RESUMEN
Migration of immune cells to sites of inflammation is a critical step in the body's response to infections but also during autoimmune flares. Chemokine receptors, members of the GPCR receptors, are instrumental in directing specific cell types to their target organs. Herein, we describe a highly potent small molecule antagonist of the chemokine receptor CCR6, which came out of fine-tuned structural elaborations from a proprietary HTS hit. Three main issues in the parent chemical series-cytotoxicity, phototoxicity, and hERG, were successfully solved. Biological characterization demonstrated that compound 45 (IDOR-1117-2520) is a selective and insurmountable antagonist of CCR6. In vivo proof-of-mechanism studies in a mouse lung inflammation model using a representative compound from the chemical class of 45 confirmed that the targeted CCR6+ cells were efficiently inhibited from migrating into the bronchoalveoli. Finally, ADMET and physicochemical properties were well balanced and the preclinical package warranted progress in the clinic.
Asunto(s)
Enfermedades Autoinmunes , Receptores CCR6 , Receptores CCR6/antagonistas & inhibidores , Receptores CCR6/metabolismo , Animales , Humanos , Enfermedades Autoinmunes/tratamiento farmacológico , Ratones , Relación Estructura-Actividad , Descubrimiento de DrogasRESUMEN
The CXCR3 chemokine receptor is a G protein-coupled receptor mainly expressed on immune cells from the lymphoid lineage, including activated T cells. Binding of its inducible chemokine ligands CXCL9, CXCL10, and CXCL11 leads to downstream signaling events and the migration of activated T cells to sites of inflammation. Herein, we report the third part of our CXCR3 antagonist program in the field of autoimmunity, culminating in the discovery of the clinical compound ACT-777991 (8a). A previously disclosed advanced molecule was exclusively metabolized by the CYP2D6 enzyme, and options to address the issue are described. ACT-777991 is a highly potent, insurmountable, and selective CXCR3 antagonist that showed dose-dependent efficacy and target engagement in a mouse model of acute lung inflammation. The excellent properties and safety profile warranted progress in the clinics.
Asunto(s)
Quimiocina CXCL10 , Receptores de Quimiocina , Animales , Ratones , Quimiocina CXCL10/metabolismo , Quimiocina CXCL9 , Receptores de Quimiocina/metabolismo , Ligandos , Transducción de Señal , Receptores CXCR3/metabolismoRESUMEN
Cenerimod is a potent, selective sphingosine 1-phosphate receptor 1 (S1P1) modulator currently investigated in a Phase IIb study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including T and B lymphocytes) in the bloodstream and inflamed tissues, making them an effective therapeutic concept for autoimmune disorders. Although the effect of S1P receptor modulators in reducing circulating lymphocytes is well documented, the precise molecular role of the S1P1 receptor on these cell types is not fully understood. In this study, the mode of action of cenerimod on human primary lymphocytes in different activation states was investigated focusing on their chemotactic behavior towards S1P in real-time, concomitant to S1P1 receptor expression and internalization dynamics. Here, we show that cenerimod effectively prevents T and B cell migration in a concentration-dependent manner. Interestingly, while T cell activation led to strong S1P1 re-expression and enhanced migration; in B cells, an enhanced migration capacity and S1P1 receptor surface expression was observed in an unstimulated state. Importantly, concomitant treatment with glucocorticoids (GCs), a frequently used treatment for autoimmune disorders, had no impact on the inhibitory activity of cenerimod on lymphocytes.
Asunto(s)
Linfocitos B/fisiología , Movimiento Celular , Lisofosfolípidos/metabolismo , Oxadiazoles/farmacología , Glicoles de Propileno/farmacología , Receptores de Esfingosina-1-Fosfato/antagonistas & inhibidores , Esfingosina/análogos & derivados , Linfocitos T/fisiología , Linfocitos B/efectos de los fármacos , Humanos , Transducción de Señal , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/metabolismo , Linfocitos T/efectos de los fármacosAsunto(s)
Lupus Eritematoso Sistémico/metabolismo , Oxadiazoles/farmacología , Glicoles de Propileno/farmacología , Receptores de Esfingosina-1-Fosfato/metabolismo , Animales , Biomarcadores , Microambiente Celular/efectos de los fármacos , Microambiente Celular/genética , Microambiente Celular/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos/métodos , Humanos , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/etiología , Lupus Eritematoso Sistémico/patología , Linfocitos/efectos de los fármacos , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Terapia Molecular Dirigida , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Investigación Biomédica TraslacionalRESUMEN
One of the most important advantages of mass spectrometry is the ability to quantify proteins and their modifications in parallel to obtain a holistic picture of the protein of interest. Here, we present a hybrid immunoaffinity targeted mass spectrometry (MS) method that combines efficient pan-antibody enrichment of a specific protein from plasma with the selectivity of high-resolution targeted MS analysis to quantitate specific proteoforms of interest. We used this approach to quantify plasma levels of the chemokine CXCL10 that has been associated with many immunological disorders such as systemic lupus erythematosus and primary Sjögren's Syndrome (pSS). The hybrid approach enabled sensitive, specific, and simultaneous quantification of total, full-length (active) CXCL101-77 and DPP4-truncated (inactive) CXCL103-77 in human plasma down to the low pg/mL level, reaching ELISA sensitivities. Samples from 30 control subjects and 34 pSS patients (n = 64) were analyzed. The ratio of CXCL101-77 to truncated CXCL103-77 was significantly increased in patients with pSS and provided the highest correlation with pSS disease activity. Therefore, this CXCL10 proteoform ratio represents an interesting exploratory disease activity biomarker to further investigate. As this strategy can be readily adapted to other plasma proteins and proteoforms of interest, we are convinced that it will lead to a more detailed understanding of proteoforms in physiology and pathology yielding more relevant biomarkers and drug targets.
