Synovial advanced glycosylation end products aggravate periprosthetic infection in diabetes by upregulating Staphylococcus aureus RNAIII

Abstract

Background

Diabetes mellitus (DM) is a high-risk factor for periprosthetic joint infection (PJI). However, the mechanism how hyperglycemia induces or promotes PJI remains unclear. This study aimed to determine how host hyperglycemia stimulates pathogenic bacteria and thus induces PJI.

Methods

The rats were divided into 4 groups: control total knee arthroplasty (TKA) group, control PJI group, DM + TKA group, and DM + PJI group namely treating with high-sugar and high-fat diet + streptozotocin (STZ), and artificially induced PJI. After 3 weeks, bone and joint local inflammatory response, micro-CT, bacterial load, and biofilm formation were analyzed. The effects of advanced glycosylation end products (AGEs) and STF-31 on the biofilm formation of Staphylococcus aureus (S. aureus) were detected by crystal violet and confocal microscopy in vitro. In addition, the mechanism of AGEs promoting biofilm formation was explored by combined proteomics and transcriptomics analysis, and the effect of AGEs on RNAIII was further detected by constructing mutants.

Results

In both humans and rats, we found more severe infectious responses in the knee joint specimens of patients and rats with DM + PJI when compared with those without PJI. Moreover, DM + PJI specimens showed higher levels of synovial AGEs and expression of glucose transporter 1 (GLUT1). AGEs exacerbated the localized infectious response of joints in vivo and promoted biofilm formation in vitro, whereas GLUT1 receptor inhibitors attenuated these effects. Finally, RNA-seq and proteomics revealed that RNAIII may be the target of AGEs acting on S. aureus. AGEs directly promoted biofilm formation by enhancing δ-hemolysin translated by RNAIII. In contrast, inhibition of RNAIII effectively attenuated AGEs-induced biofilm formation.

Conclusions

In summary, high glucose upregulates S. aureus RNAIII expression by activating the synovial GLUT1-AGEs pathway, thereby promoting S. aureus colonization and biofilm formation on the surface of articular prostheses, contributing to the onset and progression of PJI.

The translational potential of this article

Our study shows the great potential of STF-31 as a specific treatment for DM + PJI, and is expected to become a new treatment method combined with antibiotics. RNAIII may be the target of AGEs-induced Staphylococcus aureus biofilm, which provides theoretical support and experimental basis for analyzing the effect of DM on PJI.

Graphical abstract

Summary diagram. High glucose induces AGEs accumulation in FLSs via the GLUT1-AGE-RNAIII pathway which in turn stimulates S. aureus RNAIII leading to biofilm formation.

1. Introduction

Orthopedic implant infection, particularly periprosthetic joint infection (PJI), is a catastrophic consequence of orthopedic surgery.The reported incidence of PJI after primary hip and knee arthroplasty is 0.3–1.9 %, whereas the incidence of PJI after revision surgery can be as high as 5 % [1]. PJI often requires repeated surgery and long-term antibiotic use. However, in some retrospective studies, these treatments have been found to still have low cure rates and could lead to a variety of complications, eventually leading to joint dysfunction and increased mortality [[2], [3], [4], [5]].

Diabetes mellitus (DM) is one of the most common and harmful metabolic diseases in human beings [6]. Hyperglycemia or the accumulation of harmful metabolites produced by glucose metabolism, such as advanced glycation end products (AGEs), can lead to secondary complications such as diabetic nephropathy, retinopathy, vascular disease, and incision complications after surgery by inducing inflammatory and oxidative stress responses [[7], [8], [9], [10], [11]]. Patients with DM are in a state of systemic chronic inflammation, with reduced resistance and susceptibility to multi-organ infections [12]. There is also a significant correlation between an increased rate of glucose variability and an increased rate of treatment failure after second-stage revision surgery [13]. Recent studies have demonstrated a strong correlation between DM and systemic infections, as well as local tissue and organ infections, especially bone and joint infections [14]. Data have shown that the incidence of PJI in patients with DM undergoing primary joint replacement is as high as 4.3 %, which is much higher than that in patients without DM [15]. Not only does diabetic status after hip and knee arthroplasty increase the risk of PJI, but there is also a trend toward an association between preoperative blood glucose levels and the incidence of PJI [[16], [17], [18], [19]]. This strongly suggests that DM increases the risk of the development of PJI. However, whether and how DM triggers or aggravates PJI has not yet been reported.

Staphylococcus aureus (S. aureus) is one of the major and most virulent pathogens that cause PJI [20]. On the one hand, S. aureus can produce multiple toxins (e.g. hemolysin) and enzymes (e.g. serum coagulase, hyaluronidase) for its local colonization and reproduction; on the other hand, it can form a biofilm on the surface of the prosthesis, which causes immune escape and resistance to antibiotics [21]. It was found that in diabetic foot ulcer, the sustained accumulation of AGEs in wound tissue could favor local microbial colonization by enhancing biofilm formation of S.aureus [22]. Our previous study found that DM promotes the accumulation of AGEs in synovial fibroblasts (FLS), which thus lead to the stimulation of inflammation in the knee joint [23]. Therefore, we hypothesized that DM may promote S. aureus colonization in the prosthetic region by inducing the production of AGEs in synovial FLSs. In the current study, we aimed to determine whether hyperglycemia affects PJI severity and the underlying mechanisms involved, using a diet-induced DM rat model, to provide the theoretical and experimental basis for the prevention and treatment strategies of DM + PJI.

