Ethical approval for the study was obtained from the Medical Ethics Committee of the First Affiliated Hospital of Guangxi Medical University, and informed consent forms were signed by all patients and their families. Heart valves were obtained from three female patients aged 45–55 years diagnosed with RHD, who had undergone mitral valve replacement at the First Affiliated Hospital of Guangxi Medical University. Immediately after dissection, the valves were placed in pre-cooled phosphate-buffered saline, cleansed of surface blood, and then fixed in 4% paraformaldehyde (P1110, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Group A Streptococcus (GAS, American Type Culture Collection) was inoculated into Brain Heart Infusion Powder (Guangdong Huankai Microbial Technology Co., Ltd., Guangdong, China) and then incubated at 37 °C for 24 h using a rotary mixer (Fuma, Shanghai, China) at 110 r/min. Bacterial fluids were collected and fixed in 4% paraformaldehyde for 12 h, then washed five times with saline before being finally diluted to 10 $\times$ 10¹¹ CFU/mL in saline. Antigen A was prepared by emulsification using a sonicator (Sonics & Materials, Inc, Newtown, CT, USA) at the time of administration, while antigen B was prepared by mixing 1:1 with complete Freund’s adjuvant (F5881, Sigma-Aldrich, Darmstadt, Germany).

All animal experimental procedures adhered to ethical guidelines for animal care and use, approved by the First Affiliated Medical Ethics Committee of Guangxi Medical University Affiliated Hospital (Approval Number: 2023-E751-01). Twenty-four 10-week-old female Lewis rats were obtained from Beijing Viton Lihua Laboratory Animal Technology Co., Ltd. (Beijing, China) and were randomly assigned to three groups: Control, RHD, or RHD+Rs-504393 groups, each comprising eight rats. After one week of acclimatization and feeding in the Guangxi Medical University Laboratory Animal Center (SPF grade), the rats received an initial injection of 0.2 mL of antigen B (bacterial emulsion: complete Fuchs’ adjuvant = 1:1) into the footpad under 2% concentration of isoflurane. Following a week of rest, 0.5 mL of antigen B was injected subcutaneously into the abdominal wall, repeated once a week for four consecutive weeks. Additionally, 0.5 mL of antigen A (inactivated GSA) was injected subcutaneously into the abdominal wall after a one-week rest period following the completion of the antigen B injections [21, 22]. The Rs-504393 treatment group received intraperitoneal injections of 5 mg/kg of the CCR2 antagonist (Rs-504393, BD218736, Shanghai Bide Pharmaceutical Technology Co., Ltd., Shanghai, China) concurrently with the first subcutaneous injection of antigen B into the abdominal wall. This treatment was administered once daily for eight weeks. In the control group, complete Fuchs’ adjuvant was injected instead of antigen B, while physiological saline was administered in place of antigen A, in equivalent amounts and at the same sites. Upon completion of all the interventions, 1 ml of blood was collected from the tail vein. Subsequently, all rats were euthanized using intraperitoneal administration of pentobarbital sodium at a concentration of 150 mg/kg to collect the heart valves.

The collected valve tissues were fixed in 4% paraformaldehyde for 24 h, followed by embedding in paraffin, sectioning into 4 µm thick slices, and mounting onto slides. Hematoxylin and eosin (H&E) staining was employed to evaluate inflammation, while Sirius red staining was used to assess valve fibrosis. Imaging of the H&E-stained samples was conducted using a BX43 light microscope (Olympus Corporation, Tokyo, Japan) at a magnification of $\times$400, and images of the Sirius red staining samples were captured using a BX43 confocal microscope (Olympus Corporation, Tokyo, Japan) at the same magnification. For immunohistochemical staining, the valve sections were incubated with CCL2 (1:200, ab25124, Abcam, Cambridge, UK), collagen-1 (COL-1, 1:500, ab316222, Abcam), CD68 (1:200, GB113109, Servicebio, Wuhan, China), CCR2 (1:200, GB11326, Servicebio), iNOS (1:200, GB11119, Servicebio), or CD206 (1:500, GB113497, Servicebio). Add the secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1:500, ab97051, Abcam) and incubate for 30 min.