Asunto(s)
Lupus Eritematoso Sistémico , Síndrome de Sjögren , Biomarcadores , Quimiocina CXCL10/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Espectrometría de Masas , Síndrome de Sjögren/diagnóstico , Síndrome de Sjögren/genéticaRESUMEN
Regulatory T (Treg) cells are pivotal for the maintenance of peripheral tolerance by controlling self-reactive, chronic, and homeostatic T-cell responses. Here, we report that the increase in Treg-cell suppressive function observed in lymphopenic mice correlates with the degree of lymphopenia and is caused by a higher frequency of a novel subpopulation of CD103(pos) ICOS(pos) Treg cells. Though present in the thymus, CD103(pos) ICOS(pos) Treg cells are not generated there but recirculate from the periphery to that site. The acquisition and maintenance of this distinctive phenotype requires the LN microenvironment and the in situ availability of antigen. Contrary to conventional effector and other Treg cells, the cellularity of CD103(pos) ICOS(pos) Treg cells is not affected by the absence of IL-7 and thymic stroma lymphopoetin. Given their increased frequency in lymphopenia, the absolute number of CD103(pos) ICOS(pos) Treg cells remains unchanged in the periphery irrespective of a paucity of total Treg cells. We furthermore demonstrate, with cell transfers in mice, that the CD103(pos) ICOS(pos) phenotype represents a LN-specific differentiation stage arrived at by several other Treg-cell subsets. Thus, tissue-specific cues determine the overall potency of the peripheral Treg-cell pool by shaping its subset composition.
Asunto(s)
Antígenos CD/metabolismo , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Cadenas alfa de Integrinas/metabolismo , Ganglios Linfáticos/inmunología , Linfopenia/sangre , Linfopenia/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Diferenciación Celular/inmunología , Microambiente Celular , Análisis por Conglomerados , Citocinas/metabolismo , Perfilación de la Expresión Génica , Homeostasis , Interleucina-7/metabolismo , Recuento de Linfocitos , Linfopenia/metabolismo , Ratones , Transducción de Señal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfopoyetina del Estroma TímicoRESUMEN
Thymic T cell development is dependent on a specialized epithelial microenvironment mainly composed of cortical and medullary thymic epithelial cells (TECs). The molecular programs governing the differentiation and maintenance of TECs remain largely unknown. Wnt signaling is central to the development and maintenance of several organ systems but a specific role of this pathway for thymus organogenesis has not yet been ascertained. In this report, we demonstrate that activation of the canonical Wnt signaling pathway by a stabilizing mutation of beta-catenin targeted exclusively to TECs changes the initial commitment of endodermal epithelia to a thymic cell fate. Consequently, the formation of a correctly composed and organized thymic microenvironment is prevented, thymic immigration of hematopoietic precursors is restricted, and intrathymic T cell differentiation is arrested at a very early developmental stage causing severe immunodeficiency. These results suggest that a precise regulation of canonical Wnt signaling in thymic epithelia is essential for normal thymus development and function.
Asunto(s)
Diferenciación Celular/inmunología , Células Epiteliales/metabolismo , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/metabolismo , Timo/inmunología , Timo/metabolismo , beta Catenina/genética , beta Catenina/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Endodermo/citología , Endodermo/inmunología , Endodermo/metabolismo , Células Epiteliales/inmunología , Células Epiteliales/patología , Factores de Transcripción Forkhead/genética , Marcación de Gen , Inhibidores de Crecimiento/biosíntesis , Inhibidores de Crecimiento/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Transducción de Señal/genética , Transducción de Señal/inmunología , Timo/citología , Timo/patología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/biosíntesis , beta Catenina/fisiologíaRESUMEN
Signals mediated by the transforming growth factor-beta superfamily of growth factors have been implicated in thymic epithelial cell (TEC) differentiation, homeostasis, and function, but a direct reliance on these signals has not been established. Here we demonstrate that a block in canonical transforming growth factor-beta signaling by the loss of Smad4 expression in TECs leads to qualitative changes in TEC function and a progressively disorganized thymic microenvironment. Moreover, the number of thymus resident early T-lineage progenitors is severely reduced in the absence of Smad4 expression in TECs and directly correlates with extensive thymic and peripheral lymphopenia. Our observations hence place Smad4 within the signaling events in TECs that determine total thymus cellularity by controlling the number of early T-lineage progenitors.
