Multi-omics approaches reveal TNF-mediated inhibition of tissue reparative macrophages is via a gene-selective transcriptional mechanism (bulk RNA-Seq)

Anti-TNF therapies are a core anti-inflammatory approach for chronic diseases such as rheumatoid arthritis and Crohn's Disease. Previously, we and others found that TNF blocks the emergence and function of alternatively-activated or M2 macrophages involved in wound healing and tissue-reparative functions. Conceivably, anti-TNF drugs could mediate their protective effects in part by an altered balanced of macrophage activity. To understand the mechanistic basis of how TNF regulates tissue-reparative macrophages we used RNAseq, scRNAseq, ATACseq, time-resolved phospho-proteomics, gene-specific approaches, metabolic analysis and signaling pathway deconvolution. Our findings reveal that TNF controls tissue-reparative macrophage gene expression in a highly gene-specific way dependent on JNK signaling. We uncover principles of the selectively inhibition by TNF via the type 1 TNF receptor on specific populations of alternative activated macrophages. Overall design: Methods: BMDMs mRNA profiles of 8 weeks old wild-type (WT) mice were generated, prestimulated with DMSO or JNK-inhibitors and stimulated with IL4/IL13, IL4/IL13+ TNF or TNF, deep sequenced, in triplicate, using Illumina NovaSeq 6000 platform. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9). Conclusions: Our study represents the first detailed analysis of IL4/IL13 or IL4/IL13+TNF stimulated BMDMs in the presence of JNK-inhibitors, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. mRNA profiles of wild-type BMDMs prestimulated with DMSO, JNK-IN-8 or SP600125 for 1 hour and stimulated with IL4/IL13, IL4/IL13 + TNF or TNF for 6 hours