Bone marrow mesenchymal stem cell exosome-derived miR-223 regulated cellular pyroptosis of macrophage in osteomyelitis through regulating LACC1

Abstract

Osteomyelitis (OM) is a severe bone infection characterized by inflammation and tissue damage. Macrophages play a crucial role in the inflammatory response during OM, and exosomes derived from bone marrow mesenchymal stem cells (BMSCs) have been proposed as potential therapeutic agents. Previous studies suggest that miR-223, a microRNA involved in inflammatory processes, is dysregulated in OM. This study investigates the role of BMSCs-derived exosomes carrying miR-223 in regulating macrophage pyroptosis, a form of programmed cell death triggered by inflammation. Blood samples were collected from OM patients and control subjects to assess miR-223 expression. BMSCs were treated with LPS to simulate the OM environment. Exosomes were extracted from miR-223 overexpressing BMSCs and characterized. The effects of these exosomes on macrophage survival, apoptosis, and pyroptosis were assessed through CCK-8 assays, flow cytometry, TUNEL staining, ELISA, and western blotting. The miR-223-mediated regulation of Caspase-1 and LACC1 expression was evaluated using specific inhibitors and gene expression analysis. miR-223 expression was significantly reduced in OM patients and in LPS-treated BMSCs. BMSCs-derived exosomes carrying miR-223 (miR-223 exo) enhanced macrophage viability, reduced apoptosis, and mitigated LPS-induced pyroptosis by targeting the NLRP3 inflammasome and Caspase-1 expression. Co-treatment with miR-223 inhibitors and Caspase-1 inhibitors showed that miR-223 regulated macrophage survival and inflammation through Caspase-1 modulation. Further investigation revealed that miR-223 targeted LACC1 to alleviate macrophage pyroptosis, with LACC1 overexpression reversing the protective effects of miR-223. BMSCs-derived exosomes carrying miR-223 play a protective role in OM by regulating macrophage pyroptosis and inflammation. This effect is mediated through the modulation of Caspase-1 and LACC1 expression, highlighting the potential of miR-223-based therapies for OM treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-22511-3.

Introduction

Osteomyelitis (OM) is a severe orthopedic infection marked by inflammation, bone destruction, and progressive bone loss¹. Clinically, diagnosis relies on a combination of radiographic imaging (e.g., MRI, CT), microbiological culture of infected tissue, histopathology, and serological markers such as CRP, ESR, and procalcitonin^2,3. However, due to its diverse clinical manifestations and overlap with other bone disorders, early and accurate diagnosis remains challenging.Current treatments typically involve prolonged systemic antibiotic therapy alongside surgical debridement to remove necrotic bone⁴. In chronic or recurrent cases, multiple surgeries or even amputation may be required. The rise of antibiotic resistance and bacterial biofilm formation further complicate treatment and contribute to high relapse rates and poor outcomes⁵.Recent advances in basic research have shed light on the cellular and molecular mechanisms of OM. Macrophages and neutrophils are key immune cells involved in both bacterial clearance and inflammation. Studies have highlighted the roles of pro-inflammatory cytokines, inflammasome activation, and pyroptosis in disease progression⁶. Meanwhile, emerging regenerative strategies—such as mesenchymal stem cell (MSC)-based therapies, biomaterials, and exosome delivery—show promise in modulating inflammation and promoting bone repair⁷. These findings provide a foundation for developing new immunomodulatory and regenerative treatments beyond traditional antibiotics.

The pathogenesis of OM is not solely a consequence of bacterial invasion but is also profoundly influenced by the host immune response. Among immune cells, macrophages play a central and dual role—acting as early defenders against infection and as key mediators of inflammatory bone damage^8,9. Upon pathogen recognition, macrophages release pro-inflammatory cytokines and coordinate the recruitment of other immune cells. However, prolonged or dysregulated macrophage activation can exacerbate local inflammation, contributing to osteolysis, necrosis, and impaired bone remodeling^10,11. Recent evidence has highlighted macrophage pyroptosis, a caspase-1-dependent form of inflammatory programmed cell death, as a critical process in the amplification of bone inflammation. Pyroptotic macrophages release large quantities of IL-1β and IL-18, perpetuating cytokine storms and promoting tissue damage^12–14. Thus, modulating macrophage pyroptosis has emerged as a promising therapeutic strategy in OM.

Bone marrow-derived MSCs (BMSCs), have attracted increasing attention for their immunomodulatory, regenerative, and anti-inflammatory capabilities. A growing body of evidence suggests that these effects are largely mediated by BMSC-derived exosomes. Exosomes are nanoscale extracellular vesicles (30–100 nm) secreted by various cell types, including MSCs, and serve as important intercellular communicators by transferring bioactive molecules such as proteins, mRNAs, and microRNAs (miRNAs) to recipient cells^15,16.Several studies have demonstrated that BMSC-derived exosomes can regulate macrophage polarization, suppress excessive inflammation, and promote tissue repair^17–19. For instance, exosomal miR-146a from BMSCs has been shown to inhibit inflammatory responses by targeting TRAF6 and IRAK1 in colitis²⁰.MiR-21 in BMSC-derived exosomes has been reported to promote M2 macrophage polarization²¹.miR-223—a miRNA well known for its role in regulating inflammation and myeloid cell function²².However, its role in OM has not been studied. Furthermore, the effect of BMSC-derived exosomal miR-223 on macrophage pyroptosis in OM remains unexplored.Our study addresses this gap by investigating how BMSC exosomal miR-223 regulates macrophage pyroptosis via LACC1 in an OM-like inflammatory environment.

