Introduction

Paroxetine (PRX) is a selective serotonin reuptake inhibitor approved for the treatment of various psychiatric disorders such as major depressive disorder (MDD), panic disorder (PD), and post-traumatic stress disorder (PTSD)1,2,3. Recent studies have identified a novel mechanism of action of PRX within the context of drug repositioning. For instance, PRX has been shown to improve cardiac function in a myocardial infarction (MI) mouse model as an inhibitor of G-protein coupled receptor kinase 2 (GRK2)4. Mounting evidence suggests that PRX acts as a modulator of inflammation. Previous research has demonstrated that PRX modulates immune responses through the activation of JAK2/STAT3 signaling5. PRX reduces the production of TNF-α and IL-6 in mouse macrophage cells following LPS stimulation6. Moreover, PRX regulates dendritic cell activation through modulation of the PI3K/Akt/mTOR signaling pathway7, highlighting its involvement in the broader regulation of inflammatory responses. However, the mechanisms by which PRX regulates inflammation in human immune cells are yet to be understood.

Oxysterols are oxidized derivatives of cholesterol molecules formed through enzymatic or non-enzymatic oxidation reactions8. Among the known oxysterols, 27-hydroxycholesterol (27OHChol) is the most abundant in the human circulatory system and is found in inflammatory lesions9,10. This bioactive lipid has been implicated in the development of chronic inflammatory diseases due to its role in promoting metabolic inflammation11,12. Specifically, 27OHChol induces the expression of C–C motif chemokine ligand 2 (CCL2), which facilitates monocyte chemotaxis13, and enhances the expression of matrix metalloproteinases (MMPs), which contribute to tissue infiltration14,15. Additionally, it impairs phagocytic function by driving the differentiation of monocytes into mature dendritic cell (mDC)-like cells16,17. The involvement of 27OHChol in pro-inflammatory signaling highlights its importance as a target in inflammation-associated pathologies.

Despite its established role in promoting inflammation, effective pharmacological approaches to suppress 27OHChol-mediated inflammatory responses remain limited. Given the emerging evidence of PRX’s immunomodulatory properties and its involvement in the PI3K/Akt/mTOR pathway, we hypothesized that PRX can attenuate inflammation and Akt/mTORC1 signaling induced by 27OHChol. In this study, we report a new pharmacological effect of PRX on 27OHChol-driven inflammation in monocytic cells. Taken together, our findings suggest that an attentuation of Akt/mTORC1 siganling by PRX is important to regulate 27OHChol-mediated cellular responses. Furthermore, these results raise the possibility that the structural scaffold of PRX may provide a basis for the development of novel compounds targeting oxysterol-induced immune dysregulation.

Results

PRX impairs 27-hydroxycholesterol-induced CCL2 expression and migration

We investigated whether paroxetine (PRX) suppresses 27OHChol-induced CCL2 expression in monocytic cells. The PRX concentration was determined based on preliminary experiments, including the IC50 value for CCL2 mRNA suppression, assessments of cell viability and transcriptional regulation of other pro-inflammatory cytokines (Fig. 1A, Supplementary Fig. S1A, S1B). Treatment of THP-1 cells with 27OHChol increased CCL2 mRNA levels by approximately 18-fold, which was significantly reduced in a dose-dependent manner to sevenfold in the presence of 5 µM PRX (p < 0.001) (Fig. 1B). We next assessed CCL2 at the protein level. Exposure to 27OHChol elevated CCL2 secretion to 356 pg/mL, consistent with the transcript level (p < 0.001) (Fig. 1C). PRX treatment mitigated this effect, reducing secretion to 168 pg/mL in the presence of 5 µM PRX (p < 0.001). We also investigated whether other selective serotonin reuptake inhibitors like imipramine, citalopram, fluoxetine, and fluvoxamine had activity similar to paroxetine. We could not obtain data indicating their inhibitory effects on 27OHChol-induced CCL2 expression, as determined by RT-PCR (Supplementary Fig. S1C).

Fig. 1
figure 1

PRX impairs 27OHChol-induced CCL2 expression and migration of THP-1 monocytic cells. THP-1 cells (2.5 × 105 cells/mL) were serum-starved for 18 h in RPMI 1640 medium containing endotoxin-free 0.1% bovine serum albumin and then treated with 27OHChol (2 μg/mL) in the presence of the indicated concentrations of PRX for 48 h. (A) Half-maximal inhibitory concentration (IC50) of PRX on 27OHChol-induced CCL2 transcription. (B) CCL2 mRNA levels were assessed by quantitative reverse transcription-PCR. Data were normalized to GAPDH and expressed relative to untreated control. (C) Secreted CCL2 protein levels in the culture media were measured by ELISA. (D) Conditioned media were applied to monocytic cells, and migration was assessed using a chemotaxis assay. All results are representative of three independent experiments (n = 3) and expressed as mean ± standard deviation (SD). ###p < 0.001 vs control; *p < 0.05; **p < 0.01; ***p < 0.001 vs 27OHChol. The presented blots were cropped for clarity, and the corresponding original images are available in Supplementary Fig. 5.

