ABSTRACT
Genetically engineered mouse models (GEMMs) are very powerful tools to study lineage hierarchy and cellular dynamics of stem cells in vivo. Stem cell behavior in various contexts such as development, normal homeostasis and diseases have been investigated using GEMMs. The strategies to generate GEMMs have drastically changed in the last decade with the development of the CRISPR/Cas9 system for manipulation of the mammalian genome. The advantages of the CRISPR/Cas9 are its simplicity and efficiency. The bioinformatics tools available now allow us to quickly identify appropriate guide RNAs and design experimental conditions to generate the targeted mutation. In addition, the genome can be manipulated directly in the zygote which reduces the time to modify target genes compared to other technologies such as Embryonic Stem (ES) cells. Equally important is that we can manipulate the genome of any mouse background with the CRISPR/Cas9 system which omits time-consuming backcrossing processes, accelerates research and increases flexibility. Here, we will summarize basic allelic types and our standard strategies of how to generate them.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , CRISPR-Cas Systems/genetics , Genome/genetics , Mammals/genetics , Mice , RNA, Guide, Kinetoplastida/genetics , ZygoteABSTRACT
Patients with systemic sclerosis (SSc) display altered intestinal microbiota. However, the influence of intestinal dysbiosis on the development of experimental SSc remains unknown. Topoisomerase I peptide-loaded dendritic cell immunization induces SSc-like disease, with progressive skin and lung fibrosis. Breeders were given streptomycin and pups continued to receive antibiotic (ATB) until endpoint (lifelongATB). Alternately, ATB was withdrawn (earlyATB) or initiated (adultATB) during adulthood. Topoisomerase I peptide-loaded dendritic cell (no ATB) immunization induced pronounced skin fibrosis, with increased matrix (Col1a1), profibrotic (Il13, Tweakr), and vascular function (Serpine1) gene expression. Remarkably, earlyATB exposure was sufficient to augment skin Col5a1 and Il13 expression, and inflammatory cell infiltration, which included IL-13+ cells, mononuclear phagocytes, and mast cells. Moreover, skin pathology exacerbation was also observed in lifelongATB and adultATB groups. Oral streptomycin administration induced intestinal dysbiosis, with exposure limited to early life (earlyATB) being sufficient to cause long-term modification of the microbiota and a shift toward increased Bacteroidetes/Firmicutes ratio. Finally, aggravated lung fibrosis and dysregulated pulmonary T-cell responses were observed in earlyATB and lifelongATB but not adultATB-exposed mice. Collectively, intestinal microbiota manipulation with streptomycin exacerbated pathology in two distinct sites, skin and lungs, with early life being a critical window to affect the course of SSc-like disease.
Subject(s)
Dysbiosis/genetics , Gastrointestinal Microbiome/drug effects , Pulmonary Fibrosis/pathology , Scleroderma, Systemic/genetics , Scleroderma, Systemic/immunology , Streptomycin/pharmacology , Age Factors , Animals , Cells, Cultured , DNA, Bacterial/analysis , Dendritic Cells/drug effects , Disease Models, Animal , Dysbiosis/microbiology , Female , Gastrointestinal Microbiome/genetics , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Transgenic , Pulmonary Fibrosis/genetics , Random Allocation , Real-Time Polymerase Chain Reaction/methods , Risk Factors , Scleroderma, Systemic/pathology , Statistics, NonparametricABSTRACT
OBJECTIVES: Dysregulated coagulation cascade has been implicated in development of fibrosis in systemic sclerosis (SSc). Thrombin, a key mediator of the coagulation pathway, has both proinflammatory and procoagulant properties. Here, we evaluated the efficacy of oral dabigatran, a direct thrombin inhibitor, on topoisomerase I dendritic cells (TOPOIA DCs)-induced lung and skin fibrosis, an experimental model of SSc. METHODS: Mice were repeatedly immunized with TOPOIA DCs. Dabigatran was administered in food either during the onset of fibrotic (late treatment) or inflammatory (early treatment) phase. RESULTS: Early administration of dabigatran caused an aggravation of pulmonary fibrosis associated with signs of severe perivascular inflammation while late treatment was not protective when compared to the untreated TOPOIA DCs group. Thrombin was increased in lungs of TOPOIA DCs immunized group and, paradoxically, further augmented by administration of dabigatran to immunized mice. As in lungs, early and not late drug administration exacerbated skin fibrosis. Moreover, early dabigatran treatment induced a profibrotic and inflammatory skin gene expression signature with upregulated expression of Col5a1, Timp1, Tweakr, Vwf, Il6, Il33, Il4 and Ifng. CONCLUSIONS: Dabigatran aggravated lung and skin fibrosis in a TOPOIA DCs-induced model of SSc-like disease. Therefore, our results argue against the use of dabigatran to treat patients with SSc.