Herein, we employed lipopolysaccharide (LPS) to induce a controlled inflammatory response in vitro, as it is widely recognized as a classical and reliable stimulant for macrophage activation. In our study, LPS was not intended to directly replicate the full pathological process of OM, but rather to mimic key inflammatory features by involving in activating macrophages. LPS, a major component of the outer membrane of Gram-negative bacteria, activates immune responses primarily through the Toll-like receptor 4 (TLR4) signaling pathway, leading to the activation of NF-κB and the release of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6^23,24.Although Staphylococcus aureus, a Gram-positive pathogen, is the predominant cause of OM, the downstream inflammatory pathways it triggers—such as inflammasome activation and pyroptosis—are not pathogen-specific and can also be effectively induced by LPS in vitro¹³. Furthermore, recent studies have shown that LPS stimulation affects not only macrophages but also BMSCs, modulating their immunoregulatory properties and altering their exosomal content, thereby supporting the relevance of LPS in co-culture systems investigating BMSC–macrophage interactions^25–27.It is important to note that while LPS cannot fully recapitulate the complex and multifactorial nature of bacterial OM in vivo, it offers a controlled and reproducible model for examining specific components of the host immune response, particularly macrophage activation and pyroptosis. Therefore, in this study, LPS was employed as a well-characterized pro-inflammatory agent to establish an in vitro inflammatory microenvironment, allowing us to explore the regulatory role of BMSC-derived exosomal miR-223 in macrophage pyroptosis via LACC1.

In this study, we found that BMSC-derived exosomes could interact with LPS-induced macrophages. In an LPS-simulated in vitro inflammatory model of OM, exosomes mitigated macrophage pyroptosis by delivering miR-223, thereby improving macrophage viability and providing a potential therapeutic approach for OM.

Methods and methods

Specimen collection

A total of 10 patients with OM and 10 children with traumatic fractures without infection (as controls) admitted to the Children’s Hospital of Soochow University from February 2023 to February 2024 were included. The OM group consisted of 6 males and 4 females, with an average age of 93.00 ± 32.35 months, while the fracture group included 6 males and 4 females, with an average age of 78.70 ± 24.30 months. All OM patients met the diagnostic criteria for OM, and the predominant pathogen was Staphylococcus aureus. None of the patients in either group had significant underlying conditions such as diabetes or immune deficiencies.All participants provided informed consent, and the Ethics Committee of the Children’s Hospital of Soochow University approved the study (approval number: 2024CS075). For all enrolled individuals, 10 mL venous blood samples were obtained. To extract the plasma, blood is spun at 1600 g for 10 min, the supernatant is collected in a new tube, and then spun again at 12 000 g for 10 min to remove any cellular debris. Plasma is processed or cryopreserved (-80 °C) within 6 h of blood collection. Following the manufacturer’s instructions, thawed plasma samples were used to extract and purify miRNA for RT-qPCR assay using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany).

Cell culture

Human bone marrow mesenchymal stem cells (BMSCs) and human bone marrow-derived macrophages (CP-H186) were purchased from the Chinese Academy of Medical Sciences, and mouse mononuclear macrophages (RAW264.7) were purchased from American type culture collection (ATCC, Manassas, VA, USA). BMSCs and RAW264.7 were cultured in DMEM (Gibco, Grande Island, USA) supplemented with 10% FBS (Gibco, Grande Island, USA), 500 U/ml penicillin, and 500 µg/ml streptomycin (Invitrogen, Shanghai, China). While CP-H186 was cultured in 1 ml of DMEM F12 growth media (Gibco, Grande Island, USA) containing l-glutamine (2 mM), penicillin (100 units/ml), streptomycin (0.1 mg/ml), 10% FBS and 10% L-Cell Media (LCM). All cells were cultivated at 37 °C in an incubator with 5% CO2.

Cell transfection

MiR-223 inhibitors, NC inhibitors, miR-223 mimics, disordered NC mimic, LACC1 plasmid vector and Blank plasmid vector (NC-OE) were constructed by GeneChem (Shanghai, China). The sequences were listed in Supplementary Table 1. BMSCs were inoculated at 1.5 × 10⁵ cells/well in 12-well plates and then transfected with disordered NC mimic (50 nM) and miR-223 mimic (50 nM) using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocols. For RAW264.7 and CP-H186 cells, after treated with LPS (1 µg/ml) for 2 h, NC inhibitors (50 nM), miR-223 inhibitors (50 nM), miR-223 mimics (50 nM), NC mimic (50 nM), LACC1 plasmid vector (50 nM) and NC-OE (50 nM) were transfected into RAW264.7 and CP-H186 cells using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocols.

Exosome purification

Exosomes, including NC exo or miR-223 exo, were both isolated. The exosome isolation protocol was based on a previous exosome isolation method¹. BMSCs were cultured with complete DMEM containing 10% FBS (depletion of exosomes by ultracentrifugation) for 2 days. Then, exosomes were isolated from the conditioned medium of BMSCs transfected with NC mimic (NC exo) and miR-223 mimic (miR-223 exo) by ultracentrifugation, and nearly 50 ml of conditioned medium was collected. Initial spins consisted of 1000 g × 10 min, 2000 g × 10 min, and 10,000 g × 30 min. The supernatant was retained and centrifuged at 100,000 g for 70 min and then resuspended in PBS. The samples were then centrifuged at 100,000 g for an additional 70 min. The final precipitate was resuspended in 200 µl of PBS and stored at -80 °C. The final precipitate was then centrifuged at 100,000 g for an additional 70 min. Protein concentrations were assayed using the Bicinchoninic Acid (BCA) Protein Assay Reagent (Pierce, Rockford, USA) with an exosome concentration of approximately 1 mg/ml.

Transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA)

The morphology of exosomes was assessed by TEM. Drop 20 µl of exosome suspension sample onto the carbon film copper grid and leave it for 3–5 min, then use filter paper to absorb the excess liquid. Another drop of 2% phosphotungstic acid was placed on a carbon-supported membrane copper mesh for 1–2 min, excess liquid was blotted off with filter paper, air dried, and the sample was examined by TEM (TEM, HT7700, Hitachi, Tokyo, Japan).NanoSight NS300 (Cambridge, MA, USA) was used to determine the size and distribution of exosomes. The instrument was calibrated using 100 nm diameter polystyrene latex microspheres (Malvern Instruments Ltd, Malvern, UK). Exosomes were then diluted 1:1000 in PBS to a final volume of 1 ml and loaded into a 1 ml syringe for assay.

Exosome uptake experiments

After being extracted, the exosomes were co-cultured with RAW264.7 and CP-H186 cells for 48 h after being labeled with 1 µM PKH26 (red; Sigma-Aldrich, Waltham, MA, USA). Following treatment, the cells underwent two PBS washes before being fixed in 4% paraformaldehyde. Following DAPI staining, the cellular uptake of the exosomes was seen using Zeiss fluorescence microscopy (Jena, Germany).

Cell processing

To mimic inflammation-induced OM in vitro, we seeded BMSCs into 6-well plates at a density of 2.5 × 10⁵ for 24 h. Then, the cells were treated with 1 µg/mL LPS (Invitrogen, Waltham, MA, USA) for another 24 h^2,3.For the inflammation model, we used LPS (Invitrogen, Waltham, MA, USA) to treat CP-H186 and RAW264.7 to cause the inflammation model for 24 h. 10 µl NC exo or miR-223 exo (100 µg/mL, approximately 1 µg) were added after 2 h of LPS stimulation, and the experiments were terminated for testing after 24 h of treatment. Groups administered the caspase-1 inhibitor AC-YVAD-CMK (Sigma-Adrich, St. Louis, MO, USA) at a dose of 50 µM were present in the experiment and treated for 2 h.

Cell counting kit 8 (CCK-8)

CP-H186 and RAW264.7 cells from different groups were inoculated into 96-well plates at a density of 1 × 10⁴ cells /well and incubated for 24 h to allow cells to attach to the surface. Next, for one to three days, a Cell Counting Kit 8 (CCK-8; Beyotime Institute of Biotechnology, Beijing, China) solution was added. The OD values were measured at 450 nm using a microplate reader (M2009PR, Tecan Infinite, Australia) at different time points.

Flow cytometry

According to the manufacturer’s instructions, apoptosis was detected using the Annexin-V/PI Apoptosis Assay Kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA). CellQuest software (version 7.6.1; Flow Jo LLC) was used to evaluate the data that were obtained using a FACScan flow cytometer (BD Biosciences, New Jersey, USA).

Tunel staining

Tunel (terminal deoxynucleotidyl transferase) was applied to determine CP-H186 and RAW264.7 apoptosis. The results were observed using a Tunel assay kit (T2195, Solarbio, Beijing, China) and using fluorescence microscopy (Jena, Germany). Experiments followed the manufacturer’s protocol.

ELISA

Lysates were obtained by lysing CP-H186 and RAW264.7 cells using RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA). The levels of levels of IL- 1β (#SEKH-0002), TNF-α (#SEKH-0047), and IL-18 (#SEKH-0028) were detected using an ELISA kit (Solarbio, Beijing, China) according to the manufacturer’s protocol. The results were recorded at OD 450 nm absorbance using a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Immunofluorescence (IF)

CP-H186 and RAW264.7 cells from different groups were cultured in 6-well plates, fixed in 4% formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. After blocking frozen cells with 5% BSA for 30 min at room temperature, they were treated with an antibody against Caspase-1 (1:100 dilution; #PA5-87536, Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C overnight. After incubation with secondary antibody goat anti-rabbit-Fluor594 (#S0006, Affinity, Changzhou, China) for one hour at 37 °C, the cells were re-stained using DAPI (Sigma-Aldrich, St. Louis, MO, USA) for ten minutes. Next, cells were observed using fluorescence microscopy (Jena, Germany).

Dual luciferase reporter assays

Wild-type LACC1 (LACC1 3’UTR-WT) and the 3’-UTR mutant plasmid of LACC1 (LACC1 3’UTR-MUT) were synthesized by GeneChem (Shanghai, China). The construction method involved screening the binding sites between miR-223 and LACC1 using TargetScanHuman (https://www.targetscan.org/vert_80/). A 59-base pair region flanking the identified binding site was selected, and the corresponding fragment was inserted into the pMIR-REPORT Luciferase vector using homologous recombination. The resulting constructs were LACC1 3′UTR (WT) and LACC1 3′UTR (MUT). The WT sequence was: 5’-TTGTATAACTGCTTCCATGCCTCCTTCCAAACTGACTGCAAGAGAGAAATTTAGCTGTTT-3’. The MUT sequence was: 5’-TTGTATAACTGCTTCCATGCCTCCTGATCGCATGACTGCAAGAGAGAAATTTAGCTGTTT-3’. Subsequently, these vectors were co-transfected with NC mimics or miR-223 mimics into the cells using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA). Forty-eight hours after transfection, luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA).

