The Impact of TET2-Deficiency on Neutrophil Cell Signalling and Retrotransposon Expression
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Authors
Sadh, Sanathan
Date
Type
thesis
Language
eng
Keyword
CHIP , Clonal Hematopoiesis , Neutrophils , TET2 , Retrotransposons
Alternative Title
Abstract
Background: Clonal hematopoiesis of indeterminate potential (CHIP) promotes a pre-leukemic phenotype that can progress to more severe hematological malignancies such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). One of the most common CHIP drivers is mutant TET2, a gene which encodes an epigenetic regulator of the genome. Neutrophils have the second highest mutation burden in CHIP and patients with CHIP display increased susceptibility to infection. We hypothesized that Tet2-inactivation alters intracellular neutrophil signalling, which may then have the effect of inhibiting proper neutrophil function.
Methods: RNA-seq data from previously isolated murine neutrophil RNA was analyzed for differential expression. A pipeline was developed to measure the expression of retrotransposon elements (RTEs) within RNA-seq data. Additionally, a murine cell line with Tet2-/- and WT neutrophil variants was established. qRT-PCR was conducted to explore the expression of Interferon stimulated genes (ISGs) and ERVs in the Tet2-deficient neutrophils. Subsequently, intracellular flow cytometry was conducted to examine the expression of Ifitm1, an ISG of interest, and the RTE-product accumulation. Finally, an analysis of public CHIP datasets was conducted to explore whether universal patterns of modulated RTE expression were associated with CHIP.
Results: Tet2-deficient primary murine neutrophils displayed increased expression of ISGs and Interferon pathways. The expression of RTEs in Tet2-deficient neutrophils was varied; there was a focal regulation of some RTEs and a downregulation of others. Similar results were observed in the ER-Hoxb8 cell line-derived neutrophils. Flow cytometric analysis revealed that the expression of Ifitm1 was upregulated at the protein level. Subsequent intracellular flow cytometric analysis suggested a slight increase in the accumulation of RTE products in the cytoplasm of Tet2-deficient neutrophils. However, these results were not clear enough to be conclusive. An analysis of public CHIP datasets revealed that RTE expression varied based on cell type and mutation type.
Discussion: There has been some evidence to suggest that neutrophil function is compromised in Tet2-deficient patients. However, the mechanisms that would be responsible for this change are not understood. This study suggests that improper neutrophil function may be driven through the reactivation of RTE elements and subsequent sustained activation of the interferon response.
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Intellectual Property Guidelines at Queen's University
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This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.