RESUMEN
Innate lymphoid cells (ILCs) are a family of developmentally related leukocytes that rapidly secrete polarized sets of cytokines to combat infection and promote tissue repair at mucosal barriers. Among them, group 3 ILCs (ILC3s) play an important role in maintenance of the gut homeostasis by producing IL-22, and their development and function critically depend on the transcription factor RORγt. Although recent evidence indicates that RORγt+ ILC3s are reduced in the gut in the absence of the Cdc42 activator DOCK8 (dedicator of cytokinesis 8), the underlying mechanism remains unclear. We found that genetic deletion of Dock8 in RORγt+-lineage cells markedly reduced ILC3s in the lamina propria of the small intestine. By analyzing BrdU incorporation, it was revealed that DOCK8 deficiency did not affect the cell proliferation. Furthermore, when lineage marker-negative (Lin-) α4ß7+ CD127+ RORγt- fetal liver cells were cultured with OP9 stromal cells in the presence of stem cell factor (SCF) and IL-7 in vitro, RORγt+ ILC3s normally developed irrespective of DOCK8 expression. However, DOCK8-deficient ILC3s exhibited a severe defect in survival of ILC3s under the condition with or without IL-7. Similar defects were observed when we analyzed Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Thus, DOCK8 acts in cell-autonomous manner to control survival of ILC3s in the gut through Cdc42 activation.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Mucosa Intestinal/citología , Linfocitos/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Animales , Dominio Catalítico/genética , Línea Celular , Proliferación Celular/genética , Supervivencia Celular/genética , Citocinas/metabolismo , Activación Enzimática/inmunología , Factores de Intercambio de Guanina Nucleótido/genética , Células HEK293 , Humanos , Interleucina-7/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Factor de Células Madre/metabolismoRESUMEN
Dedicator of cytokinesis 8 (DOCK8) is a guanine nucleotide exchange factor (GEF) for Cdc42. In humans, homozygous or compound heterozygous deletions in DOCK8 cause a combined immunodeficiency characterized by various allergic diseases including food allergies. Although group 2 innate lymphoid cells (ILC2s) contribute to the development of allergic inflammation by producing interleukin (IL)-5 and IL-13, the role of ILC2s in DOCK8 deficiency has not been fully explored. With the use of cytometry by time-of-flight (CyTOF), we performed high-dimensional phenotyping of intestinal immune cells and found that DOCK8-deficient (Dock8-/-) mice exhibited expansion of ILC2s and other leukocytes associated with type 2 immunity in the small intestine. Moreover, IL-5- and IL-13-producing cells markedly increased in Dock8-/- mice, and the majority of them were lineage-negative cells, most likely ILC2s. Intestinal ILC2s expanded when DOCK8 expression was selectively deleted in hematopoietic cells. Importantly, intestinal ILC2 expansion was also observed in Dock8VAGR mice having mutations in the catalytic center of DOCK8, thereby failing to activate Cdc42. Our findings indicate that DOCK8 is a negative regulator of intestinal ILC2s to inhibit their expansion via Cdc42 activation, and that deletion of DOCK8 causes a skewing to type 2 immunity in the gut.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/inmunología , Inmunidad Innata , Intestino Delgado/inmunología , Linfocitos/inmunología , Animales , Eliminación de Gen , Factores de Intercambio de Guanina Nucleótido/genética , Intestino Delgado/citología , Intestino Delgado/metabolismo , Linfocitos/citología , Ratones Endogámicos C57BLRESUMEN
DOCK8 is a Cdc42-specific guanine-nucleotide exchange factor that is essential for development and functions of various subsets of leukocytes in innate and acquired immune responses. Although DOCK8 plays a critical role in spatial control of Cdc42 activity during interstitial leukocyte migration, the mechanism remains unclear. We show that the DOCK homology region (DHR)-1 domain of DOCK8 binds specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and is required for its recruitment to the plasma membrane. Structural and biochemical analyses reveal that DOCK8 DHR-1 domain consists of a C2 domain-like core with loops creating the upper surface pocket, where three basic residues are located for stereospecific recognition of phosphoinositides. Substitution of the two basic residues, K576 and R581, with alanine abolished PI(4,5)P2 binding in vitro, ablated the ability of DOCK8 to activate Cdc42 and support leukocyte migration in three-dimensional collagen gels. Dendritic cells carrying the mutation exhibited defective interstitial migration in vivo. Thus, our study uncovers a critical role of DOCK8 in coupling PI(4,5)P2 signaling with Cdc42 activation for immune regulation.