Quantitative reverse transcription PCR(RT-PCR)

RNA was extracted using Trizol reagent (TaKaRa Biotech, Tokyo, Japan) according to the manufacturer’s protocol, and then reverse transcribed to cDNA using the miRNA Reverse Transcription Kit. miR-223 expression was determined using the Hairpin-itTM miRNA qPCR kit (GenePharma, Shanghai, China) and the ABI PRISM 7000 tool (Applied Biosystems, Waltham, MA, USA). U6 was used as an endogenous control. The primers used in this study were as follows: miR-223 (human): forward, 5′-AGCTGGTGTTGTGAATCAGGCCG-3′, and reverse 5′-TGGTGTCGTGGAGTCG-3′; U6 (human): forward, 5′-CTCGCTTCGGCAGCACA-3′, and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; miR-223 (mouse): forward, 5′-TGTCAGTTTGTCAAA-3′, and reverse 5′-CAGTGCGTGTCGTGGAGT-3′; U6 (mouse): forward, 5′-GCTTCGGCAGCACATATACTAAAAT-3′, and reverse, 5′-CGCTTCAGAATTTGCGTGTCAT-3′; LACC1 (human): forward, 5′-CTCACGCTGGTTGGAAAG-3′, and reverse 5′-GAAGATTATGAAATGCCTC-3′; GAPDH (human): forward, 5′-ATCATCAGCAATGCCTCC-3′, and reverse, 5′-CATCACGCCACAGTTTCC-3; LACC1 (mouse): forward, 5′-AAAATGTTCGCAGGTTGG-3′, and reverse 5′-ATTCAGGCTCCTTCTTCC-3′; GAPDH (mouse): forward, 5′-CCTTCCGTGTTCCTACCC-3′, and reverse, 5′-AAGTCGCAGGAGACAACC-3.

Relative expression was calculated from the cycling threshold (Ct) using the following equation: relative expression = 2^{−(SΔCt−CΔCt)}.

Western blot (WB)

Using RIPA protease inhibitor lysis buffer, total protein was extracted from exosomes and cells. After that, the samples were centrifuged for 30 min at 4 °C at 12,000 rpm to exclude any samples that couldn’t be lysed. Protein samples were electrophoretically transferred to polyvinylidene difluoride membranes after being separated on SDS-PAGE gels. In PBS Tween-20 (PBST) buffer, 5% skim milk was used to block the membranes. Primary antibodies were incubated on the membranes at 4 °C. Then, the membrane was washed and incubated with horseradish peroxidase secondary antibody (1:5000, abcam, Cambridge, UK) at room temperature for 2 h. GAPDH was used as an internal reference. The bands were visualized using ECL (Beyotime Biotechnology, Shanghai, China).Primary antibodies used in the study is as follows: anti- CD63 (1:1000, #ab271286, Abcam, Cambridge, UK), anti-TSG101 (1:2000, # ab125011, Abcam, Cambridge, UK), anti-GM130 (1:1000, #ab52649, Abcam, Cambridge, UK), anti-NLRP3 (1:1000, #ab263899, Abcam, Cambridge, UK), anti-Cleaved GSDMD (1:1000, #36425, CST, Danvers, Massachusetts, USA), anti-Cleaved caspase-1 (1:1000, #4199, CST, Danvers, Massachusetts, USA), anti- LACC1 (1:2000, #27895-1-AP, Proteintech, Chicago, IL, USA) and anti-GAPDH (1:3000, #AF7021, Affinity, Changzhou, China).

Statistical analysis

All data were analyzed using GraphPad Prism software 8.0 (San Diego, CA, USA). All experiments were repeated 3 times and expressed as mean ± standard deviation (SD). Comparisons between two groups were analyzed for significance by the Student’s t-test, while multiple comparisons were tested for statistically significant differences by one-way ANOVA with Dunnett’s multiple comparison test. P < 0.05 was considered significant.

Results

MiR-223 is underexpressed in OM and its exosomes-derived BMSCs

To explore the abnormally expressed miRNAs in OM, we first collected blood samples from 10 OM patients and 10 patients with traumatic fractures and without infection (as controls)( Fig. 1A). We found that miR-223 expression was decreased in OM patients than in controls (Fig. 1B). Since exosomes can carry miRNAs for intercellular genetic information interaction, we speculated that BMSCs-derived exosomes might carry miR-223 to play a role in the OM microenvironment. Therefore, we treated BMSCs with LPS to mimic an OM environment. However, it was found that miR-223 was lowly expressed in LPS-treated BMSCs (Fig. 1C). At the same time, we found no significant difference in miR-223 expression between BMSCs and macrophages under normal conditions (Figure S7). It suggested that the absence of miR-223 might aggravate the OM process while increasing miR-223 expression might alleviate OM. Accordingly, we constructed BMSCs cells that overexpressed miR-223 by transfecting mimics. Then, exosomes extracted from miR-223 overexpressed BMSCs (miR-223 exo), normal BMSCs (NC exo) by gradient centrifugation. Unsurprisingly, RT-qPCR results found that miR-223 was highly expressed in miR-223 exo compared to NC exo (Fig. 1D).Subsequently, we also characterized both exosomes. TEM was used to analyze the morphology of the exosomes, and it was found that both exosomes were round and had a double membrane structure (Fig. 1E). In addition, NTA showed a size distribution of both exosomes from 100 to 200 nm (Fig. 1F). The exosome-positive markers CD63 and TSG101 were expressed in both exosomes, while there was no expression in BMSCs lysates. None of the exosome-negative marker GM130 was expressed in exosomes, only in lysates (Fig. 1G).

Fig. 1.

Identification and characterization of BMSCs derived exosomes. (A) The schematic diagram of this study. (B) The mRNA expression of miR-223 in OM and control blood samples was analyzed by RT-qPCR. Relative mRNA levels were compared in two groups with Student’s t-test, **P < 0.01. (C) The mRNA expression of miR-223 in LPS treated BMSCs was analyzed by RT-qPCR. Relative mRNA levels were compared in two groups with Student’s t-test, ***P < 0.001. (D) The mRNA expression of miR-223 in miR-NC exo and miR-223 exo was analyzed by RT-qPCR. Relative mRNA levels were compared in two groups with Student’s t-test, **P < 0.01. (E) Morphology of exosomes under TEM. Scale bar: 100 nm. (F) Histogram of the diameter and concentration of exosomes determined by NTA. (G) The protein expression of CD63, TSG101 and GM130 in exosomes determined by WB. (H) IF staining for exosomes uptake by macrophages. Scale bar: 100 μm. MiR-NC exo means exosomes extracted from BMSCs transfected with disordered NC mimic; miR-223 exo means exosomes extracted from BMSCs transfected with miR-223 mimics. LPS means BMSCs treated with LPS.

