Near-infrared light-driven metabolic reprogramming of synoviocytes for the treatment of rheumatoid arthritis

Abstract

Rheumatoid arthritis is a common autoimmune disease characterized by chronic synovial inflammation and joint destruction, primarily driven by an imbalanced cellular metabolism and inflammatory microenvironment. While gene therapy offers a promising therapeutic approach, its effectiveness is limited by the challenges of non-specific gene expression in healthy tissues. Here, we develop a gene delivery system (namely APPC), in which near-infrared (NIR)-responsive gold nanorods are coated with chondroitin sulfate-modified polyethyleneimine to facilitate the heat-responsive targeted delivery of heme oxygenase 1 (HO-1) gene. The APPC shows favorable transfection efficiency due to its targeting ability and significantly facilitates HO-1 expression under NIR irradiation. The combination of APPC/pHO-1 and NIR can effectively reprogram the cellular metabolism and repolarize the macrophages and fibroblast-like synoviocytes, thereby inhibiting inflammation by suppressing glycolysis. Meanwhile, APPC can specifically enhance the HO-1 expression in inflamed tissues through NIR-mediated the activation of heat shock protein 70 promoter, ensuring the precise gene expression via photothermal conversion. In a collagen-induced arthritis model, APPC/pHO-1 under NIR irradiation exhibits potent therapeutic efficacy, restoring the articular microenvironmental homeostasis and mitigating the symptoms of rheumatoid arthritis. These findings highlight the potential of APPC/pHO-1 nanoparticles in the gene therapy of rheumatoid arthritis and other inflammatory diseases.

Fig. 1. Elevated glycolysis in RA patients and CIA mice.

a, b GO and KEGG enrichment analysis of differentially expressed genes between the synovium macrophages of RA patients and healthy individuals. c Expression of glycolysis-related genes, PFKFB1, PFKP and LDHA in RA patients. d Expression of OXPHOS-related genes, TXNRD2, ATP6V0A1 and NDUFV1 in RA patients. e HMOX1 gene expression in RA patients. f, g Expression level of GLUT1 and HK2 in the arthritic joints of CIA mice detected by immunohistochemistry. Scale bar: 50 μm. h The phenotype of macrophages in the synovial tissues of CIA mice was determined by the immunofluorescence staining of F4/80, CD206 and INOS. Nuclei, blue (DAPI); F4/80, green; CD206, red; INOS, violet. Scale bar: 10 μm. i The FLS phenotype in the synovial tissues of CIA mice was determined by the immunofluorescence staining of CAD11 and MMP9. Nuclei, blue (DAPI); MMP9, green; CAD11, red. Scale bar: 10 μm. a, b The size of the dots denotes the number of genes in this term. The color of the dots is determined by the Benjamini-Hochberg adjusted log₁₀(P values), highlighting the statistical significance of observed variations. Benjamini-Hochberg adjusted P-values are determined by a two-tailed Fisher’s exact test. c–e Data are presented as mean value ± SD, and statistical significance is measured by unpaired two-tailed Student’s t-test. f–i Representative images of three biologically independent experiments with similar results are shown. Source data are provided as Source Data files.

Fig. 2. Preparation and characterization of APPC.

a Synthetic route of APPC. b, c Hydrodynamic diameter and zeta potential of AR, APP, APPC and APPC/pHO-1, respectively. d TEM images of AR, APP and APPC. Scale bar: 50 nm. e UV-vis absorption spectra of AR, APP and APPC. f Temperature change curves of PBS, AR, APP and APPC under NIR irradiation (2.0 W/cm²) over time. g Temperature change curves of APPC (200 μg/mL) under various NIR radiation intensities. h Temperature change curves of different APPC concentrations under NIR irradiation (2.0 W/cm²) over time. i TEM and EDS mapping of APPC/pHO-1 nanoparticles. Scale bar: 25 nm. j, k XPS spectra of AR, APP, APPC and APPC/pHO-1. (l, m) The binding and condensation ability of APP and APPC with pHO-1 at different w/w ratios. Data are presented as mean value ± SD (b, c, f–h, n = 3 independent experiments). i, l, m Representative images of three biologically independent experiments with similar results are shown. Source data are provided as Source Data files.

