Molecular Determinants of Neutrophil Extracellular Vesicles That Drive Cartilage Regeneration in Inflammatory Arthritis

Abstract

Objective: This study was undertaken to establish the potential therapeutic profile of neutrophil-derived extracellular vesicles (EVs) in experimental inflammatory arthritis and associate pharmacological activity with specific EV components, focusing on microRNAs.

Methods: Neutrophil EVs were administered intra-articularly through a prophylactic or therapeutic protocol to male C57BL/6 mice undergoing serum-transfer-induced inflammatory arthritis. Transcriptomic analysis of knees was performed on joints following EV administration, naive and arthritic mice (untreated; n = 4/group) and EV-treated diseased mice (intra-articular administration) with contralateral (vehicle-treated; n = 8/group). Comparison of healthy donor and patients with rheumatoid arthritis (RA) neutrophil EVs was performed.

Results: EVs afforded cartilage protection with an increase in collagen-II and reduced collagen-X expression within the joint. To gain mechanistic insights, RNA sequencing of the arthritic joints was conducted. A total of 5,231 genes were differentially expressed (P < 0.05), with 257 unique to EV treatment. EVs affected key regenerative pathways involved in joint development, including Wnt and Notch signaling. This wealth of genomic alteration prompted to identify microRNAs in EVs, 10 of which are associated with RA. As a proof of concept, we focused on miR-455-3p, which was detected in both healthy donor and RA EVs. EV addition to chondrocyte cultures elevated miR-455-3p and exerted anticatabolic effects upon interleukin-1β stimulation; these effects were blocked by actinomycin or miR-455-3p antagomir.

Conclusion: Neutrophils from patients with RA yielded EVs with composition, efficacy, and miR-455-3p content similar to those of healthy volunteers, suggesting that neutrophil EVs could be developed as an autologous treatment to protect and repair joint tissue of patients affected by inflammatory arthritides.

Figure 1

Long‐term chondroprotective properties of neutrophil EVs. K/BxN serum was injected i.p. at day 0, 2, 13, and 24 (arrows) to induce arthritis (STIA). Mice were treated on day 3 intra‐articularly with 1.0 × 10⁵ EV in one knee and vehicle in contralateral knee (open triangle) and disease profile was monitored over 30 days (A and B). (A) Disease profile (score out of 12 for inflamed digits, paws, and wrists). Data are mean ± SD, 5 mice per group. (B) Characterization of cartilage breakdown from vehicle‐treated knees at indicated timepoints of the protocol shown in panel A; representative images of safranin O‐stained knee joints, used to measure proteoglycan content. (C) Day 30 safranin O‐stained images of arthritic mice treated with vehicle or EVs and (D) quantification and (E) OARSI score (sum of all compartments) for structural integrity (*P < 0.05 nonparametric t‐test, Wilcoxon matched‐pairs). (F–I) Day 30 staining for collagen type‐II and collagen type‐X, expressed as intensity within the extracellular matrix and percentage of total (DAPI+) cells. Data are mean ± SD, n = 8 mice per group. (J) STIA was conducted as in A, knee width measurements in EV‐treated (EV) and contralateral vehicle‐treated (V) measured as percentage of baseline at day 3. Data are mean ± SD, n = 8 mice per group. (K–M) Day 30 histological scores for inflammation (0–3), bone resorption (0–5), and osteophyte formation. EV, extracellular vesicle; i.p., intraperitoneal; OARSI, Osteoarthritis Research Society International; STIA, serum‐transfer–induced arthritis.