Full size image

A functional chemotaxis assay showed that supernatants isolated after treatment with 27OHChol alone enhanced THP-1 cell migration to 54,000 cells (p < 0.001). Treatment with PRX, however, reduced cell migration in a concentration-dependent manner. In particular, the number of migrated cells was reduced to 12,000 when they were exposed to supernatants isolated after treatment with 27OHChol in the presence of 5 µM PRX (p < 0.001) (Fig. 1D). Collectively, these results suggest that PRX reduces THP-1 monocytic cell chemotaxis by downregulating 27OHChol-induced CCL2 expression.

PRX suppresses MMP-9 activity

To determine whether PRX affects matrix remodeling enzymes, we examined its impact on MMP-9 expression. Treatment with 27OHChol upregulated MMP-9 transcription by threefold (p < 0.001). PRX, however, attenuated this induction in a concentration-dependent manner. The 27OHChol-induced MMP-9 transcription was reduced to almost basal level in the presence of 5 µM PRX (p < 0.001) (Fig. 2A). Gelatin zymography also revealed increased MMP-9 enzymatic activity following 27OHChol stimulation. The MMP-9 activity, however, was progressively suppressed by PRX treatment (Fig. 2B). These data indicate that PRX inhibits both the transcription and proteolytic activity of MMP-9 induced by 27OHChol.

Fig. 2
figure 2

PRX suppresses MMP-9 activity. THP-1 cells were serum-starved as described in Fig. 1. and treated with 27OHChol (2 μg/mL) and the indicated concentrations of PRX for 48 h. (A) MMP-9 mRNA levels were measured by quantitative reverse transcription-PCR. Data were normalized to GAPDH and presented relative to control. (B) Gelatinolytic activity in culture supernatants was assessed by zymography. Clear zones indicate MMP-9 activity. Data represent mean ± SD of three independent biological replicates (n = 3). ###p < 0.001 vs control; ***p < 0.001 vs 27OHChol. Original full-length gels are presented in Supplementary Fig. 4.

Full size image

PRX downregulates mature dendritic cell (mDC) marker expression and partially restores phagocytic function

We next explored whether PRX influences 27OHChol-induced differentiation of monocytes into mDC-like cells and affects phagocytic capacity. Treatment with 27OHChol increased the expression of mDC surface markers, including CD80, CD83, and CD88. PRX, however, reduced the expression of these markers in a concentration-dependent manner (Fig. 3A, Supplementary Fig. S2). CD80 expression returned to the basal level in the presence of 5 µM PRX (from 11.76% to 4.92%). CD83 and CD88 were also reduced, from 22.65% to 13.82% and from 32.01% to 14.09%, respectively. PRX also changed transcript levels of CD80, CD83, and CD88 in a consistent with protein levels. (Fig. 3B).

Fig. 3
figure 3figure 3

PRX downregulates mDC markers and restores phagocytic activity. THP-1 cells were serum-starved and treated with 27OHChol (2 μg/mL) and the indicated concentrations of PRX for 48 h. (A) Surface expression of CD80, CD83, and CD88 was analyzed by flow cytometry using FITC-conjugated antibodies. (B) mRNA levels of the CD molecules were measured by quantitative reverse transcription-PCR. Data were normalized to GAPDH and presented relative to control. (C) For phagocytosis assays, cells were incubated with FITC-conjugated dextran (0.5 mg/mL) for 30 min, and uptake was analyzed by flow cytometry. Bar graphs represent the mean ± SD from three independent biological replicates (n = 3). ###p < 0.001 vs control; *p < 0.05; **p < 0.01; ***p < 0.001 vs 27OHChol.

Full size image

Phagocytic activity, assessed by FITC-dextran uptake, was impaired following stimulation with 27OHChol (from 11.62% to 3.70%) (Fig. 3C). This impairment was partially restored in the presence of PRX in a concentration-dependent manner (from 3.11% to 5.64%). Treatment with 250 nM phorbol 12-myristate 13-acetate (PMA), used as a positive control, fully recovered endocytosis to the basal level (10.21%). These findings suggest that PRX suppresses 27OHChol-induced dendritic cell-like differentiation and restores compromised phagocytic function in monocytic cells.