Total RNA was extracted from both valve tissues and cells using TRIzol® reagent (15596026CN, Thermo Fisher Scientific, Inc., Waltham, MA, USA), following the manufacturer’s guidelines. The cDNA first-strand synthesis kit (YFXM0009, Nanjing Yifeixue Biotechnology Co., Ltd., Nanjing, China) was then employed to reverse-transcribe the total RNA into cDNA, according to the manufacturer’s protocol. Subsequently, real time-quantitative polymerase chain reaction (RT-qPCR) was performed using 2$\times$ SYBR Green qPCR Master Mix (YFXM0001, Nanjing Yifeixue Biotechnology Co., Ltd., Nanjing, China). Using the 2^{-Δ⁢Δ⁢Ct} method, the results were normalized to the relative expression of each target gene, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or $\beta$-actin as reference genes. Table 1 summarized the primer sequences used in this study.

Valve tissues or cells were lysed in RIPA buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and individual samples (30 mg of protein) were then prepared for electrophoresis on 7.5–15% sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis (PAGE) gels, followed by transfer to polyvinylidene fluoride (PVDF) membranes (ISEQ00005, EMD Millipore, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk in TBST (T1082, Beijing Solarbio Science & Technology Co., Ltd, Beijing, China) for 1 h at room temperature, washed three times, and subsequently incubated at 4 °C overnight with the following specific primary antibodies: anti-CCR2 (1:1000, ab223366, Abcam, Cambridge, UK), anti-CCR2 (1:1000, 16153-1-AP, Proteintech, Wuhan, China), anti-CCL2 (1:2000, ab25124, Abcam), anti-$\alpha$-smooth muscle actin ($\alpha$-SMA) (1:10,000, ab124964, Abcam), anti-COL1A1 (1:1000, ab316222, Abcam), anti-COL3A1 (1:1000, ab184993, Abcam), anti-CD86 (1:1000, CY5238, Abways, Beijing, China), anti-GAPDH (1:10,000, ab181602, Abcam), and Tubulin (1:5000, 11224-1-AP, Proteintech). After washing, the membranes were incubated for 1 h with an HRP-coupled secondary antibody (1:10,000, ab97051, Abcam). The final bands were visualized using an ECL chemiluminescence kit (P2200, Suzhou New Saimei Biotechnology Co., Ltd., Suzhou, China). Quantify the grayscale values of the bands corresponding to the final target protein using Image J software 1.53t (National Institutes of Health, Bethesda, MD, USA).

Tissue samples were rapidly frozen in liquid nitrogen and processed using the indirect immunofluorescence technique. Frozen sections were stained overnight at 4 °C in a humidified room with primary antibodies targeting anti-CD68 (1:2000, GB113109, Servicebio, Wuhan, China), anti-CCR2 (1:200, GB11326, Servicebio), anti-CCL2 (1:200, ab25124, Abcam, Cambridge, UK), anti-CD45 (1:300, GB115428, Servicebio), anti-CD31 (1:2000, ab182981, Abcam), and anti-s100a4 (1:2000, GB11397, Servicebio). Following elution, the sections underwent sequential reactions with fluorescent secondary antibodies (1:1000, GB23303, Servicebio) for 50 min, shielded from the light. After a final rinsing step, DAPI dye was briefly applied briefly to the tissue sections, which were then dehydrated, sealed, and examined under light microscopy. For cellular immunofluorescence, cells were initially cultured in 48-well plates until the intervention concluded. Samples were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% PBST (PBS containing 0.1% Triton X-100 [9002-93-1, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China]), and blocked with 1% bovine serum albumin (BSA, GC305006, Servicebio). Subsequently, cells were treated with anti-CCR2 and anti-CCL2 antibodies, incubated overnight at 4 °C, washed, and exposed to fluorescent secondary antibodies in the dark for 30 min. After additional washing, 4${}^{\prime }$,6-diamidino-2-phenylindole (DAPI) stain was applied for 10 min, followed by a final rinse and examination under an optical microscope.

Human monocytic leukemia cells were sourced from Wuhan Punosai Life Science and Technology Co., Ltd. All cell lines underwent validation by STR profiling and tested negative for mycoplasma contamination. Cells were cultured in a humidified incubator at 37 °C with 5% CO₂. Initially, THP-1 cells were induced into M0 adherent morphology by treating them with 100 ng/mL phorbol 12-myristate 13-acetate (PMA, P8139, Sigma-Aldrich, Darmstadt, Germany) for 48 hours. The cells were then divided into four groups for different interventions: NC group, LPS group (100 ng/mL), CCL2 group (100 ng/mL), and LPS+CCL2 group, with LPS (100 ng/mL) serving as the positive control. Finally, cell lysates analyzed using RT-qPCR and Western blotting to assess the expression of CCR2 and CCL2, supplemented by immunofluorescence.

