type mice during the course of NTN, indicating their functional importance. Notably, naïve Foxp3CrexT-betfl/fl mice, lacking Treg1 cells, showed spontaneous skewing toward Th1 immunity. Furthermore, absence of Treg1 cells resulted in aggravated NTN with selectively dysregulated renal and systemic Th1 responses. Detailed analyses of Treg cells from Foxp3CrexT-betfl/fl mice revealed unaltered cytokine production and suppressive capacity. However, in competitive cotransfer experiments, wild-type Treg cells outcompeted T-bet-deficient Treg cells in terms of population expansion and expression levels of Foxp3, indicating that T-bet expression is crucial for general Treg fitness. Additionally, T-bet-deficient Treg cells lacked expression of the Th1-characteristic trafficking receptor CXCR3, which correlated with significant impairment of renal Treg infiltration. In summary, our data indicate a new subtype of Treg cells in cGN. These Treg1 cells are characterized by activation of the transcription factor T-bet, which enhances the overall fitness of these cells and optimizes their capacity to downregulate Th1 responses by inducing chemokine receptor CXCR3 expression.
Asunto(s)
Glomerulonefritis/inmunología , Proteínas de Dominio T Box/fisiología , Linfocitos T Reguladores/fisiología , Células TH1/fisiología , Animales , Masculino , Ratones , Receptores CXCR3/fisiologíaRESUMEN
Cells expressing both the regulatory T cell (Treg)-inducing transcription factor Foxp3 and the Th17 transcription factor RORγt have been identified (biTregs). It is unclear whether RORγt(+)Foxp3(+) biTregs belong to the Th17-specific Treg17 cells, represent intermediates during Treg/Th17 transdifferentiation, or constitute a distinct cell lineage. Because the role of biTregs in inflammatory renal disease is also unknown, we studied these cells in the nephrotoxic nephritis (NTN) model of acute crescentic GN. Induction of NTN resulted in rapid renal and systemic expansion of biTregs. Notably, analyses of the biTreg expression profile revealed production of both anti-inflammatory (IL-10, IL-35) and proinflammatory (IL-17) cytokines. Additionally, biTregs expressed a signature of surface molecules and transcription factors distinct from those of Th17 cells and conventional Tregs (cTregs), and biTregs were identified in Treg17-deficient mice. Finally, fate reporter and cell transfer studies confirmed that biTregs are not Treg/Th17 transdifferentiating cells. Therapeutic transfer of biTregs suppressed the development of nephritis to an extent similar to that observed with transferred cTregs, but in vitro studies indicated different mechanisms of immunosuppression for biTregs and cTregs. Intriguingely, as predicted from their cytokine profile, endogenous biTregs displayed additional proinflammatory functions in NTN that were abrogated by cell-specific deletion of RORγt. In summary, we provide evidence that RORγt(+)Foxp3(+) biTregs are a novel and independent bifunctional regulatory T cell lineage distinct from cTregs, Treg17 cells, and Th17 cells. Furthermore, biTregs appear to contribute to crescentic GN and hence may be novel therapeutic targets.
Asunto(s)
Factores de Transcripción Forkhead/fisiología , Glomerulonefritis/etiología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/fisiología , Linfocitos T Reguladores/fisiología , Animales , Linaje de la Célula , Masculino , RatonesRESUMEN
The ultimate goal of tissue engineering is the generation of implants similar to native tissue. Thus, it is essential to utilize physiological stimuli to improve the quality of engineered constructs. Numerous publications reported that mechanical stimulation of small-sized, non-perfusable, tissue engineered cardiac constructs leads to a maturation of immature cardiomyocytes like neonatal rat cardiomyocytes or induced pluripotent stem cells/embryonic stem cells derived self-contracting cells. The aim of this study was to investigate the impact of mechanical stimulation and perfusion on the maturation process of large-scale (2.5×4.5cm), implantable cardiac patches based on decellularized porcine small intestinal submucosa (SIS) or Biological Vascularized Matrix (BioVaM) and a 3-dimensional construct containing neonatal rat heart cells. Application of cyclic mechanical stretch improved contractile function, cardiomyocyte alignment along the stretch axis and gene expression of cardiomyocyte markers. The development of a complex network formed by endothelial cells within the cardiac construct was enhanced by cyclic stretch. Finally, the utilization of BioVaM enabled the perfusion of the matrix during stimulation, augmenting the beneficial influence of cyclic stretch. Thus, this study demonstrates the maturation of cardiac constructs with clinically relevant dimensions by the application of cyclic mechanical stretch and perfusion of the starter matrix. STATEMENT OF SIGNIFICANCE: Considering the poor endogenous regeneration of the heart, engineering of bioartificial cardiac tissue for the replacement of infarcted myocardium is an exciting strategy. Most techniques for the generation of cardiac tissue result in relative small-sized constructs insufficient for clinical applications. Another issue is to achieve cardiomyocytes and tissue maturation in culture. Here we report, for the first time, the effect of mechanical stimulation and simultaneous perfusion on the maturation of cardiac constructs of clinical relevant dimensions, which are based on a perfusable starter matrix derived from porcine small intestine. In response to these stimuli superior organization of cardiomyocytes and vascular networks was observed in contrast to untreated controls. The study provides substantial progress towards the generation of implantable cardiac patches.
Asunto(s)
Matriz Extracelular/química , Implantes Experimentales , Miocardio , Miocitos Cardíacos , Estrés Mecánico , Animales , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Miocardio/citología , Miocardio/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Systemic lupus erythematosus (SLE) is a complex and potentially fatal autoimmune disorder. Although Th17 cells are thought to be central mediators of SLE, mechanisms underlying their counter regulation remain largely unknown. To help define this, we studied the function of the newly defined Stat3-dependent Th17-specific regulatory T cells (Treg17). Treg-specific deletion of Stat3 was achieved by generating Foxp3(Cre) × Stat3(fl/fl) mice and SLE was induced by intraperitoneal injection of pristane. Lack of Treg17 cells in these mice caused selectively enhanced peritoneal Th17 inflammation. Importantly, Treg17 deficiency also resulted in aggravated pulmonary vasculitis with increased percentages of Th17 cells and significantly higher mortality. Similarly, 4 and 9 months after pristane injection, analysis of renal and systemic immunity showed overshooting Th17 responses in the absence of Treg17 cells, associated with the aggravation of lupus nephritis. Expression of the Th17 characteristic trafficking receptor CCR6 was strikingly reduced on Tregs of Foxp3(Cre) × Stat3(fl/fl) mice, resulting in impaired renal Treg infiltration. Thus, Stat3-induced Treg17 cells are novel antiinflammatory mediators of SLE. One mechanism enabling Treg17 cells to target pathogenic Th17 responses is shared expression of the chemokine receptor CCR6.
Asunto(s)
Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Receptores CCR6/inmunología , Factor de Transcripción STAT3/metabolismo , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Animales , Femenino , Inmunoglobulinas/sangre , Glomérulos Renales/patología , Lupus Eritematoso Sistémico/inducido químicamente , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Recuento de Linfocitos , Ratones , Peritonitis/inmunología , Receptores CCR6/análisis , Factor de Transcripción STAT3/genética , Tasa de Supervivencia , Linfocitos T Reguladores/química , Terpenos , Células Th17/química , Vasculitis/inmunologíaRESUMEN
IL-6 can mediate proinflammatory effects, and IL-6 receptor (IL-6R) blockade as a treatment for inflammatory diseases has entered clinical practice. However, opposing effects of IL-6 have been observed in models of GN. Although IL-6 is proinflammatory in murine lupus nephritis, protective effects have been observed for IL-6 in the nephrotoxic nephritis (NTN) model of acute crescentic GN. In light of the potential dangers of IL-6-directed treatment, we studied the mechanisms underlying the contradictory findings in GN. IL-6 can signal through the membrane-bound IL-6R, which is expressed only on hepatocytes and certain leukocytes (classic), or through the soluble IL-6R, which binds the ubiquitously expressed gp130 (alternative). Preemptive treatment of mice with anti-IL-6R or anti-IL-6 worsened NTN, whereas selective blockade of alternative IL-6 signaling by the fusion protein sgp130Fc did not. FACS analysis of mouse spleen cells revealed proinflammatory macrophages express the highest levels of IL-6Rα, and in vitro treatment with IL-6 blocked macrophage proliferation. Furthermore, proinflammatory macrophages were expanded during inflammation in IL-6(-/-) mice. Late application of anti-IL-6 after establishment of adaptive nephritogenic immunity was sufficient to aggravate NTN within 2.5 days, a period when macrophages are active. Finally, NTN was aggravated in mice with macrophage-specific impairment of IL-6 classic signaling, coincident with enhanced macrophage proliferation and accumulation in the kidney. Our data thus reveal a novel mechanism in which IL-6-mediated dampening of macrophage activation protects tissues from overshooting immune responses. This finding has important implications for potential IL-6-directed therapies and supports the careful choice of recipient patients and timing.