Colchicine Inhibits Chemokine Production in Monosodium Urate–Stimulated Innate Immune Cells

Abstract

Objective

Colchicine is a well‐established, widely available drug used to treat inflammatory diseases driven by the innate immune system, most notably gout. Although much research has focused on neutrophils, the first responders in a gout flare, monocytes and lymphocytes, also play critical roles in sustaining the inflammatory response. However, the impact of colchicine on these cell types remains poorly understood. The objective of this study was to assess the in vitro effects of colchicine on primary peripheral blood mononuclear cells (PBMCs) stimulated with monosodium urate (MSU) crystals.

Methods

PBMCs or monocytes from healthy volunteers were isolated and cultured with a one‐hour preincubation of colchicine, followed by a 24‐hour stimulation with MSU crystals and lipopolysaccharide. Supernatants were collected for cytokine quantification and chemotaxis assay, whereas cells were processed for RNA isolation, metabolic analysis, reactive oxygen species (ROS) activity, and phagocytosis.

Results

Colchicine reduced the secretion of several chemokines, notably MCP‐1, whereas no inhibitory effect on the release of proinflammatory cytokines such as interleukin‐1β (IL‐1β), IL‐6, and tumor necrosis factor (TNF) was observed. Colchicine also reduced MCP‐1 and TNF, but not IL‐1β and IL‐6, production in monocytes. Both MCP‐1 messenger RNA expression and intracellular protein concentrations were inhibited by colchicine, suggesting an effect at transcriptional level. Lower chemokine production is responsible for reduced chemoattraction of monocytes. Moreover, colchicine attenuated ROS production and phagocytic activity in monocytes, alongside suppressing glycolytic metabolism and oxidative phosphorylation.

Conclusion

Colchicine's anti‐inflammatory mechanisms in monocytes and PBMCs consist mainly of reduction of chemokine production and chemoattraction accompanied by changes in cellular metabolism and attenuated ROS production.

INTRODUCTION

Gout is the most common form of inflammatory arthritis in adults. Its incidence is steadily increasing over recent decades, rising by approximately 22.5% in the last 30 years, particularly impacting lower socioeconomic communities, ¹ underlining the yet unsatisfactory understanding of disease prevention, risk factors, and treatment. Gout has several risk factors, most important being hyperuricemia, which leads to the chronic deposition of monosodium urate (MSU) crystals in the synovium of peripheral joints. These deposits trigger recurrent, painful flares driven by local recruitment and activation of innate immune cells. ² If left untreated, ongoing inflammation and MSU crystal accumulation result in the formation of large tophi, ultimately causing joint deformation and destruction. Moreover, persistent hyperuricemia and MSU crystals can sustain systemic low‐grade inflammation, increasing the risk of major cardiovascular events and severely affecting patients’ quality of life. ³

The gout flare is initiated by resident macrophages and monocytes that require a two‐signal system to activate the assembly of the NLRP3 inflammasome to prevent unregulated activation and unwanted host damage. The first signal originates from engagement of the Toll‐like receptor 4 (TLR4) and TLR2 and activation of the NFκB pathway, leading to pro–interleukin‐1β (IL‐1β) synthesis. The second signal is composed by the phagocytosis of MSU crystals, which induces the assembly of the NLRP3 inflammasome, leading to activation of caspase‐1 that in turn cleaves pro–IL‐1β to its bioactive form. ⁴ IL‐1β signal then induces the release of other proinflammatory cytokines and chemokines (IL‐6, MCP1, TNFSF14, prostaglandins and reactive oxygen species [ROS]) and the recruitment of neutrophils and other immune cells, propagating the inflammation. ⁵