PRX regulates Akt/mTORC1 downstream activity

We further investigated whether PRX interferes with the Akt/mTORC1 pathway. After treatment with 27OHChol for 40 min, phosphorylation of Akt, S6, and 4E-BP1 was markedly increased (Fig. 4A). Rapamycin (Rapa), a potent mTOR inhibitor, blocked phosphorylation of S6 and 4E-BP1 with minimal impact on Akt phosphorylation, while PRX inhibited phosphorylation of Akt, S6 and 4E-BP1 (Fig. 4A). Increased phosphorylation of Akt, S6, and 4E-BP1 was sustained up to 4 h post-treatment with 27OHChol. PRX also inhibited their phosphorylation (Fig. 4B). Phosphorylation of S6 and 4E-BP1, but not of Akt, persisted at 48 h post-treatment with 27OHChol. PRX suppressed the phosphorylation of S6 and 4E-BP1, consistent with the inhibition observed with Rapa (Fig. 4C). This sustained suppression at the 48 h time point aligns with the endpoints employed in the aforementioned assays of Figs. 1, 2, 3, supporting the prolonged anti-inflammatory effect of PRX through modulation of the Akt/mTORC1 pathway.

Fig. 4
figure 4

PRX suppresses phosphorylation of Akt, S6, and 4E-BP1. THP-1 cells were serum-starved and treated with 27OHChol (2 μg/mL) in the presence of PRX or rapamycin (Rapa) (0.01 μM). Total and phosphorylated forms of Akt, S6, and 4E-BP1 were analyzed by Western blotting at three post-treatment time points: 40 min (A), 1–4 h (B), and 48 h (C). Results are representative of three independent biological replicates (n = 3). The presented blots were cropped for clarity, and the corresponding original images are available in Supplementary Fig. 3.

Full size image

Discussion

27OHChol has emerged as a key endogenous mediator in the pathogenesis of chronic inflammatory diseases. Previous studies have demonstrated that 27OHChol promotes vascular inflammation and atherogenesis by activating ERα-dependent signaling, leading to endothelial dysfunction, macrophage infiltration, and foam cell formation18. Additionally, 27OHChol has been implicated in intestinal fibrosis and epithelial barrier disruption in inflammatory bowel disease (IBD), where it induces oxidative stress and epithelial-mesenchymal transition (EMT) via β-catenin signaling19. Moreover, accumulating evidence indicates that 27OHChol alters macrophage phenotype and function, enhancing monocyte recruitment and cytokine production that perpetuate adipose tissue inflammation and metabolic dysregulation20. Given its widespread effects on chronic inflammatory diseases, we sought to identify the drugs to resolve the 27OHChol-induced inflammatory pathways. Based on these findings, we investigated whether PRX, a selective serotonin reuptake inhibitor with reported immunomodulatory properties, can attenuate the proinflammatory responses induced by 27OHChol in monocytic cells.

This study demonstrates that PRX suppresses multiple inflammatory phenotypes induced by 27OHChol, particularly through regulation of monocyte chemotactic signaling. CCL2 is a critical chemokine that drives monocyte/macrophage recruitment in numerous inflammatory conditions21,22. MMP-9 also contributes to disease progression by modulating extracellular matrix remodeling, immune cell trafficking, and cytokine regulation15,23. Our findings show that PRX reduces 27OHChol-induced CCL2 expression, inhibits monocyte migration, and suppresses both the expression and activity of MMP-9. These results suggest that PRX may modulate monocyte dynamics and dampen inflammation by targeting key chemotactic and matrix-remodeling factors. While CCL2 appears to be a principal mediator of monocyte trafficking, further studies are needed to determine whether additional chemotactic factors are also regulated by PRX.

Previous research has shown that 27OHChol induces the differentiation of monocytes into mature dendritic cell (mDC)-like cells17, in part through upregulation of tight junction proteins such as Zonula occludens-1 (ZO-1), which are not typically expressed in monocytes24. This hyperactivation of DC-like phenotypes may contribute to sustained inflammation and tissue damage in chronic disease contexts25. In this study, PRX treatment reduced the surface expression of mDC markers CD80, CD83, and CD88, all of which were elevated by 27OHChol. Furthermore, PRX partially restored phagocytic function, which is typically suppressed during mDC differentiation26. Given that JAK/STAT signaling has been shown to suppress DC maturation and that PRX enhances this pathway5, these results suggest that PRX may interfere with 27OHChol-induced mDC differentiation through JAK/STAT-dependent mechanisms.

