RESUMO
Retinoblastoma is a childhood tumor of the retina that is caused mostly by biallelic inactivation of the tumor suppressor gene RB1. To generate a research resource, we abrogated expression of RB1 in H9 hESCs by CRISPR/Cas9 induced deletion of the RB1 promoter, either on one or on both alleles. This enables studies on the role of RB1 loss during differentiation, for example in differentiation towards neural retina. The generation of three isogenic lines per deletion state enables validation of phenotypic results in independent clonal lines.
Assuntos
Células-Tronco Embrionárias Humanas , Neoplasias da Retina , Retinoblastoma , Criança , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Regiões Promotoras Genéticas/genética , Neoplasias da Retina/genética , Retinoblastoma/genética , Proteínas de Ligação a Retinoblastoma/genética , Proteína do Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The urothelium of the urinary bladder represents the first line of defense. However, uropathogenic E. coli (UPEC) damage the urothelium and cause acute bacterial infection. Here, we demonstrate the crosstalk between macrophages and the urothelium stimulating macrophage migration into the urothelium. Using spatial proteomics by MALDI-MSI and LC-MS/MS, a novel algorithm revealed the spatial activation and migration of macrophages. Analysis of the spatial proteome unravelled the coexpression of Myo9b and F4/80 in the infected urothelium, indicating that macrophages have entered the urothelium upon infection. Immunofluorescence microscopy additionally indicated that intraurothelial macrophages phagocytosed UPEC and eliminated neutrophils. Further analysis of the spatial proteome by MALDI-MSI showed strong expression of IL-6 in the urothelium and local inhibition of this molecule reduced macrophage migration into the urothelium and aggravated the infection. After IL-6 inhibition, the expression of matrix metalloproteinases and chemokines, such as CX3CL1 was reduced in the urothelium. Accordingly, macrophage migration into the urothelium was diminished in the absence of CX3CL1 signaling in Cx3cr1gfp/gfp mice. Conclusively, this study describes the crosstalk between the infected urothelium and macrophages through IL-6-induced CX3CL1 expression. Such crosstalk facilitates the relocation of macrophages into the urothelium and reduces bacterial burden in the urinary bladder.