Fig. 3. In vitro transfection efficiency of APPC.

a Bio-TEM images of RAW264.7 cells after the transfection of APPC/pHO-1 nanoparticles. The red arrows indicated the presence of APPC in the cytoplasm. Scale bar: 1 μm. b Fluorescence images of LPS-stimulated RAW264.7 cells after the transfection of APP/pEGFP-N3 and APPC/pEGFP-N3 for 48 h. Scale bar: 200 μm. c The CD44 expression in normal and LPS-stimulated RAW264.7 cells. Nuclei, blue (DAPI); CD44, red. Scale bar: 10 μm. d Endocytosis of APP and APPC in normal and LPS-stimulated RAW264.7 cells. Nuclei, blue (DAPI); APP or APPC, green (FITC). Scale bar: 20 μm. e Endocytosis of APP and APPC in LPS-stimulated RAW264.7 cells after the CS shielding. Nuclei, blue (DAPI); APP or APPC, green (FITC). Scale bar: 20 μm. f Intracellular biodistribution of APPC/pHO-1 in LPS-stimulated RAW264.7 cells. Nuclei, blue (DAPI); APPC, green (FITC); lysosomes, red (Lyso Tracker Red). The white arrows represented the overlap of green and red fluorescence, indicating that the nanoparticles were entrapped in endosomes. Scale bar: 10 μm. a–f Representative images of three biologically independent experiments with similar results are shown.

Fig. 4. Inhibition of glycolysis and inflammation by pHO-1 delivery.

a The expression level of HO-1 in LPS-stimulated RAW264.7 cells after the pHO-1 transfection. Representative blots of three independent experiments with similar results are shown. b, c ECAR and OCR of LPS-stimulated RAW264.7 cells after the pHO-1 transfection. Data are presented as mean ± SD (n = 5 independent samples). d–f Relative mRNA levels of Glut1, Hk2 and Ldha in LPS-stimulated RAW264.7 cells after the pHO-1 transfection. g The macrophage phenotype after the pHO-1 transfection. Nuclei, blue (DAPI); CD206, green; INOS, red. Scale bar: 10 μm. Representative images of three biologically independent experiments with similar results are shown. h, i Relative mRNA levels of Il1b and Il10 in LPS-stimulated RAW264.7 cells after the pHO-1 transfection. LPS- represents the RAW264.7 cells without lipopolysaccharide stimulation, and LPS+ groups represent the RAW264.7 cells treated with lipopolysaccharide (0.1 μg/ml) for 24 h (d–f, h, i). Data are presented as mean ± SD (d–f, h, i, n = 3 independent experiments), and one-way ANOVA with LSD test is used for statistical analysis. Source data are provided as Source Data files.

Fig. 5. The transcriptional analysis of macrophages after the transfection of APPC/pHO-1.

a GO enriched pathways of DEGs between normal (Control) and LPS-stimulated RAW264.7 cells. b Activation of signaling pathways related to glycolysis in LPS-stimulated RAW264.7 cells. c GO enriched pathways of DEGs in LPS-stimulated RAW264.7 cells after the transfection of APPC/pHO-1. d Activation of signaling pathways related to OXPHOS in APPC/pHO-1 group. e Expression of genes related to glycolysis, macrophage polarization and inflammation in LPS-stimulated RAW264.7 cells after the pHO-1 transfection. Data are presented as mean ± SD (n = 3 independent samples), and one-way ANOVA with LSD test is used for statistical analysis. f IPA analysis of cell signaling pathways containing DEGs between two groups, colored by Z-score. A positive Z-score indicated activation, and a negative Z-score indicated inhibition. g Illustration of HO-1’s inhibitory effects on glycolysis and its anti-inflammatory mechanism. a, c The size of the dots represents the number of genes in this term. The sizes of the dots are governed by the Benjamini-Hochberg adjusted-log₁₀(P values), highlighting the statistical significance of observed variations. Benjamini-Hochberg adjusted-P values are obtained by a two-tailed Fisher’s exact test. Source data are provided as Source Data files.

Fig. 6. Inhibition of glycolysis and proliferative capacity through pHO-1 delivery.

a The expression level of HO-1 in TNF-α-stimulated FLSs after the pHO-1 transfection. Representative blots of three independent experiments with similar results are shown. b, c Relative mRNA levels of Glut1 and Hk2 in TNF-α-stimulated FLSs after the pHO-1 transfection, respectively. TNF-α- represents the FLS cells without TNF-α stimulation, and TNF-α+ groups represent the FLS cells treated with TNF-α (0.1 μg/ml) for 24 h. d Representative images for the wound healing of FLSs under different treatments at 0, 24, and 48 h. Scale bar: 200 μm. Representative images of three biologically independent experiments with similar results are shown. e The statistical quantification of scratch width of FLSs under various treatments at different time. f The illustration of HO-1’s inhibitory effect on the proliferation of FLSs. g Schematic representation of the impact of RAW264.7 cells on the physiology of FLSs. h Schematic representation of the influence of FLSs on RAW264.7 cells. i, j Relative mRNA level of Glut1 and Mmp9 in FLSs after the stimulation with supernatants of RAW264.7 cells under different treatments. k Relative mRNA level of Glut1 in RAW264.7 cells following the stimulation with supernatants from FLSs under various conditions. Data are presented as mean ± SD (b, c, e and i–k, n = 3 independent experiments), and one-way ANOVA with LSD test is used for statistical analysis. Source data are provided as Source Data files.