The THP-1 cells were seeded at a density of 1 $\times$ 10⁶ cells per well in a six-well plate and treated with PMA (Sigma) for 48 h to induce differentiation into M0 macrophages. To suppress CCR2 expression, three siRNAs provided by a commercial company (Hanbio Biotechnology Co.Ltd, Shanghai, China) were used following their transfection instructions. Transfection efficiency was confirmed initially (Supplementary Fig. 1), and subsequently, one of the siRNAs was selected for further experiments (5${}^{\prime }$-CAGGAAUCAUCUUUACUAATT-3${}^{\prime }$; 5${}^{\prime }$-UUAGUAAAGAUGAUUCCUGTT-3${}^{\prime }$). The sequence of si-NC is (5${}^{\prime }$-UCUCCGAACGUCACGUTT-3${}^{\prime }$; 5${}^{\prime }$-ACUGACACGUUCGGAGAATT-3${}^{\prime }$). Following treatment with 100 ng/mL PMA for 48 h, siRNA was used to silence CCR2 expression, and the cells were then randomly divided into four groups: NC group, si-NC group, si-NC+CCL2 group, and si-CCR2+CCL2 group for transfection. After completing the transfection, cells were further treated with CCL2 (100 ng/mL). Finally, subsequent experiments were conducted.

The concentration of CCL2 in rat serum and levels of Interleukin-12A (IL-12A), IL-6, and IL-1$\beta$ in cell supernatants were measured using enzyme-linked immunosorbent assay (ELISA) kits (GEH0009, GEH0001, GEH0002, Servicebio, Wuhan, China). Each sample was tested in triplicate, and the absorbance of each well at 450 nm was measured using an enzyme reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer’s protocol.

The number of animals used in the in vivo experiments was denoted as “n”. For the in vitro investigations, “n” represented the number of experiments conducted. Statistical analyses were performed using GraphPad Prism 9.5 (GraphPad Software, San Diego, CA, USA) and SPSS 27 (IBM-SPSS Statistics, Chicago, IL, USA). Data were expressed as mean $\pm$ SEM, and differences among three or more groups were analyzed using ANOVA. Statistical significance was set at p 0.05.

The co-expression of CD68, CCR2, and COL1A revealed by immunofluorescence is shown in (Supplementary Fig. 2A). CCL2 was also expressed in leukocytes marked with CD45, endothelial cells marked with CD31, and fibroblasts marked with s100a4 (Supplementary Fig. 2B). These findings provide evidence for the presence of CCR2⁺ macrophages in the valves of RHD patients, notably in close proximity to fibrin. The expression of CCL2 on endothelial cells and fibroblasts suggests a potential mechanism for recruiting CCR2⁺ macrophages from the bloodstream to the valve site.

Key observations included a notable increase in the infiltration of inflammatory cells within RHD rat valves, as evidenced by HE stains (Fig. 1A), and exacerbation of valve fibrosis in the RHD group, observed in Sirius red polarized light images (Fig. 1B). Remarkably, these adverse changes were significantly mitigated following administration of Rs-504393. Analysis via RT-qPCR (Fig. 2A) and western blot (Fig. 2C) in RHD rats revealed heightened activation of the CCL2/CCR2 axis, elevated levels of pro-inflammatory and pro-fibrotic cytokines (TNF-$\alpha$, IL-6, TGF-$\beta$, CCL2, CCR2, CD68, COL1A1, and COL3A1), and increased expression offibrotic markers such as COL1A, COL3A, and $\alpha$-SMA. Treatment with Rs-504393 effectively attenuated these alterations. These findings underscore the activation of the CCL2/CCR2 axis in RHD pathology. Moreover, CCR2 antagonists demonstrated therapeutic efficacy by effectively blocking the CCL2/CCR2 axis, thereby ameliorating inflammatory infiltration and valvular fibrosis in RHD rats. Importantly, there were no statistically significant differences in weight changes observed among the experimental groups.

Fig. 1.

Pathological staining in each rat group. (A) Inflammatory changes in the valves of the NC group, RHD group, and RHD+Rs-504393 treatment group were observed through HE staining, with images captured at the original magnification of $\times$400, scale bar = 50 µm, n = 6. (B) Upper Image: Sirius Red staining captured under polarized light displays the expression of type I collagen fibers (COL1A, red or yellow light) and type III collagen fibers in the NC group, RHD group, and RHD+Rs-504393 treatment group, with images captured at the original magnification of $\times$400, scale bar = 50 µm, n = 6. Lower Image: Statistical data of the COL3A/COL1A ratio for each group, where a higher ratio indicates a greater degree of fibrosis. Data are presented as mean $\pm$ standard deviation. ***p 0.001. NC, negative control; RHD, rheumatic heart disease; HE, hematoxylin-eosin staining.

