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. 2021 Apr 12;12(1):2174.
doi: 10.1038/s41467-021-22454-z.

Targeted apoptosis of macrophages and osteoclasts in arthritic joints is effective against advanced inflammatory arthritis

Caifeng Deng  1   2 , Quan Zhang  3   4 , Penghui He  1 , Bin Zhou  2   5 , Ke He  2   5 , Xun Sun  1 , Guanghua Lei  6   7   8 , Tao Gong  9 , Zhirong Zhang  1
Affiliations

Targeted apoptosis of macrophages and osteoclasts in arthritic joints is effective against advanced inflammatory arthritis

Caifeng Deng et al. Nat Commun. .

Abstract

Insufficient apoptosis of inflammatory macrophages and osteoclasts (OCs) in rheumatoid arthritis (RA) joints contributes toward the persistent progression of joint inflammation and destruction. Here, we deliver celastrol (CEL) to selectively induce apoptosis of OCs and macrophages in arthritic joints, with enzyme-responsive nanoparticles (termed PRNPs) composed of RGD modified nanoparticles (termed RNPs) covered with cleavable PEG chains. CEL-loaded PRNPs (CEL-PRNPs) dually target OCs and inflammatory macrophages derived from patients with RA via an RGD-αvβ3 integrin interaction after PEG cleavage by matrix metalloprotease 9, leading to increased apoptosis of these cells. In an adjuvant-induced arthritis rat model, PRNPs have an arthritic joint-specific distribution and CEL-PRNPs efficiently reduce the number of OCs and inflammatory macrophages within these joints. Additionally, rats with advanced arthritis go into inflammatory remission with bone erosion repair and negligible side effects after CEL-PRNPs treatment. These findings indicate potential for targeting chemotherapy-induced apoptosis in the treatment of advanced inflammatory arthritis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Pathological features differ between rats with early and advanced arthritis.
a OCs-induced bone erosion and macrophages-mediated synovial inflammation in advanced arthritis. Representative micro-CT images of ankle joints showing bone erosion levels in ankle joints from normal rats, AIA rats with early-stage arthritis, and AIA rats with late-stage arthritis. Immunohistochemical analyses of the TRAP (tartrate-resistant acid phosphatase)-stained OCs, CD68+ synovial macrophages, and MMP9 (matrix metalloproteinase 9) expression in the joint tissues from rats in each group (n = 3). Scale bar = 100 μm. b, c Quantitative micro-CT analyses of bone mineral density (BMD) and bone surface density (BS/BV). Data represent the mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test.
Fig. 2
Fig. 2. Preparation and characterization of CEL-PRNPs.
a Schematic illustration of CEL-PRNPs preparation. CEL celastrol, MMP9 matrix metalloproteinase 9, CEL-RNPs CEL-loaded RGD peptide-modified poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, CEL-PRNPs CEL-loaded MMP9-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles. b Representative size distribution image of CEL-PRNPs (n = 3 independent samples). c TEM image of CEL-PRNPs (n = 3 independent samples). Scale bar = 100 nm. d The serum stability of CEL-PRNPs during 24 h incubation with 10% FBS at 37 °C and cumulative CEL release from CEL-PRNPs in PBS at 37 °C. Data represent mean ± SD (n = 3 independent samples).
Fig. 3
Fig. 3. Increased apoptosis of OCs and LPS-activated macrophages caused by RGD-mediated endocytosis of PRNPs.
a Confocal images of cellular uptake in OCs and LPS-activated macrophages (n = 3 independent samples). Scale bar = 50 μm. b Flow cytometric analysis of OCs and LPS-activated macrophages apoptosis induced by CEL-RNPs, CEL-RNPs, or CEL-PRNPs with or without the presence of MMP9 for 24 h at the CEL concentration of 100 ng/mL. c Confocal images showing JC-1 assay of LPS-activated macrophages treated with CEL-RNPs, CEL-RNPs, or CEL-PRNPs with or without the presence of MMP9 for 24 h at the CEL concentration of 100 ng/mL (n = 3 independent samples). Scale bar = 50 μm. d, e Quantitative cellular uptake C6-loaded NPs, C6-loaded RNPs, or C6-loaded PRNPs on OCs (d) and LPS-activated macrophages (e) after 1 h incubation at the C6 concentration of 50 ng/mL. Data represent mean ± SD (n = 3 independent samples). Statistical significance was determined by a two-sided Student’s t test. f, g Quantitative analysis for the apoptosis of OCs (f) and LPS-activated macrophages (g) by CEL-NPs, CEL-RNPs, or CEL-PRNPs. Data represent mean ± SD (n = 3 independent samples). Statistical significance was determined by a two-sided Student’s t test. OCs osteoclasts, LPS lipopolysaccharide, C6 coumarin 6, DAPI 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, MMP9 matrix metalloproteinase 9, NPs poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, RNPs RGD peptide-modified PLGA nanoparticles, PRNPs MMP9-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
Fig. 4
Fig. 4. Increased apoptosis of OCs and synovial macrophages derived from patients with late-stage RA by PRNPs.
a Confocal images of the cellular uptake on OCs and synovial macrophages (n = 3 independent samples). Scale bar = 50 μm. b–d Quantitative analysis of the cellular uptake of C6-loaded NPs, RNPs or PRNPs on OCs (b), synovial macrophages (c), and HUVECs (d) after 1-h incubation at the C6 concentration of 50 ng/mL. Data represent mean ± SD (n = 3 independent samples). Statistical significance was determined by a two-sided Student’s t test. e, f Quantitative analysis for the apoptosis of OCs (e) and synovial macrophages (f) by CEL-NPs, CEL-RNPs, or CEL-PRNPs. Data represent mean ± SD (n = 3 independent samples). Statistical significance was determined by a two-sided Student’s t test. OCs osteoclasts, C6 coumarin 6, DAPI 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride, MMP9 matrix metalloproteinase 9, HUVECs human umbilical vein endothelial cells, NPs poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, RNPs RGD peptide-modified PLGA nanoparticles, PRNPs MMP9-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
Fig. 5
Fig. 5. PRNPs selectively accumulate in inflamed joints of rats with advanced arthritis.
a Ex vivo DiD fluorescence images showing the biodistribution of NPs, RNPs, and PRNPs in AIA rats with advanced arthritis (A, heart; B, liver; C, spleen; D, lung; E, kidney; F, Blood; G, arthritic joint) at 24 h post injection. b In vivo DiD fluorescence images showing the arthritic joint distribution of free DiD, and DiD-loaded NPs, RNPs, and PRNPs in AIA rats with a unilateral inflamed joint at 24 h post injection. c Ex vivo DiD fluorescence images in the inflamed joints and un-inflamed joints from AIA rats with a unilateral inflamed joint at 24 h post injection with free DiD, and DiD-labeled NPs, RNPs, or PRNPs. d The statistical graphs of the fluorescence intensity of inflamed joints based on the semi-quantitative analysis of the ex vivo fluorescence images after i.v. administration of free DiD or DiD-labeled nanoparticles. Data represent mean ± SD (n = 6 inflamed joints from 3 independent animals). Statistical significance was determined by two-sided Student’s t test. e The statistical graphs of the fluorescence intensity of major organs from AIA rats with advanced arthritis after i.v. administration of free DiD or DiD-labeled nanoparticles. Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. f The statistical graphs of the fluorescence intensity of inflamed joints and un-inflamed joints from AIA rats with a unilateral inflamed joint after i.v. administration of free DiD or DiD-labeled nanoparticles. Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. DiD 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine, 4-chlorobenzenesulfonate salt, DiD-NPs DiD labeled poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, DiD-RNPs DiD-labeled RGD peptide-modified PLGA nanoparticles, DiD-PRNPs DiD-labeled matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
Fig. 6
Fig. 6. PRNPs are selectively distributed in OCs and inflammatory macrophages in arthritic joints of rats with advanced arthritis.
Confocal images showing the distribution of different DiD formulations in synovial macrophages (a) and OCs (b) in inflamed joints. Macrophages and OCs were determined by immunofluorescence analysis of CD68 and CD51 (green fluorescence), respectively. (Scale bar = 25 μm) (n = 3 independent animals). DiD 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl indodicarbocyanine, 4-chlorobenzenesulfonate salt, DiD-NPs DiD labeled poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, DiD-RNPs DiD-labeled RGD peptide-modified PLGA nanoparticles, DiD-PRNPs DiD-labeled matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
Fig. 7
Fig. 7. CEL-PRNPs reduce the number of OCs and inflammatory macrophages in joints of rats with advanced arthritis.
a TUNEL immunofluorescence staining in ankle joints from AIA rats receiving the indicated treatment (Scale bar = 200 μm) (n = 5 independent animals). b Immunohistochemical analyses of the TRAP-stained OCs and CD68-stained synovial macrophages in the joint tissues from rats receiving the indicated treatment (Scale bar = 100 μm) (n = 5 independent animals). c RANKL/OPG ratio in arthritic joints, IL-1β secretion in blood, and TNF secretion in blood from rats receiving the indicated treatment. Data represent mean ± SD (n = 5 independent animals). Statistical significance was determined by a two-sided Student’s t test. d Detection of IL-1β, TNF, OCN, and ALP expression levels in arthritic joints in different groups. Arthritic joints in different groups were stained with IL-1β, TNF, and OCN antibodies, respectively. ALP was stained light–dark in arthritic joints from different groups (Scale bar = 100 μm) (n = 5 independent animals). CEL celastrol CEL-NPs CEL-loaded poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles, TUNEL TdT-mediated dUTP nick end labeling, TRAP tartrate-resistant acid phosphatase, RANKL receptor of activator of NF-kB ligand, OPG osteoprotegerin, IL-1β interleukin-1 β, TNF tumor necrosis factor, OCN osteocalcin, ALP alkaline phosphatase.
Fig. 8
Fig. 8. Therapeutic efficacy of CEL-PRNPs in rats with advanced arthritis.
a The schematic illustration of CEL-PRNPs treatment. b, c Ankle diameter (b) and paw thickness (c) of AIA rats were recorded every other day during the treatment period. Data represent mean ± SD (n = 7 independent animals). Statistical significance was determined by a two-sided Student’s t test. d Representative photographs of hindlimbs at the endpoint of the experiment from different treatment groups (Scale bar = 10 mm); histopathology evaluation of ankle joints was identified using H&E (scale bar = 200 µm), safranin-O and toluidine blue staining (scale bar = 100 µm) (n = 5 independent animals). i.v. intravenous, anti-TNF anti-TNF (tumor necrosis factor) antibody, CEL celastrol, CEL-NPs CEL-loaded poly (d, l-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles, H&E hematoxylin-eosin.
Fig. 9
Fig. 9. CEL-PRNPs reverse bone erosion in rats with advanced arthritis.
a Representative micro-CT images of the ankle joints at the endpoint of the experiment from different treatment groups in therapeutic efficacy study (n = 3 independent animals). b Representative micro-CT images of the trabecular in bone and the reconstructed trabecular structure (n = 3 independent animals). c, d Quantitative micro-CT analysis of BMD (c) and BS/BV (d) of the ankle joints at the endpoint of the experiment. Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. e–g Quantitative micro-CT analysis of trabecular number (Tb.N) (e), trabecular bone thickness (Tb.Th) (f), and decreasing trabecular separation (Tb.Sp) (g). Data represent mean ± SD (n = 3 independent animals). Statistical significance was determined by a two-sided Student’s t test. Anti-TNF anti-TNF (tumor necrosis factor) antibody, CEL celastrol, CEL-NPs CEL-loaded poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles, CEL-RNPs CEL-loaded RGD peptide-modified PLGA nanoparticles, CEL-PRNPs CEL-loaded matrix metalloproteinase 9 (MMP9)-cleavable polyethylene glycol (PEG)- and RGD peptide-modified PLGA nanoparticles.
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References

    1. Smolen JS, et al. Rheumatoid arthritis. Nat. Rev. Dis. Prim. 2018;4:18001. doi: 10.1038/nrdp.2018.1. - DOI - PubMed
    1. Yeo L, et al. Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis. Ann. Rheum. Dis. 2016;75:763–771. doi: 10.1136/annrheumdis-2014-206921. - DOI - PMC - PubMed
    1. Bijlsma JWJ, Boers M, Saag KG, Furst DE. Glucocorticoids in the treatment of early and late RA. Ann. Rheum. Dis. 2003;62:1033–1037. doi: 10.1136/ard.62.11.1033. - DOI - PMC - PubMed
    1. Benito MJ, Veale DJ, FitzGerald O, van den Berg WB, Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis. Ann. Rheum. Dis. 2005;64:1263–1267. doi: 10.1136/ard.2004.025270. - DOI - PMC - PubMed
    1. Schett G, Gravallese E. Bone erosion in rheumatoid arthritis: mechanisms, diagnosis and treatment. Nat. Rev. Rheumatol. 2012;8:656–664. doi: 10.1038/nrrheum.2012.153. - DOI - PMC - PubMed

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