Colchicine is a widely available, inexpensive, orally administered alkaloid derived from the plant Colchicum autumnale. It is widely used in diseases characterized by inappropriate activation of innate immunity, such as gout, familial Mediterranean fever, Behçet disease, and cardiovascular disorders. ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ , ¹¹ Although colchicine exerts multiple effects, many of its mechanisms remain incompletely understood. Its primary target is intracellular tubulin monomers, preventing microtubule polymerization. ¹² Microtubules are essential components of cellular cytoskeleton, involved in cell migration, intracellular trafficking, secretion, and division. Disruption of tubulin has been shown to inhibit inflammasome assembly, resulting in reduced secretion of inflammatory cytokines. ⁴ Most studies on colchicine's anti‐inflammatory effects have focused on neutrophils because they are the first and most abundant immune cells recruited during gout flares. In neutrophils, colchicine inhibits key inflammatory processes including chemotaxis, extravasation, superoxide production, and the release of proinflammatory cytokines. ¹³ , ¹⁴ , ¹⁵ However, monocytes and lymphocytes also infiltrate joints during gout flares and contribute significantly to inflammation. Their activation can perpetuate a state of low‐grade systemic inflammation, increasing cardiovascular risk in patients with gout. ¹⁶ Despite the well‐documented systemic benefits of colchicine during and after gout flares, its mechanisms of action on monocytes and other peripheral blood mononuclear cells (PBMCs) remain poorly understood. This study therefore aims to investigate the in vitro effects of colchicine on PBMCs and monocytes, particularly under stimulation with MSU crystals and lipopolysaccharide (LPS), to deepen our understanding of its immunomodulatory role in gout.

MATERIALS AND METHODS

Human PBMC and monocyte isolation and culture

For in vitro studies, buffy coats from healthy adult male and female donors were obtained after written informed consent (Sanquin). PBMC isolation was performed by different density centrifugation over Ficoll‐Paque (GE Healthcare) and three consecutive washes with pyrogen‐free cold phosphate‐buffered saline (PBS). This was followed by monocyte isolation with hyperosmotic Percoll (Sigma) density gradient centrifugation and one wash with PBS. Cells obtained were resuspended in complete medium, defined as RPMI 1640, Dutch modified (Invitrogen), and supplemented with 5 mg/mL gentamicin (Centraform), 2 mM GlutaMAX (Gibco), and 1 mM pyruvate (Gibco). The PBMCs and monocytes were counted using a Sysmex XN‐450 hemocytometer and brought to 1 × 10⁶/mL RPMI. To ensure purity, Percoll‐isolated monocytes were left to adhere to polystyrene flat‐bottom plates (Corning) for one hour at 37°C and 5% CO₂ and then washed once with warm PBS. PBMC monocytes were cultured under standard cell culture conditions (37°C and 5% CO₂) with RPMI supplemented with 10% human pooled serum.

Viability assays (cytotoxicity)

CytoTox 96 Non‐Radioactive Cytotoxicity Assay (Promega) was used to assess cell viability through lactate dehydrogenase (LDH) measurements, following the manufacturer's instructions.

Cytokine measurement

Cytokine production was determined in supernatants using commercial enzyme‐linked immunosorbent assay (ELISA) kits for human IL‐1β, tumor necrosis factor (TNF), IL‐6, IL‐8, IL‐1Ra, and MCP‐1 following the manufacturer's instructions (R&D Systems). Intracellular cytokines were measured after cell lysis with Triton X‐100.

Reverse transcription quantitative polymerase chain reaction

Healthy donor monocytes were treated with different concentrations of colchicine and stimulated (as previously described) for four hours. They were then collected, and messenger RNA (mRNA) was immediately isolated with NucleoSpin RNA Midi kit for RNA purification (Macherey‐Nagel), following the manufacturer's instructions, and eluted in 20 μL of H₂O. The total RNA was reverse transcribed to complementary DNA (cDNA) using iScript cDNA Synthesis Kit (Bio‐Rad Laboratories), following manufacturer's instructions. Relative expression was determined using SYBR Green (ThermoFisher Scientific) on an Applied Biosystems StepOnePlus Real‐Time polymerase chain reaction system. For gene expression analysis, the relative amounts of mRNA were quantified using the ΔΔCt method. β‐2 Microglobulin was used as the internal control gene for data normalization. Primer pairs used are reported in Supplementary Table 1.