2. Materials and methods

2.1. Chemicals and regents

S. aureus strain Newman was obtained from the Microbiology Laboratory, Institute of Medical Research, Wuhan University. STZ (CAS No. S0130) was obtained from Sigma–Aldrich Co., Ltd. (StLouis, MO, USA). Isoflurane was purchased from Baxter Healthcare Co., Ltd. (Deerfield, IL, USA). Anti-GLUT1 (No. 21829-1-AP) were purchased from Proteintech Technology (Wuhan, China). Anti-AGEs (No. bs-1158R) was acquired from Bioss (Beijing, China). STF-31 (CAS No. 724741-75-7) was obtained from MedChemExpress (Shanghai, China). Advanced glycation end products (AGE-BSA, No. 2221-BSA) and BSA (No. 2221-10) were purchased from BioVision (Milpitas, CA, USA). Rat α1-AGP (Alpha-1-Acid Glycoprotein) ELISA Kit (CAS No. ELK5247) and Rat IL-6 (Interleukin 6) ELISA Kit were purchased from ELK Biotechnology CO., Ltd (Wuhan, China). SYTO 9/PI Live/Dead Bacterial Double Stain Kit (CAS No. MX4234-80T) was purchased from Maokang Biotechnology CO., Ltd (Shanghai, China). RNAIII-inhibiting peptide (CAS No. HY-P1452A) was acquired from MedChemExpress (Shanghai, China). Crystal violet staining solution (CAS No. BL802A) was purchased from Biosharp Biotechnology CO., Ltd (Hefei, China).

2.2. Human synovial tissue acquisition

Periprosthetic synovial tissues were extracted intraoperatively from patients with chronic PJI after hip and knee arthroplasty in one- or two-staged revision surgeries and fixed in 4 % paraformaldehyde (PFA) solution. Synovial tissue was extracted with the informed consent of the patients, and the study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (approval number: 2023176).

2.3. Bacterial preparation

S. aureus strain Newman was streaked on agar plates and incubated overnight at 37 °C for 24 h. Individual colonies were taken from the agar plates spliced into LB broth and incubated at 37 °C for 12 h with shaking. The overnight bacteria were transferred 1:100 to LB broth and incubated for 4 h with shaking. After centrifugation to remove the supernatant, the inoculum was resuspended in PBS as 2 × 10⁷ CFU.

2.4. Animal models

Sixty male Wistar rats (220 ± 5g) were purchased from Vital River Company. These animals were housed in the Animal Experimental Center of Wuhan University under controlled environmental conditions, in separate ventilated cages, room temperature: 18–22 °C; Humidity: 40–60 %; Light cycle: 12 h light–dark cycle, food and water freely available. All protocols concerning the animal experiment were approved by the Animal Experiment Ethics Committee of Wuhan University School of Medicine. All animal experimental procedures were performed following the Guide for the Care and Use of Laboratory Animals of the Chinese Animal Welfare Committee (license number: WP20210583).

After one week of acclimatization, 60 rats were randomly divided into two groups: 30 rats in the control group and 30 rats in the diabetic group. The control group was given a basic diet, and the diabetic group was given a high-glucose and high-fat diet (basal chow + 10 % lard + 20 % sucrose + 2.5 % cholesterol + 0.5 % sodium cholate) for 4 weeks, and at the end of the 4 weeks, the rats were fasted for 8 h and given an intraperitoneal injection of STZ (40 mg/kg), and then a blood sample was collected by vein from the tail for measurement of fasting blood glucose concentration after 72 h. Blood samples were collected intravenously 72 h later from the tail to measure the fasting blood glucose concentration, and rats with fasting blood glucose ≥11.1 mmol/L were considered hyperglycemic and were selected for the experiment [24].

Based on a previous rat PJI surgical model [25], Orthopedic grade titanium screws were selected to simulate clinical knee arthroplasty and prosthesis implantation in the marrow of the femur. All surgeries were performed in a sterile surgical area using an aseptic technique and sterile instruments and materials. Anesthesia was administered using a 2 % pentobarbital sodium intraperitoneal injection, the knee was shaved and sterilized with iodine and the surgical area was covered aseptically. A 2 cm longitudinal incision was made along the midline of the right knee to expose the knee joint. A 1.4 mm drill was used to enter the femoral canal and a screwdriver was inserted retrogradely to place the prosthesis (diameter, 1.6 mm, length, 8 mm), the patella was reset, and the joint capsule was closed with a 4-0 suture. Half of each of the DM group and the control group were randomly selected for intra-articular injections of 50 μl of 2 × 10⁷ CFU S. aureus, and the other half were injected with 50 μl of saline postoperatively as a control. The rats were divided into 4 groups: control + total knee arthroplasty (TKA) (CT), control + PJI (CP), DM + TKA (DT), and DM + PJI (DP).

2.5. X-ray

One week after surgery, radiographic X-ray images were captured using a Bruker Xtreme BI system to assess the positioning of the prosthesis in the right hind limb of the rats. Subsequently, at the three-week postoperative stage, additional X-ray images of the right hind limb were obtained to evaluate the presence of osteolysis and bone absorption surrounding the prosthesis. The X-ray images were acquired by IP266 X-ray (Germany).

2.6. Mirco-CT and data analysis

Following the extraction of the implant, the distal femurs were scanned using SkyScan 1276 micro-CT (Bruker, Germany). The scanning data were reconstructed using NRecon (V1.7.0.4, SkyScan) and subsequently analyzed the three-dimensional structural parameters using DataViewer (V1.5.6.2, SkyScan), CTAn (V1.18.4, SkyScan) and CTVol (V2.0, SkyScan). These parameters encompass bone volume/tissue volume (BV/TV), trabecular Thickness (Tb. Th), and trabecular separation (Tb. Sp).

2.7. Scanning electron microscope (SEM)

The prostheses were carefully removed and placed in an electron microscope fixative. Tissue blocks were washed with 0.1 M PB (pH 7.4) three times, for 15 min each time. The prosthesis blocks were transferred into 1 % OsO₄ in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. The prosthesis blocks were washed in 0.1M PB (pH 7.4) three times, for 15 min each time, and placed in 50 %, 60 %, 70 %, 80 %, 90 %, 100 % ethanol for 15 min. The samples were then dried using a critical point dryer. The specimens were attached to metallic stubs using carbon stickers and were sputter-coated with gold for 30 s. Finally, the cells were observed under an SEM (HITACHI, Japan).

