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USP15 regulates type I interferon response in vivo and is required for pathogenesis of microbial and autoimmune neuroinflammation

ABSTRACT: Genes and pathways in which inactivation dampens tissue inflammation present new opportunities for understanding the pathogenesis of common human inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis and multiple sclerosis. We identified a mutation in the gene encoding the deubiquitination enzyme USP15 (Usp15L749R) that protected mice against both experimental cerebral malaria (ECM) induced by Plasmodium berghei and experimental autoimmune encephalomyelitis (EAE). Combining immunophenotyping and RNA sequencing in brain (ECM) and spinal cord (EAE) revealed that Usp15L749R-associated resistance to neuroinflammation was linked to dampened type I interferon responses in situ. In hematopoietic cells and in resident brain cells, USP15 was coexpressed with, and functionally acted together with the E3 ubiquitin ligase TRIM25 to positively regulate type I interferon responses and to promote pathogenesis during neuroinflammation. The USP15-TRIM25 dyad might be a potential target for intervention in acute or chronic states of neuroinflammation.

ORGANISM(S): Mus musculus

PROVIDER: GSE79702 | GEO | 2016/10/10

SECONDARY ACCESSION(S): PRJNA316741

REPOSITORIES: GEO

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Project description:Purpose: the goal of this study is to investigate the consequences of ubiquitin specific protease 15 (USP15) deletion on gene expression in mouse LSK hematopoietic progenitors. Methods: mRNA profiles of 8 weeks-old wild-type (WT) and ubiquitin specific protease 15 knockout (Usp15−/−) mice were generated by deep sequencing using Illumina Hiseq2500. The sequence reads that passed quality filters Alignments of the sequence reads that passed quality filters were performed using TopHat2.1, Genome build 38 and Ensembl gtf version 77 and genecounts have been generated using Itreecount. https://github.com/NKI-GCF/itreecount Results: We assigned about 30 million reads per sample uniquely to a gene of the mouse reference genome (mm10) We identified 21,219 genes in the LSKs of WT and Usp15−/− mice using TopHat2.1 in combination with Itreecount. https://github.com/NKI-GCF/itreecount. Distinct LSK-specific expressed genes (such as Eng, Tek, the MlI receptor and the Kit receptor) are identified. Comparison of normalized gene expression data for Usp15-/- versus WT LSK confirmed the loss of Usp15. Apart from Usp15, almost no other significantly deregurated genes were detected using a treshold of fold change ≥1.5 and p value <0.05, suggesting the maintenance of an overall stable identity of the cellular compartment in Usp15-/- mice. Conclusions: Our results represent the first detailed analyis of the consequences of USP15 deletion on gene expression in hematopoietic populations such as LSKs progenitors by genome wide expression profiling in WT and Usp15-/- mice. RNAseq of two freshly isolated biological replicas of sorted LSKs from 8 weeks old Usp15-/- animals confirmed the loss of Usp15, while showing almost no significant other up or down regulated genes among the 21,219 genes identified in Usp15-/- LSKs. We conclude that young adult hematopoietic stem and progenitor cells (LSKs) perpetuated a stable gene expression program regardless of the homozygous deletion of USP15.

Project description:Purpose: the goal of this study is to investigate the consequences of ubiquitin specific protease 15 (USP15) knockdown on gene expression in KBM7 and K562 leukemia cells. Methods: KBM7 or K562 cells were transfected with USP15 ( siGENOME Human USP15 (9958) siRNA-SMART pool M-006066-01) or control (Ctrl) siRNAs (siGENOME Non-Targeting siRNA Control Pool#2 D-001206-14-05, Dharmacon). Cells were transfected using Lipofectamine RNAiMAX Reagent from Life Technologies following the manufacturer’s instructions and assayed at 72 hrs after transfection in Western blotting, RNA-seq and qRT-PCR. Western blotting confirmed knokdown of USP15 protein. mRNA profiles were generated by deep sequencing using Illumina Hiseq2500. Alignments of the sequence reads that passed quality filters were performed using TopHat2.1, Genome build 38 and Ensembl gtf version 77 and genecounts have been generated using Itreecount. https://github.com/NKI-GCF/itreecount. qRT–PCR validation was performed using SYBR Green assays.The amount of target, normalized to an endogenous reference (HPRT), was calculated by 2-DDCT. Results: We assigned about 30 million reads per sample uniquely to a gene of the human reference genome (hg38). We identified 26,227 genes in KBM7 and 28,329 genes in K562 cells using TopHat2.1 in combination with Itreecount (https://github.com/NKI-GCF/itreecount) workflow. Comparison of normalized gene expression data resulted in a total of 657 and 330 differential expressed genes between USP15 KD vs Ctrl in KBM7 and K562 cells (in two separate experiments), respectively, with a treshold of logFC_C1 ≥1. and adj.PVal_C1 value <0.05. Downregulation of USP15 was confirmed by Western blotting and by qRT-PCR. Ingenuity pathway analysis of USP15-dependent genes in the KBM7 and K562 datasets (cut-off logFC > 1; adjusted P value <0.05) uncovered activation of inflammation-related pathways, which involve JAK/STAT and PI3K signal transduction. In K562, we also measured significant down-modulation of TGF-beta signaling. Expression of a set of 17 genes was assessed by RT-qPCR in three independent experiments per cell line and altered expression was validated. Overlap between Top 50 significantly enriched Ingenuity Upstream Regulators in each cell line RNAseq datasets was found. The Upstream Regulators were calculated based on differential gene expression between USP15 and control siRNAs. The nine overlapping terms include: Ifnar, IRF3, STAT1, Interferon alpha, IFNG, CD40LG, IFNB1, TNF and poly rI:rC-RNA. Conclusions: Our results represent the first detailed analyis of the consequences of USP15 depletion on gene expression in malignant hematopoietic populations such as KBM7 and K562 chronic myeloid leukemia cells by genome wide expression profiling in USP15 KD vs Ctrl cells. We confirmed downregulation of USP15 and validated altered expression of a set of 17 genes by qRT-PCR. Ingenuity pathway analysis identified 657 and 330 differentially regulated genes in KBM7 and K562, respectively. The changes in gene expression, though not extensive, indicate that RNAi of USP15 led to activation of inflammation-related pathways, which involve JAK/STAT and PI3K signal transduction in both cell lines. In K562, we also measured significant down-modulation of TGF-beta signaling, which is required for USP15 pro-oncogenic role in human glioma cells (Eichhorn et al., 2012). Regulation of inflammatory signals and TGF-beta are relevant both in normal HSC and in malignant development (Blank and Karlsson, 2015).

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USP15 regulates type I interferon response in vivo and is required for pathogenesis of microbial and autoimmune neuroinflammation

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