Fig. 2.

Changes in the expression levels of pro-inflammatory cytokines, fibrosis markers, and macrophage markers in valve tissues after Rs-504393 treatment. (A) RT-qPCR was employed to detect mRNA levels of TNF-$\alpha$, IL-6, TGF-$\beta$, CCL2, CCR2, CD68, COL1A1, and COL3A1 in valve tissues. (B) CCL2 levels in rat serum were assessed using ELISA. (C) Upper panel: Western blotting analysis of the expression levels of CCL2, CCR2, COL1A1, COL3A1, and $\alpha$-SMA in valve tissues. Lower panel: Relative expression levels in each group were analyzed using ImageJ. Data are presented as mean $\pm$ SD, n = 6. ns: Not Statistically Significant, *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. RT-qPCR, Quantitative reverse transcription polymerase chain reaction; TNF-$\alpha$, tumor necrosis factor-$\alpha$; IL-6, Interleukin 6; TGF-$\beta$, Transforming growth factor-$\beta$; CCL2, C-C motif chemokine ligand 2; CCR2, C-C chemokine receptor type 2; COL1A1, collagen type I alpha 1; COL3A1, collagen type III alpha 1; $\alpha$-SMA, $\alpha$-smooth muscle actin; ELISA, enzyme-linked immunosorbent assay.

Protein blot analysis (Fig. 2C), ELISA (Fig. 2B), RT-qPCR (Fig. 2A) and immunohistochemistry (Fig. 3) conducted on the valves of RHD rats showed a significant upregulation in CCL2 expression and a notable increase in macrophage numbers compared to controls. Treatment with Rs-504393 resulted in a marked reduction in the total count of CD68-labeled macrophages, specifically decreasing both CCR2⁺ and M1-type macrophages. Statistical analysis of the immunohistochemistry showed a minimal change in the number of M2-labeled macrophages, potentially attributable to the acute nature of this experimental model. These findings collectively suggest that Rs-504393 treatment attenuates inflammation and fibrosis in rheumatic rat valves by reducing the infiltration of CCR2⁺ macrophages.

Fig. 3.

Immunohistochemistry reveals infiltration of different macrophage subtypes in each group. Upper Panel: Immunohistochemical expression of CCL2, CD68, CCR2, iNOS, and CD206 in the NC group, RHD group, and RHD+Rs-504393 treatment group. Images were captured at the original magnification of $\times$400, scale bar = 50 µm. Lower Panel: Relative positive areas analyzed through Image-pro plus 6.0 (Media Cybernetics, Rockville, MD, USA). Data are presented as mean $\pm$ standard deviation, n = 6. ns: Not Statistically Significant, ***p 0.001, ****p 0.0001. iNOS, inducible nitric oxide sythase.

Various concentrations of CCL2 were applied to intervene on M0 macrophages for 24h, and the optimal concentration that maximized CCR2 expression was determined to be 100 ng/mL based on the CCK8 assay (Fig. 4A) and RT-qPCR experiment (Fig. 4B). RT-qPCR (Fig. 4C) and flow cytometry (Fig. 4D) demonstrated elevated levels of inflammatory factors (CCL2, IL-6, TNF-$\alpha$, CCR2, and CD86), along with increased expression of CCR2 and the M1 macrophage marker CD86 in both the LPS and CCL2 groups compared to levels observed in the NC group. Furthermore, CCL2 amplified the pro-inflammatory and M1 polarization effects induced by LPS. Immunofluorescence analysis (Fig. 4E,F and Supplementary Fig. 3A,B) corroborated these findings, showing consistent increases in CCR2 and CCL2 expression. Specifically, fluorescence intensity of both CCR2 and CCL2 was higher in the LPS and CCL2 groups compared to the NC group. Moreover, fluorescence intensity was further elevated in the LPS+CCL2 group compared to both the LPS alone and CCL2 alone groups.

Fig. 4.

