Actividad de la hormona de crecimiento en el desarrollo de enfermedades autoinmunes

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-12-2013Durante años se han establecido diversos efectos del sistema neuroendocrino sobre el desarrollo y función del sistema inmune. Sin embargo el particular efecto de las hormonas somatotrópicas, ha sido poco estudiado, restringiéndose muchas veces ese análisis a su papel en la hiperactivación de células linfoides o en la producción de citoquinas. En este proyecto hemos estudiado el efecto de la hormona de crecimiento en el desarrollo de la diabetes tipo 1. Está enfermedad autoinmune que afecta a más de 20 millones de personas en el mundo se caracteriza por una insuficiente producción de insulina por parte de las células β, lo que provoca el acúmulo de glucosa en circulación. La línea de ratones NOD (Non-obese diabetic) desarrolla T1D de manera espontánea y es uno de los modelos más empleados para el estudio de esta enfermedad. En nuestro laboratorio, sobre el fondo genético NOD, hemos desarrollado una línea de ratones transgénicos para GH bovina (NOD-bGH), que permita estudiar como la presencia de niveles constantes de GH circulante influye en el desarrollo de la diabetes tipo 1. Los ratones NOD-bGH son completamente resistentes a la patología. El estudio del mecanismo responsable de esa resistencia implica una menor apoptosis y mayor proliferación de las células β en los ratones NOD-bGH y un efecto de la hormona sobre la presentación antigénica que explica la no detección de autoanticuerpos en estos ratones, sobre la polarización de macrófagos que en estos ratones son fundamentalmente M2 antiinflamatorios, sobre las células T reguladoras donde detectamos que la GH incrementa los niveles de FoxP3 y la capacidad de supresión y finalmente sobre la plasticidad de las células Th17 al inhibir su transformación hacia células Th1. Estos resultados vinculan a la GH con un papel esencial en la respuesta inmunitaria y la identifican como una diana de interés para el tratamiento de la diabetes tipo 1.Various neuroendocrine effects on development of the immune system have been known for decades. Nonetheless, the individual effect of somatotropic hormones has been little studied, and analysis is often restricted to its role in lymphoid cell hyperactivation and cytokine production. In this project, we analyzed the effect of growth hormone (GH) on the development of type 1 diabetes (T1D). T1D is an autoimmune disease that affects more than 20 million people worldwide; it is characterized by insufficient insulin production by pancreatic β cells, which results in the deleterious accumulation of circulating glucose. The non-obese diabetic (NOD) mouse strain develops spontaneous T1D and is one of the most widely used animal models for the research on this disease. In our laboratory, we developed a transgenic mouse strain for bovine GH (NOD-bGH) on the NOD genetic background, which allows study of how constant levels of circulating GH influence T1D development. NOD-bGH mice are completely resistant to the disease; we therefore studied the mechanism responsible for this phenomenon. We found mechanisms that lead this resistance in NOD-bGH mice that involves lower apoptosis and increased proliferation of β cells compared to control littermates. The absence of autoantibodies in the mice, explained as a GH effect on antigen presentation. Macrophage polarization in NOD-bGH mice leads to an essentially anti-inflammatory M2 phenotype. Finally, we show plasticity of non-pathogenic Th17 cells that avoid conversion into pathogenic Th1 cells. These results identify a key role for GH in the immune response and pinpoint this hormone as a target of interest for treatment of type 1 diabetes

Altered gut microbiota activate and expand insulin B15-23-Reactive CD8+ T-Cells

Insulin is a major autoantigen in type 1 diabetes, targeted by both CD8 and CD4 T-cells. We studied an insulin-reactive T-cell receptor (TCR) alpha-chain transgenic non-obese diabetic (NOD) mouse on a TCRCα and proinsulin2 (PI2)-deficient background, designated as A22Cα-/-PI2-/-NOD mice. These mice develop a low incidence of autoimmune diabetes. To test the role of gut microbiota on diabetes development in this model system, we treated the A22Cα-/-PI2-/-NOD mice with enrofloxacin, a broad-spectrum antibiotic. The treatment led to male mice developing accelerated diabetes. We found that enrofloxacin increased the frequency of the insulin-reactive CD8+ T-cells and activated the cells in the Peyer’s patches (PP) and pancreatic lymph nodes (PLNs), together with induction of immunological effects on the antigen-presenting cell populations. The composition of gut microbiota differed between the enrofloxacin-treated and untreated mice and also between the enrofloxacin-treated mice that developed diabetes, compared with those that remained normoglycemic. Our results provide evidence that the composition of the gut microbiota is important for determining the expansion and activation of insulin-reactive CD8+ T-cells

Adoptive transfer of mRNA-Transfected T cells redirected against diabetogenic CD8 T cells can prevent diabetes

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that β2 microglobulin (β2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15–23 (InsB15–23) to the N terminus of β2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15–23/β2m/CD3-ζ mRNA was activated by an InsB15–23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15–23/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206–214 (IGRP206–214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15–23/β2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes

Novel engineered B lymphocytes targeting islet-specific T cells inhibit the development of type 1 diabetes in non-obese diabetic Scid mice

