Mesenchymal Stem Cells (MSCs) are effective therapeutic agents enhancing the repair of injured tissues mostly through their paracrine activity. Increasing evidences show that besides the secretion of soluble molecules, the release of extracellular vesicles (EVs) represents an alternative mechanism adopted by MSCs. Since macrophages are essential contributors toward the resolution of inflammation, which has emerged as a finely orchestrated process, the aim of the present study was to carry out a detailed characterization of EVs released by human adipose derived‐MSCs to investigate their involvement as modulators of MSC anti‐inflammatory effects inducing macrophage polarization. The EV‐isolation method was based on repeated ultracentrifugations of the medium conditioned by MSC exposed to normoxic or hypoxic conditions (EVNormo and EVHypo). Both types of EVs were efficiently internalized by responding bone marrow‐derived macrophages, eliciting their switch from a M1 to a M2 phenotype. In vivo, following cardiotoxin‐induced skeletal muscle damage, EVNormo and EVHypo interacted with macrophages recruited during the initial inflammatory response. In injured and EV‐treated muscles, a downregulation of IL6 and the early marker of innate and classical activation Nos2 were concurrent to a significant upregulation of Arg1 and Ym1, late markers of alternative activation, as well as an increased percentage of infiltrating CD206pos cells. These effects, accompanied by an accelerated expression of the myogenic markers Pax7, MyoD, and eMyhc, were even greater following EVHypo administration. Collectively, these data indicate that MSC‐EVs possess effective anti‐inflammatory properties, making them potential therapeutic agents more handy and safe than MSCs. stem cells translational medicine 2017 Stem Cells Translational Medicine 2017;6:1018–1028

SummaryPicolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-y-treated murine macrophages . We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation ofiNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN -'Y and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid-but not LPS-induced CAT activity by more than 70% . Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-y or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRB . Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-y activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression . These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Peripheral blood monocytes migrate to and accumulate in hypoxic areas of inflammatory and tumor lesions. To characterize the molecular bases underlying monocyte functions within a hypoxic microenvironment, we investigated the transcriptional profile induced by hypoxia in primary human monocytes using high-density oligonucleotide microarrays. Profound changes in the gene expression pattern were detected following 16 h exposure to 1% O2, with 536 and 677 sequences showing at least a 1.5-fold increase and decrease, respectively. Validation of this analysis was provided by quantitative RT-PCR confirmation of expression differences of selected genes. Among modulated genes, 74 were known hypoxia-responsive genes, whereas the majority were new genes whose responsiveness to hypoxia had not been previously described. The hypoxic transcriptome was characterized by the modulation of a significant cluster of genes with immunological relevance. These included scavenger receptors (CD163, STAB1, C1qR1, MSR1, MARCO, TLR7), immunoregulatory, costimulatory, and adhesion molecules (CD32, CD64, CD69, CD89, CMRF-35H, ITGB5, LAIR1, LIR9), chemokines/cytokines and receptors (CCL23, CCL15, CCL8, CCR1, CCR2, RDC1, IL-23A, IL-6ST). Furthermore, we provided conclusive evidence of hypoxic induction of CCL20, a chemoattractant for immature dendritic cells, activated/memory T lymphocytes, and naive B cells. CCL20 mRNA up-regulation was paralleled by increased protein expression and secretion. This study represents the first transcriptome analysis of hypoxic primary human monocytes, which provides novel insights into monocyte functional behavior within ischemic/hypoxic tissues. CCL20 up-regulation by hypoxia may constitute an important mechanism to promote recruitment of specific leukocyte subsets at pathological sites and may have implications for the pathogenesis of chronic inflammatory diseases.

Myeloid precursors can be grown in vitro in the presence of specific growth factors; however, their expansion is limited by a competing process of terminal differentiation. Proto-oncogenes seem to be involved in cellular proliferation and/or differentiation and may also play a role in the myelopoietic process. Murine myeloid precursors which are grown in vitro with growth factors respond with augmented self-renewal upon infection with recombinant retroviruses carrying the v-myc or v-src oncogenes, suggesting a synergism or complementation between some viral oncogenes (v-onc) and certain growth factors. We now show that the combination of two v-onc genes (raf and myc) induces the selective proliferation of monocytic cells from fresh murine bone marrow (BM) in the absence of a specific growth factor supplement. Depending on the culture conditions these cells can either differentiate and cease to proliferate or grow continuously, thus mimicking the alternative pathways that can be followed by committed BM stem cells in vivo.