TLR7/8 stress response drives histiocytosis in SLC29A3 disorders
Data files
Jun 13, 2023 version files 53.07 GB
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CombineAlignments.IonXpress_025.fastq
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CombineAlignments.IonXpress_026.fastq
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CombineAlignments.IonXpress_027.fastq
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CombineAlignments.IonXpress_029.fastq
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CombineAlignments.IonXpress_031.fastq
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CombineAlignments.IonXpress_032.fastq
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CombineAlignments.IonXpress_033.fastq
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CombineAlignments.IonXpress_038.fastq
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CombineAlignments.IonXpress_039.fastq
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CombineAlignments.IonXpress_044.fastq
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CombineAlignments.IonXpress_045.fastq
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CombineAlignments.IonXpress_046.fastq
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CombineAlignments.IonXpress_WT_Cla_014.fastq
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CombineAlignments.IonXpress_WT_Cla_015.fastq
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CombineAlignments.IonXpress_WT_Pat_011.fastq
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CombineAlignments.IonXpress_WT_Pat_012.fastq
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CombineAlignments.IonXpress_WT_Pat_013.fastq
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README.md
Abstract
Loss-of-function mutations in the lysosomal nucleoside transporter SLC29A3 cause lysosomal nucleoside storage and histiocytosis: phagocyte accumulation in multiple organs. However, little is known about the mechanism by which lysosomal nucleoside storage drives histiocytosis. Herein, histiocytosis in Slc29a3–/– mice was shown to depend on Toll-like receptor 7 (TLR7), which senses a combination of nucleosides and oligoribonucleotides (ORNs). TLR7 increased phagocyte numbers by driving the proliferation of Ly6Chi immature monocytes and their maturation into Ly6Clow phagocytes in Slc29a3–/– mice. Downstream of TLR7, FcRγ and DAP10 were required for monocyte proliferation. Histiocytosis is accompanied by inflammation in SLC29A3 disorders. However, TLR7 in nucleoside-laden splenic macrophages failed to activate inflammatory responses. Enhanced production of pro-inflammatory cytokines was observed only after stimulation with ssRNAs, which would increase lysosomal ORNs. Patient-derived monocytes harboring the G208R SLC29A3 mutation showed enhanced survival and proliferation in a TLR8 antagonist-sensitive manner. These results demonstrated that TLR7/8 responses to lysosomal nucleoside stress drive SLC29A3 disorders.
Methods
Ly6Clow and Ly6Chigh splenic monocytes were obtained by FACS sorting from WT, Slc29a3−/−, and Slc29a3−/− Tlr7−/− mice. Total RNA was extracted using RNeasy Mini Kits (Qiagen, Germany), and the quality of RNA was evaluated using the Agilent Bioanalyzer device (Agilent Technologies, Santa Clara, CA). The samples with RIN (RNA Integrity Number) value of more than 7.3 were subjected to library preparation. RNA-seq libraries were prepared with 1 ng of total RNA using an Ion AmpliSeq Transcriptome Mouse Gene Expression kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The libraries were sequenced on Ion Proton using an Ion PI Hi-Q Sequencing 200 kit and Ion PI Chip v3 (Thermo Fisher Scientific). The FASTQ files were generated using AmpliSeqRNA plug-in v5.2.0.3 in the Torrent Suite Software v5.2.2 (Thermo Fisher Scientific) and analyzed by ROSALIND (https://rosalind.bio/, OnRamp Bioinformatics, USA), which is a cloud-based bioinformatics software. Raw reads were trimmed using Cutadapt, and quality scores were assessed using FastQC2. Reads were aligned to the Mus musculus genome build mm10 using the STAR aligner. Individual sample reads were quantified using HTseq and normalized via relative log expression (RLE) using the DESeq2 R library. DEseq2 was used to determine the fold changes and p-values. Genes showing more than a 1.5-fold change in expression (p < 0.05) were considered to be significantly altered. To interpret gene expression profiles, gene set enrichment analysis (GSEA) was performed using MSigDB hallmark gene sets to explore the pathways associated with SLC29A3 deficiency. Enriched pathways with FDR-adjusted p-values lower than 0.05 are shown in Fig. 3B.