October 2017 | Volume 8 | Article 1253 1 ORIGINAL RESEARCH published: 03 October 2017 doi: 10.3389/fmmu.2017.01253 Frontiers in Immunology | www.frontiersin.org Edited by: Uday Kishore, Brunel University London, United Kingdom Reviewed by: Kushagra Bansal, Harvard Medical School, United States Paola Italiani, Consiglio Nazionale Delle Ricerche (CNR), Italy *Correspondence: Humam Kadara hk94@aub.edu.lb; Georges M. Bahr georges.bahr@balamand.edu.lb † These authors have contributed equally to this work. Specialty section: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology Received: 28 April 2017 Accepted: 20 September 2017 Published: 03 October 2017 Citation: Bazzi S, El-Darzi E, McDowell T, Modjtahedi H, Mudan S, Achkar M, Akle C, Kadara H and Bahr GM (2017) Defning Genome-Wide Expression and Phenotypic Contextual Cues in Macrophages Generated by Granulocyte/ Macrophage Colony-Stimulating Factor, Macrophage Colony-Stimulating Factor, and Heat-Killed Mycobacteria. Front. Immunol. 8:1253. doi: 10.3389/fmmu.2017.01253 Defining Genome-Wide Expression and Phenotypic Contextual Cues in Macrophages Generated by Granulocyte/Macrophage Colony-Stimulating Factor, Macrophage Colony-Stimulating Factor, and Heat-Killed Mycobacteria Samer Bazzi 1,2 , Emale El-Darzi 3 , Tina McDowell 4 , Helmout Modjtahedi 1 , Satvinder Mudan 5 , Marcel Achkar 6 , Charles Akle 7 , Humam Kadara 8 * † and Georges M. Bahr 3 * † 1 Faculty of Science, Engineering and Computing, School of Life Sciences, Kingston University, Kingston upon Thames, United Kingdom, 2 Faculty of Sciences, University of Balamand, Al Kurah, Lebanon, 3 Faculty of Medicine and Medical Sciences, University of Balamand, Al Kurah, Lebanon, 4 Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States, 5 St George’s University of London, Imperial College, London and The Royal Marsden Hospital, London, United Kingdom, 6 Clinical Laboratory, Nini Hospital, Tripoli, Lebanon, 7 Immodulon Therapeutics Ltd., Uxbridge, United Kingdom, 8 Faculty of Medicine, Department of Biochemistry and Molecular Genetics, American University of Beirut, Beirut, Lebanon Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecu- lar underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefned. To fll this void, we sought to perform immunophenotyping, chemokine/cytokine release analysis and genome-wide characterization of monocyte- derived macrophages (MDM) in which monocytes were originally isolated from healthy donors and differentiated by HK M. obuense (Mob-MDM) relative to macrophage colony-stimulating factor (M-MDM) and granulocyte/macrophage colony-stimulating factor (GM-MDM). Immunophenotyping and cytokine release analysis revealed down- regulated surface expression of CD36, decreased spontaneous release of CCL2 and increased spontaneous secretion of CCL5, CXCL8/IL-8, IL-6, and TNF-α in Mob-MDM relative to M-MDM and GM-MDM. Analysis of cytostatic activity showed that Mob-MDM exhibited similar growth inhibitory effects on immortalized and malignant epithelial cells compared with GM-MDM but at an elevated rate relative to M-MDM. To understand global cues in Mob-MDM, we performed comparative RNA-sequencing (RNA-Seq) analysis of Mob-MDM relative to GM-MDM and M-MDM (n = 4 donors). Clustering analysis underscored expression profles (n = 256) that were signifcantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/ cytokines and their receptors, enzymes and transcriptions factors. Topological func- tional analysis of these profles identifed pathways and gene sets linked to Mob-MDM