The mechanistic basis for these effects appears to involve the Akt/mTOR pathway. mTOR is a central regulator of immune cell metabolism and differentiation27,28,29, functioning through two complexes: mTORC1 and mTORC230. Among them, mTORC1 regulates the phosphorylation of S6K and 4E-BP1, thereby controlling protein synthesis and cell growth30. Our previous work demonstrated that the PI3K/Akt axis, upstream activator of mTORC1, plays critical role in 27OHChol-mediated activation of monocytic cells31. We have also shown that the mTOR inhibitor rapamycin attenuates 27OHChol-induced responses in THP-1 cells32. Consistent with these findings, the present study shows that PRX reduces the phosphorylation of Akt, S6, and 4E-BP1, suggesting that PRX interferes with 27OHChol-induced mTORC1 signaling. Previous reports have linked mTORC1 activation to the expression of CCL233,34, supporting the idea that PRX may suppress CCL2 and other inflammatory mediators via this pathway. However, given that Akt is not the sole regulator of mTORC1 and also plays diverse roles in cellular metabolism and immune cell function28,29, it is plausible that PRX exerts its effects through additional mechansims beyond Akt/mTORC1 axis inhibition. Therefore, our data support the involvement of the Akt/mTORC1 axis, further studies are warranted to elucidate alternative pathways modulated by PRX.

In conclusion, this study demonstrates that PRX attenuates 27OHChol-induced responses in monocytic cells by modulating key pathways associated with chemotaxis, matrix remodeling, dendritic cell differentiation, and mTORC1 signaling. While these findings highlight the potential of PRX as an immunomodulatory agent, it is important to note that THP-1 cells are leukemic in origin and harbor multiple genetic mutations35, limiting their representation of primary human monocytes. Further validation using in vivo or primary cell-based models will be essential to assess the translational relevance of our findings. Given the established role of 27OHChol in metabolic inflammation11, PRX may offer a novel approach for targeting chronic inflammatory diseases. Moreover, our findings raise the possibility that the structural scaffold of PRX could serve as a foundation for developing new compounds that inhibit oxysterol-driven immune signaling. Optimization of such analogs may improve specificity and reduce off-target effects, paving the way for a new class of immunomodulatory agents.

Materials and methods

Cell culture and treatment

The THP-1 human monocytic cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and routinely cultured in a humidified incubator (5% CO2, 37 °C) using RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Upon reaching passage number 9, the cells were washed once with warm phosphate-buffered saline (PBS), resuspended in growth medium containing endotoxin-free 0.1% bovine serum albumin (BSA), and seeded in a 60 mm dish (2.5 × 105 cells/ml). After 18 h of serum starvation, the cells were treated with or without 27OHChol and PRX under the specified conditions.

Reagents

27OHChol was purchased from Sigma-Aldrich (St. Louis, MO, USA). PRX hydrochloride was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies against Akt, p-Akt (Ser473), S6, p-S6 (Ser240/244), 4E-BP1, p-4E-BP1 (Ser65) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against CD80, CD83, CD88, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated from the cells using TRIzol reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Reverse transcription was performed on the total RNA using Moloney Murine Leukemia Virus reverse transcriptase at 42 °C for 1 h. Synthesized cDNA was used to perform PCR or quantitative real-time PCR. Non-quantitative PCR was conducted using HS prime Taq DNA polymerase (Genetbio, Daejeon, South Korea) on a GeneAtlas Thermal Cycler (Astec Bio, Fukuoka, Japan). The thermal cycling conditions were as follows: 95 °C for 10 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s. The PCR products were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide for 30 min, and images were captured using Printgraph CMOS 1 (ATTO SupportingLifeResearch, Tokyo, Japan). The forward and reverse primers used were as follows: GAPDH(500 bp), 5’-GAGTCAACGGATTTGGTCCT-3’(forward) and 5’-TGTGGTCATGAGTCCTTCCA-3’(reverse); GAPDH(226 bp), 5’-GAAGGTGAAGGTCGGAGT-3’(forward) and 5’-GAAGATGGTGATGGGATTTC-3’(reverse) CCL2, 5’-TCTGTGCCTGCTGCTCATAG-3’(forward) and 5’-CAGATCTCCTTGGCCACAAT-3’(reverse);