Olink proteomics

Supernatant protein concentrations of stimulated PBMCs were analyzed using the Olink Target 96 Inflammation panel, which consists of 96 unique proteins involved in inflammatory pathways (Supplementary Table 2). Processing and quality assessment of proteomics data were performed using the Olink NPX manager software. The data were transformed and normalized to Olink's NPX value (a relative protein quantification unit on a log2 scale, where a difference of 1 NPX equates to a doubling of protein concentration). Further downstream data processing was performed using R version 4.5 (2025).

Seahorse XF analysis

Percoll monocytes were seeded in quintuple in XF96 microplates (200,000 cells/well; Agilent Technologies) in RPMI 1640 medium supplemented with GlutaMAX. Cells were left to adhere for one hour and then washed with 200 μL of PBS and supplemented with RPMI + 10% human pooled serum. Cells were either left unstimulated or treated with LPS (10 ng/mL) and MSU (0.3 mg/mL) with or without 50 nM of colchicine for 24 hours at 37°C and 5%CO₂. The medium was changed to “assay medium” 45 to 60 minutes before running the assay, during which cells were incubated in ambient CO₂ concentration at 37°C. For mitochondrial stress testing, the assay medium (Agilent Seahorse XF, RPMI medium, pH 7.4) was supplemented with l‐glutamine (2 mM), d‐glucose (11 mM), and pyruvate (1 mM). The mitochondrial stress test was used to measure mitochondrial parameters. The inhibitors used were 1 μM oligomycin A (Sigma‐Aldrich), 1 μM Carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone (FCCP) (Sigma‐Aldrich), 2.5 μM rotenone (Sigma‐Aldrich), and 1.25 μM antimycin A (Sigma‐Aldrich). Oxygen consumption rate (OCR) measurements were used to calculate ATP‐linked respiration (basal OCR − OCR after oligomycin) and spare respiratory capacity (OCR after FCCP − basal OCR). Glycolytic stress tests were used to measure glycolytic parameters. The inhibitors used were 11 mM glucose (Sigma‐Aldrich), 1 μM oligomycin A (Sigma‐Aldrich), and 22 mM 2‐deoxy‐d‐glucose (Sigma‐Aldrich). Extracellular acidification rate (ECAR) measurements were used to calculate baseline glycolysis, glycolysis, maximal ECAR, and glycolytic reserve.

ROS assay

A luminol‐based ROS assay was conducted to quantify the ROS produced by activated monocytes after 24‐hour incubation in the presence or the absence of colchicine. Percoll‐isolated monocytes were plated in a white, flat‐bottom 96‐well plate, 1 × 10⁵ cells/well, and allowed to adhere for one hour. Each well was washed with warm PBS. Each condition was prepared in four technical replicates, including 0, 50, and 500 nM of colchicine. After 24 hours of incubation, the medium is changed, and 20 μL of luminol‐working solution is added. This includes luminol (Sigma, A8511) (0.177 mg/mL) and 0.5% bovine serum albumin (BSA) dissolved in Hank's balanced salt solution. Finally, 20 μL of opsonized zymosan (Sigma, Z4250) or MSU crystals is added to activate the cells. Immediately, the plate is read in the Biotek Synergy HT, detecting 425‐nm luminescence for one hour.

Phagocytosis assay

Monocytes were cultured in the presence of different concentrations of colchicine (0, 50, and 500 nM). After 24 hours, the cells were restimulated with Incucyte pHrodo Green Zymosan Bioparticles (Sartorius) (1 mg/mL) and incubated for 24 hours in the Incucyte S3 (Sartorius). Measurements were taken every 15 minutes in the first and last hour of stimulation and every one hour between these times. Fluorescence per cell count was calculated by dividing total green object integrated intensity (green calibration units (GCU) × μm²/image) by phase object count (per image).

