DOI: 10.5061/dryad.j0zpc86kn
title: TLR7/8 stress response drives histiocytosis in SLC29A3 disorders

description: 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.
description: Ly6C<sup>low</sup> and
 Ly6C<sup>high</sup> splenic monocytes were obtained by FACS
 sorting from WT, <em>Slc29a3<sup>−/−</sup></em>,
 and <em>Slc29a3</em><sup><em>−/−</em>
 </sup><em>Tlr7<sup>−/−</sup></em> 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
 &lt; 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.