Quantitative real-time PCR was performed using SYBR green Q-PCR Master Mix (Smart gene, Seoul, South Korea) on a CFX Duet Real time PCR System (Bio-Rad Laboratories, Hercules, CA, USA). The thermal cycling conditions were as follows: 95 °C for 3 min, 40 cycles at 95 °C for 10 s and 60 °C for 30 s. The 2−ΔΔCt method was used to calculate relative gene expression to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The forward and reverse primers used were as follows: GAPDH, 5’-GAAGGTGAAGGTCGGAGT-3’(forward) and 5’-GAAGATGGTGATGGGATTTC-3’(reverse); CCL2, 5’-CAGCCAGATGCAATCAATGCC-3’(forward) and 5’-TGGAATCCTGAACCCACTTCT-3’(reverse); MMP-9, 5’-GCACGACGTCTTCCAGTACC-3’(forward) and 5’-CAGGATGTCATAGGTCACGTAGC-3’(reverse). CD80, 5’-GCAGGGAACATCACCATCCA-3’(forward) and 5’-TCACGTGGATAACACCTGAACA-3’(reverse); CD83, 5’-TCCTGAGCTGCGCCTACAG-3’(forward) and 5’-GCAGGGCAAGTCCACATCTT-3’(reverse); CD88, 5’-GTGGTCCGGGAGGAGTACTTT-3’(forward) and 5’-GCCGTTTGTCGTGGCTGTA-3’(reverse).

Enzyme-linked immunosorbant assay (ELISA)

The cell culture supernatant was collected by centrifugation (1300 rpm, 5 min). The level of secreted CCL2 protein in the supernatant was measured using BD OptEIA ELISA kit (BD Biosciences, Franklin Lakes, NJ, USA) as per the manufacturer’s instructions.

Chemotaxis assay

The migration of the THP-1 cells was evaluated using Transwell Permeable Supports (Costar, Cambridge, MA, USA) as previously described36. Briefly, THP-1 cells were washed once with warm PBS and resuspended in growth media containing 0.1% BSA. Transwell chambers were filled with 600 μl of supernatant, and the cells were loaded onto the upper chamber of 5 μm-pore polycarbonate inserts (5 × 105 cells / 100 μl). After incubation (37 °C, 2 h), the number of cells that migrated to the bottom chamber was counted using Vi-cell XR counter (Beckman Coulter, Indianapolis, IN, USA).

Flow cytometric analysis

Following treatment with 27OHChol and PRX, the cells were washed once with ice-cold PBS. The cells were then incubated with flow cytometry buffer (2 mM EDTA and 0.1% BSA in PBS) containing FITC-conjugated antibodies against CD80, CD83, CD88 diluted to 1:100. After incubation in a dark room (4 °C, 4 h), the cells were washed once with ice-cold PBS and fixed in 1% paraformaldehyde (PFA). The fluorescence intensity of the cells was analyzed using a CytoFLEX Flow cytometer (Beckman Coulter) according to the manufacturer’s instructions.

Endocytosis assay

For endocytosis analysis, the treated cells were stained with 0.5 mg/ml FITC-conjugated dextran (37 °C, 30 min). After incubation, the cells were washed twice with ice-cold PBS and resuspended in 1% PFA. The amount of dextran taken up by the cells was assessed by measuring fluorescence intensity using a CytoFLEX Flow cytometer (Beckman Coulter).

Gelatin zymography

Gelatinolytic activity of secreted gelatinase in the supernatant was evaluated by zymography as previously described37. Briefly, the collected supernatant was concentrated 20-fold using Vivaspin 2 Centricon (Satorius, Göttingen, Germany) according to the manufacturer’s instructions. Proteins in the concentrate were separated by 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) containing 0.15% gelatin. After incubation in an activation buffer (50 mM Tris–HCl pH 7.7, 0.2 M NaCl, 5 mM CaCl2) at 37 °C for 18 h, the gel was stained with 0.5% Coomasie brilliant blue R-250. Areas of degradation appeared as clear bands against the stained blue background and visualized using Printgraph CMOS 1 (ATTO Supporting Life Research).

Western blot analysis

To analyze the intracellular protein levels, the cells were lysed using PRO-PREP protein extraction solution containing protease inhibitors (iNtRON Biotechnology, Seoul, South Korea). The protein concentration was quantified using a BCA assay, and the samples were denatured in SDS-containing buffer (95 °C, 5 min). The samples were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes. After blotting, the membranes were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and incubated with the primary antibody (4 °C, 18 h). Next, the membranes were washed three times with TBS-T, incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h, and washed three times with TBS-T. The bands were detected using chemiluminescent reagent, and the images were captured using Amersham Imager 600 (GE healthcare Life Sciences, PA, USA).

Statistical analysis

GraphPad Prism 5 software (GraphPad Software Inc., CA, USA) was used for statistical analysis. Statistical significance between variables was confirmed by One-way ANOVA, followed by Tukey’s multiple comparison tests. Data were presented as mean ± standard deviation (SD) as indicated in the figure legends.