To further investigate the biological role of exosomes derived from BMSCs, it is important to assess whether these exosomes can be internalized by immune cells such as macrophages, which play a key role in inflammation and tissue repair. Our results found that exosomes secreted by BMSC could be internalized by macrophages CP-H186 and RAW264.7. The exosomes were labeled by PKH26 staining and then co-cultured with CP-H186 and RAW264.7, and it was found that the exosomes could be internalized into both macrophages and distributed in the cytoplasm (Fig. 1H). The above results indicated that the two BMSCs-derived exosomes were successfully constructed and could be taken up and internalized by macrophages, and these exosomes could be used for the next step of research.

BMSCs-derived exosomes inhibited macrophage proliferation and pyroptosis by carrying miR-223

We further explored the role of BMSC-derived exosomes in the OM microenvironment by utilizing LPS-treated macrophages to model the immune response. The results demonstrated that co-culturing macrophages with either miR-223 exosomes or NC exosomes led to an increase in miR-223 mRNA expression, with miR-223 exosomes having a more pronounced effect (Fig. 2A). CCK-8 assays revealed that miR-223 exo improved macrophage viability more effectively than NC exo after LPS treatment (Fig. 2B). Flow cytometry and TUNEL staining showed that miR-223 exo reduced LPS-induced apoptosis in macrophages, with a more pronounced effect compared to NC exo (Fig. 2C-E). These results suggest that BMSCs-derived exosomes, particularly miR-223 exo, enhance macrophage survival by reducing apoptosis.

Fig. 2.

BMSCs-derived exosomes promoted macrophage proliferation and inhibited apoptosis by carrying miR-223. (A) The mRNA expression of miR-223 in LPS-induced macrophages treated with miR-NC exo or miR-223 exo was analyzed by RT-qPCR. Relative mRNA levels were compared in different groups by one-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001. (B) The cell viability of LPS-induced macrophages treated with miR-NC exo or miR-223 exo was detected by CCK-8 assay. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01. (C, D) Apoptosis of LPS-induced macrophages treated with NC exo or miR-223 exo was detected by flow cytometry. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. (E) Apoptosis of LPS-induced macrophages treated with N miR-NC exo or miR-223 exo was detected by Tunel, Scale bar: 100 μm. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. MiR-NC exo means exosomes extracted from BMSCs transfected with disordered NC mimic; miR-223 exo means exosomes extracted from BMSCs transfected with miR-223 mimics. LPS means macrophages (CPH-168 or RAW264.7) treated with LPS.

Cellular pyroptosis is a type of programmed cell death caused by inflammatory attack. Pyroptosis consists of a caspase-1-dependent process that leads to rapid cell lysis and release of inflammatory contents. Then we explored the effect of BMSCs-derived exosomes on pyroptosis. ELISA showed that LPS stimulated macrophages to release high levels of IL-1β, TNF-α, and IL-18, but NC exo and miR-223 exo reduced this release, with miR-223 exo being more effective (Fig. 3A). LPS also increased pyroptosis markers (cleaved Gasdermin D, cleaved Caspase-1, and NLRP3), but both exosome treatments reversed this effect, with miR-223 exo showing a stronger impact (Fig. 3B, Figure S1A,1B). Immunofluorescence confirmed that miR-223 exo reduced Caspase-1 expression compared to LPS or LPS + NC exo (Fig. 3C). These findings suggest that BMSCs-derived exosomes, particularly miR-223 exo, can reduce LPS-induced pyroptosis and inflammation.

Fig. 3.

BMSCs-derived exosomes inhibited macrophage pyroptosis by carrying miR-223. (A) The levels of inflammatory product IL- 1β, TNF-α, and IL-18 in cell lysates from LPS-induced macrophages treated with miR-NC exo or miR-223 exo was analyzed by ELISA. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. (B) The protein expression of Cleaved Gasdermin D, Cleaved Caspase-1, and NLRP3 in LPS-induced macrophages treated with miR-NC exo or miR-223 exo was analyzed by WB. (C) The expression of Caspase-1 in LPS-induced macrophages treated with miR-NC exo or miR-223 exo was analyzed by IF, Scale bar: 100 μm. MiR-NC exo means exosomes extracted from BMSCs transfected with disordered NC mimic; miR-223 exo means exosomes extracted from BMSCs transfected with miR-223 mimics. LPS means macrophages (CPH-168 or RAW264.7) treated with LPS.

BMSCs-derived exosomes inhibit the expression of molecules related to macrophage pyroptosis by carrying miR-223

It is well known that the activation of the NLRP3 inflammasome promotes cellular pyroptosis. In this study, we explored whether miR-223 exo alleviate macrophage pyroptosis by regulating the NLRP3 inflammasome. QS-21, an NLRP3 activator, was introduced into the study. We found that miR-223 exo restored cell viability reduced by LPS, while QS-21 reversed this effect (Figure S2A). Flow cytometry and TUNEL staining results revealed that miR-223 exo decreased LPS-induced macrophage apoptosis, whereas QS-21 increased apoptosis that was reduced by miR-223 exo (Figure S2B,2 C). ELISA results indicated that QS-21 enhanced the release of inflammatory factors that were diminished by miR-223 exo (Figure S2D). Additionally, pyroptosis proteins were downregulated by miR-223 exo, and QS-21 reversed this change (Figure S2E, S3A,3B). These findings suggested that BMSC exosomes carrying miR-223 ameliorated LPS-induced macrophage pyroptosis by modulating the activity of the NLRP3 inflammasome.