Monocyte chemotaxis assay

Monocytes were isolated from fresh blood of three healthy donors, following informed consent and ethical approval by the Medical Ethical Committee Oost Nederland (NL84281.091.23). Monocyte purity at >95% was achieved through an initial isolation of PBMCs through different density centrifugation over Ficoll‐Paque, followed by Pan Monocyte Isolation (Miltenyi Biotec). The chemotaxis assay was conducted using the Incucyte S3 (Sartorius), including the Incucyte Clearview 96‐well Microplate for Chemotaxis. For this purpose, the insert of the plate was coated on both sides with fibronectin (5 μg/mL) + 0.1% BSA in Dulbecco's PBS for at least one hour. Then, 5,000 monocytes were added in the top insert and allowed to attach for 30 minutes. The chemoattractant used in this experiment were the supernatants of PBMCs (n = 6) stimulated with LPS + MSU without colchicine or with 50 or 500 nM of colchicine. The chemotaxis plate was incubated in the Incucyte for 24 hours, with a scan interval of once an hour. The results were analyzed using the Incucyte software for chemotaxis assays.

Statistical analyses

Unless otherwise indicated, all data are presented with the mean, and the error bars represent the SEM. For all experiments except the analyses of Olink, results were performed in GraphPad Prism (version 9.4.0, GraphPad Software). One‐way analysis of variance or Wilcoxon matched pairs signed rank tests were used to compute P values. P values <0.05 (two‐tailed) were regarded as statistically significant. Olink results were analyzed in R‐Studio (version 2025.09.0+387). When exact P values are not provided in the figures, statistical significance is indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant.

Ethics approval and consent to participate

For in vitro studies, buffy coats from healthy adult donors were obtained after written informed consent (Sanquin). Furthermore, fresh blood from healthy adults was obtained after written informed consent approved by the Medical Ethical Committee Oost Nederland (NL84281.091.23).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

RESULTS

Colchicine treatment prevents the secretion of chemokines and some inflammatory cytokines from PBMCs stimulated with MSU

To elucidate the mechanisms underlying colchicine‐induced immunosuppression during a gout flare, we employed an in vitro model using isolated PBMCs from healthy donors (n = 6). Cells were pretreated for 24 hours with increasing concentrations of colchicine (10, 50, and 500 nM), and cytotoxicity was assessed using an LDH assay (Supplementary Figure 1A), confirming the absence of toxic effects at all doses. Following pretreatment, both colchicine‐treated and untreated cells were stimulated for 24 hours with a combination of LPS to activate TLR4 (signal 1: activation of transcription) and MSU crystals (signal 2: inflammasome activation). Supernatants were then collected and analyzed using the Olink 96 inflammatory panel to profile changes in the secretion of inflammatory mediators (Figure 1A).

Figure 1.

Proteomic assessment of Colch effect on inflammatory cytokines and chemokines secretion after PBMC stimulation. (A) Schematic representation of the in vitro stimulation model, in which healthy donor PBMCs (n = 6) were isolated and cultured for 24 hours, with a one‐hour preincubation of Colch at different concentrations (0, 10, 50, and 500 nM), followed by stimulation with LPS (10 ng/μL) and MSU crystals (0.3 mg/mL). (B) Heatmap displaying the relative protein expression (mean NPX) increase (red) and decrease (blue) in the supernatant of Colch‐treated compared to untreated PBMCs. Only significantly increased or decreased proteins (adjusted P value <0.05) are shown. (C) Volcano plot showing the top differentially expressed proteins between stimulated cells, treated with and without colchicine (500 nM), where blue dots indicate adjusted P value <0.05. (D) Boxplots of NXP values of IL‐18, TNF, MCP‐1, and CXCL10 across all conditions. Colch, colchicine; IL, interleukin: LPS, lipopolysaccharide; MSU, monosodium urate; NPX, Normalized Protein Expression; PBMC, peripheral blood mononuclear cell; TNF, tumor necrosis factor.

After quality control filtering, 42 proteins were excluded due to low concentrations, resulting in the detection of 54 proteins overall. The results from different treatments show a clear separation of the principal component (PC) plots, with PC2 showing the clearest difference (Supplementary Figure 1B). Stimulation of PBMCs without colchicine treatment induced a pronounced increase in inflammatory protein secretion (Supplementary Figure 1C). Treatment with the lowest colchicine concentration (10 nM) did not significantly alter cytokine secretion compared to untreated controls. In contrast, exposure to 50 and 500 nM colchicine resulted in noticeable changes in the secretion profile (Figure 1B). At 50 nM, TGF‐α secretion was significantly elevated, marking the onset of colchicine's modulatory effects. At 500 nM, a broader range of proteins was affected: proinflammatory mediators including IL‐1α, IL‐18, IL‐6, and VEGF, as well as anti‐inflammatory cytokines such as IL‐10 and PD‐L1, were up‐regulated. Conversely, several chemokines (MCP‐1, MCP‐2, MCP‐3, CCL19, and CCL21) and the inflammatory cytokine TNF were down‐regulated (Figure 1C). This pattern indicates that the immunomodulatory effects of colchicine on PBMC cytokine secretion become evident beginning at the 50 nM concentration after 24 hours of treatment (Figure 1D).

PBMCs treated with colchicine produce and secrete less chemokines, which reduces the chemoattraction of monocytes

From the initial exploration of overall inflammatory protein secretion, it emerged that colchicine strongly affects the secretion of both cytokines and chemokines. These results were confirmed with an ELISA assay on a further 12 healthy controls (Figure 2). Similarly to the previous setup, the healthy control PBMCs were preincubated with different concentrations of colchicine and stimulated for 24 hours with a combination of LPS and MSU crystals. Supernatants were used to measure secretion of inflammatory proteins of interest. First, we observed that production of proinflammatory cytokines IL‐1β, IL‐6, and IL‐8 did not significantly decrease but stayed unvaried when stimulated PBMCs were treated with colchicine at 50 nM concentration. On the other hand, these cytokines were significantly increased when PBMCs were treated with higher colchicine concentrations (500 nM). TNF protein secretion was not affected by colchicine treatment, whereas IL‐1Ra protein secretion was decreased only by treatment with 500 nM colchicine. Most significantly, MCP‐1 secretion was inhibited by the colchicine treatment at both 50 and 500 nM concentrations (Figure 2A). Intracellular IL‐1β concentrations did not change by colchicine treatment, whereas intracellular MCP‐1 was significantly decreased (Figure 2B). The lack of chemokines in the supernatant of stimulated PBMCs treated with colchicine has functional consequences, such as the decreased recruitment of monocytes to the site of inflammation. This was confirmed by a chemotaxis assay in which the previous supernatants were used as a chemoattractant (Figure 2C). Clearly, supernatants of PBMCs treated with colchicine induces a significantly lower chemotaxis.

Figure 2.

Colchicine affects cytokine and chemokine secretion of peripheral blood mononuclear cell, affecting their ability to induce monocyte chemoattraction. (A) Quantification of extracellular content of IL‐1β, IL‐6, TNF, and MCP‐1, after 24‐hour stimulation with LPS and MSU, in the presence of colchicine (50 and 500 nM). (B) Quantification of intracellular IL‐1β and MCP‐1 after 24‐hour stimulation with LPS and MSU, in the presence of colchicine (50 and 500 nM). (C) Chemotaxis assay of monocytes, chemoattracted by the supernatants of peripheral blood mononuclear cells (n = 6) stimulation with LPS and MSU, treated with colchicine (50 nM [orange] and 500 nM [red]) or without (black) over 24 hours. Results are shown as area under the curve and time course of chemotaxis. Mean ± SEM, one‐way analysis of variance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. IL, interleukin; LPS, lipopolysaccharide; MSU, monosodium urate; ns, not significant; TNF, tumor necrosis factor.

Monocytes treated with colchicine produce less chemokines, less ROS, and phagocytosis

We repeated the previous experimental setup using monocytes isolated from healthy donors (Figure 3A). Consistent with our earlier findings, colchicine did not alter the release of IL‐1β and IL‐6 in MSU‐ and LPS‐stimulated monocytes, whereas significant inhibition was observed in the secretion of TNF and MCP‐1 (Figure 3B). To determine whether colchicine's effects were due to inhibition of protein secretion or transcription, we assessed mRNA expression levels after four hours of stimulation with MSU/LPS. Although mRNA levels of TNF and IL‐1β remained unchanged, MCP‐1 mRNA expression was significantly reduced (Figure 3C), suggesting that colchicine selectively suppresses MCP‐1 transcription. Additionally, colchicine modulated other key monocyte functions by decreasing ROS production (Figure 3D) and reducing phagocytic activity in response to zymosan stimulation (Figure 3E). These findings indicate that colchicine exerts anti‐inflammatory effects on monocytes through selective transcriptional regulation and functional impairment.

Figure 3.

Colchicine affects monocytes effector function, including cytokine transcription and secretion, ROS production, and phagocytosis. (A) Schematic representation of the experimental setup. Monocytes are isolated from healthy donor buffy coats and preincubated with colchicine (50–500 nM) for one hour and stimulated with LPS alone or LPS + MSU crystals. After four hours, cells are processed for RNA detection, whereas supernatants are collected after 24 hours of stimulation. (B) Cytokine production after 24‐hour stimulation, including IL‐1β, TNF, IL‐6, and MCP‐1 (n = 9). (C) mRNA expression of cytokines IL‐1β, TNF, and MCP‐1 after four‐hour stimulation (n = 6). (D) ROS production after zymosan stimulation of colchicine‐treated monocytes, represented as AUC of a one‐hour measurement (n = 6). (E) Phagocytosis capacity of zymosan‐coated beads by colchicine‐treated monocytes, represented as AUC of 24‐hour measurement (n = 6). Mean ± SEM, one‐way analysis of variance, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AUC, area under the curve; IL, interleukin; LPS, lipopolysaccharide; mRNA, messenger RNA; MSU, monosodium urate; ns, not significant; ROS, reactive oxygen species; TNF, tumor necrosis factor.

Colchicine treatment on activated monocytes decreases cellular metabolism

To better understand colchicine's impact on the innate immune system's metabolic activity, we performed metabolic assays on MSU and LPS‐stimulated monocytes in the absence or the presence of colchicine. After 24 hours of colchicine treatment, both glycolysis and oxidative phosphorylation were significantly reduced (Figure 4A) (Figure 4B). Although baseline glycolytic rates remained similar between treated and untreated cells, colchicine decreased both glycolysis (cells capacity to use glucose for glycolysis) and glycolytic capacity (the maximum rate of glycolysis, induced by blocking the mitochondrial ATP synthesis by oligomycin) in monocytes (Figure4C. In terms of oxygen consumption, colchicine did not affect nonmitochondrial oxygen consumption; however, basal respiration, maximal respiration, proton leak, and ATP‐linked respiration were all significantly diminished in colchicine‐treated monocytes (Figure 4D). These findings demonstrate that colchicine affects cellular metabolism by dampening both glycolytic and mitochondrial respiratory functions, which likely contributes to its anti‐inflammatory effects by reducing the energy availability required for immune cell activation and function.

Figure 4.

Colchicine affects monocyte metabolism by reducing both mitochondrial respiration and glycolysis. Overview of the (A) glycolytic stress test and (B) mitochondrial stress test on monocytes treated with colchicine and stimulated for 24 hours with LPS and MSU crystals. (C) Calculated ECAR parameters, and (D) calculated OCR parameters (n = 9), mean ± SEM, *P < 0.05, ** < 0.01. Wilcoxon signed‐ranked test. Colch, colchicine; 2‐DG, 2‐deoxy‐d‐glucose; ECAR, extracellular acidification rate; FCCP, carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone LPS, lipopolysaccharide; MSU, monosodium urate; non‐mit, nonmitochondrial; ns, not significant; OCR, oxygen consumption rate.

DISCUSSION

In the present study, we examined the in vitro effects of colchicine on healthy human PBMCs and monocytes upon stimulation with MSU crystals to better understand the underlying mechanisms involved in the resolution of a gout flare. So far, it has been generally accepted that colchicine's main mechanism of action is a blockade of tubulin polymerization, affecting primary inflammasome assembly. In this study, we identified that for primary human PBMCs and monocytes, one of the key mechanisms of anti‐inflammatory activity of colchicine is the inhibition of chemokine production, preventing the further recruitment of inflammatory cells to the gout‐induced inflamed joint. We show that colchicine treatment alters the effector function of monocytes by significantly reducing the transcription and secretion of chemokines such as MCP‐1, as well as inhibiting ROS production, phagocytosis, and cellular metabolism. The secretion of inflammatory cytokines (IL‐1β, IL‐6, and TNF) was not decreased by colchicine treatment of PBMCs, whereas only TNF production was moderately decreased when treating monocytes.