Expression of cytokines and M1 phenotype after intervention with recombinant CCL2 (100 ng/mL) and LPS (100 ng/mL) in THP-1 cells. (A) CCK8 assay assessing the impact on cell viability when THP-1 cells in the M0 state were intervened with different concentrations of recombinant CCL2 for 24 hours, n = 4. (B) RT-qPCR measuring the expression levels of CCR2 in M0 state THP-1 cells intervened with different concentrations of recombinant CCL2, n = 4. (C) RT-qPCR detecting the mRNA levels of CCL2, IL-6, TNF-$\alpha$, CCR2, and CD86 in THP-1 cells intervened with recombinant CCL2 (100 ng/mL), LPS (100 ng/mL), and CCL2+LPS, n = 4. (D) Flow cytometry measuring the average fluorescence intensity of CD86 in each intervention group, n = 4. (E) Immunofluorescence showing the fluorescence intensity of CCR2 (green light) in each intervention group, images were captured at the original magnification of $\times$200, scale bar = 200 µm, n = 3. (F) Immunofluorescence showing the fluorescence intensity of CCL2 (green light) in each intervention group, images were captured at the original magnification of $\times$200, scale bar = 200 µm, n = 3. Data are presented as mean $\pm$ SD. ns: Not Statistically Significant, *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001. LPS, lipopolysaccharide; MFI, mean fluorescence intensity; CCK8, cell counting kit-8.

RT-qPCR analysis (Fig. 5A) showed that the mRNA expression levels of signature indicators of M1-type macrophages (including IL-6, TNF-$\alpha$, and CD86) and CCL2/CCR2 were increased in the si-NC+CCL2 group compared to the si-NC group. Conversely, the mRNA expression levels of IL-6, TNF-$\alpha$, CD86, and CCL2/CCR2 were significantly reduced in the si-CCR2+CCL2 group compared to the si-NC+CCL2 group. Immunofluorescence analysis (Fig. 5B,C and Supplementary Fig. 3C,D) further confirmed that the fluorescence intensity of CCL2 and CCR2 was lower in the si-CCR2+CCL2 group compared to the si-NC+CCL2 group.

Fig. 5.

Expression of inflammatory cytokines and M1 phenotype molecules after knockdown of CCR2 in THP-1 cells using siRNA, followed by intervention with CCL2. (A) RT-qPCR detecting the mRNA levels of CCL2, IL-6, TNF-$\alpha$, CCR2, and CD86 in THP-1 cells with siRNA-CCR2 knockdown intervened with recombinant CCL2 (100 ng/mL), n = 4. (B) Immunofluorescence showing the fluorescence intensity of CCR2 (green light) in each intervention group, images were captured at the original magnification of $\times$200, scale bar = 200 µm, n = 3. (C) Immunofluorescence showing the fluorescence intensity of CCL2 (green light) in each intervention group, images were captured at the original magnification of $\times$400, scale bar = 200 µm, n = 3. Data are presented as mean $\pm$ SD. ns: Not Statistically Significant, *p 0.05, **p 0.01, ***p 0.001. si-NC, small interfering-negative control; si-CCR2, small interfering-C-C chemokine receptor type 2.

The ELISA results from cell supernatants showed elevated levels of inflammatory factors such as IL-12A, IL-6, and IL-1$\beta$ in the supernatants of the si-NC+CCL2 group compared to the si-NC group. Conversely, after silencing CCR2, the levels of these inflammatory factors decreased in the supernatants of the si-CCR2+CCL2 group (Fig. 6A). This indicates that silencing CCR2 expression in macrophages significantly inhibits the pro-inflammatory effect of CCL2 protein on macrophages. Additionally, protein blotting experiments revealed increased expression levels of CCL2, CCR2, and CD86 in macrophages of the si-NC+CCL2 group compared to the si-NC group. Following CCR2 silencing, the expression of these proteins was significantly reduced in the si-CCR2+CCL2 group compared to the si-NC+CCL2 group (Fig. 6B). These results demonstrate that silencing CCR2 expression in macrophages not only prevents macrophage polarization towards M1 but also reduces the expression of CCL2/CCR2 proteins.

Fig. 6.

Silencing CCR2 in THP-1 cells followed by treatment with CCL2 was conducted to assess the levels of inflammatory cytokines in the cell supernatants and the expression of CCL2, CCR2, and CD86 in THP-1 cells. (A) ELISA was used to measure the levels of IL-12A, IL-6, and IL-1$\beta$ in the cell supernatants of each group, n = 4. (B) Western blotting was performed to analyze the protein expression levels of CCL2, CCR2, and CD86 in each group, n = 3. Data are presented as mean $\pm$ SD. ns: Not Statistically Significant, *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001.