Differential effects of GM-CSF and M-CSF on long-interval LPS tolerance in human macrophages

Macrophage tolerance to lipopolysaccharide (LPS) is characterized by reduced production of pro-inflammatory cytokines upon repeated stimulation. Impaired tolerance of circulating monocytes has been described in chronic inflammatory diseases, including atherosclerosis, systemic sclerosis and type 2 diabetes mellitus, which are associated with elevated levels of macrophage growth factors, cytokines and chemokines. This suggests that the inflammatory microenvironment may modulate macrophage tolerization.

The aim of this study was to evaluate the effects of GM-CSF, M-CSF and selected cytokines and chemokines on the development of macrophage tolerance to LPS. CD14 + monocytes were isolated from peripheral blood of healthy donors and cultured with growth factors and cytokines, and exposed to one or two consecutive LPS stimulations at different rest intervals. Cytokine secretion, surface marker expression and cellular morphology were analyzed.

GM-CSF–differentiated macrophages showed higher TNF secretion in response to LPS and a tendency toward higher IL-6 secretion, while retaining the ability to develop long-lasting tolerance. In contrast, M-CSF-derived macrophages exhibited only short-term tolerance and progressively regained responsiveness with longer rest intervals, accompanied by renewed cytokine and CCL2 production and cluster formation. Among mediators tested between LPS stimulations, IL-1β emerged as a potent tolerance-breaking self-amplified factor, while other factors promoted tolerization to varying degrees.

In conclusion, GM-CSF and M-CSF are associated with distinct temporal patterns of long-interval LPS tolerance in human macrophages. Under the tested in vitro conditions, GM-CSF was associated with maintenance of long-interval LPS tolerance, whereas M-CSF-differentiated macrophages progressively regained responsiveness as the interval between stimulations increased. Collectively, macrophage tolerization is a highly plastic process regulated by multiple soluble mediators present in the inflammatory microenvironment.

We thank Viktoria Alekseevna Sarkisova from the Engelhardt Institute of Molecular Biology, RAS, for providing antibodies. We also acknowledge the Center of Collective Usage of the Institute of Gene Biology. The experiments assessing the effects of growth factors on macrophage function were supported by the Russian Science Foundation (Grant 22-15-00273-П).

Experiments testing recombinant cytokines were supported by the Ministry of Science and Higher Education of the Russian Federation (Project # FGFU-2025-0006). These authors contributed equally: Yegor S. Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, 8 Baltiyskaya Street, Moscow, 125315, Russia Yegor S.