2.8. Tissue CFU assay

After the euthanasia of the rats, the implants, along with the surrounding bone and soft tissues, were obtained under sterile conditions. The collected bone and soft tissues were subsequently homogenized using a sterile tissue grinder. Meanwhile, the prostheses were immersed in PBS comprising 0.3 % Tween 20, subjected to sonication for 15 min, and then vortexed for 1 min. A 100 μl aliquot from each sample was inoculated onto agar plates and kept at 37 °C for 24 h to determine the bacterial load through colony counting.

2.9. Hematoxylin eosin (H&E) staining

Human synovial tissues and intact joint tissues from rats were fixed in 4 % paraformaldehyde (PFA) solution for 48–72 h and embedded in paraffin for subsequent H&E staining. Tissues were fixed in paraffin sections, dehydrated, and embedded in paraffin. The paraffin-embedded samples were then cut into 5-μm serial sections, stained with hematoxylin dye for 5 min, and washed with water. Sections were soaked in ammonia and washed with water. The sections were stained with 1 % eosin and washed with water. Ten different fields of view were selected for each sample and all images were captured using a Nikon NIS Elements BR optical microscope (Nikon, Tokyo, Japan).

2.10. Immunohistochemical staining (IHC)

Human synovial tissues and intact rat joint tissues were fixed in 4 % paraformaldehyde solution for 48 h and processed by paraffin embedding. Paraffin samples were used for morphological staining analysis in 5 μm sections. After dewaxing, rehydration, and antigen repair, paraffin sections were treated with ethylenediaminetetraacetic acid (EDTA) antigen repair buffer (pH 8.0). The BSA was used to seal the previously added primary antibodies, which were diluted at a ratio of anti-AGE (1:200 dilution) and anti-HIF-1α (1:200 dilution). The DAB staining kit was used (GeneTech Company, Ltd., Shanghai, China). All images were captured using a Nikon NIS Elements BR optical microscope (Nikon). ImageJ software (version 8.0; US National Institutes of Health, Bethesda, MD, USA) was used to analyze the IHC results. The staining intensity was determined by measuring the IOD of each sample in 10 different fields of view.

2.11. Crystalline violet staining

Non-adherent cultures were removed from each inoculated well and 200 μl of sterile PBS was gently added to the biofilm while minimizing biofilm disturbance. The plate was gently shaken for a few seconds to remove loose and non-adhered cells from the biofilm. The buffer was discarded, the methanol was fixed for 5 min, the methanol was discarded, and the 96-well plate was dried. Then, 200 μl of 1 % (w/v) crystal violet solution was added to each well and incubated at room temperature for 30 min. The crystal violet solution was removed, and the cells were rinsed three times with PBS. Then, 200 μl of methanol was added to each well to elute the crystal violet,. Then, the absorbance value was measured at 600 nm with an enzyme counter.

2.12. Confocal laser scanning microscope (CLSM)

First, 2.5 μl of PI and 2.5 μl of SYTO 9 stock solution were added to 1 ml of sterile 0.9 % (w/v) NaCl, then blown and mixed, and added to a confocal dish and incubated at room temperature away from light for 30 min. 100 μl of 0.9 % (w/v) NaCl solution were slowly added after removing the liquid. During CLSM imaging, SYTO9 emits green fluorescence to identify live microorganisms with intact membranes, whereas PI emits red fluorescence and stains dead bacteria with damaged membranes. Image acquisition was performed using a Leica confocal laser scanning microscope (Leica Instruments, New York, USA). Biofilms were observed with a 63 × lens, horizontal planimetry of the biofilm was captured, and a 3D projection of the biofilm was reconstructed. Each experiment was repeated three times independently. The quantitative parametric structure of the biofilm was quantified by looking at the total biofilm and the live cells represented by green fluorescence, the dead cells represented by red fluorescence, and the maximum thickness of the biofilm (μm) was determined directly from the confocal 3D images.

2.13. In vitro co-culture of FLS metabolites with S. aureus

Rats were sacrificed under isoflurane anesthesia and synovial tissues were isolated under aseptic manipulation. The synovial tissues were completely minced under aseptic conditions and then digested with type IV collagenase and incubated in a 37 °C incubator for 8 h. Tissues were resuspended in DMEM containing 10 % FBS and centrifuged at 1200 g for 5 min. Primary cells were inoculated at a density of 1 × 10⁵ cells/ml. Subsequently, experiments were performed using third to fourth-generation cells. To simulate the physiological relationship between the synovial membrane and S. aureus in the joint cavity and to study the effect of metabolites released by FLS on the colonization of S. aureus on the prosthesis surface to form a biofilm, we established an in vitro co-culture system of the host cells and bacterial biofilm. The FLSs were treated with different concentrations of glucose (10 and 30 mM) or other intervening agents for 72 h. The synovial cell culture medium was collected and co-cultured with S. aureus at rest in an incubator at 37 °C for 48 h. The experimental groups of co-culture included FLS+10 mM glucose, FLS+30 mM glucose, FLS+10 mM glucose+0.5 μM STF-31, FLS+30 mM glucose+0.5 μM STF-31, FLS+30 mM glucose+0.5 μM STF-31 + 50 ug/ml AGEs-BSA.The concentrations of STF-31 and AGEs were referenced to previous study [26]. For the in vitro experiments, similar results were obtained in six independent experiments and each experiment was repeated three times.

2.14. RNA-seq library construction and sequencing

RNA was extracted from the samples using the TRIzol method (Tiangen, Beijing, China) and an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was used to accurately detect RNA integrity. Libraries were constructed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina. Bacterial RT-qPCR experiments were performed to validate the RNA-seq data. Genes significantly upregulated or downregulated in response to AGEs intervention. These genes were selected because they encode a range of virulence factors, including those related to the promotion of bacterial colonization and biofilm formation. Total RNA was extracted using TRIzol reagent (Tiangen) and translated into cDNA using PrimeScript RT premix (TaKaRa, Dalian, China) according to the manufacturer's instructions. cDNA was then used as a template to assess the transcript levels of biofilm-associated genes by RT-qPCR with paired primers and 16S rRNA as an internal reference.

2.15. Label free proteomics

The sample was taken into an ultrafiltration tube and centrifuged at 14000 g for 15 min. After discarding the permeate, the ultra-filtered sample was put into a 1.5 ml centrifuge tube and balanced with DB buffer (8 M Urea, 100 mM TEAB, pH 8.5). The protein solution was reduced with 10 mM DTT for 1 h at 56 °C, and subsequently alkylated with sufficient IAM for 1 h at room temperature in the dark. Gene Ontology (GO) and InterPro (IPR) functional analysis were conducted using the interproscan program against the non-redundant protein database (including Pfam, PRINTS, ProDom, SMART, ProSite, PANTHER, and the databases of COG (Clusters of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used to analyze the protein family and pathway. DEPs were used for Volcanic map analysis, cluster heat map analysis and enrichment analysis of GO, IPR and KEGG.

2.16. Statistical analysis

Prism (GraphPad Software, La Jolla, CA, USA, version 8.0) was used for all data analyses. All data values shown are presented as mean ± S.E.M. For in vitro experiments, using the single factor analysis of variance (ANOVA) analysis data of different drug concentrations, and multiple comparisons after the inspection. If the homogeneity of variance was consistent, an unpaired t-test was used to compare the two groups. P < 0.05 was considered statistically significant.

3. Results

3.1. DM stimulated the systematic inflammatory reaction in rats with PJI caused by S. aureus

To determine whether hyperglycemia affects PJI severity in DM rats, we used a diet-induced DM rat model supplemented with a high-glucose, high-fat diet before and during infection (Fig. 1a). We established a PJI surgical model by exposing the rat knee joint to femoral titanium screw implantation and intra-articular injection of S. aureus (Fig. 1b), and postoperative X-ray was used to verify the prosthesis position and the initial initiation of osteolysis (Fig. 1c). Our study found that rats in the DP (DM and PJI) group exhibited more pronounced signs of infection and significantly more weight loss than those in the control CP (PJI) group, regardless of dietary supplementation (Fig. 1d, P < 0.05). Blood glucose was maintained at a higher level after modeling in the diabetic group, and there was no significant difference in blood glucose levels between the DP and DT (DM and total knee arthroplasty [TKA]) groups (Fig. 1e, P < 0.05). To observe local infection in rats, we observed changes in knee joint skin temperature using infrared imaging and found that the local joint temperature in the DP group increased more significantly than that in the CP group. Three weeks after the operation, the knee joints of rats in the CP and DP groups showed obvious swelling, and some rats showed wound ulceration and sinus formation (Fig. 1f, g, P < 0.05). The results of serum inflammatory markers showed that the expression of α1-acid glycoprotein levels was more elevated in the DP group compared with the CP group (Fig. 1h, P < 0.05), whereas there was no significant change in the expression levels of IL-6 (Fig. 1i, P > 0.05). These results indicated that DM may accelerate the course and severity of PJI.

Fig. 1.

Establishment and evaluation of a rat model of DM combined with PJI. (a) Schematic diagram of the experimental time. (b) Prosthesis implanted in the femoral side of the knee joint. (c) Postoperative X-ray photographs. (d) Weight changes of rats in each group during the modeling period. (e) Changes in blood glucose of rats in each group during the modeling period. (f) Infrared imaging of rats 3 weeks after the operation. (g) Local skin temperature of the knee joint. (h) ELISA for the measurement of serum IL-6. (i) ELISA for the measurement of serum α1-AGP. n = 8, Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no signifince.

3.2. DM enhanced local inflammation of the knee in rats with PJI caused by S. aureus

After determining the overall effect of diabetes on PJI rats, we investigated the effect of hyperglycemia on bone erosion in PJI rats. First, we evaluated the histological assessment of synovial tissue samples from rats and clinical patients. In our clinical specimens, we found that PJI patients with comorbid DM had significantly more synovial inflammatory infiltrate and a significantly higher synovitis index than patients with regular PJI (Fig. 2a, P < 0.01). In addition, we found a similar phenomenon in rat specimens we observed the histopathological changes in the synovium of rats in each group postoperatively and found that compared with the CT group, the synovium of the CP, DT, and DP groups exhibited different degrees of mesenchymal cell hyperplasia, increased number of cell layers, and inflammatory cell infiltration. The most significant synovial inflammatory manifestations were found in the DP group, with significantly more neovascularization and a significantly higher synovitis histological score (Fig. 2b, P < 0.01). Subsequently, based on the histological results, we performed an imaging evaluation. The micro-CT images and analyses showed a significant decrease in bone volume (BV/TV, Tb. Th, and Tb. Sp) and a significant increase in bone tunnel diameter in the DP group compared with the DT and CP groups (Fig. 2c–g, P < 0.05). These results suggest that diabetes may make PJI-induced intra-articular soft tissue lesions and bone erosion more severe. Next, we investigated the role of hyperglycemia in the colonization of S. aureus on the prosthesis and the formation of biofilms on the prosthesis by performing a count of colony-forming units (CFU) on the prosthesis, bone tissue, and soft tissue as well as the formation of biofilms on the prosthesis. We observed a large distribution of S. aureus surrounded by a large number of erythrocytes and leukocytes in both the CP and DP groups using scanning electron microscopy (SEM), in addition to the denser fibrin structure and biofilm formation observed in the DP group (Fig. 2h). Subsequently, we extracted bacteria from the bone tissue, synovial tissue, and prosthesis for overnight culture. The DP group showed significantly more bacterial colonization than the CP group by counting the prosthesis, bone, and soft tissues. No significant bacterial colonization was found in the CT and DT groups (Fig. 2i–l, P < 0.05). These results suggest that DM promotes S. aureus colonization of intra-articular tissues and biofilm formation on prosthetic surfaces.

Fig. 2.

DM exacerbates PJI-induced tissue erosion and biofilm formation. (a) Representative H&E images and synovitis score of human joint synovium. (b) Representative H&E images and synovitis score of rat femur and joint synovium. (c) Representative μCT images of each group. (d-f) μCT quantitative analysis of bone loss (including BV/TV, Tb. Th, Tb. Sp). (g) Measurement of bone tunnel diameter. (h) Microorganisms on the surface of titanium screws observed by scanning electron microscope at high magnification; red arrows indicate S. aureus organisms. (i) Representative images of microbiological cultures of prostheses, bone, and soft tissues from each group of animals. (j) Prosthetic CFU counts. (k) Bone tissue CFU counts. (l) Soft tissue CFU counts. n = 6, Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no significance.

3.3. Hyperglycemia induces AGEs accumulation through up-regulating glucose transporter 1 (GLUT1) in FLS in vitro

Studies have shown that AGEs are associated with many signaling pathways that contribute to the inflammatory state and disease development. Therefore, we hypothesized that AGEs play an important role in DM + PJI. We examined the content of AGEs in synovial tissues from clinical patients using IHC and found that their expression was significantly elevated in patients with DM + PJI (Fig. 3a–d, P < 0.01). Similarly, IHC results showed a significant increase in AGEs content in rat synovial tissue in the DT group compared to that in the CT group. AGEs protein expression was significantly higher in the DP group than in the CP group. Interestingly, there was a smaller amount of AGEs deposition in the CP group compared to the CT group (Fig. 3b–e, P < 0.05). In addition, we demonstrated in vitro that AGEs expression levels were elevated in FLSs treated with 30 and 40 mM Glu high glucose (Fig. 3c–f, P < 0.01). These results suggest that the dual effects of DM and S. aureus can lead to the accumulation of AGEs in the synovium. Previous studies identified GLUT1 as an important regulator in biogenesis of AGEs. GLUT1 plays an important role in glucose transport. We confirmed that glucose induces the accumulation of AGEs in FLSs by upregulating GLUT1 expression. Therefore, we hypothesized that AGEs induce bacterial growth and biofilm formation in FLSs via GLUT1. Immunohistochemistry (IHC) results showed that GLUT1 levels in the synovial tissue were significantly higher in patients with DM than in patients without DM (Fig. 3g–k, P < 0.01). Immunofluorescence results showed that the synovial tissue GLUT1 expression level in the DP group was significantly higher than that in the CP group, and that the synovial tissue GLUT1 expression level in the DT group was significantly higher than that in the CT group (Fig. 3h–l, P < 0.01). In vitro, we found that GLUT1 mRNA expression was significantly elevated in the 30 and 40 mM Glu group compared to that in the 10 mM Glu group after treating FLSs with different concentrations of glucose for 72 h (Fig. 3i–m, P < 0.01). In addition, we found that the expression of AGEs induced at high glucose concentrations was significantly inhibited after administration of the GLUT1 inhibitor STF-31 (Fig. 3j–n, P < 0.01). These results suggest that upregulation of GLUT1 promotes elevated AGEs expression in patients with DM + PJI.

Fig. 3.

Regulation of AGEs accumulation by GLUT1 in FLSs. (a) Representative IHC images of AGEs in human synovial tissue. (b) IOD values of AGEs in human FLSs, scale bar 50 μm. (c) Representative IHC images of AGEs in rat synovial tissue. (d) IOD values of AGEs in rat FLSs, scale bar 50 μm. (e) IF staining after high glucose treatment. (f) IOD values of AGEs expression in FLSs. (g) Representative image of GLUT1 immunohistochemistry in patient synovial tissue. (h) Representative images of GLUT1 immunofluorescence in rat synovial tissue. (i) GLUT1 IF staining in synovial cells after 10, 20, 30, and 40 mM Glu treatment. (j) IF staining of STF-31 FLSs added after high glucose treatment. (k) IOD histogram of GLUT1 in clinical patient synovial tissue. (l) IOD histogram of GLUT1 in rat synovial tissue. (m) GLUT1 IOD histogram and mRNA expression in synovial cells. (n) Relative expression of AGEs IOD in synovial cells. n = 6, Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no significance.

3.4. High glucose promotes S. aureus biofilm formation by activating the synovial GLUT1-AGEs pathway

To investigate whether high glucose or AGEs directly promoted S. aureus colonization on the prosthetic surface in the articular cavity, we further examined the surface area and thickness of biofilm formation by crystal violet staining and SYTO9/PI staining. In vitro, we divided S. aureus into four groups that were treated with the different concentration of AGEs-bovine serum albumin (AGEs-BSA) (0, 50, 100, and 200 μg/ml) and then examined by crystal violet staining. We observed a significant increase in S. aureus biofilm surface area after the addition of AGEs in a concentration-dependent manner (Fig. 4a and b, P < 0.05). In addition, confocal microscopy revealed a stepped increase in biofilm surface area and thickness in the AGEs-treated group after SYTO9/PI staining (Fig. 4c–f, P < 0.05). These results suggest that AGEs promote biofilm formation in vitro. To uncover the role of the synovium in the colonization and biofilm formation of S. aureus on the prosthesis surface in a high-glucose environment, we used metabolites from cultured FLSs to treat S. aureus in vitro to investigate the effect of GLUT1 expression in FLSs on biofilm formation by S. aureus under high-glucose stimulation. The culture was divided into seven groups: a medium for untreated FLS (C30-STF, C30, and C-STF) and a medium for treated FLS (STF, 30-STF, 30, 30+STF + AGE). Crystalline violet staining showed that the addition of the GLUT1 inhibitor STF-31 effectively inhibited the growth of biofilms in a high-glucose environment and in high-glucose metabolites of FLSs, and quantification at an absorbance of 600 nm showed that biofilm formation was effectively reduced by approximately 50 % with the addition of STF-31 (Fig. 4g–j, P < 0.05). A similar phenomenon was observed by confocal microscopy, where green fluorescence coverage and biofilm thickness were reduced after the addition of STF-31. Interestingly, the addition of AGEs alongside inhibition of GLUT1 expression re-observed the extensive growth of biofilms (Fig. 4h, i, k, l, P < 0.05).