The effects of colchicine on IL‐1β are complex and highly context dependent. Although colchicine is well known for its anti‐inflammatory properties, its impact on IL‐1β production can vary, resulting in either increases or decreases, depending on the cellular environment and specific experimental conditions. ⁴ , ¹⁷ , ¹⁸ In our study, colchicine treatment of PBMCs stimulated with MSU and LPS led to an increase in both IL‐1β and IL‐6 secretion. Conversely, treatment of isolated monocytes under the same stimulation conditions did not alter IL‐1β or IL‐6 secretion but resulted in a moderate reduction of extracellular TNF release. Stimulation with LPS and MSU activates the NLRP3 inflammasome in monocytes, a key pathway for IL‐1β maturation and release. ¹⁷ Although colchicine has been reported to inhibit NLRP3 inflammasome activation, ⁴ our findings revealed no clear decrease in IL‐1β production or secretion. This aligns with previous studies in which LPS‐stimulated monocytes treated with varying colchicine doses showed an increase in IL‐1β and a decrease in TNF production. ¹⁸ , ¹⁹ The differing responses between PBMCs and isolated monocytes may be due to the presence of multiple cell populations within PBMCs, which could react to microtubule disruption by releasing preformed inflammatory mediators. This hypothesis is supported by a reduction in IL‐1Ra production, which acts as a negative feedback regulator of active IL‐1β. ²⁰ Therefore, the increased extracellular IL‐1β detected in our PBMC cultures may include both cleaved (active) and pro–IL‐1β forms, accounting for the complex cytokine profile observed.

In this study, we highlighted the central role of chemokine suppression by colchicine treatment. Previously, similar effects of colchicine have been observed in different contexts. For instance, in patients with acute coronary syndrome, colchicine treatment was associated with reduced MCP‐1 concentrations in coronary arteries as well as upon in vitro stimulations. ²¹

Furthermore, in familial Mediterranean fever, in which colchicine is a key drug for disease control, patients were identified by marked reduction of chemokine production as compared to controls. ²² These studies point toward the clear possibility that colchicine influences chemokine production and chemoattraction as a key mechanism of action of the drug, prompting us to investigate it further. Our proteomic analysis initially identified several chemokines that were significantly down‐regulated in the supernatant of PBMCs treated with colchicine and stimulated with MSU and LPS. These included MCP‐1, MCP‐2, and MCP‐3, which are key monocyte chemoattractant, ²³ as well as CCL23, which attracts lymphocytes, monocytes, and neutrophils, ²⁴ and CCL19, an immunologic homeostasis chemokine that recruits inflammatory T cells and dendritic cells. ²⁵ Focusing on MCP‐1, we confirmed that its secretion was reduced in both stimulated PBMCs and monocytes treated with colchicine. We initially hypothesized that the reduction was due to impaired exocytosis caused by colchicine‐induced microtubule depolymerization. ²⁶ However, the significant decrease in intracellular MCP‐1 suggests that this is not the primary mechanism. Recognizing that monocytes are the main source of MCP‐1 in PBMCs, we also assessed gene transcription and protein production and confirmed that colchicine inhibits MCP‐1 at the RNA level. This finding clearly demonstrates the baseline and exclusive impact that colchicine exerts on chemokines. Finally, we demonstrated that reduced chemokine secretion by immune cells significantly impairs monocyte chemoattraction. Supernatants from colchicine‐treated PBMCs induced markedly less monocyte migration than those from untreated cells, consistent with their chemokine deficiency. Given that MCP‐1 is elevated in both serum and synovium of patients with gout compared to healthy controls, ²⁷ it is likely that colchicine helps suppress gout flares also by diminishing monocytes’ ability to recruit other immune cells to the joint.

