RESUMEN
The tumor microenvironment contains high concentrations of TGFß, a crucial immunosuppressive cytokine. TGFß stimulates immune escape by promoting peripheral immune tolerance to avoid tumoricidal attack. Small-molecule inhibitors of TGFßR1 are a prospective method for next-generation immunotherapies. In the present study, we identified selective 4-aminoquinoline-based inhibitors of TGFßR1 through structural and rational-based design strategies. This led to the identification of compound 4i, which was found to be selective for TGFßR1 with the exception of MAP4K4 in the kinase profiling assay. The compound was then further optimized to remove MAP4K4 activity, since MAP4K4 is vital for proper T-cell function and its inhibition could exacerbate tumor immunosuppression. Optimization efforts led to compound 4s that inhibited TGFßR1 at an IC50 of 0.79 ± 0.19 nM with 2000-fold selectivity against MAP4K4. Compound 4s represents a highly selective TGFßR1 inhibitor that has potential applications in immuno-oncology.
Asunto(s)
Aminoquinolinas/farmacología , Descubrimiento de Drogas , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptor Tipo I de Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Aminoquinolinas/síntesis química , Aminoquinolinas/química , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/inmunología , Estructura Molecular , Proteínas Serina-Treonina Quinasas/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/inmunología , Relación Estructura-ActividadRESUMEN
Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFß1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFß1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFß1 signaling require initial interaction of regDCs EXO with TGFß1R followed by internalization of regDCs EXO with TGFß1-TGFß1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFß1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.
Asunto(s)
COVID-19/inmunología , Células Dendríticas/inmunología , Exosomas/inmunología , Proteoma/inmunología , SARS-CoV-2/inmunología , Animales , Ratones , Proteómica , Receptor Tipo I de Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
OBJECTIVE: Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts. METHODS: Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates. RESULTS: The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor ß under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05). CONCLUSION: These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.
Asunto(s)
Fibroblastos/metabolismo , Macrófagos/inmunología , Esclerodermia Sistémica/genética , Piel/metabolismo , Adulto , Antígenos CD/inmunología , Antígenos de Diferenciación Mielomonocítica/inmunología , Diferenciación Celular , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Técnicas de Cocultivo , Femenino , Fibrosis/genética , Fibrosis/inmunología , Fibrosis/metabolismo , Antígenos HLA-DR/inmunología , Humanos , Inmunofenotipificación , Interleucina-6/genética , Interleucina-6/inmunología , Lectinas Tipo C/inmunología , Leucocitos Mononucleares , Activación de Macrófagos , Macrófagos/metabolismo , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/inmunología , Persona de Mediana Edad , Monocitos/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/inmunología , Receptores de Superficie Celular/inmunología , Factor de Transcripción STAT3/metabolismo , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Piel/patología , Transcriptoma , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunologíaRESUMEN
Asthma is one of the most common chronic diseases affecting both children and adults. We report a genome-wide association meta-analysis of 69,189 cases and 702,199 controls from Iceland and UK biobank. We find 88 asthma risk variants at 56 loci, 19 previously unreported, and evaluate their effect on other asthma and allergic phenotypes. Of special interest are two low frequency variants associated with protection against asthma; a missense variant in TNFRSF8 and 3' UTR variant in TGFBR1. Functional studies show that the TNFRSF8 variant reduces TNFRSF8 expression both on cell surface and in soluble form, acting as loss of function. eQTL analysis suggests that the TGFBR1 variant acts through gain of function and together with an intronic variant in a downstream gene, SMAD3, points to defective TGFßR1 signaling as one of the biological perturbations increasing asthma risk. Our results increase the number of asthma variants and implicate genes with known role in T cell regulation, inflammation and airway remodeling in asthma pathogenesis.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/genética , Asma/genética , Antígeno Ki-1/genética , Receptor Tipo I de Factor de Crecimiento Transformador beta/genética , Linfocitos T/inmunología , Regiones no Traducidas 3'/genética , Remodelación de las Vías Aéreas (Respiratorias)/inmunología , Asma/inmunología , Eosinófilos , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Islandia , Antígeno Ki-1/inmunología , Antígeno Ki-1/metabolismo , Recuento de Leucocitos , MicroARNs/metabolismo , Polimorfismo de Nucleótido Simple/inmunología , Sitios de Carácter Cuantitativo/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/inmunología , Receptor Tipo I de Factor de Crecimiento Transformador beta/metabolismo , Reino UnidoRESUMEN
B cells in germinal centers (GCs) cycle between light zone (LZ) and dark zone (DZ). The cues in the GC microenvironment that regulate the transition from LZ to DZ have not been well characterized. In Peyer's patches (PPs), transforming growth factor-ß (TGFß) promotes IgA induction in activated B cells that can then differentiate into GC B cells. We show here that TGFß signaling occurs in B cells in GCs and is distinct from signaling that occurs in activated B cells in PPs. Whereas in activated B cells TGFß signaling is required for IgA induction, in the GC it was instead required for the transition from LZ to DZ. In the absence of TGFß signaling, there was an accumulation of LZ GC B cells and reduced antibody affinity maturation likely due to reduced activation of Foxo1. This work identifies TGFß as a microenvironmental cue that is critical for GC homeostasis and function.