RESUMO
Although Notch signaling has been proposed as a therapeutic target for type-2 diabetes, liver steatosis, and atherosclerosis, its direct effect on pancreatic islets remains unknown. Here, we demonstrated a function of Dll4-Notch signaling inhibition on the biology of insulin-producing cells. We confirmed enhanced expression of key Notch signaling genes in purified pancreatic islets from diabetic NOD mice and showed that treatment with anti-Dll4 antibody specifically abolished Notch signaling pathway activation. Furthermore, we showed that Notch inhibition could drive proliferation of ß-islet cells and confer protection from the development of STZ-induced diabetes. Importantly, inhibition of the Dll4 pathway in WT mice increased insulin secretion by inducing the differentiation of pancreatic ß-islet cell progenitors, as well as the proliferation of insulin-secreting cells. These findings reveal a direct effect of Dll4-blockade on pancreatic islets that, in conjunction with its immunomodulatory effects, could be used for unmet medical needs hallmarked by inefficient insulin action.
Assuntos
Secreção de Insulina/genética , Receptor Notch4/genética , Animais , Diferenciação Celular , Camundongos , Camundongos Endogâmicos NOD , Transdução de SinaisRESUMO
Delta-like ligand 4 (Dll4)-Notch signaling is essential for T cell development and alternative thymic lineage decisions. How Dll4-Notch signaling affects pro-T cell fate and thymic dendritic cell (tDC) development is unknown. We found that Dll4 pharmacological blockade induces accumulation of tDCs and CD4(+)CD25(+)FoxP3(+) regulatory T cells (T(reg) cells) in the thymic cortex. Both genetic inactivation models and anti-Dll4 antibody (Ab) treatment promote de novo natural T(reg) cell expansion by a DC-dependent mechanism that requires major histocompatibility complex II expression on DCs. Anti-Dll4 treatment converts CD4(-)CD8(-)c-kit(+)CD44(+)CD25(-) (DN1) T cell progenitors to immature DCs that induce ex vivo differentiation of naive CD4(+) T cells into T(reg) cells. Induction of these tolerogenic DN1-derived tDCs and the ensuing expansion of T(reg) cells are Fms-like tyrosine kinase 3 (Flt3) independent, occur in the context of transcriptional up-regulation of PU.1, Irf-4, Irf-8, and CSF-1, genes critical for DC differentiation, and are abrogated in thymectomized mice. Anti-Dll4 treatment fully prevents type 1 diabetes (T1D) via a T(reg) cell-mediated mechanism and inhibits CD8(+) T cell pancreatic islet infiltration. Furthermore, a single injection of anti-Dll4 Ab reverses established T1D. Disease remission and recurrence are correlated with increased T(reg) cell numbers in the pancreas-draining lymph nodes. These results identify Dll4-Notch as a novel Flt3-alternative pathway important for regulating tDC-mediated T(reg) cell homeostasis and autoimmunity.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/fisiologia , Diabetes Mellitus Tipo 1/prevenção & controle , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Secretases da Proteína Precursora do Amiloide/deficiência , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Anticorpos/farmacologia , Western Blotting , Proteínas de Ligação ao Cálcio , Primers do DNA/genética , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Genes MHC da Classe II/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Microscopia Confocal , Análise de Sequência com Séries de Oligonucleotídeos , Pâncreas/patologia , Reação em Cadeia da Polimerase , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/citologia , Timo/imunologia , Tirosina Quinase 3 Semelhante a fms/genética , Tirosina Quinase 3 Semelhante a fms/metabolismoRESUMO
The essential role of the Delta-like ligand 4 (Dll4)-Notch signaling pathway in T-lymphocyte development is well established. It has been shown that specific inactivation of Dll4 on thymic stromal cells during early post-natal development leads to a deregulation in T-cell differentiation. However, whether ongoing Dll4-Notch signaling is required for T-cell development in the adult thymus is unknown. The use of anti-Dll4 Abs allowed us to confirm and expand previous studies by examining the kinetics and the reversibility of Dll4-Notch signaling blockade in T-cell development in adult mice. We found that anti-Dll4 treatment reduced thymic cellularity after 7 days, as a consequence of a developmental delay in T-cell maturation at the pro-T-cell double negative 1 (CD4(-) CD8(-) c-kit(+) CD44(+) CD25(-) ) stage, leading to decreased numbers of immature double-positive (CD4(+) CD8(+) ) T cells without affecting the frequency of mature single positive CD4(+) and CD8(+) thymocytes, while promoting alternative thymic B-cell expansion. This cellular phenotype was similarly observed in both young adult and aged mice (>1.5 years), extending our understanding of the ongoing role for Dll4-Notch signaling during T-cell development in the adult thymus. Finally, after cessation of Dll4 Ab treatment, thymic cellularity and thymocyte subset ratios returned to normal levels, indicating reversibility of this phenotype in both adult and aged mice, which has important implications for potential clinical use of Dll4-Notch inhibitors.