ROS are among the inflammatory mechanisms that induce local damage and perpetration of the inflammatory environment in the arthritic joints. ²⁸ Here, we demonstrated that colchicine effectively reduces both ROS production and phagocytosis in monocytes stimulated with zymosan, a potent activator of these processes. Although previous studies have shown colchicine inhibits ROS production in endothelial cells ²⁹ and neutrophils, ¹² , ³⁰ , ³¹ its effects on monocytes had not been explored. Moreover, the phagocytosis of MSU crystals by leukocytes, a key pathogenic feature of gout, was reported early on to be inhibited by colchicine, due also to decreased microtubule assembly. ¹⁷ , ³² Our study confirms that colchicine similarly suppresses these mechanisms in monocytes. Because both phagocytosis and ROS generation require substantial energy, monocytes increase both glycolysis and oxidative phosphorylation upon LPS and MSU stimulation. ³³ We therefore wanted to understand whether the high energy consumption caused by LPS and MSU stimulation could be blunted by colchicine treatment. We observed an overall reduction in both glycolytic activity and oxidative phosphorylation following colchicine treatment. These metabolic effects align with previous findings in which colchicine decreased mitochondrial membrane potential. ³⁴ Overall, colchicine effectively blunts cellular metabolism, possibly contributing to decreased monocyte ROS production and phagocytosis capabilities.

This study also has some limitations. The in vitro setting aimed to mimic the synovial environment during a gout flare but may not fully reflect the complexity of a systemic disease. Therefore, our findings could be validated on synovial immune cells of patients with gout, assessed both before and after colchicine treatment. Second, the colchicine concentrations used in our experiments were relatively high and not achievable in blood through standard oral administration, although local concentrations in the tissue may differ. Lower concentrations tested did not yield measurable effects, highlighting a challenge in translating these results directly to clinical settings. However, emerging drug delivery technologies, such as nanoparticles and microspheres, hold promise for enabling targeted colchicine delivery at higher local concentrations. Such approaches could improve therapeutic efficacy while minimizing systemic toxicity in the future.

In summary, our findings extend the current understanding of colchicine's anti‐inflammatory actions by showing that it suppresses monocyte chemokine production, metabolism, ROS production, and phagocytosis. These results suggest that colchicine's therapeutic efficacy may rely not only on its known effects on neutrophils and inflammasome inhibition but also on restraining monocyte‐driven inflammation and further recruitment of immune cells.

AUTHOR CONTRIBUTIONS

All authors contributed to at least one of the following manuscript preparation roles: conceptualization AND/OR methodology, software, investigation, formal analysis, data curation, visualization, and validation AND drafting or reviewing/editing the final draft. As corresponding author, Dr Merlo Pich confirms that all authors have provided the final approval of the version to be published and takes responsibility for the affirmations regarding article submission (eg, not under consideration by another journal), the integrity of the data presented, and the statements regarding compliance with institutional review board/Declaration of Helsinki requirements.

ADDITIONAL DISCLOSURES

Drs Netea and Joosten are scientific co‐founders of and have equity in Trained Therapeutix Discovery, Lemba, and Salvina.

Supporting information

Supplementary Figure 1 Cytotoxicity assay and supplementary analysis of Olink assay. (A) LDH assay to measure cell cytotoxicity of treatment and stimulation. (B) Olink proteins PCA plot, identifying the separation of clusters based on colchicine treatment. Box plot of PC2 that significantly differentiates the supernatants of PBMCs treated with different concentrations of colchicine. (C) Vulcano plots showing from left to right, the differentially expressed protein of unstimulated vs MSL+LPS stimulated PBMCs; stimulated vs stimulated and 10nM colchicine treated PBMCs; stimulated vs stimulated and 50nM colchicine treated PBMCs.

Supplementary Table 1: Primers used in RTqPCR performed on monocytes treated with or without colchicine and stimulated with LPS and MSU.

Supplementary Table 2: Olink 96 inflammation pannel, detectable proteins.

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