Asunto(s)
Proteína Smad4/metabolismo , Linfocitos T/citología , Linfocitos T/metabolismo , Timo/citología , Timo/metabolismo , Animales , Secuencia de Bases , Quimiocinas/genética , ADN Complementario/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Homeostasis , Masculino , Ratones , Ratones Mutantes , Ratones Transgénicos , Embarazo , Transducción de Señal , Proteína Smad4/deficiencia , Proteína Smad4/genética , Linfocitos T/inmunología , Timo/inmunología , Transcripción GenéticaRESUMEN
The thymus constitutes the primary lymphoid organ responsible for the generation of naive T cells. Its stromal compartment is largely composed of a scaffold of different subsets of epithelial cells that provide soluble and membrane-bound molecules essential for thymocyte maturation and selection. With senescence, a steady decline in the thymic output of T cells has been observed. Numeric and qualitative changes in the stromal compartment of the thymus resulting in reduced thymopoietic capacity have been suggested to account for this physiologic process. The precise cellular and molecular mechanisms underlying thymic senescence are, however, only incompletely understood. Here, we demonstrate that TGF-beta signaling in thymic epithelial cells exerts a direct influence on the cell's capacity to support thymopoiesis in the aged mouse as the physiologic process of thymic senescence is mitigated in mice deficient for the expression of TGF-beta RII on thymic epithelial cells. Moreover, TGF-beta signaling in these stromal cells transiently hinders the early phase of thymic reconstitution after myeloablative conditioning and hematopoietic stem cell transplantation. Hence, inhibition of TGF-beta signaling decelerates the process of age-related thymic involution and may hasten the reconstitution of regular thymopoiesis after hematopoietic stem cell transplantation.
Asunto(s)
Envejecimiento/fisiología , Células Epiteliales/fisiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Regeneración , Timo/fisiología , Factor de Crecimiento Transformador beta/fisiología , Animales , Ratones , Transducción de Señal , Células del Estroma , Timo/citologíaRESUMEN
BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a multiorgan autoimmune disorder caused by mutations in AIRE, the autoimmune regulator gene. Though recent studies concerning AIRE deficiency have begun to elucidate the molecular pathogenesis of organ-specific autoimmunity in patients with APS-1, the autoantigen responsible for hypoparathyroidism, a hallmark of APS-1 and its most common autoimmune endocrinopathy, has not yet been identified. METHODS: We performed immunoscreening of a human parathyroid complementary DNA library, using serum samples from patients with APS-1 and hypoparathyroidism, to identify patients with reactivity to the NACHT leucine-rich-repeat protein 5 (NALP5). Subsequently, serum samples from 87 patients with APS-1 and 293 controls, including patients with other autoimmune disorders, were used to determine the frequency and specificity of autoantibodies against NALP5. In addition, the expression of NALP5 was investigated in various tissues. RESULTS: NALP5-specific autoantibodies were detected in 49% of the patients with APS-1 and hypoparathyroidism but were absent in all patients with APS-1 but without hypoparathyroidism, in all patients with other autoimmune endocrine disorders, and in all healthy controls. NALP5 was predominantly expressed in the cytoplasm of parathyroid chief cells. CONCLUSIONS: NALP5 appears to be a tissue-specific autoantigen involved in hypoparathyroidism in patients with APS-1. Autoantibodies against NALP5 appear to be highly specific and may be diagnostic for this prominent component of APS-1.
Asunto(s)
Autoanticuerpos/sangre , Autoantígenos/inmunología , Hipoparatiroidismo/diagnóstico , Glándulas Paratiroides/inmunología , Poliendocrinopatías Autoinmunes/inmunología , Autoanticuerpos/análisis , Autoantígenos/genética , Biomarcadores/análisis , Biomarcadores/sangre , ADN Complementario/análisis , Biblioteca de Genes , Humanos , Hipoparatiroidismo/etiología , Hipoparatiroidismo/inmunología , Proteínas Mitocondriales , Proteínas Nucleares , Glándulas Paratiroides/química , Poliendocrinopatías Autoinmunes/complicaciones , ARN Mensajero/análisisRESUMEN
Haematopoietic precursors have to undergo a complex series of maturational steps in the thymus before they exit into the periphery as functional T lymphocytes. Thymic stroma cells, the majority being of epithelial origin, provide the functional partners for the maturational progression along this differentiation pathway. Here we review some of the molecular and cellular mechanisms that account for thymus organogenesis and discuss a strategy to use thymic epithelial precursor cells for the regeneration of the thymic microenvironment.
RESUMEN
The systemic administration of keratinocyte growth factor (KGF) enhances T-cell lymphopoiesis in normal mice and mice that received a bone marrow transplant. KGF exerts protection to thymic stromal cells from cytoablative conditioning and graft-versus-host disease-induced injury. However, little is known regarding KGF's molecular and cellular mechanisms of action on thymic stromal cells. Here, we report that KGF induces in vivo a transient expansion of both mature and immature thymic epithelial cells (TECs) and promotes the differentiation of the latter type of cells. The increased TEC numbers return within 2 weeks to normal values and the microenvironment displays a normal architectural organization. Stromal changes initiate an expansion of immature thymocytes and permit regular T-cell development at an increased rate and for an extended period of time. KGF signaling in TECs activates both the p53 and NF-kappaB pathways and results in the transcription of several target genes necessary for TEC function and T-cell development, including bone morphogenetic protein 2 (BMP2), BMP4, Wnt5b, and Wnt10b. Signaling via the canonical BMP pathway is critical for the KGF effects. Taken together, these data provide new insights into the mechanism(s) of action of exogenous KGF on TEC function and thymopoiesis.
Asunto(s)
Proliferación Celular , Células Epiteliales/inmunología , Factor 7 de Crecimiento de Fibroblastos/inmunología , Linfopoyesis/inmunología , Linfocitos T/inmunología , Timo/inmunología , Animales , Proteínas Morfogenéticas Óseas/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Epiteliales/citología , Femenino , Factor 7 de Crecimiento de Fibroblastos/farmacología , Linfopoyesis/efectos de los fármacos , Ratones , FN-kappa B/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/citología , Timo/citología , Proteína p53 Supresora de Tumor/inmunologíaRESUMEN
Acute graft-versus-host disease (aGVHD) impairs thymus-dependent T-cell regeneration in recipients of allogeneic bone marrow transplants through yet to be defined mechanisms. Here, we demonstrate in mice that MHC-mismatched donor T cells home into the thymus of unconditioned recipients. There, activated donor T cells secrete IFN-gamma, which in turn stimulates the programmed cell death of thymic epithelial cells (TECs). Because TECs themselves are competent and sufficient to prime naive allospecific T cells and to elicit their effector function, the elimination of host-type professional antigen-presenting cells (APCs) does not prevent donor T-cell activation and TEC apoptosis, thus precluding normal thymopoiesis in transplant recipients. Hence, strategies that protect TECs may be necessary to improve immune reconstitution following allogeneic bone marrow transplantation.
Asunto(s)
Apoptosis/inmunología , Trasplante de Médula Ósea/inmunología , Endotelio/inmunología , Enfermedad Injerto contra Huésped/inmunología , Linfocitos T/inmunología , Timo/inmunología , Enfermedad Aguda , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/patología , Endotelio/patología , Interferón gamma/deficiencia , Interferón gamma/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Recuperación de la Función/inmunología , Linfocitos T/patología , Timo/patología , Quimera por Trasplante/inmunología , Trasplante HomólogoRESUMEN
Haematopoietic precursors have to undergo a complex series of maturational steps in the thymus before they exit into the periphery as functional T lymphocytes. Thymic stroma cells, the majority being of epithelial origin, provide the functional partners for the maturational progression along this differentiation pathway. Here we review some of the molecular and cellular mechanisms that account for thymus organogenesis and discuss a strategy to use thymic epithelial precursor cells for the regeneration of the thymic microenvironment.
Asunto(s)
Organogénesis , Regeneración , Linfocitos T/inmunología , Timo/citología , Autoantígenos , Células Epiteliales/citología , Células Madre Hematopoyéticas/citología , Humanos , Transducción de Señal , Células del Estroma/citología , Linfocitos T/citología , Timo/inmunologíaRESUMEN
T cell development and selection require the fully mature and diverse epithelial microenvironment of the thymus. Acquisition of these characteristics is dependent on expression of the forkhead (also known as winged-helix) transcription factor FoxN1, as a lack of functional FoxN1 results in aberrant epithelial morphogenesis and an inability to attract lymphoid precursors to the thymus primordium. However, the transcriptional control of Foxn1 expression has not been elucidated. Here we report that secreted Wnt glycoproteins, expressed by thymic epithelial cells and thymocytes, regulate epithelial Foxn1 expression in both autocrine and paracrine fashions. Wnt molecules therefore provide regulatory signals critical for thymic function.