Laura M. Merlo Pich (1), Calin D. Popa (2,3), Leo A.B. Joosten 1,4), Mihai G. Netea (1,5)
Affiliation(s):
1. Department of Internal Medicine, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands.
2. Department of Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands
3. Department of Rheumatology, Sint Maartenskliniek, Nijmegen, the Netherlands
4. Department of Medical Genetics, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania
5. Department for Immunology and Metabolism, Life and Medical Sciences Institute (LIMES), University of Bonn, 53115 Bonn, Germany
Background: Colchicine, a millennia-old drug that inhibits microtubule assembly, has proven effective in treating IL-1b-driven diseases such as gout. Neutrophils are the main effector cell type in gout flares, and it is known that colchicine inhibits their capacity to secrete inflammatory mediators. Nonetheless, monocytes and differentiated macrophages are also relevant for the pathophysiology of gout and yet, the individual interaction of colchicine with these cell types is still not fully elucidated. A better understanding of the mechanisms and the extent of colchicine’s effect on primary human monocytes might therefore be crucial to inform the development of new therapeutic targets and potential alternative treatments for gout and related inflammatory conditions.
Objectives: To assess the in vitro effects of colchicine on primary peripheral blood mononuclear cells (PBMCs) and monocytes, stimulated with MSU crystals, as well as the effects of colchicine on macrophage differentiation.
Methodology: PBMCs were isolated from healthy volunteers (n=12) using Ficoll-Paque density gradient centrifugation. Monocytes were subsequently separated through hyper-osmotic density gradient centrifugation. The isolated cells were cultured with a 1-hour pre-incubation of colchicine (50nM-500nM), followed by a 24-hour stimulation with monosodium urate (MSU) crystals and lipopolysaccharide (LPS). Supernatants were collected for cytotoxicity assessment and cytokine quantification, while the cells were processed for RNA isolation and analyzed using a Seahorse metabolic assay. In parallel, monocytes were differentiated into macrophages by culturing them with either GM-CSF or M-CSF for six days in the presence or absence of colchicine (1nM-50nM). Macrophage differentiation and marker expression were assessed using flow cytometry.
Findings: The stimulation of monocytes with MSU crystals and LPS in presence of colchicine inhibits the release of IL-1β, IL-6, TNF and MCP-1. The mRNA expression of these cytokines is decreased in presence of colchicine, in a dose-dependent manner. On the other hand, when the same conditions are applied to PBMCs, the production of inflammatory mediators such as IL-1β, IL-6, TNF and IL-8 was not affected by colchicine treatment. In contrast, the production of the chemokine MCP-1 was strongly inhibited. Colchicine treatment in monocytes also reduced oxygen consumption and cellular glycolysis. Colchicine also affects monocyte to macrophage differentiation, in both GM-CSF and M-CSF differentiated cells. Colchicine treatment induced a higher expression of CD86 and a lower expression of CD206 and CD163 as well as CD14 and CD16.
Significance: Our findings reveal novel insights into colchicine's anti-inflammatory mechanism of action on monocytes and PBMCs during gout flares. By highlighting differential impacts on cytokine production, metabolism, and macrophage marker expression, we provide evidence for a new understanding of colchicine's therapeutic potential beyond current clinical applications.