Abstract
Heat-killed (HK) Mycobacterium obuense (NCTC13365) is currently being evaluated in the clinic as an immunotherapeutic agent for cancer treatment. Yet, the molecular underpinnings underlying immunomodulatory properties of HK M. obuense are still largely undefined. To fill 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 downregulated 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 profiles (n = 256) that were significantly modulated in Mob-MDM versus both M-MDM and GM-MDM including, among others, chemokines/cytokines and their receptors, enzymes and transcriptions factors. Topological functional analysis of these profiles identified pathways and gene sets linked to Mob-MDM phenotype including nitric oxide production, acute phase response signaling and microbe recognition pathways as well as signaling cues mediated by the proinflammatory cytokine, interferon-gamma, and the intracellular pattern recognition receptor, nucleotide-binding oligomerization domain-containing protein 2. Taken together, our study highlights molecular immune phenotypes and global signaling cues in Mob-MDM that may underlie immunomodulatory properties of HK M. obuense. Such properties could be of valuable use in immunotherapy approaches such as adoptive cell therapy against cancer.
Highlights
Macrophages (Mφ) are key members of the mononuclear phagocytic system with fundamental roles in the development, repair and homeostasis of tissues [1, 2]
Our analysis revealed that M-monocyte-derived macrophages (MDM) displayed higher surface expression levels (% and mean fluorescence intensity (MFI) of positive cells) of the M2-like markers CD14, CD36, CD163, and CD195, relative to GM-MDM and Mob-MDM, which both exhibited comparable levels of CD195 (Figures 2A,B)
We noted that CD206 expression was significantly (P < 0.05) higher in GM-MDM compared to M-MDM and Mob-MDM (Figures 2A,B)
Summary
Macrophages (Mφ) are key members of the mononuclear phagocytic system with fundamental roles in the development, repair and homeostasis of tissues [1, 2]. Tissue Mφ are sustained through either local proliferation of cells or the recruitment of blood monocytes which in turn differentiate into Mφ [3, 4] Based on their activation/polarization state, Mφ are broadly classified into M1 and M2 types which represent the polar states of a functional continuum [5]. Despite the fact that GM-CSF was originally defined as a hematopoietic growth factor, several reports have demonstrated that monocytes differentiated into Mφ in the presence of GM-CSF (unpolarized GM-MDM) spontaneously release inflammatory cytokines/chemokines, whereas unpolarized M-MDM either do not release or release some of these molecules at significantly lower levels [16, 22, 23]. The role of GM-CSF has been well described in several inflammatory diseases, including rheumatoid arthritis and experimental autoimmune encephalomyelitis [24, 25]
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