Fig. 7. In vivo photothermal conversion of APPC/pHO-1-mCherry to activate the gene expression.

a Representative thermal images of mice limbs after the intramuscular injection of different nanoparticles and subsequent NIR irradiation. b Ex vivo expression of mCherry in muscles of C57BL/6 N mice observed at 12, 24 and 48 h post-administration. c The mCherry expression in irradiated muscles at 48 h post-administration. Nuclei, blue (DAPI); mCherry, red. Scale bar: 20 μm. d Representative thermal images of mice abdomen after the intravenous injection of different nanoparticles and subsequent NIR irradiation at the liver area. e Ex vivo expression of mCherry in the liver of C57BL/6 N mice observed at 12, 24, and 48 h post-administration. f The mCherry expression in the irradiated liver of C57BL/6 N mice at 48 h post-administration. Nuclei, blue (DAPI); mCherry, red. Scale bar: 100 μm. a–f Representative images of three biologically independent animals with similar results are shown.

Fig. 8. Biodistribution of APPC/pHO-1 in CIA mice.

a Ex vivo biodistribution of free TOTO-3-labelled pHO-1, APP/TOTO-3-labelled pHO-1 and APPC/TOTO-3-labelled pHO-1 in CIA mice after the intravenous injection for 1, 4 and 24 h. b Representative images for the distribution of nanoparticles in the synovium of mice. Nuclei, blue (DAPI); TOTO-3, red. Scale bar: 50 μm. c Ex vivo expression of free pHO-1-mCherry, APP/pHO-1-mCherry and APPC/pHO-1-mCherry in CIA mice at 12, 24 and 48 h post-administration. d The mCherry expression in macrophages and FLSs in the synovium after NIR irradiation. Nuclei, blue (DAPI); F4/80, green; CAD11, yellow; mCherry, red. Scale bar: 25 μm. (a–d) Representative images of three biologically independent animals with similar results are shown.

Fig. 9. Therapeutic efficiency of APPC/pHO-1 nanoparticles in CIA mice model.

a The therapeutic strategy in CIA mice. b The macroscopic observation to assess the swelling severity of soft tissue at Day 35 post-adjuvant induction. c The representative micro-CT images of arthritis mice after different treatments at Day 35 post-adjuvant induction. d H&E staining of the arthritis ankles of CIA mice. Scale bar: 100 μm. The red region represented the bone erosion (bottom left) and infiltration of synovial tissues (bottom right). Scale bar: 50 μm. e, f Articular cartilages of ankle joints were identified by Safranin O/Fast Green staining and Toluidine blue, respectively. Scale bar: 50 μm. b–f Representative images of five biologically independent animals with similar results are shown.

Fig. 10. The APPC/pHO-1 nanoparticles regulated the glycolysis to induce the anti-inflammatory and anti-proliferative effect in CIA mice.

a–c Levels of HO-1, GLUT1, and HK2 in the arthritic joints of CIA mice detected by IHC. Scale bar: 50 μm. d Effect of APPC/pHO-1 on the phenotype of macrophages in the synovial tissues of CIA mice, assessed by the immunofluorescence staining of CD206 and INOS. Nuclei, blue (DAPI); F4/80, green; CD206, red; INOS, violet. Scale bar: 10 μm. e Effect of APPC/pHO-1 on the phenotype of FLSs in the synovial tissues of CIA mice, assessed by the immunofluorescence staining of CAD11 and MMP9. Nuclei, blue (DAPI); MMP9, green; CAD11, red. Scale bar: 20 μm. f, g The mRNA level of Il1b and Il10 in the arthritic joints of CIA mice. Data are presented as mean value ± SD (n = 5 independent animals), and one-way ANOVA with LSD test is selected for statistical analysis. h The schematic presentation of APPC/pHO-1 intravenously injected into CIA mice. Following the injection, the nanoparticles could be accumulated in inflamed joints via ELVIS effect and further captured by the synoviocytes via CD44 receptor. Under NIR irradiation, HSF monomers in synoviocytes aggregated into trimers, which translocated to the nucleus to bind to the HSP70 promoter and subsequently triggered the HO-1 expression, leading to the suppression of inflammation by inhibiting glycolysis to ultimately attenuate the symptoms of RA. a–e Representative images of five biologically independent animals with similar results are shown. Source data are provided as Source Data files.