In atherosclerotic lesions, macrophages are exposed to CSFs and various microenvironmental cues, which ultimately drive their polarization state. We studied the expression of different CSFs in artery specimen and cultured vascular cells and assessed whether concurrent stimulation (CS) of monocytes with CSF1 and polarizing cytokines generated macrophages (CSM1 and CSM2) that were phenotypically and functionally different from classically polarized M1 and M2 macrophages. We also assessed the influence of acetylsalicylic acid (ASA) on the capacity of polarized macrophages to stimulate T‐cell proliferation. CSF1 was the most prominent CSF expressed in arteries and cultured vascular cells. M1 and CSM1 macrophages differed in CD86 and CD14 expression, which was up‐regulated respectively down‐regulated by LPS. M2 and CSM2 macrophages were phenotypically similar. Cyclooxygenase expression was different in CSM1 (COX‐1− and COX‐2+ after LPS stimulation) and CSM2 (COX‐1+ and COX‐2−) macrophages. TNFα production was more pronounced in CSM1 macrophages, whereas IL‐10 was produced at higher levels by CSM2 macrophages. Proliferation of allogeneic T cells was strongly supported by CSM2, but not by CSM1 polarized macrophages. Although ASA did not affect anti‐CD3/CD28‐mediated proliferation, it significantly reduced CSM2 and CSM1‐mediated T‐cell proliferation. Supernatants of LPS‐stimulated CSM2 but not of CSM1 macrophages could overcome the inhibition by ASA. Hence, we demonstrate that CSM1 and CSM2 macrophages are phenotypically and to some extent functionally distinct from classically polarized M1 and M2 macrophages. CSM2 macrophages produce a COX‐1‐dependent soluble factor that supports T‐cell proliferation, the identity hereof is still elusive and warrants further studies.

Evidence suggests that the anti‐inflammatory nucleoside adenosine can shape immune responses by shifting the regulatory (Treg)/helper (Th17) T‐cell balance in favour of Treg. Since this observation is based on in vivo and in vitro studies mostly confined to murine models, we comprehensively analysed effects of adenosine on human T‐cells. Proliferation, phenotype and cytokine production of stimulated T‐cells were assessed by flow cytometry, multiplex assay and ELISA, gene expression profiling was determined by microarray. We found that the pan‐adenosine agonist 5′‐N‐ethylcarboxamidoadenosine (NECA) skews human CD3+ T‐cell responses towards non‐inflammatory Th17 cells. Addition of NECA during T‐cell activation increased the development of IL‐17+ cells with a CD4+ RORγt+ phenotype and enhanced CD161 and CD196 surface expression. Remarkably, these Th17 cells displayed non‐inflammatory cytokine and gene expression profiles including reduced Th1/Th17 transdifferentiation, a stem cell‐like molecular signature and induced surface expression of the adenosine‐producing ectoenzymes CD39 and CD73. Thus, T‐cells cultured under Th17‐inducing conditions together with NECA were capable of suppressing responder T‐cells. Finally, genome‐wide gene expression profiling revealed metabolic quiescence previously associated with non‐pathogenic Th17 cells in response to adenosine signalling. Our data suggest that adenosine induces non‐inflammatory Th17 cells in human T‐cell differentiation, potentially through regulation of metabolic pathways.