IntroductionIn this study, we report a novel therapeutic approach using B lymphocytes to attract islet-specific T cells in the non-obese diabetic (NOD) mouse model and prevent the development of autoimmune diabetes. Rather than using the antibody receptor of B cells, this approach utilizes their properties as antigen-presenting cells to T cells.MethodsPurified splenic B cells were treated with lipopolysaccharide, which increases regulatory B (Breg) cell function, then electroporated with mRNA encoding either chimeric MHC-I or MHC-II molecules covalently linked to antigenic peptides. Immunoregulatory functions of these engineered B cells (e-B cells) were tested by in vitro assays and in vivo co-transfer experiments with beta-cell-antigen-specific CD8+ or CD4+ T cells in NOD.Scid mice, respectively.ResultsThe e-B cells expressing chimeric MHC-I-peptide inhibited antigen-specific CD8+ T-cell cytotoxicity in vitro. The e-B cells expressing chimeric MHC-II-peptide induced antigen-specific CD4+ T cells to express the regulatory markers, PD-1, ICOS, CTLA-4, Lag3, and Nrp1. Furthermore, e-B cells encoding the chimeric MHC-I and MHC-II peptide constructs protected NOD.Scid mice from autoimmune diabetes induced by transfer of antigen-specific CD8+ and CD4+ T cells.DiscussionMHC–peptide chimeric e-B cells interacted with pathogenic T cells, and protected the host from autoimmune diabetes, in a mouse model. Thus, we have successfully expressed MHC–peptide constructs in B cells that selectively targeted antigen-specific cells, raising the possibility that this strategy could be used to endow different protective cell types to specifically regulate/remove pathogenic cells

Proinsulin expression shapes the TCR repertoire but fails to control the development of low-avidity insulin-reactive CD8+ T cells

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8+ T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8+ T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23–reactive TRBV19+CD8+ T cells and causes diabetes; 2) insulin B15-23–reactive TRBV19+CD8+ T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8+ T-cell compartment; 4) a biased repertoire of insulin-reactive CD8+ T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8+ T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes

Transferencia de genes de resistencia a antibióticos en aislados de Escherichia coli provenientes de comunidades remotas de la provincia de Esmeraldas

Fifty one antibiotic resistant strains of commensal Escherichia coli were isolated from human fecal samples obtained from San Agustin, Playa de Oro communities of the Esmeraldas province. The strains were resistant to different antibiotics among which Ampicillin (AMP), Tetracycline (TE), Trimethoprim-sulfamethoxazol (SXT) were the most common resistance in recollected strains. The majority of the antibiotic resistance (92%) was transferable by conjugation and 88% of the resistant isolates were able to pass resistance to SXT, TE, and AMP to an E.coli K12 resistant to nadilixic acid.Cincuenta y un aislados de Escherichia coli comensal resistentes a antibióticos fueron obtenidos de muestras fecales humanas provenientes de las comunidades de San Agustín y Playa de Oro de la provincia de Esmeraldas. Las bacterias aisladas fueron resistentes a diferentes antibióticos como a Ampicilina (AMP), Tectracilina (TE) y Trimethoprim-sulfamethoxazol (SXT). Estas fueron las resistencias más comunes encontradas en las cepas analizadas y fueron seleccionadas para ser analizadas en mayor detalle

Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8 +

NOD mice, a model strain for human type 1 diabetes, express proinsulin (PI) in the thymus. However, insulin-reactive T cells escape negative selection, and subsequent activation of the CD8+ T-cell clonotype G9C8, which recognizes insulin B15-23 via an αβ T-cell receptor (TCR) incorporating TRAV8-1/TRAJ9 and TRBV19/TRBJ2-3 gene rearrangements, contributes to the development of diabetes. In this study, we used fixed TRAV8-1/TRAJ9 TCRα-chain transgenic mice to assess the impact of PI isoform expression on the insulin-reactive CD8+ T-cell repertoire. The key findings were: 1) PI2 deficiency increases the frequency of insulin B15-23–reactive TRBV19+CD8+ T cells and causes diabetes; 2) insulin B15-23–reactive TRBV19+CD8+ T cells are more abundant in the pancreatic lymph nodes of mice lacking PI1 and/or PI2; 3) overexpression of PI2 decreases TRBV19 usage in the global CD8+ T-cell compartment; 4) a biased repertoire of insulin-reactive CD8+ T cells emerges in the periphery regardless of antigen exposure; and 5) low-avidity insulin-reactive CD8+ T cells are less affected by antigen exposure in the thymus than in the periphery. These findings inform our understanding of the diabetogenic process and reveal new avenues for therapeutic exploitation in type 1 diabetes

Adoptive Transfer of mRNA-Transfected T Cells Redirected against Diabetogenic CD8 T Cells Can Prevent Diabetes

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that β2 microglobulin (β2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15–23 (InsB15–23) to the N terminus of β2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15–23/β2m/CD3-ζ mRNA was activated by an InsB15–23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15–23/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206–214 (IGRP206–214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15–23/β2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes