DOI: 10.5061/dryad.ksn02v7fn
title: An inherited mtDNA mutation remodels inflammatory cytokine responses in macrophages and in vivo

description: Impaired mitochondrial bioenergetics in macrophages can drive
 hyperinflammatory cytokine production, but whether this is caused by
 inherited mtDNA mutations is unknown. Here, we address this important
 question using a multi-omic approach that integrates super-resolution
 imaging and metabolic analyses to profile macrophages from a mouse model
 of mitochondrial disease caused by a heteroplasmic mutation
 (m.5019A&gt;G) in the mitochondrial tRNA for alanine. These
 m.5019A&gt;G macrophages exhibit defects in respiratory chain
 complexes and oxidative phosphorylation (OxPhos) due to decreased
 intra-mitochondrial translation. To adapt to this metabolic stress,
 mitochondrial fusion, reductive glutamine metabolism, and aerobic
 glycolysis are all increased. Upon inflammatory activation, type I
 interferon (IFN-I) release is enhanced, while the production of
 pro-inflammatory cytokines and oxylipins are restrained in
 m.5019A&gt;G macrophages. Finally, an in vivo endotoxemia model using
 m.5019A&gt;G mice reveal elevated IFN-I levels and sickness behaviour.
 In conclusion, our study identifies an unexpected imbalance in innate
 immune signalling in response to a pathogenic mtDNA mutation, with
 important implications for the progression of pathology in patients with
 mtDNA diseases. Data from this study that is included in this Dryad
 submission is as follows: 1. Label-free proteomic analysis of
 non-stimulated (non-stim) or lipopolysacchride (LPS)-stimulated (6 h) WT
 and m.5019A&gt;G murine bone marrow-derived macrophages (BMDMs). Five
 to seven biological replicates per condition. 2. RNA sequencing of
 non-stim or LPS-stimulated (1 h) WT and m.5019A&gt;G murine bone
 marrow-derived macrophages (BMDMs). Three biological replicates per
 condition.
description: Proteomic analysis Cell pellets were lysed,
 reduced and alkylated in 50 µl of 6 M Gu-HCl, 200 mM Tris-HCl pH 8.5, 10
 mM TCEP, 15 mM chloroacetamide by probe sonication and heating to 95ºC for
 5 mins. Protein concentration was measured by a Bradford assay and
 initially digested with LysC (Wako) with an enzyme to substrate ratio of
 1/200 for 4 h at 37 ºC. Subsequently, the samples were diluted 10-fold
 with water and digested with porcine trypsin (Promega) at 37ºC overnight.
 Samples were acidified to 1% TFA, cleared by centrifugation (16,000 g at
 RT) and approximately 20 µg of the sample was desalted using a Stage-tip.
 Eluted peptides were lyophilized, resuspended in 0.1% TFA/water and the
 peptide concentration was measured by A280 on a nanodrop instrument
 (Thermo). The sample was diluted to 2 µg/ 5 µl for subsequent
 analysis. The tryptic peptides were analyzed on a
 Fusion Lumos mass spectrometer connected to an Ultimate Ultra3000
 chromatography system (both Thermo Scientific, Germany) incorporating an
 autosampler. 2 µg of de-salted peptides were loaded onto a 50 cm emitter
 packed with 1.9 µm ReproSil-Pur 200 C18-AQ (Dr Maisch, Germany) using a
 RSLC-nano uHPLC systems connected to a Fusion Lumos mass spectrometer
 (both Thermo, UK). Peptides were separated by a 140 min linear gradient
 from 5% to 30% acetonitrile, 0.5% acetic acid. The mass spectrometer was
 operated in DIA mode, acquiring a MS 350-1650 Da at 120k resolution
 followed by MS/MS on 45 windows with 0.5 Da overlap (200-2000 Da) at 30k
 with a NCE setting of 27. Raw files were analysed and
 quantified by searching against the Uniprot <em>Mus
 Musculus</em> data base using DIA-NN 1.8 (https://github.com/vdemichev/DiaNN). Library-free search was selected, and the precursor ion spectra were generated from the FASTA file using the deep learning option. Default settings were used throughout apart from using “Robust LC (high precision)”. In brief, Carbamidomethylation was specified as fixed modification while acetylation of protein N-termini was specified as variable. Peptide length was set to minimum 7 amino acids, precursor FDR was set to 1%.  Subsequently, missing values were replaced by a normal distribution (1.8 π shifted with a distribution of 0.3 π) in order to allow the following statistical analysis.  Protein-wise linear models combined with empirical Bayes statistics are used for the differential expression analyses. We use the Bioconductor package limma to carry out the analysis as previously described<sup>67</sup>. Heatmaps were generated using Morpheus software from the Broad Institute. RNA sequencing RNA isolation was carried using RNeasy^® ^Plus kit (74136, Qiagen) following manufacturer’s suggestions and eluted RNA was purified using RNA Clean & Concentrator Kits (Zymo Research). RNA-seq samples libraries were prepared by Cambridge Genomic Services (CGS) using TruSeq Stranded mRNA (Illumina) following the manufacturer’s description. For the sequencing, the NextSeq 75 cycle high output kit (Illumina) was used and samples spiked in with 1% PhiX. The samples were run using NextSeq 500 sequencer (Illumina). Differential Gene Expression Analysis was done using the counted reads and the R package edgeR version 3.26.5 (R version 3.6.1) for the pairwise comparisons.
