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Identification of the networks that regulate human monocytic myeloid-derived suppressor cell differentiation into inflammatory macrophages

ABSTRACT: Background: Monocytic myeloid-derived suppressor cells (mMDSC) support immune evasion of tumors by blocking tumoricidal T and NK cell response. Efforts to reverse mMDSC-mediated immunosuppression identified that the TLR7/8 agonist R848 induces their maturation into tumoricidal macrophages Purpose: The factors that determine whether human mMDSC differentiate into MACinflam or MACsuppress are unclear. To investigate this issue, the role of cytokines and genes activated following R848 treatment was examined. Methods: After 4 hour stimulation, cells from stimulated mMDSC were stored in RNA protect (Qiagen, Frederick, MD). Total RNA was isolated using the RNeasy micro kit (Qiagen) and RNA quality was assessed using an Agilent 2200 TapeStation. mRNA libraries were generated using the Smart-Seq ultra-low input kit (Clontech) and sequenced using a HiSeq2500 sequencer using IlluminaTruSeq v4 chemistry with 125bp paired-end reads. Sequences were aligned to the human (hg19) reference genome. Genes that were differentially expressed compared to untreated samples were identified using CLC genomics workbench (version 10). Results: TNFa combined with IL-6 effectively supports the differentiation of mMDSC into tumoricidal macrophage by activating expression of conserved set of genes. Mechanistically, transcription factors NF-KB and STAT4 emerge as important regulators and potential targets to modify mMDSC behavior.

ORGANISM(S): Homo sapiens

PROVIDER: GSE105142 | GEO | 2017/10/19

SECONDARY ACCESSION(S): PRJNA414921

REPOSITORIES: GEO

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Project description:Purpose: Pulmonary tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has long been associated with the development of lung adenocarcinoma (LUAD) and sarcoidosis. These chronic lung diseases share histopathological similarities that challenge differential diagnosis. Methods: Human lung tissues were used for total RNA extraction with RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA purity was assessed using a Nanodrop ND-2000 (Thermo Scientific), and each RNA sample had an A260:A280 ratio above 1.8 and an A260:A230 ratio above 2.0. RNA integrity was evaluated using the Agilent 2200 TapeStation (Agilent Technologies), and each sample had the RNA Integrity Number (RIN) above 7.0. The ribosomal RNAs (rRNAs) were removed using Ribo-Zero rRNA Removal Kits (Illumina). RNA libraries were then constructed by NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) and evaluated using the Agilent 2200 TapeStation and Qubit® 2.0 (Life Technologies). Sequencing was performed by RiboBio Co., Ltd. on an Illumina HiSeq 3000 machine with paired-end 150-bp reads. The adaptors and low quality bases assessed using FASTQC 0.11.3 were trimmed by Trimmomatic 0.39 using the following options: TRAILING:20, MINLEN:235 and CROP:235. Trimmed reads were then aligned to the ensembl 79 (GRCh38.p2) reference genome using STAR 2.4.2a. FeatureCounts 1.6.2 was subsequently employed to convert aligned short reads into read counts for each sample. Genes with less than ten counts in two or more samples were removed. The data were then analyzed using R 3.4.4 and DEseq2 1.18.1. Differentially expressed genes of each disease group were identified using Wald statistics test, with fold-change > 2 and Benjamini–Hochberg (BH)-adjusted P < 0.1 as compared to NC group. Results: These findings provide novel pathogenic links and molecular markers for better understanding and differential diagnosis of pulmonary TB, LUAD and sarcoidosis.

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Identification of the networks that regulate human monocytic myeloid-derived suppressor cell differentiation into inflammatory macrophages

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