We have previously confirmed that BMSCs-derived exosomes can regulate Caspase-1 expression in macrophages, then we further investigated that this effect is mediated by miR-223 in the pyroptosis process.To investigate the role of miR-223 in macrophage pyroptosis, we co-treated LPS-induced macrophages with a miR-223 inhibitor and Caspase-1 inhibitor AC-YVAD-CMK. RT-qPCR showed that miR-223 inhibition reduced miR-223 expression, while Caspase-1 inhibition increased it, with co-treatment showing a stronger increase (Fig. 4A). MiR-223 knockdown reduced macrophage viability, while Caspase-1 inhibition increased it. However, co-treatment reversed the effect of Caspase-1 inhibition alone (Fig. 4B). Flow cytometry and TUNEL staining showed that miR-223 knockdown promoted apoptosis, while Caspase-1 inhibition reduced it; co-treatment increased apoptosis, which was reduced by Caspase-1 inhibition (Fig. 4C, D). These findings suggest miR-223 promotes macrophage proliferation and inhibits apoptosis via Caspase-1.We also examined the impact on inflammatory factor release. MiR-223 inhibition increased, while Caspase-1 inhibition decreased inflammatory release. Co-treatment led to higher release than Caspase-1 inhibition alone (Fig. 5A, B). Pyroptosis markers (Cleaved Gasdermin D, Caspase-1, NLRP3) were elevated by miR-223 inhibition, but co-treatment reversed this effect (Fig. 5C, S4A,4B). Caspase-1 fluorescence intensity was consistent with these findings, showing increased intensity with miR-223 knockdown and decreased with Caspase-1 inhibition (Fig. 5D). These results confirm that miR-223 also regulates the Caspase-1 expression of pyroptosis.

Fig. 4.

MiR-223 regulated macrophage proliferation and apoptosis through caspase1-dependent expression. (A) The mRNA expression of miR-223 in LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK analyzed by RT-qPCR. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. (B) The cell viability of LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was detected by CCK-8 assay. One-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001. (C) Apoptosis of LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was detected by flow cytometry. (D) Apoptosis of LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was detected by Tunel, Scale bar: 100 μm. MiR-223-IN means macrophages (CPH-168 or RAW264.7) transfected with miR-223 inhibitor; AC means macrophages (CPH-168 or RAW264.7) treated with AC-YVAD-CMK. LPS means macrophages (CPH-168 or RAW264.7) treated with LPS.

Fig. 5.

MiR-223 regulated macrophage pyroptosis through caspase1-dependent expression. (A, B) The levels of inflammatory product IL-1β, TNF-α, and IL-18 in cell lysates from LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was analyzed by ELISA. One-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001. (C) The protein expression of Cleaved Gasdermin D, Cleaved Caspase-1, and NLRP3 in LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was analyzed by WB. (D) The expression of Caspase-1in LPS-induced macrophages treated with miR-223 inhibitor or AC-YVAD-CMK was analyzed by IF, Scale bar: 100 μm. MiR-223-IN means macrophages (CPH-168 or RAW264.7) transfected with miR-223 inhibitor; miR-NC means macrophages (CPH-168 or RAW264.7) transfected with NC inhibitor; AC means macrophages (CPH-168 or RAW264.7) treated with AC-YVAD-CMK. LPS means macrophages (CPH-168 or RAW264.7) treated with LPS.

BMSCs-derived exosome miR-223 regulates macrophage pyroptosis by targeting LACC1

It is well known that miRNAs can bind to the 3’-UTR sites of downstream genes to regulate the transcription process of genes. We firstly predicted the binding site of miR-223 by TargetScan and found that miR-223 could bind to the 3’-UTR position of LACC1. Subsequently, we explored the expression of LACC1 in macrophages. The results showed that LPS induced a significant increase in mRNA and protein expression of LACC1 in macrophages (Fig. 6A, B,S5A). The results of the dual luciferase reporter assay demonstrated that, in 293T co-transfected with wild-type LACC1, miR-223 mimics dramatically decreased luciferase activity, but not the 3’-UTR mutant of LACC1 (Fig. 6C). This result suggested that miR-223 may perform its function by binding to LACC1.

Fig. 6.

Molecular mechanism of exosome-derived miR-223 on macrophage pyroptosis. (A) The mRNA expression of LACC1 in LPS-induced macrophages was analyzed by RT-qPCR. Relative mRNA levels were compared in two groups with Student’s t-test, ***P < 0.001. (B) The protein expression of LACC1 in LPS-induced macrophages was analyzed by WB. (C) Luciferase activities were measured by a dual luciferase reporter in 293T cells co-transfected with luciferase reporter plasmids with wild type 3’UTR of LACC1 or mutant 3’UTR of LACC1 and miR-223 mimics or NC. One-way ANOVA with Dunnett’s multiple comparison test, ***P < 0.001, ns: non-statistically significant. (D) The mRNA expression of LACC1 in LPS-induced macrophages treated with miR-223 mimics or LACC1 OE was analyzed by RT-qPCR. One-way ANOVA with Dunnett’s multiple comparison test, *P < 0.05, **P < 0.01, ***P < 0.001. (E) The protien expression of LACC1 in LPS-induced macrophages treated with miR-223 mimics or LACC1 OE was analyzed by WB. (F) The cell viability of LPS-induced macrophages treated with miR-223 mimics or LACC1 OE was detected by CCK-8 assay. One-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001. (G) The levels of inflammatory product IL- 1β, TNF-α, and IL-18 in cell lysates from LPS-induced macrophages treated with miR-223 mimics or LACC1 OE was analyzed by ELISA. One-way ANOVA with Dunnett’s multiple comparison test, **P < 0.01, ***P < 0.001. (H) The protein expression of Cleaved Gasdermin D, Cleaved Caspase-1, and NLRP3 in LPS-induced macrophages treated with miR-223 mimics or LACC1 OE was analyzed by WB.LPS + miR-NC + NC-OE means macrophages (CPH-168 or RAW264.7) transfected with NC mimic and NC-OE after LPS treatment; LPS + miR-223 + NC-OE means macrophages (CPH-168 or RAW264.7) transfected with miR-223 mimic and NC-OE after LPS treatment; LPS + miR-NC + LACC1 means macrophages (CPH-168 or RAW264.7) transfected with NC mimic and LACC1-OE after LPS treatment; LPS + miR-223-NC + LACC1 means macrophages (CPH-168 or RAW264.7) transfected with miR-223 mimic and LACC1-OE after LPS treatment.

Subsequently, we transfected both miR-223 mimics and the LACC1 plasmid vector into macrophages to investigate the role of the miR-223/LACC1 axis in regulating macrophage pyroptosis. The results revealed that the overexpression of miR-223 significantly reduced LACC1 expression in LPS-treated macrophages, while the transfection of the overexpressed LACC1 plasmid significantly increased LACC1 expression (Fig. 6D, E,S5B). CCK-8 assays indicated that overexpression of miR-223 enhanced the viability of LPS-treated macrophages, whereas LACC1 decreased cell viability (Fig. 6F). Additionally, overexpression of miR-223 diminished the release of inflammatory factors, while LACC1 reversed this effect (Fig. 6G). WB results indicated that overexpression of miR-223 reduced the expression of pyroptosis-associated proteins, whereas LACC1 counteracted this reduction (Fig. 6H, S6A,6B). These findings suggested that the alleviation of pyroptosis by miR-223 in LPS-induced macrophages was mediated through the regulation of LACC1 expression.

Discussion

OM is an orthopedic disease of bone tissue and bone marrow infection caused primarily by microbial pathogens. Macrophages, as important cells in the immune microenvironment of the disease, also play an integral role in the bacterial infection process. And recent studies have found that exosomes derived from mesenchymal stem cells are involved in the regulation of immune cells²⁸. Therefore, the present study also explored the interaction of BMSCs-derived exosomes with LPS-induced macrophages.

It is well known that exosomes can participate in cell-to-cell communication by transferring miRNAs, thereby influencing gene expression in recipient cells²⁹. MSC-derived exosomes have demonstrated immunomodulatory functions in orthopedic diseases, particularly through macrophage regulation. For example, adipose stem cell–derived exosomes modulated macrophage M1/M2 polarization via the miR-451a/MIF axis, promoting immune homeostasis and bone healing³⁰. Similarly, BMSC-derived exosomes promoted tendon–bone healing by delivering miR-23a-3p to facilitate macrophage polarization from M1 to M2 phenotype³¹.Moreover, accumulating evidence reveals that miRNAs play important roles in regulating inflammation in OM³². For instance, miR-155 is reported to be highly upregulated and promotes apoptosis in LPS-stimulated MC3T3-E1 cells³³. Additionally, miR-24 has been shown to alleviate inflammation in Staphylococcus aureus-induced OM by modulating CHI3L1³⁴. Furthermore, METTL3 accelerates OM progression induced by SpA through regulating m6A methylation modification of miR-320a³⁵. Another study by Ding Ran et al. demonstrated that overexpression of the long non-coding RNA KCNQ1OT1 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells infected with Staphylococcus aureus by sponging miR-29b-3p, suggesting that regulating KCNQ1OT1 expression may serve as a therapeutic strategy to improve OM³⁶ .These findings support a broader framework in which miRNAs act as crucial regulators of macrophage function and inflammation in the context of OM.

In this study, we focused on miR-223, a microRNA enriched in exosomes derived from BMSCs³⁷. miR-223 has been shown to possess potent anti-inflammatory and protective functions in various cell types. For instance, in osteoarthritis (OA), miR-223 promotes chondrogenic differentiation of BMSCs and inhibits NLRP3 inflammasome expression, demonstrating its anti-inflammatory and immunomodulatory potential³⁸. Additionally, miR-223 regulates TRIM37 expression in chondrocytes, influencing their proliferation and cell cycle³⁹. In diabetes-related OA, miR-223 upregulates peroxisomal function, inducing chondrocyte apoptosis and exacerbating pathological changes in OA⁴⁰. These studies highlight the critical role of miR-223 in immune regulation and tissue repair, particularly in bone and cartilage.Although the role of miR-223 in various cell types has been well-documented, our study specifically investigates its impact in macrophages via exosomes derived from BMSCs. Previous studies have shown that miR-223 exerts anti-inflammatory protective effects in organs such as the liver and lungs^41,42. However, its role in OM remains underexplored. In our study, miR-223 expression was significantly downregulated in both OM patient samples and LPS-stimulated macrophages, suggesting its involvement in OM pathogenesis. Consistent with this, our in vitro experiments demonstrated that BMSC-derived exosomes carrying miR-223 (miR-223 exo) significantly enhanced macrophage survival and reduced cell death under LPS stimulation. These findings suggest that miR-223 may protect macrophages from pathogen-induced damage, thereby helping to maintain immune function and tissue integrity during infection.

Beyond apoptosis, macrophage death in response to pathogenic stimuli often involves pyroptosis, a caspase-1–dependent form of inflammatory cell death^43,44. In our study, LPS induced the release of pro-inflammatory cytokines and upregulation of pyroptosis markers in macrophages. Remarkably, co-culture with miR-223 exo reduced these inflammatory responses and suppressed pyroptosis-related protein expression. This is in line with previous studies showing the anti-pyroptotic effects of miR-223. For instance, miR-223 overexpression was shown to inhibit NLRP3 inflammasome activation and M1 polarization, thereby reducing macrophage pyroptosis⁴⁵. It also attenuated coxsackievirus-induced myocardial injury by suppressing inflammation in macrophages⁴⁶. In the context of infectious bone disease, miR-223 suppressed pro-inflammatory cytokine production in Mtb-infected myeloid cells by dampening NF-κB signaling⁴⁷.Considering the detrimental effects of pyroptosis—including sustained inflammation, osteonecrosis, and disruption of bone remodeling⁴⁸—inhibition of macrophage pyroptosis by miR-223 further highlights its potential as a therapeutic target in OM. Thus, our study provides strong evidence that miR-223 contributes to osteoprotection by limiting inflammatory damage in macrophages.

Mechanistically, we confirmed that miR-223 directly binds to the 3’-UTR of LACC1, a pro-inflammatory factor in macrophages known to promote NF-κB signaling and exacerbate immune responses^49,50. Suppression of LACC1 by miR-223 represents a novel anti-inflammatory axis, potentially explaining the reduced pyroptosis and cytokine release observed in our study.Furthermore, we investigated the relationship between miR-223 and caspase-1, a key mediator of pyroptosis. We found that caspase-1 inhibitors improved macrophage survival and decreased pyroptosis, whereas knockdown of miR-223 negated these protective effects. Previous reports have similarly demonstrated that miR-223 can indirectly downregulate caspase-1 expression, thereby reducing pyroptotic responses^50,52. These findings collectively support the idea that miR-223 protects macrophages by modulating the LACC1/NF-κB and caspase-1–dependent pyroptotic pathways, adding to the growing body of literature that positions miRNAs as key immunoregulators in OM (Fig. 7).

Fig. 7.

Schematic illustration of the regulatory role of BMSC-derived exosomal miR-223 in macrophage pyroptosis during OM via modulation of LACC1.This diagram depicts the proposed mechanism by which exosomes secreted from BMSCs deliver miR-223 to macrophages. The transferred miR-223 downregulates the expression of LACC1, thereby suppressing macrophage pyroptosis and alleviating the inflammatory response associated with OM.

Limitation and conclusion

Although this study provides preliminary evidence for the potential therapeutic role of miR-223 in OM, several limitations warrant further investigation. First, the lack of in vivo validation is a significant limitation. While the in vitro findings are promising, they must be confirmed through animal models to better understand the therapeutic efficacy, safety, and adaptability of exosome-based treatments in a more complex biological context. In vivo studies are crucial for assessing the role of exosomes in modulating immune responses and tissue repair in OM.Additionally, exosomes, as natural carriers, encapsulate various genetic materials (including miRNAs, mRNAs, and proteins) that may collectively contribute to their therapeutic effects. Therefore, future research should explore the comprehensive mechanisms of exosomes, particularly their role in regulating inflammation and other immune responses in OM. Although this study primarily focused on the effect of miR-223 on macrophages, the potential contributions of other exosomal components to immune modulation and inflammation have not been fully explored. Future studies should assess the involvement of additional factors in BMSC-derived exosomes and their potential roles in OM pathogenesis. Moreover, while this study concentrated on the role of macrophages in OM, interactions between macrophages and other bone marrow-derived cells (such as BMSCs and osteoblasts) were not explored. Future research should investigate the complex interactions between these cell types, which would provide a more comprehensive understanding of the immune and repair processes in OM. The interplay between BMSCs, osteoblasts, and macrophages may significantly impact immune regulation and tissue repair, suggesting that such interactions should be explored to optimize therapeutic strategies.Finally, the similarities and differences in mechanisms between acute (RAW264.7) and chronic (CPH-168) inflammation models were not sufficiently addressed. Differences in immune environments, cytokine secretion, and immune cell functions in acute versus chronic inflammation may affect the response to miR-223. Future studies should investigate the differential roles of miR-223 in these models, particularly the regulatory mechanisms underlying acute and chronic immune responses.

In conclusion, this study explored the effects and mechanisms of BMSCs-derived exosomes on LPS-induced macrophages. The results revealed that BMSCs-derived exosomes played a macrophage-protective role to inhibit the apoptosis and pyroptosis process in LPS-induced macrophages by carrying miR-223. Protecting macrophages from inflammation-mediated pyroptosis, on the one hand, strengthend the phagocytosis of pathogenic microorganisms by macrophages as the first barrier, and on the other hand, reduced the damage of inflammatory storms to bone tissue. Mechanistically, exosome-secreted miR-223 exerts anti-inflammatory and anti-pyroptosis effects by binding and targeting LACC1 expression. Thus BMSCs-derived exosomes carrying miR-223 might provide a strategy as a clinical treatment for OM.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

OM

OM

BMSCs

Bone marrow mesenchymal stem cells

miRNAs

microRNAs

Exo

Exosomes

TEM

Transmission electron microscopy

NTA

Nanoparticle tracking analysis

IF

Immunofluorescence

CCK-8

Cell Counting Kit 8

RT-PCR

Quantitative Reverse Transcription PCR

WB

Western blot

SD

Standard deviation

Author contributions

TZ, JZ, and FS contributed to the study’s conception, design, and data collection. XDW, YFZ, and JW contributed to data collection and statistical analysis. TZ and XDW write the draft of the manuscript. All authors commented on previous versions. YFW finished the critical revision and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (82172520), Suzhou Science and Technology Development Program (Basic Research - Medical Applied Basic Research) Project (SKY2023060), and Suzhou Medical College Clinical Science and Technology High-end Platform and Translation Base Construction Project (ML13101423).

Data availability

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

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures performed in this study were reviewed and approved by the Ethics Committee of Children’s Hospital of Soochow University (2024CS075).

Human ethics and consent to participate declarations

The study was performed in accordance with the Declaration of Helsinki and the International Conference on Harmonisation Good Clinical Practice guidelines. The protocol and all modifications were approved by relevant ethics committees and regulatory authorities. All patients provided written informed consent.