Fig. 4.

AGEs promote biofilm formation. (a) Microscopic observation of crystalline violet to measure biofilm surface area after treatment with different concentrations of AGEs (0, 50, 100, and 200 μg/ml). (b) OD600 measurement of biofilm formation using the crystal violet method. (c) Confocal biofilm 2D image after AGEs treatment after SYTO9/PI staining. (d) Biofilm fluorescence coverage of surface area as a percentage. (e) Confocal biofilm 3D image after AGEs treatment. (f) Biofilm 3D thickness determination. (g) Crystal violet staining to observe biofilm formation. (h) Confocal biofilm 2D image after STF-31 intervention. (i) Confocal biofilm 3D image after STF-31 intervention. (j) Crystalline violet quantification to observe the formation of synovial metabolites of high glucose-treated synovium versus biofilm formation after treatment with high glucose medium. (k) Biofilm coverage surface area percentage. (l) Biofilm 3D thickness measurement. n = 3 for CLSM and n = 6 for crystalline violet. Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no significance.

3.5. Intra-articular injection of shGLUT1 attenuated the progression of DM + PJI in rats

To investigate the role of GLUT1 in FLSs and its potential effect on the resulting biofilm infection in DM + PJI rats, we sifted out shRNA that could effectively inhibit the expression of GLUT1 in rat synovial tissues (Fig. S1a and b). We injected shGLUT1 into the knee joint cavity of rats with DM + PJI and observed its effect on S. aureus biofilm formation. We observed that shGLUT1 significantly reduced synovial tissue GLUT1 expression in DM + PJI (Figs. S1c, d, P < 0.05). In addition, we observed a lower accumulation of AGEs in the synovial tissue (Figs. S1e, f, P < 0.05). The results of serum inflammatory marker analysis showed that intra-articular injection of AGEs and shGLUT1 did not affect systemic inflammatory changes (Fig. 5a, P > 0.05). We also observed that the body weights of rats in the shGLUT1 group and shGLUT1 + AGEs group showed the same decreasing trend as those in the DM group, but there was no difference in the degree of reduction (Fig. 5b, P > 0.05). Histological assessment revealed that the degree of synovial inflammation was reduced after the inhibition of GLUT1, whereas injection of exogenous AGEs increased the degree of synovial inflammation (Fig. 5c–f, P < 0.05).

Fig. 5.

GLUT1 silencing slows the progression of DM + PJI. (a) Serum IL-6 in rats. (b) Changes in body weight of rats. (c) H&E staining of rat synovial tissues. (d) Microorganisms on the surface of the titanium screw are observed at high magnification by SEM. Red arrows indicate S. aureus biofilms. (e) Each representative CT image. (f) Synovitis scores for each group. (g) CFU count of prosthesis. (h–i) μCT quantitative analysis of bone loss (including BV/TV, Tb. Th). n = 6, Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no significance.

Following intra-articular injection of shGLUT1 on the colonization of S. aureus on the prosthetic surface, intra-articular injection of AGEs promoted the adhesion of S. aureus to the prosthetic surface, whereas intra-articular injection of shGLUT1 into the articular cavity of DM + PJI rats attenuated the colonization of S. aureus on the prosthesis (Fig. 5d–g, P < 0.05). We evaluated the changes in bone volume in the distal femur of rats using μCT and found that the distal femur with local injection of AGEs in the joint showed significant osteolysis and resorption, enlargement of bone tunnels, destruction of the microstructure of bone trabeculae and thinning, and thinning of the bone cortex compared with that of the PJI group. However, intra-articular injection of shGLUT1 into the joint cavity of rats with DM + PJI partially improved the destruction of the distal femur induced by AGEs (Fig. 5e–h, i, P < 0.05). This result suggests that the intra-articular injection of shGLUT1 can effectively delay the progression of DM + PJI and the formation of biofilms on the prosthesis.

3.6. AGEs enhance bacterial virulence and biofilm formation by upregulating RNAIII-hld

To investigate the potential mechanism by which AGEs promote biofilm formation in S. aureus, we used liquid chromatography-mass spectrometry to compare the secreted proteome of S. aureus before and after treatment with AGEs in vitro. Kyoto Encyclopedia of analysis revealed that S. aureus infection and ribosome and quorum-sensing pathways, which are closely related to biofilm formation, were upregulated after AGEs treatment (Fig. 6a). Gene Ontology analysis revealed a significant increase in hemolysis by symbionts of host erythrocytes and cytolysis in other organisms after AGEs treatment. This suggests that hemolysins may play an important role in this process (Fig. 6b). The expression of hld in the biofilm supernatant after AGEs treatment was detected by ELISA, and it was found that the expression of hld in the AGEs-treated group was significantly higher than that in the control group (Fig. 6c, P < 0.01). Next, we used RNA-seq to compare the transcriptome profiles of biofilms before and after AGEs intervention in vitro and found that compared with the control group, 203 genes were up-regulated and 182 genes were down-regulated during biofilm formation in the AGEs-treated group (Fig. 6d). In addition, six groups of differentialy expressed genes (DEGs) were analyzed by clustering to show similarities in gene expression levels and patterns (Fig. 6e).

Fig. 6.

RNAIII-hld is involved in AGEs-induced biofilm formation. (a) KEGG pathway enrichment analysis of differential proteins. (b) Functional GO enrichment analysis of differential proteins. (c) The percentage of hemolysis in the biofilm supernatant was detected by ELISA. (d) Volcano plot of differential genes, DEGs are shown in red (increased expression), green (decreased expression), and blue (not significant). (e) Heatmap of DEGs. Upregulated DEGs in the untreated (C) and treated (A) groups after 48 h of treatment are shown in red, and downregulated DEGs are shown in blue. (f) RT-qPCR verification. The upregulated and downregulated genes are selected for validation in RNA-Seq results. (g) Crystal violet method is used to observe the inhibition of biofilm by RNAIII inhibiting peptide at (0, 50, 100, and 200 μM). (h) Crystal violet is used to measure the inhibitory effect of Newman-ΔRNAIII strain on AGEs-induced biofilm formation. (i) Crystalline violet quantification to observe the biofilm formation. (j) Biofilm coverage surface area percentage. n = 6, Mean ± S.E.M. ∗P < 0.05, ∗∗P < 0.01, n.s, no significance.

Subsequently, to understand the mechanism in biofilm formation in S. aureus after AGEs intervention, we selected 12 genes related to bacterial colonization and biofilm formation identified by transcriptome analysis and further confirmed the expression of DEGs using RT-qPCR. We found six genes with significantly up-regulated expression levels (Fig. 6f). Among the genes promoting biofilm formation and virulence, emp played an important role in biofilm formation as an extracellular matrix-binding protein. The transcriptome sequencing results showed that hemolysin encoded by hld, which plays an important role in biofilm formation, was significantly elevated in AGEs-treated biofilms, The result was consistent with the secreted proteome results. Then, we confirmed elevated expression of hld by measuring the proteins secreted into the medium by AGEs-treated S. aureus. The hld gene is included in the RNAIII transcript and acts as an effector molecule of the agr system. RT-qPCR results showed that the levels of hld and RNAIII were significantly higher in the AGEs group, suggesting that AGEs may promote biofilm formation by inducing the expression of RNAIII associated with the QS system (Fig. 6f, P < 0.01). It was observed that the formation of S. aureus biofilm could be inhibited after knockdown of RNAIII by OD 600 nm measurement, while more obvious biofilm formation was not observed in the ΔRNAIII + AGEs group compared to ΔRNAIII. Further we observed that knockdown of RNAIII effectively inhibited AGEs-induced biofilm formation by comparing the ΔRNAIII + AGEs group with the Newman + AGEs group (Fig. 6g–i, P < 0.01). Through confocal microscopy, we observed that knockdown of RNAIII effectively inhibited AGEs-induced bacterial adhesion, and the surface area coverage of biofilm formation was significantly reduced, in close agreement with crystal violet staining (Fig. 6h–j, P < 0.01). These results suggest that AGEs can act directly on S. aureus RNAIII and that knockdown of RNAIII can effectively inhibit the effect of AGEs on biofilm formation.

4. Discussion

4.1. DM promotes PJI progression

Currently, PJI remains a clinical challenge. It is characterized by complex interactions between microbes and host immunity [27]. PJI often occur with a very low bacterial load, and these bacteria can adhere to joint prostheses to form biofilms [28]. Clinical studies have shown that the biofilm properties and antimicrobial sensitivity of Staphylococcus in PJI are closely related to the prognosis of patients [29]. However, the indicators of systemic inflammation in blood may be normal at low infection levels [[30], [31], [32]]. Local biomarkers are more sensitive than systemic biomarkers in diagnosing PJI, such as the alpha-defensin in synovial fluid [33]. The challenge for PJI does not only derive from the diagnostic aspect but more significantly from the therapeutic dilemma. Most current bacterial research has focused on infections caused by planktonic bacteria, while there are limited instruments available for the eradication of persistent biofilm infections.

DM is an important independent risk factor for PJI, with a clear association between DM and PJI and increased revision rates [34]. One study reported that DM plays a major role in the increase in PJI recovery time [18]. In addition, diabetic hyperglycemia provides a special environment for bacterial infections around joint prostheses, which in turn affects bacterial proliferation, colonization, virulence, and drug resistance. However, little is known about the mechanisms through which DM causes or exacerbates PJI progression. By establishing a rat model of DM + PJI, we found that the knee joints of DM + PJI rats were swollen and the local skin temperature was elevated, whereas individual rats showed skin breakdown and sinus tract formation. Our study also found that both the local and systemic manifestations of PJI were more obvious in individuals with DM than in those without DM, which is consistent with the above findings [35]. PJI, in combination with DM, is associated with severe intra-articular osteomyelitis, bone defects, synovial tissue hyperplasia, and increased synovial inflammation, which may be related to increased bacterial colonization of the bone, soft tissue, and prostheses. This study reveals the potential mechanisms by which DM promotes the progression of PJI and provides an experimental basis for the early diagnosis, prevention, and treatment of DM + PJI.

4.2. GLUT1-AGEs mediates DM + PJI bacterial colonization

Planktonic bacteria colonize and form biofilms in vivo, and in addition to changes in the bacteria themselves, changes in the local temperature, pH, and nutrients in the host also play a crucial role in the formation, maturation, and dissociation of the biofilm [36]. Biofilm formation is the main reason for the persistence of PJI, and materials commonly used in orthopedic surgery, including stainless steel, titanium, polymethylmethacrylate cement, cobalt chromium, and other polymer surfaces, are susceptible to biofilm adherence [37]. Mottal et al. found that the concentration of antibiotics required to eradicate infections in patients with diabetic foot infections caused by S. aureus was much higher than the minimum inhibitory concentration (MIC) and difficult to achieve in the clinical setting [38]. It has also been found that a high-glucose environment in DM is more favorable for bacterial biofilm formation [39,40]. Therefore, the eradication of bacterial colonization and biofilms remains a great challenge in the treatment of PJI, especially DM + PJI.

Synovial performance plays a specific role in PJIs [41]. FLSs, non-immune cells in the synovial tissue, are the major cell population of the synovial tissue lining [42]. In general, under physiological conditions, the lining of the synovium consists of one to three layers of cells, and its thickness can increase to 10–15 layers when activated by inflammatory stimulation [43]. In previous studies, we demonstrated high glucose-stimulated HIF-1α-GLUT1-AGEs signaling in FLSs, followed by activation of ERS and release of pro-inflammatory factors from the synovium to exacerbate intra-articular inflammation [23]. We have found in animal models that not only does hyperglycemia lead to the accumulation of AGEs in the synovium, but also that PJI has been found to cause the deposition of AGEs. This vicious circle may lead to further exacerbation of the disease. We similarly identified a role for AGEs in PJI and demonstrated that they promote bacterial adhesion to prosthetic surfaces and biofilm formation in vivo, and cause an increase in biofilm surface area and thickness in vitro. AGEs can be regulated by GLUT1 in FLSs, and we also observed a significant reduction in the surface area and thickness of S. aureus biofilms induced by FLS metabolites after the addition of GLUT1 inhibitor STF-31 in vitro. Local injection of shGLUT1 into joints retarded biofilm formation on prostheses in vivo. Furthermore, the direct effect of AGEs on biofilm formation was stronger than that of glucose. Therefore, controlling localized AGEs in the joints may be an effective and feasible approach for patients with PJI + DM.

Biofilm growth induced by high glucose was effectively alleviated by GLUT1 inhibition, and biofilm growth inhibited by GLUT1 could be restored by the addition of exogenous AGE-BSA. This suggests that glucose in FLSs may lead to the accumulation of AGEs via the GLUT1-AGEs axis, thereby exacerbating intra-articular bacterial colonization. Furthermore, we found that AGEs were more favorable than glucose for promoting biofilm formation in vitro. This suggests that the accumulation of AGEs in the synovium may be mainly responsible for bacterial adherence and biofilm formation on the surface of implants in patients with DM + PJI. Our study suggests that, while focusing on the colonization and biofilm formation of PJI-causing organisms in the host, attention to the effects of the host microenvironment and underlying disease conditions on the colonization properties of the causative organisms is also important for the treatment of bacterial infections, including PJI. The effects of AGEs in the DM + PJI process are widespread and can affect both the host and bacteria.

4.3. AGEs promote colonization and biofilm formation by upregulating the RNAIII-hld pathway in S. aureus

Bacteria usually cause disease through the surgical implantation process or blood transport. During the biofilm colonization phase, due to the host tissue microenvironment or immune mechanisms, the bacteria alter their gene expression patterns, leading to more persistent bacterial infections and decreasing their responsiveness to drugs such as antibiotics [44]. This behavior of changing gene or protein expression patterns is also one of the ways pathogens adapt to the environment. The formation of AGEs caused by diabetes can have persistent effects on cells and microorganisms in tissues. Some studies suggest that GlmS is involved in the regulation of S. aureus virulence factors and biofilm by regulating the activity of sigB [45]. Consequently, in this study, we hypothesized that AGEs influence the colonization ability of S. aureus by regulating gene expression. We found that δ-hemolysin was significantly elevated in secretory proteins of AGEs-treated S. aureus by secretory proteomics analysis. Further analysis by transcriptome sequencing revealed the enrichment of pathways related to hemolysin synthesis and upregulation of the expression of hld, a gene regulating hemolysin synthesis, and its regulator, RNAIII. It is reported that various genes including RNAIII and hld play a crucial role in staphylococcal pathogenesis, including biofilm formation, proliferation, and immune escape [46], and activation of RNAIII is responsible for higher production of δ-hemolysin [47]. In a clinical study, hld was present in all PJI clinical isolates, suggesting that it plays a crucial role in bacterial survival and pathogenicity [48]. To assess the effect of RNAIII on AGEs-induced biofilm formation, RNAIII-deficient mutants were prepared for in vitro evaluation. In our study, inhibition of RNAIII reversed AGEs-mediated adhesion and biofilm formation by S. aureus, suggesting that AGEs promote bacterial colonization and biofilm formation through RNAIII-hld-hemolysin.

However, this study still has some limitations. First, in order to exclude the influence of other bacterial participatory infections in a long-term diabetes-susceptible state, we focused our study on localized infections in the preoperative period rather than on hematogenous or late infections. Secondly, concentration of relevant inflammatory markers and metabolites in the synovial fluid of rats were not detected, because of the faliure in the collecting of the synovial fluid in the knee joint cavity of rats. We intend to address this limitation through additional larger animal and preclinical studies. Finally, the combined host-bacteria treatment in this study is still in the animal stage and further clinically relevant studies are still needed.

5. Conclusion

DM is closely related to the progression and prognosis of PJI. Our study demonstrates that hyperglycemia affects the intra-articular microenvironment through the GLUT1-AGEs pathway and reveals a central role of RNAIII in AGEs-mediated expression of virulence factors and biofilm formation by S. aureus. At the same time, we believe that combined targeted therapies against the host and bacteria are a promising new direction for future research and treatment of PJI.

Author contributions

Tianyu Dai and Qingxian Li designed and performed the research, analyzed the data and prepared the manuscript. Yinxian Wen and Liaobin Chen designed the research and revised the manuscript. Yingying Pu provided S. aureus and designed the research. Wang Hui also revised the manuscript. Hebin Liao analyzed the data.

Funding

The study was granted by the National Natural Science Foundation of China (No. 81673524, 81603214, 81673490, 81972036, 82402852), the Key Research and Development Project of Hubei province (No. 2020BCA071), the National Natural Science Foundation of China (31970089, T2125002), Science Fund for Distinguished Young Scholars of Hubei Province (2022CFA077), and China Postdoctoral Science Foundation (No. 2023M742931).

Declaration of competing of interest

The authors have declared no conflict of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: