Oleic acid alleviates the pathogenesis of lupus by suppressing the activation of TLR signaling pathways through the STAT3/IL-10 axis

Abstract

Toll-like receptors (TLRs) play a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE) by regulating the activation and differentiation of immune cells. Oleic acid (OA), the predominant monounsaturated fatty acid found in adipose tissue, is known to regulate various biological processes. However, the regulatory role of OA in TLR signaling pathways activation and SLE pathogenesis remains unclear. In this study, we reveal that OA treatment significantly alleviates symptoms in both MRL/lpr mice and imiquimod (IMQ)-induced lupus model mice, as evidenced by reduced splenomegaly, decreased anti-dsDNA antibody levels, ameliorated renal pathological damage, and diminished glomerular deposits of IgG and IgM. Notably, OA treatment not only suppresses the activation of immune cells, including B cells, macrophages, dendritic cells, and T cells, but also inhibits the differentiation of B cells and T cells in vivo. Importantly, OA effectively inhibits TLRs-mediated activation of immune cells by blocking the MAPK pathway and NF-κB pathway. Mechanistically, OA promotes IL-10 expression in B cells by activating STAT3, and IL-10 can significantly inhibit the activation of TLR pathways. Collectively, our findings reveal that OA mitigates lupus pathogenesis by targeting TLR pathways through the STAT3/IL-10 axis, indicating that OA may be a promising treatment candidate for SLE.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-34755-0.

Introduction

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by loss of self-tolerance, production of pathogenic autoantibodies, and deposition of immune complexes in various tissues^1,2. As is known, hyper-activated immune cells are widely involved in the occurrence and development of SLE. B cells play a central role in SLE pathogenesis by producing autoantibodies, presenting antigens, secreting cytokines, and activating T cells^3–5. Aberrantly activated macrophages and dendritic cells exacerbate tissue damage and inflammation through the release of reactive oxygen species (ROS), proteolytic enzymes, and pro-inflammatory cytokines^6,7. Each T cell subset function (such as effector, helper, memory or regulatory function) is critically involved in the development of systemic autoimmunity and organ inflammation in SLE⁸. Given the crucial role of excessive activation of immune cells in the progression of SLE, it is urgently necessary to clarify the specific regulatory mechanisms of the abnormal differentiation and activation of immune cells.

Toll-like receptors (TLRs) are key regulators of innate immunity, recognizing pathogen-associated molecular patterns and initiating downstream signaling through MyD88-dependent pathways, leading to activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) cascades and subsequent production of pro-inflammatory mediators^9,10. Substantial evidence demonstrates that TLR signaling pathways contribute to SLE pathogenesis by regulating abnormal immune cell activation and differentiation^11,12. In B cells, TLR signaling pathways promotes plasma cell activation, exacerbating tissue damage and disease severity^13–15. Similarly, TLR-activated macrophages release pro-inflammatory mediators and exhibit impaired clearance of apoptotic debris, further promoting autoimmunity and accelerating disease progression¹⁶. Following TLR activation, dendritic cells generate large amounts of type I interferons (IFN-I) and inflammatory cytokines, engaging autoreactive lymphocytes and amplifying the autoimmune response^17,18. These findings highlight the crucial role of the TLR signaling pathways in the pathogenesis of SLE, and targeted inhibition of TLR pathway activation can significantly alleviate the onset of SLE. However, the regulatory mechanism of TLR pathway activation remains not fully understood.

Oleic acid (OA) is the main monounsaturated fatty acid in adipose tissue and is involved in regulating the functions of various types of cells. It has been shown that OA can influence cell survival, proliferation, and metastatic potential through its participation in cell membrane architecture and oxidative phosphorylation (OXPHOS). In terms of immunological regulation, OA enhances mitochondrial respiration in B cells and facilitates immune effector protein trafficking, thereby augmenting antimicrobial defense mechanisms. Moreover, OA contributes significantly to anti-tumor immunity and metabolic homeostasis by regulating intraepithelial T-cell metabolism^19–22. In addition, OA can also promote the differentiation of regulatory T cells (Treg cells) and exhibits anti-inflammatory properties²³. However, the role of OA in modulating the activation of the TLR signaling pathway and the pathogenesis of SLE remains poorly understood.

In the present study, we found that OA significantly ameliorated disease severity and reduced immune cell activation in both MRL/lpr lupus mice and imiquimod-induced lupus model mice. OA inhibited TLRs-mediated activation of immune cells by blocking the MAPK and NF-κB pathways. Mechanistically, OA directly activated STAT3 and then promoted the expression of IL-10, which could negatively regulate the activation of TLR pathways. Collectively, the above results indicate that OA can inhibit TLR-mediated immune cell activation and alleviate lupus pathogenesis by activating the STAT/IL-10 signaling axis, indicating that OA maybe used for the treatment of SLE.

Materials and methods

Animals

Female C57BL/6 mice (6–8 weeks, weighting 17 ~ 18 g) and MRL-fas^lpr (MRL/lpr) mice (10 weeks, weighting 33 ~ 38 g) were obtained from Pengyue Animal Breeding Co., Ltd. and Jiangsu Aniphe Biolaboratory, Inc., respectively. Interleukin (IL)−10 gene-deficient mice on a C57BL/6 background were obtained from Cyagen Biosciences Inc. All animals were maintained in a sterile environment with strictly regulated conditions.

