Fractional factorial design to investigate stromal cell regulation of macrophage plasticity

Abstract

Understanding the regulatory networks which control specific macrophage phenotypes is essential in identifying novel targets to correct macrophage mediated clinical disorders, often accompanied by inflammatory events. Since mesenchymal stromal cells (MSCs) have been shown to play key roles in regulating immune functions predominantly via a large number of secreted products, we used a fractional factorial approach to streamline experimental evaluation of MSC mediated inflammatory macrophage regulation. Our macrophage reprogramming metrics, human bone marrow MSC attenuation of macrophage pro-inflammatory M1 TNFα secretion and simultaneous enhanced expression of the M2 macrophage marker, CD206, were used as analysis endpoints. Objective evaluation of a panel of MSC secreted mediators indicated that PGE2 alone was sufficient in facilitating macrophage reprogramming, while IL4 only provided partial reprogramming. Inhibiting stromal cell PGE2 secretion with Indomethacin, reversed the macrophage reprogramming effect. PGE2 reprogramming was mediated through the EP4 receptor and indirectly through the CREB signaling pathway as GSK3 specific inhibitors induced M1 macrophages to express CD206. This reprogramming pathway functioned independently from the M1 suppression pathway, as neither CREB nor GSK3 inhibition reversed PGE2 TNF-α secretion attenuation. In conclusion, fractional factorial experimental design identified stromal derived PGE2 as the factor most important in facilitating macrophage reprogramming, albeit via two unique pathways.

Hypoxia impairs mesenchymal stromal cell-induced macrophage M1 to M2 transition

“…Primary macrophages were differentiated from monocytes isolated from human whole blood and treated with GM-CSF to acquire the M1 phenotype 13, 14, 16 . In general, macrophages secrete very little cytokine in the absence of a stimulus 6, 17 .…”

“…Conventionally, monocytes that migrate into the wound differentiate into the pro-inflammatory M1 macrophages due to the presence of interferon (IFN)-γ, LPS or GM-CSF 6, 13, 16 . These M1 macrophages are pro-inflammatory; they clear the wound from debris and pathogens and produce high levels of pro-inflammatory mediators such as TNF-α, IL-12, IL-23, and IL-6 and produce low levels of the anti-inflammatory IL-10 2, 3, 16 .…”

“…Primary peripheral blood-derived macrophages were isolated from peripheral blood as previously described 13 . Briefly, human whole blood was purchased from the Blood Center of New Jersey (East Orange, NJ) and mononuclear cells were isolated by density gradient centrifugation.…”

“…Then cells were washed with phosphate buffered saline (PBS) to remove non-adherent cells and cultured in fresh medium supplemented with 5ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF; R&D, Minneapolis, MN) for 7 days. CD14 + monocytes stimulated with GM-CSF produce a subset of macrophages expressing M1 phenotype 13, 14 . After 7 days in culture, cells were detached from the flasks using trypsin-EDTA (Life Technologies) and 1 × 10 6 cells/cryovial were frozen in 1 ml advanced RPMI 1640 medium with 40% v/v FBS, 1% pen-strep, 4mM L-glutamine, and 10% dimethyl sulfoxide (DMSO).…”

RNA Interference Screening Reveals Host CaMK4 as a Regulator of Cryptococcal Uptake and Pathogenesis

“…CREB activation is also involved in expression and signaling of the C-type lectin receptors Dectin-1 and CD206/mannose receptor (24,25). We observed a significant reduction in cells positive for TLR2, Dectin-1, and CD206 across multiple phagocytic cell types during infection in CaMK4 Ϫ/Ϫ mice compared to WT mice (Fig.…”