description: # An inherited mtDNA mutation remodels inflammatory cytokine responses in
 macrophages and in vivo This README file was generated by DR ### DATA
 & FILE OVERVIEW File list:
 Results_gc_length_corrected_WT_NS__VS__5019_NS.csv;
 Results_gc_length_corrected_WT_LPS_1h__VS__5019_LPS_1h.csv;
 WT1_NS_S1_R1_001.fastq.gz; WT2_NS_S2_R1_001.fastq.gz;
 WT3_NS_S3_R1_001.fastq.gz; WT1_LPS_1h_S7_R1_001.fastq.gz;
 WT2_LPS_1h_S8_R1_001.fastq.gz; WT3_LPS_1h_S9_R1_001.fastq.gz;
 5019A_NS_S4_R1_001.fastq.gz; 5019B_NS_S5_R1_001.fastq.gz;
 5019C_NS_S6_R1_001.fastq.gz; 5019A_LPS_1h_S10_R1_001.fastq.gz;
 5019B_LPS_1h_S11_R1_001.fastq.gz; 5019C_LPS_1h_S12_R1_001.fastq.gz;
 DIA_proteomics_report.pg_matrix.tsv.xlsx Relationship between files, if
 important: Analysed RNA sequencing datasets (CPM) and the fastq files of
 raw reads for each sample; DIA proteomics (LFQ intensity) ###
 DATA-SPECIFIC INFORMATION
 **Results_gc_length_corrected_WT_NS__VS__5019_NS.csv** Total: Conditions:
 Number of conditions: 2 WT NS 5019 NS Number of samples/biological
 replicates: 6 3 3 Sample key: * WT1_NS: Wildtype non-stimulated * WT2_NS:
 Wildtype non-stimulated * WT3_NS: Wildtype non-stimulated * 5019A_NS: 5019
 non-stimulated * 5019B_NS: 5019 non-stimulated * 5019C_NS: 5019
 non-stimulated
 **Results_gc_length_corrected_WT_LPS_1h__VS__5019_LPS_1h.csv** Total:
 Conditions: Number of conditions: 2 WT LPS 1h 5019 LPS 1h Number of
 samples/biological replicates: 6 3 3 Sample key: * WT1_LPS_1h: Wildtype
 LPS-stimulated 1 hour * WT2_LPS_1h: Wildtype LPS-stimulated 1 hour *
 WT3_LPS_1h: Wildtype LPS-stimulated 1 hour * 5019A_LPS_1h: 5019
 LPS-stimulated 1 hour * 5019B_LPS_1h: 5019 LPS-stimulated 1 hour *
 5019C_LPS_1h: 5019 LPS-stimulated 1 hour Variable key: * GeneID: Ensembl
 gene ID * GeneName: Common gene name * logFC: log 2 fold change * logCPM:
 log 2 average abundance across samples (counts per million) * LR: log
 ratio statistic * PValue: P-value for the differential expression test. *
 FDR: The adjusted p-value for multiple testing
 **DIA_proteomics_report.pg_matrix.tsv.xlsx** Total: Conditions: Number of
 conditions: 4 WT NS 5019 NS WT LPS 6h 5019 LPS 24h Number of
 samples/biological replicates: 22 6 7 4 5 Sample key: * WT1_NS: Wildtype
 non-stimulated * WT2_NS: Wildtype non-stimulated * WT3_NS: Wildtype
 non-stimulated * WT4_NS: Wildtype non-stimulated * WT5_NS: Wildtype
 non-stimulated * WT6_NS: Wildtype non-stimulated * WT1_LPS: Wildtype
 LPS-stimulated 6 hours * WT2_LPS: Wildtype LPS-stimulated 6 hours *
 WT3_LPS: Wildtype LPS-stimulated 6 hours * WT4_LPS: Wildtype
 LPS-stimulated 6 hours * 5019A_NS: 5019 non-stimulated * 5019B_NS: 5019
 non-stimulated * 5019C_NS: 5019 non-stimulated * 5019D_NS: 5019
 non-stimulated * 5019E_NS: 5019 non-stimulated * 5019F_NS: 5019
 non-stimulated * 5019G_NS: 5019 non-stimulated * 5019A_LPS: 5019
 LPS-stimulated 6 hours * 5019B_LPS: 5019 LPS-stimulated 6 hours *
 5019C_LPS: 5019 LPS-stimulated 6 hours * 5019D_LPS: 5019 LPS-stimulated 6
 hours * 5019E_LPS: 5019 LPS-stimulated 6 hours Variables key: *
 Protein.Group: Codes of each identified protein from Uniprot database *
 Protein.Ids: Codes of each identified protein from Uniprot database *
 Protein.Names: Codes of each identified protein from Uniprot database *
 Genes: Common gene name of identified proteins *
 First.Protein.Description: Full name length of protein