For evaluation of OA treatment in MRL/lpr lupus mice, animals were divided randomly into two identical groups (n = 6/group): (1) a vehicle group given vehicle (10 mL/kg) via intraperitoneal injection, and (2) an OA treatment group treated with OA working solution (10 mL/kg). The preparation method of OA working solution is as follows: first, OA (purchased from Selleck) was initially dissolved in DMSO to create a clear stock solution with a concentration of 34 mg/mL; to prepare 1 mL of the OA working solution, 50 µL of the clear stock solution was added to 400 µL of PEG300, mixed thoroughly until clear, followed by the addition of 50 µL of Tween 80 to the mixture, which was then mixed again until clear; subsequently, 500 µL of PBS was added to reach a final volume of 1 mL. The working solution was prepared for immediate use. The injection dosage for mice was set at 10 mL/kg, equating to 17 mg/kg. Correspondingly, the vehicle control comprised 5% DMSO, 40% PEG300, 5% Tween 80, and 50% PBS. Both groups received their respective injections three times weekly. All animal experiments followed institutional care protocols. After a 4-week therapy phase, mice were euthanized with CO₂ for analyses. All animal experiments were conducted in compliance with the protocols approved by the Animal Care Committee of Jining Medical University. All animal experiments complied with the ARRIVE guidelines.

To establish the imiquimod (IMQ)-induced lupus mouse model, researchers applied 1.5 mg of 5% IMQ (Sichuan Pharmaceutical Co., Ltd.) to the right ears of 8-week-old female C57BL/6 mice. This treatment was administered three times per week over an eight-week period. To assess OA’s therapeutic impact, mice were assigned mice to five experimental cohorts (n = 6/group): (A) vehicle group; (B) 17 mg/kg OA group; (C) IMQ + vehicle group; (D) IMQ + 8.5 mg/kg OA group; and (E) IMQ + 17 mg/kg OA group. Mice from group B and E were injected with the OA working solution mentioned above (10 mL/kg), mice from group D were injected with the OA working solution diluted with PBS at a ratio of 1:1(10 mL/kg). Following an 8-week treatment period, mice were treated as MRL/lpr lupus mice.

Antibodies and reagents

The following antibodies were used for immunoblotting: Cell Signaling Technology, anti-mouse p38, p-p38, Erk, p-Erk, JNK, p-JNK, p65, and p-p65 antibodies; Abcam, anti-mouse IL-10 antibody; Beyotime Institute of Biotechnology, anti-mouse GAPDH antibody. For immunoblotting, HRP-conjugated secondary antibodies were used at a dilution of 1:3000. Flow cytometry analyses utilized the following antibodies from Biolegend: FITC-conjugated anti-mouse B220 and CD4; PE-conjugated anti-mouse CD40, GL7, PD-1, and CD44; APC-conjugated anti-mouse CD86, CD95, CXCR5, and CD138; BV421-conjugated anti-mouse CD69 and CD62L; and the anti-mouse IL-10 antibody was purchased from Abcam. Immunofluorescence microscopy was performed using goat anti-mouse IgG and IgM antibodies labeled with Alexa Fluor 488 (Invitrogen, USA) or C3/C3a Rabbit Polyclonal Antibody (Beyotime, Nantong, China).

Isolation and culture of primary immune cells

Production of macrophages and dendritic cells from bone marrow

Bone marrow-derived cells were isolated aseptically from femurs and tibiae of C57BL/6 mice. BMDMs underwent 7-day differentiation in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 100 ng/mL recombinant murine GM-CSF (Peprotech). Concurrently, BMDCs were differentiated in RPMI 1640 medium with 10% FBS, 1% penicillin-streptomycin, 20 ng/mL GM-CSF, and 1 ng/mL IL-4 (Peprotech) for an identical duration. Post-differentiation, cells were pretreated with OA at indicated concentrations for 12 h, followed by stimulation with 100 ng/mL LPS (Sigma), 1 µg/mL R848 (MCE), or 1 µM CpG-1826 (Invitrogen) for an additional 12 h before harvest. Cells were cultured at 37℃ in a 5% CO₂ humidified incubator.

Isolation of splenic B cells

Spleens from C57BL/6 mice were mechanically dissociated to generate single-cell suspensions. To isolate B cells, erythrocytes were first lysed using a specialized buffer. The remaining cells underwent negative selection using BD Biosciences’ Mouse B Cell Isolation Kit for the isolation procedure. This method consistently produced highly pure B cell populations, exceeding 95% purity. Isolated B cells were propagated in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin under standard culture conditions.

Cell viability assessment

B cells, BMDMs, and BMDCs were cultured in 96-well plates, and exposed to varying concentrations of OA (0, 25, 50, 100, and 200 µM) for a 24-hour incubation period. Following treatment regimens, cell viability was quantitatively assessed using Cell Counting Kit-8 (CCK-8), adhering to the manufacturer’s guidelines, with assessments conducted at four time points (0.5, 1, 2, and 4 h post-exposure). Viability was compared to untreated controls.

H&E staining

First, kidney tissues were fixed in 4% paraformaldehyde solution. Next, the tissues were dehydrated in a series of graded ethanol solutions before paraffin embedding for sectioning. The sections were mounted on slides and stained with hematoxylin and eosin (H&E) to facilitate visualization under a Nikon light microscope.

Immunofluorescence staining

Kidney tissue sections embedded in paraffin were sequentially treated with xylene for deparaffinization, followed by ethanol rehydration, and antigen retrieval was subsequently performed. To block non-specific binding, 1% BSA in PBS was applied for 1 h at room temperature. Sections were cultured for 12 h at 4 °C with Alexa Fluor 488-conjugated goat anti-mouse antibodies. After washing with TPBS for 5 min three times, sections were counterstained with DAPI and mounted using an anti-fade mounting medium that contains an autofluorescence quenching agent. Fluorescence micrographs were obtained using the Olympus fluorescence microscope.

Quantitative real-time PCR

RNA was isolated utilizing TRIzol reagent (Invitrogen) and reverse transcribed to complementary DNA (cDNA) employing the RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative real-time PCR (qPCR) was performed using SYBR Green PCR Master Mix (Vazyme Biotech), with relative quantification calculated via the 2^−ΔΔCt algorithm.

Flow cytometry

Single-cells were incubated with fluorochrome-labeled antibodies for 30 min at 4 °C in light-protected conditions. Following washing with PBS containing 1% FBS, cell samples were acquired on the BD FACSVerse™ instrument in 200 µl PBS. Appropriate isotype controls were used for each antibody to establish gating parameters, and specific cell populations were identified for independent analysis.

Enzyme-linked immunosorbent assay

Mouse IL-12p40 and TNF-α cytokine concentrations were determined using BioLegend’s ELISA kits. Antibody-coated plates underwent overnight sensitization at 4 °C and were blocked with Assay Diluent at 37 °C for 1 h, then incubated with samples and standards at 37 °C for 2 h. After washing, antibody-HRP was added for 30 min, and then TMB substrate solution was treated for 15 min, and the reaction was stopped and read at 450 nm by a BioTek microplate reader. Each sample was analyzed in duplicate.

The IL-10 levels of the serum were determined using commercially available kits (Cat#JL20242, Jianglai Biotechnology Co., Ltd., China). The concentrations of IL-10 in serum were detected and determined the absorbance at 450 nm according to the manufacturer’s instructions. Serum IgG against dsDNA in both MRL/lpr mice and mice with lupus treated by IMQ was assessed with Bethyl Laboratories’ kit, and absorbance at 450 nm was measured by the manufacturer’s guidelines via a BioTek microplate reader. Each sample was analyzed in duplicate.

Serum levels of serum creatinine (Cr) and blood urea nitrogen (BUN) determination

The Cr and BUN levels of the serum were determined using commercially available kits (Cat# C011-2-1, Cat# C013-3-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The levels of Cr and BUN were detected and determined the absorbance at 546 nm and 340 nm according to the manufacturer’s instructions.

Immunoblotting analysis

Equal amounts of the protein samples were separated by 10% SDS-PAGE, transferred onto PVDF membranes with glycine transfer buffer at 100 V for 1 h, and given a thorough blocking session with 5% BSA in TBST for 2 h. The primary antibodies were applied overnight at 4 °C, followed by HRP-conjugated secondary antibodies (1:5000) for 2 h. Immunoreactive proteins were identified with the ECL detection kit (Thermo, USA) and quantified via densitometric analysis.

RNA-seq analysis

Total RNA was isolated from murine B cells with TRIzol reagent. Samples were sequenced by Genesky Technologies on an Illumina HiSeq 2500 system under 150 bp paired-end mode. Protein-coding genes exhibiting an average expression ≥ 2 RPKM (reads per kilobase per million mapped reads) in at least one experimental condition were included for further study. Differential gene expression analysis was carried out using the edgeR package. Significantly differentially expressed genes were then used for KEGG pathway enrichment analysis²⁴.

Statistical analysis

Results are expressed as mean ± SEM, with each condition tested in triplicate across at least three separate trials. Statistical comparisons were carried out using Shapiro-Wilk test and Dunnett test for either independent-sample t-tests or one-way ANOVA, depending on the experimental design. Statistical significance was established at a P value < 0.05; **P < 0.01; ***P < 0.001;****P < 0.0001.

Results

OA dramatically reduces symptoms in MRL/lpr lupus mice

Given the effects of OA on immunomodulation in various disease contexts, and considering its unexplored therapeutic potential in SLE, we first evaluated its efficacy in the MRL/lpr mouse model. Spleen enlargement serves as a visual indicator of immune hyperactivity, while anti-dsDNA antibody levels correlate with autoimmune intensity. Compared with vehicle-treated controls, OA-treated MRL/lpr mice showed considerably reduced splenomegaly (Fig. 1A) and weight (Fig. 1B), along with decreased serum anti-dsDNA antibody concentrations (Fig. 1C). As shown (Fig. 1D and Fig. S1A), compared with vehicle, OA treatment significantly reversed characteristic pathological changes in MRL/lpr mice, including glomerular enlargement, segmental mesangial hyperplasia, and inflammatory cell infiltration. Importantly, immunofluorescence staining demonstrated significantly reduced renal accumulation of IgG, IgM and C3 immune complexes in OA-treated MRL/lpr mice compared to vehicle controls (Fig. 1E and Fig. S1B-D). In addition, as shown in Fig. S1E-F, compared with vehicle, OA treatment significantly reversed the elevated levels of Cr and BUN in serum from MRL/lpr mice. These results indicate that OA can significantly alleviate the condition of MRL/lpr lupus mice.

Fig. 1.

OA significantly mitigates the progression of disease in MRL/lpr lupus mice. A Representative splenic morphology, B splenic weights, C serum anti-dsDNA antibody levels, D renal histopathology (H&E staining) in MRL/lpr mice treated with OA (17 mg/kg, intraperitoneal) or vehicle. Scale bars: 100 μm and 50 μm. magnification: 100× and 200×. E renal IgG and IgM deposition in MRL/lpr mice treated with OA (17 mg/kg, intraperitoneal) or vehicle. Scale bars: 20 μm. magnification: 400×. Data represent means ± SEM from three independent experiments (n = 6 mice/group). Statistical significance: **p < 0.01 (two-tailed Student’s t-test).

OA inhibits the activation and differentiation of immune cells from MRL/lpr lupus mice

We next investigated the effects of OA on the activation and differentiation states of immune cells within spleen and lymph node tissues derived from MRL/lpr mice. Compared with vehicle-treated controls, OA-treated MRL/lpr mice exhibited markedly reduced levels of CD86 (Fig. 2A, B) and CD69 (Fig. 2C, D) expression on B220⁺ B cells in the spleen, as well as the proportions of GC B cells (Fig. 2E, F) and plasma cells (Fig. 2G, H). Moreover, compared with vehicle, OA treatment significantly reduced the CD86 and CD40 expression on splenic macrophages (Fig. 2I-L) and dendritic cells (Fig. 2M-P). Additionally, similar phenomenons were observed in mesenteric lymph nodes (mLNs) of MRL/lpr mice following OA treatment; that is, compared with vehicle-treated controls, OA-treated MRL/lpr mice exhibited fewer GC B cells (Fig. S2A, B) and plasma cells (Fig. S2C, D), reduced levels of CD86 (Fig. S2E, F) and CD69 (Fig. S2G, H) expression on B220⁺ B cells, and reduced levels of CD86 and CD40 expression on macrophages (Fig. S2I-L) and dendritic cells (Fig. S2M-P) in the mLNs.

Fig. 2.

OA modulates immune cell differentiation and activation in the spleens of MRL/lpr mice. A–H Surface expression of CD86 (A, B) and CD69 (C, D) on B220⁺ B cells, and flow cytometric analysis of germinal center B cells (GL7⁺) within B220⁺ B cell populations (E, F), plasma cells (CD138⁺) (G, H), and I–L Flow cytometric analysis of CD86 (I, J) and CD40 (K, L) expression on F4/80⁺ macrophages. M–P Flow cytometric analysis of CD86 (M, N) and CD40 (O, P) expression on CD11c⁺ dendritic cells. Data represent means ± SEM from three independent experiments (n = 6 mice/group). Statistical significance: **p < 0.01 and ***p < 0.001 (two-tailed Student’s t-test).

Given the central importance of T cells in lupus development, we further examined OA’s impact on T cell populations. Compared with vehicle-treated controls, OA-treated MRL/lpr mice exhibited markedly reduced proportions of follicular helper T (Tfh) cells within both the spleen (Fig. S3A, B) and mLNs (Fig. S3C, D). Of note, a significant reduction in splenic memory CD4⁺ T cell populations was observed in the spleen (Fig. S3E, F) of OA-treated MRL/lpr mice, compared to vehicle-treated controls. Concurrently, compared with vehicle-treated controls, OA-treated MRL/lpr mice exhibited markedly reduced CD69 expression on CD4⁺ T cells in both splenic tissue and mLNs (Fig. S3G-J) These findings collectively demonstrate that OA dramatically inhibits the abnormal activation of the immune system in MRL/lpr lupus mice.

OA alleviates disease progression in TLR7 agonist IMQ-induced lupus mice

To study the effect of OA on the pathogenesis of IMQ-induced lupus in mice, we conducted systematic evaluations. Compared to vehicle-treated controls, OA treatment showed considerably reduced splenomegaly (Fig. 3A) and weight (Fig. 3B), along with decreased serum anti-dsDNA antibody concentrations (Fig. 3C), reflecting decreased autoimmune activity. As shown (Fig. 3D and Fig. S4A), compared with vehicle, OA treatment significantly reversed characteristic pathological changes in IMQ mice, including glomerular enlargement, segmental mesangial hyperplasia, and inflammatory cell infiltration. Importantly, immunofluorescence staining demonstrated significantly reduced renal accumulation of IgG, IgM and C3 immune complexes in OA-treated IMQ mice compared to vehicle controls (Fig. 3E and Fig. S4B-D). In addition, as shown in Fig. S4E-F, compared with vehicle, OA treatment significantly reversed the elevated levels of Cr and BUN in serum from IMQ-induced lupus mice. These results collectively demonstrate that OA substantially alleviates disease occurrence and development in the IMQ-induced lupus model.

Fig. 3.

OA significantly alleviates the progression of disease in IMQ-induced lupus mice. A Representative splenic morphology, B splenic weights, C serum anti-dsDNA antibody levels, D renal histopathology (H&E staining) in IMQ-induced lupus mice treated with OA (8.5 and 17 mg/kg, intraperitoneal) or vehicle. Scale bars: 100 μm and 50 μm. magnification: 100× and 200×. E renal IgG and IgM deposition in IMQ-induced lupus mice treated with OA (8.5 and 17 mg/kg, intraperitoneal) or vehicle. Scale bars: 20 μm. magnification: 400×. Data represent means ± SEM from three independent experiments (n = 7 mice/group). Statistical significance: **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t-test or ANOVA tests).

OA inhibits the activation and differentiation of immune cells from IMQ-induced lupus mice

Compared with vehicle-treated controls, OA-treated IMQ mice exhibited markedly reduced levels of CD86 (Fig. 4A, B) and CD69 (Fig. 4C, D) expression on B220⁺ B cells, as well as the proportions of GC B cells (Fig. 4E, F) and plasma cells (Fig. 4G, H) in spleens. Moreover, compared with vehicle, OA treatment significantly reduced the CD86 and CD40 expression on splenic macrophages (Fig. 4I-L) and dendritic cells (Fig. 4M-P). Additionally, similar phenomenons were observed in mesenteric lymph nodes (mLNs) of IMQ mice following OA treatment; that is, compared with vehicle-treated controls, OA-treated IMQ mice exhibited fewer GC B cells (Fig. S5A, B) and plasma cells (Fig. S5C, D), reduced levels of CD86 (Fig. S5E, F) and CD69 (Fig. S5G, H) expression on B220⁺ B cells.

Fig. 4.

OA regulates immune cell differentiation and activation in the spleens of IMQ-induced lupus mice. H Flow cytometric analysis of CD86 (A, B) and CD69 (C, D) expression on B220⁺ B cells, germinal center B cells (B220⁺GL7⁺CD95⁺) within B220⁺ B cell populations (E, F), and plasma cells (CD138⁺) (G, H). I–L Flow cytometric analysis of CD86 (I, J) and CD40 (K, L) expression on F4/80⁺ macrophages. M-P Flow cytometric analysis of CD86 (M, N) and CD40 (O, P) expression on CD11c⁺ dendritic cells. Data represent means ± SEM from three independent experiments (n = 7 mice/group). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed Student’s t-test or ANOVA tests).

Additionally, OA could also inhibit the activation and differentiation of CD4⁺ T cells in IMQ mice. Compared with vehicle-treated controls, OA-treated IMQ mice exhibited markedly reduced CD69 expression on CD4⁺ T cells in both splenic tissue and mLNs (Fig. S6A-D). Concurrently, compared with vehicle-treated controls, OA-treated IMQ mice exhibited markedly reduced proportions of Tfh cells within both the spleen (Fig. S6E, F) and mLNs (Fig. S6G, H). Of note, a significant reduction in splenic memory CD4⁺ T cell populations was observed in the spleen (Fig. S6I, J) of OA-treated IMQ mice compared to vehicle-treated controls. These findings collectively demonstrate that OA dramatically inhibits the abnormal activation of the immune system in IMQ-induced lupus mice.

OA inhibits the TLRs-mediated activation of B cells in vitro

To explore the molecular mechanism underlying the immunosuppressive effects of OA observed in vivo, we next investigated whether OA can directly modulate TLRs-mediated activation of immune cells in vitro. First, we focused on whether OA can modulate the TLRs-mediated activation of B cells in vitro. Murine splenic naïve B cells were treated with various OA concentrations for 24 h, and the viability of B cells was analyzed by the CCK8 assay. As shown (Fig. S7), OA concentrations below 200 µM showed no impact on the viability of B cells; therefore, subsequent experiments utilized OA concentrations not exceeding 200 µM.

Murine naïve B cells were stimulated with varying concentrations of OA (0, 25, 50, 100, and 200 µM) for 12 h, followed by treated with LPS (TLR4 agonist), R848 (TLR7 agonist), and CpG-1826 (TLR9 agonist) for 24 h. As anticipated, OA treatment effectively reversed the upregulations of CD86 (Fig. 5A-C), CD69 (Fig. 5D-F), and CD40 (Fig. S8) expression induced by LPS, R848, and CpG-1826 in a concentration-dependent manner. Moreover, OA treatment could also significantly inhibit the expression of IL-12 (Fig. 5G-I) and TNF-α (Fig. 5J-L) induced by LPS, R848, and CpG-1826 in B cells. As is known, TLR signaling regulates immune cell activation primarily through downstream MAPK and NF-κB pathways. Intriguingly, OA treatment could significantly inhibit the phosphorylation levels of Erk, JNK, p38, and p65 proteins in R848-activated B cells (Fig. 5M). Taken together, these results strongly indicate that OA can directly inhibit the TLRs-mediated activation of B cells.

Fig. 5.

OA attenuates TLR-mediated B cell activation in vitro. B cells were pretreated with varying OA concentrations (0, 25, 50, 100, and 200 µM) for 12 h prior to stimulation with LPS (100 ng/ml), R848 (1 µg/ml), or CpG-1826 (1 µM). A-F Flow cytometric assessment of CD86 expression on B cells following stimulation with LPS (A), R848 (B), and CpG-1826 (C), and CD69 expression on B cells following stimulation with LPS (D), R848 (E), and CpG-1826 (F). G-L qPCR analysis of IL-12 (G-I) and TNF-α (J-L) mRNA expression in B cells treated with OA (0, 25, 50, and 100 µM) and subsequently stimulated with LPS, R848, or CpG-1826. M Immunoblot analysis of Erk, JNK, p38, and p65 phosphorylation levels in B cells following R848 stimulation for 30 and 60 min, with GAPDH serving as loading control. Data represent means ± SEM from three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t-test or ANOVA tests).

OA inhibits the TLRs-mediated activation of BMDMs and BMDCs in vitro

Next, we also investigated the effects of OA on TLRs-mediated activations of macrophage and dendritic cell. OA concentrations below 200 µM showed no impact on the viability of BMDMs and BMDCs; therefore, subsequent experiments utilized OA concentrations not exceeding 200 µM (Fig. S7). As expected, OA treatment dramatically reduced LPS-, R848-, and CpG-1826-induced expression of CD86 and CD40 on BMDMs (Fig. 6A-F). Moreover, OA treatment significantly inhibited the secretion of IL-12p40 (Fig. 6G) and TNF-α (Fig. 6H) by LPS-, R848-, and CpG-1826-activated BMDMs in a concentration-dependent, as well as the mRNA expression of IL-12 (Fig. S9A-C) and TNF-α (Fig. S9D-F). Notably, OA significantly inhibited R848-triggered phosphorylations of Erk, JNK, p38, and p65 in BMDMs (Fig. 6I).

Fig. 6.

OA suppresses TLR-mediated BMDMs activation in vitro. F Flow cytometric analysis of CD86 expression on BMDMs following stimulation with LPS (A), R848 (B), and CpG-1826 (C), and CD40 expression on BMDMs following stimulation with LPS (D), R848 (E), and CpG-1826 (F). G and H ELISA quantification of IL-12 p40 (G) and TNF-α (H) in cell culture supernatants. I Immunoblot analysis of Erk, JNK, p38, and p65 phosphorylation following R848 stimulation for 30 and 60 min, with GAPDH as loading control. Data represent means ± SEM from three independent experiments. Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t-test or ANOVA tests).

As observed in BMDMs, OA treatment substantially inhibited the expression of CD86 and CD40 on LPS-, R848-, and CpG-1826-activated BMDCs (Fig. S10A-F). As expectedly, OA treatment significantly inhibited the secretion of IL-12p40 (Fig. S10G) and TNF-α (Fig. S10H) by LPS-, R848-, and CpG-1826-activated BMDCs in a concentration-dependent, as well as the mRNA expression of IL-12 (Fig. S9G-I) and TNF-α (Fig. S9J-L). Notably, OA significantly inhibited R848-triggered phosphorylations of Erk, JNK, p38, and p65 in BMDCs (Fig. S10I). Collectively, these results establish that OA can directly inhibit TLRs-mediated activation of BMDMs and BMDCs in vitro.

OA promotes IL-10 expression by activating STAT3 in B cells

Given the broad inhibitory effect of OA on TLR pathways, we hypothesized that it might function by inducing a negative regulator. To explore the potential mechanism, murine naïve B cells were treated with OA for 6 h and subsequently performed RNA-seq to assess changes in gene expression. The results showed that a total of 177 genes were upregulated and 401 genes were downregulated by OA (Fig. 7A). KEGG pathway enrichment analysis revealed that these differentially expressed genes were predominantly enriched in pathways such as cytokine–cytokine receptor interaction and oxytocin signaling (Fig. 7B). Interestingly, we noticed that OA treatment could markedly upregulate the expression of IL-10 in B cells (Fig. 7C). Consistently, this finding was validated by qPCR (Fig. 7D) and flow cytometry (Fig. 7E, F) analyses, which confirmed a significant upregulation of IL-10 expression by OA. What’s more, we also found that the content of IL-10 in the serum of lupus mice treated with OA was significantly higher than that of lupus mice treated with vehicle (Fig. S11), indicating that OA can indeed promote the expression of IL-10 in vitro and in vivo.

Fig. 7.

OA upregulates IL-10 expression in B cells via the STAT3 signaling pathway. A RNA-seq analysis of murine splenic B cells revealed 177 upregulated and 401. downregulated genes. B KEGG pathway enrichment analysis of differentially expressed genes. C IL-10 mRNA expression was markedly increased in OA-treated B cells. D Murine splenic B cells were treated with OA for 6–12 h, and then the expression of IL-10 was analyzed by qPCR. E, F Murine splenic B cells were treated with OA for 24 h, and then the expression of IL-10 was analyzed by FACS. G Immunoblot analysis of STAT3 phosphorylation levels in B cells following OA stimulation for 15, 30, 60, 120, and 240 min, with actin serving as loading control. H-J Murine naive B cells were treated with different concentrations of STAT3 inhibitor Stattic (5 and 10 µM) for 2 h, followed by OA treatment (200 µM). The mRNA level of IL-10 was analyzed by qPCR (H) at 6 h and the protein level of IL-10 was analyzed by FACS (I, J) at 12 h. Data represent means ± SEM from three independent experiments. Statistical significance: **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t-test or ANOVA tests).

Since previous studies have reported that STAT3 is a key transcription factor regulating IL-10 expression, we hypothesized that OA may promotes the IL-10 expression by activating STAT3. Murine naïve B cells were treated with OA at different time points and then the phosphorylation of STAT3 was analyzed. As shown (Fig. 7G), OA can indeed induce STAT3 phosphorylation in B cells. Next, we investigated the role of STAT3 in OA-mediated expression of IL-10. As shown in Fig. 7H-J, the STAT3 inhibitor Stattic could significantly reverse OA-induced expression of IL-10. In summary, our findings indicate that OA upregulates IL-10 expression in B cells by activating the STAT3 signaling pathway.

IL-10 is essential for OA to inhibit TLRs-mediated activation of B cells

It is well established that IL-10 exerts broad immunosuppressive effects. We next investigated that whether IL-10 can inhibit the TLRs-mediated activation of B cells. Murine naïve B cells were pretreated with recombinant IL-10 for 2 h and then stimulated with LPS, R848, or CpG-1826. As expected, IL-10 could significantly inhibit the upregulation of CD86 (Fig. 8A, B) and CD69 (Fig. 8C, D) expression mediated by TLRs, as well as the expressions of IL-6 (Fig. 8E) and TNF-α (Fig. 8F). To confirm that whether OA can inhibit the TLRs-mediated activation of B cells by upregulating IL-10 expression, naïve B cells, isolated from WT and IL-10^−/− mice, were pretreated with OA for 12 h and then stimulated with LPS, R848, or CpG-1826. Firstly, we confirmed again that IL-10 negatively regulates the TLRs-mediated activation of B cells, as IL-10 deficiency lead to higher levels of CD86 (Fig. 8G, H) and CD69 (Fig. 8I, J) expression mediated by TLRs, as well as the expressions of IL-6 (Fig. 8K) and TNF-α (Fig. 8L). More importantly, as shown in Fig. 8G-L, IL-10 deficiency could markedly reverse the inhibitory function of OA on the TLRs-mediated upregulation of CD86 and CD69 expression, as well as the expressions of IL-6 and TNF-α. Notably, IL-10 deficiency could markedly reverse the inhibitory function of OA on R848-triggered phosphorylations of Erk, JNK, p38, and p65 in B cells (Fig. 8M). Collectively, these results establish that IL-10 is necessary for OA to exert its inhibitory function on TLRs-mediated activation of B cells.

Fig. 8.

IL-10 is essential for OA-mediated suppression of TLR-triggered B cell activation. A–F Murine splenic B cells were pretreated with recombinant IL-10 (20 ng/ml) for 2 h prior to stimulation with LPS, R848, or CpG-1826. The surface expression of CD86 (A, B) and CD69 (C, D) was analyzed by FACS at 24 h. The expression of IL-6 (E) and TNF-α (F) was analyzed by qPCR at 6 h. G–M Murine splenic B cells isolated from WT and IL-10^−/− mice were treated with OA (200 µM) for 12 h, followed by stimulation of TLR agonists. The surface expression of CD86 (G, H) and CD69 (I, J) was analyzed by FACS at 24 h. The expression of IL-6 (K) and TNF-α (L) was analyzed by qPCR at 6 h. M Immunoblot analysis of Erk, JNK, p38, and p65 phosphorylation following R848 stimulation for 30 min. Data represent means ± SEM from three independent experiments. Statistical significance: *p < 0.005, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (two-tailed Student’s t-test or ANOVA tests).

Collectively, our study delineates a novel mechanistic pathway that OA alleviates the pathogenesis of lupus by suppressing the activation of TLR signaling pathways through the STAT3/IL-10 axis (Fig. 9). This natural fatty acid emerges as a highly viable treatment option for SLE, offering a potential breakthrough in disease management.

Fig. 9.

Oleic acid alleviates the pathogenesis of lupus by suppressing the activation of TLR signaling pathways through the STAT3/IL-10 axis.

Discussion

Lipid metabolism significantly influences the pathogenesis of SLE, emerging as a pivotal modulator of immune activity. Although oleic acid (OA) plays essential metabolic roles and has garnered attention in the context of autoimmune disorders, its modulatory effects on TLR-mediated autoimmune responses, particularly in systemic lupus erythematosus (SLE), are not yet fully elucidated. Our study reveals that OA effectively attenuates the activation of B cells, macrophages, and dendritic cells, while also blocking the differentiation of GC B cells and plasma cells, thereby mitigating disease progression in lupus murine models. Mechanistically, we depicted a signaling axis in which OA promotes IL-10 production by activating STAT3, thereby inhibiting TLRs-mediated activation of B cells and alleviating the progression of SLE.

In vivo experiments demonstrated that OA reduced lupus progression in MRL/lpr mice and suppressed IMQ-induced autoimmunity by inhibiting the activation and differentiation of B cells. Mechanistically, OA likely inhibits B cell differentiation through suppression of NF-κB signaling activation, a pathway with relevance to various autoimmune conditions²⁵. It is well established that T cell activation and differentiation significantly influence tumor development and inflammatory processes. Previous research has demonstrated that OA can enhance tumor antigen presentation and anti-cancer immunity by modulating the activation of T cells and the differentiation of regulatory T cells^20,26,27. Thus, it is necessary to investigate the mechanisms underlying OA-mediated T cell modulation. Interestingly, we examined the effects of OA on T cell activation and differentiation in lupus mice and observed that OA significantly reduced the activation of CD4⁺ T cells and the proportions of follicular helper T cell and memory CD4⁺ T cells in lupus-prone mice. We speculate that OA may, on the one hand, affect T cell activation and differentiation by regulating the activation and function of B cells, macrophages and DCs; on the other hand, it may be involved in the pathogenesis of lupus by directly regulating T cell activation and differentiation. Notably, multiple studies have reported that OA can regulate the differentiation of T cell subsets, such as Th1, Th17, Treg, and thereby participate in the occurrence and development of immune-related diseases^28–30. However, the effect of OA on the differentiation of Tfh cells has not been reported. Therefore, we believe that the regulatory effect of OA on the differentiation of Tfh cells deserves in-depth study in order to confirm whether OA directly regulates the activation and differentiation of T cells to participate in the pathogenesis of lupus or indirectly participates in the pathogenesis of lupus through antigen-presenting cells such as B cells, macrophages and DCs.

