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Next generation sequencing of thymic dendritic cells after damage

ABSTRACT: The thymus, which is the primary site of T cell development, is particularly sensitive to insult but also has a remarkable capacity for repair. However, the mechanisms orchestrating regeneration are poorly understood and delayed repair is common after cytoreductive therapies. Here, we demonstrate a trigger of thymic repair, centered on detecting the loss of dying thymocytes which are abundant during steady-state T cell development. Specifically, apoptotic thymocytes suppressed production of the regenerative factors IL-23 and BMP4 TAM receptor signalling and downstream activation of the Rho-GTPase Rac1, the intracellular pattern recognition receptor NOD2, and micro-RNA-29c. We found that the intracellular pattern recognition receptor NOD2, via induction of microRNA-29c, suppressed levels of the regenerative factors IL-23 and BMP4, in DCs and ECs, respectively. Transcriptome analysis in highly purified DCs after damage revealed highly similar inflammatory gene signatures and enrichment for NOD2 expression

ORGANISM(S): Mus musculus

PROVIDER: GSE183056 | GEO | 2021/12/01

REPOSITORIES: GEO

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Project description:The successful repair and renewal of alveolar epithelial cells are critical steps in prohibiting the accumulation of myofibroblasts and deposition of extracellular matrix in pulmonary fibrogenesis. MicroRNAs (miRNAs) are multi-focal regulators involved in the lung repair process. The contribution of miRNAs to epithelial maintenance and renewal is incompletely understood. We provide evidence that miR-29c on type 2 alveolar epithelial cells (AEC2s) are important for inhibiting AEC2 apoptosis and promoting AEC2 renewal, thus restraining the degree of fibrosis. MiR-29c was lower in AEC2s from lungs of idiopathic pulmonary fibrosis (IPF) individuals than from healthy lungs. Epithelial cells overexpressing miR-29c showed higher proliferative rate and viability. MiR-29c was protective against epithelial apoptosis by targeting Foxo3a. Both overexpression of miR-29c conventionally and AEC2s specifically led to less fibrosis and better recovery. Furthermore, loss of miR-29c in AEC2s resulted in higher apoptosis and reduced epithelial renewal than in control animals. Thus, miR-29c maintains epithelial integrity and promotes recovery from lung injury, attenuating lung fibrosis in mice. The miR-29c overexpression mouse epithelial cell line was generated based on the strategy described before38. MiR-29c lentiviral construct was generated as follows: a region of 131 nucleotides containing pre-mmu-miR-29c was amplified from mouse genomic DNA by using the following primers: 5’ MIR-29c: GTCGGTTAACATCTCTTACACAGG and 3’ MIR-29c: ACACCTCGAGGATCCTGAGGCTGGT. They were then cloned into the HpaI/XhoI sites of a pSico vector62, named pSico-miR-29c. Viral particles were produced by calcium phosphate–mediated transfection into 293T cells as described63. Lentiviral supernatants were collected 48 hours after transfection, passed through a 0.22-μm filter, and used directly to infect MLE 12 cells. GFP-positive cells were sorted 2-3 days after infection and resulted in MLE 12-SicoGFP and MLE 12-miR-29cGFP cells. Recombinant adenoviral stocks expressing Cre recombinase were purchased from the Gene Transfer Vector Core facility of University of Iowa College of Medicine (Iowa City, IA). Infections of GFP-positive cells were performed by using 100 plaque-forming units of virus per cell. Four days after adenovirus infections, the GPF-negative cells were sorted from GFPpos infected with adeno-Cre as Mlesico and Mle29c cells.

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Next generation sequencing of thymic dendritic cells after damage

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