Concurrent stimulation of monocytes with CSF1 and polarizing cytokines reveals phenotypic and functional differences with classical polarized macrophages

Abstract

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.

Lipopolysaccharide Tolerance in Human Primary Monocytes and Polarized Macrophages

“…We have recently reported that concurrent stimulation of monocytes with CSF-1 and IFNγ or IL-4 results in polarized macrophages (M (IFNγ) and M (IL-4) , respectively) that differ phenotypically and functionally from classically polarized macrophages (M1 and M2) [23]. We therefore assessed to what extent LPS tolerance can be induced in such polarized macrophages and if this was affected by DMI.…”

“…Human CD14 + cells were polarized to M (IFNγ) and M (IL-4) as previously described [23]. In brief, for M (IFNγ) and M (IL-4) polarization, CD14 + cells were concurrently stimulated for 6 days with 10 ng/mL of colony-stimulating factor 1 (CSF-1) and 100 ng/mL of IFN-γ or 10 ng/mL IL-4 (all from PeproTech, Hamburg, Germany, cat.no.300-25; 300-2; 200-04, respectively).…”

Macrophage plasticity: signaling pathways, tissue repair, and regeneration

“…At present, the classification of macrophages into M1 and M2 types has been widely adopted in scientific research. 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 Through this extreme classification, macrophages are divided according to the functional differences of proinflammatory bactericidal and anti‐inflammatory and anti‐inflammatory prorepair. 17 , 18 , 19 While this dichotomy provided a valuable framework for understanding macrophage heterogeneity, recent advances in single‐cell technologies and systems biology approaches have revealed a spectrum of activation states that extend beyond the M1/M2 paradigm.…”