Both in vitro and in vivo experiments demonstrate that OA suppresses activation and differentiation of the immune cells by inhibiting the activation of TLRs, thereby effectively alleviating the progression of SLE. To explore the mechanism by which OA regulates the activation of the TLR pathway, we analyzed the gene expression regulated by OA using RNA-seq and found that OA significantly upregulated the expression of IL-10 mRNA in B cells. As is known, IL-10 is a well-characterized cytokine that plays a crucial role in regulating the proliferation and differentiation of various immune cells, suppressing the activation of B cells and macrophages, and limiting inflammatory responses³¹. Therefore, we speculate that OA may exert extensive immunomodulatory effects by regulating the expression of IL-10. In addition, given that STAT3 is a key transcription factor regulating the expression of IL-10, we further investigated and found that OA alleviates SLE by suppressing the activation and differentiation of B cells through the STAT3/IL-10 axis. Our research innovatively revealed the molecular mechanism by which OA regulates TLRs-mediated immune cell activation and lupus pathogenesis.

Of note, it is worth noting that there is still some controversy over the regulatory effect of IL-10 on the TLR pathways. Some studies have found that IL-10 can down-regulate key linker molecules in the TLR signaling pathways, including MyD88 and IRAK1, while inhibiting the nuclear expression of NF-κB transcription factors (c-Rel and Rel-B) as well as IRF-3 and IRF-8³². In this study, we also found that IL-10 significantly inhibited the activation of the TLR signaling pathways in B cells, but the specific molecular mechanism by which IL-10 inhibits the activation of the TLR signaling pathways was not fully clarified. Meanwhile, we recognize that IL-10 also plays an important immunomodulatory role in other immune cells such as macrophages and dendritic cells. In future research, we will further investigate whether OA is involved in the pathogenesis of SLE by regulating the STAT3/IL-10 axis of other immune cells. Not only that, in subsequent experiments, we will systematically explore the molecular regulatory mechanism of IL-10 on the activation of the TLR signaling pathways in different immune cells.

Numerous studies have shown that immune cells can be activated by multiple pathways, including TLR, IFN and other signaling pathways³³. However, our investigation concentrated primarily on TLRs-mediated regulation of immune cell activation and differentiation and their contribution to SLE development. Both in vivo and in vitro studies have found that OA significantly inhibits the activation of immune cells and improves the pathogenesis of SLE by suppressing the TLR signaling pathways. These insights enhance understanding of aberrant TLR pathways activation and suggest novel therapeutic approaches for SLE and related autoimmune conditions. Additionally, TLRs has also been linked to the pathogenesis of several immune-related disorders, including rheumatoid arthritis and viral infections. Consequently, our findings establish a foundation for targeting TLR signaling in autoimmune disease management. While acknowledging TLR signaling’s importance, we recognize it may not represent the exclusive mechanism, which necessitates further experimental exploration. Therefore, the regulatory effect of OA on other signaling pathways such as IFN is worthy of in-depth study.

It is well known that lipid metabolism is involved in regulating the occurrence and development of various diseases. Both our and others’ studies have found abnormal serum lipid metabolism in SLE patients, and there is a significant correlation between SLE disease activity and cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL)^34,35. However, the exact pathogenesis of metabolic disorders in SLE remains not fully understood. Notably, several studies have explored the impact of lipid mediators on the pathogenesis of SLE. Treatment with lysophosphatidic acid (LPA) prevents microglial activation and depression-like behaviours in a murine model of neuropsychiatric lupus³⁶. Treatment with LPA improves glomerulonephritis through the suppression of macrophage activation in lupus mice³⁷. Sphingosine-1-Phosphate (S1P) promotes macrophage M1 polarization through NLRP3 inflammasome activation in lupus nephritis³⁸. Hence, it becomes evident that lipid metabolites assume distinct roles in the onset and progression of lupus. We consider it highly significant to delve deeply into the research concerning the function of metabolites in the pathogenesis of lupus.

Notably, despite the demonstrated therapeutic effects of OA in lupus-prone mice, several critical factors warrant further investigation prior to clinical application. These factors include dose-response relationships, long-term efficacy evaluation, and comprehensive safety profiling. Future studies should aim to determine the optimal dosing regimens for OA in humans, assess the outcomes of prolonged treatment, and explore potential synergistic interactions with currently available therapeutic agents. Such research endeavors would significantly enhance the understanding of OA’s therapeutic potential in the management of SLE.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Yuxin Hu : Methodology, Writing – original draft. Yangzhe Gao: Conceptualization. Yucai Xiao: Software. Tianqi Zhao: Validation. Jiakun Liu: Formal analysis. Zhengyi Zhang: Resources. Shuo Zhao: Data curation. Huabao Xiong: Supervision. Yonghong Yang: Visualization. Guanjun Dong: Funding acquisition, Writing – review & editing. Lu Yu: Methodology, Writing– review & editing.

Funding

This work was supported by National Natural Science Foundation of China (82471834, 82071824), Tai Shan Young Scholar Foundation of Shandong Province (tsqn202211234), Shandong Provincial Natural Science Foundation (ZR2024MH279), Shandong Provincial Youth Innovation Technology Support Program (2021KJ074), Shandong Medical and Health Science and Technology Program (202402020725), and Jining medical university high-level scientiffc research project cultivation plan (JYGC2022KJ005).

Data availability

Data will be provided upon request.

Declarations

Competing interests

The authors declare no competing interests.