Figure 2

Therapeutic and translational effects of neutrophil EVs. (A and B) K/BxN STIA was induced as in Figure 1A. Mice were treated intra‐articularly with 1.0 × 10⁵ EV (EV) or PBS vehicle (V) on day 20 and experiment terminated at day 30. (A) Representative images for safranin O staining of knee sections. (B and C) Quantitative data for safranin O staining within the knee sections and OARSI score for structural integrity of knee section (*P < 0.05 nonparametric t‐test, Wilcoxon matched‐pairs). (D–I) Human articular chondrocytes translation model. Chondrocytes (1 × 10⁶) were mixed with a collagen type‐I scaffold gel and either 1.0 × 10⁵ EV (EV) or vehicle (V) before subcutaneous injection onto six‐week‐old nude mice. (C) Two weeks following implantation, cartilage nodules were harvested and sectioned through at 100‐μm intervals before toluidine blue staining. Images for representative nodule shown. (D) Analyses of the central sections of the nodules for intensity of the proteoglycan stain and size of the differentiated cartilage area and representative images and cumulative data. For each single nodule three central sections were used in the analysis. Scale bar = 500 μm. (E) Representative images for collagen type‐II staining within the nodules and data for staining intensity. Cumulative data are mean ± SEM, n = 8 mice per group. *P < 0.05, Mann‐Whitney test. EV, extracellular vesicle; OARSI, Osteoarthritis Research Society International; PBS, phosphate buffered saline; STIA, serum‐transfer–induced arthritis.

Figure 3

Transcriptomic analysis of EV‐induced changes in the joint cartilage. STIA mice were treated with either 1.0 × 10⁵ EVs or vehicle intra‐articular at day 3. At day 10, tibial and femoral cartilage surface, together with the subchondral bone, was harvested and RNA was extracted for sequencing. (A) Experimental design included nonarthritic (naive) and diseased (arthritic) mice (untreated), n = 4 knees/group, and EV‐treated diseased mice (intra‐articular administration) with contralateral control (vehicle‐treated) knees, n = 8 knees/group (paired). Venn diagrams presenting the total number of significantly altered genes in each comparison group. Groups were compared as disease vs naive or EV‐treated vs contralateral vehicle control: 153 genes altered by EV treatment were also altered by disease, whereas 257 genes were unique to EV treatment. Significantly altered genes were ranked by greatest fold change, and the top genes are displayed for each comparison group. For the genes regulated by disease and also counterregulated by the treatment, the top differentially regulated genes (up‐regulated in treatment with a significantly regulated decrease in disease or vice versa) have been displayed. (B) Functional analysis of processes, functions, and pathways for each of the comparisons (disease vs naive and EV‐treated vs nontreated) was conducted using Panther Classification System. CCKR, cell cycle‐related kinase; EGF, epidermal growth factor; EV, extracellular vesicle; MF, molecular function; PDGF, platelet‐derived growth factor; STIA, serum‐transfer–induced arthritis; TGF, transforming growth factor.

Figure 4

Investigation of miR content within neutrophil EVs. Seven distinct preparations of neutrophil EVs were analyzed using Thermofisher miRNA 4.1 GeneChip. (A) Heat map of the top 100 miRs (listed from higher‐to‐lower) shows consistency across donors. (B) Most abundant miR (ie, expression >1) within the EVs form a network with their target genes (network visual analytics platform miRNet 2.0). (C) Enriched pathways from panel B: colors are matched to highlight specific pathway involved in joint repair and disease. (D) Target genes related to rheumatoid arthritis were separated from panel B and displayed in a network in association with their targeting miRs which have been detected within the EVs. EV, extracellular vesicle; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAPK, mitogen‐activated protein kinase; miR, microRNA; mTOR, mechanistic target of rapamycin; TGF, transforming growth factor.

Figure 5

miR‐455‐3p is contained in EVs and exerts anticatabolic properties on chondrocytes. (A) Quantification of miR‐455‐3p in human neutrophils and their EVs by real‐time PCR. EVs (3 × 10⁷) from endothelial cells and platelets were also included for comparison. (B) Human articular chondrocytes were pretreated for 30 minutes with or without actinomycin D, plus or minus neutrophil EVs (3 × 10⁷), and miR‐455‐3p levels were measured after 24 hours. Cells were harvested, and microRNA extracted for reverse transcription and real‐time PCR. Data are reported as fold change from vehicle control (dotted line) *P < 0.05 vs EV Mann‐Whitney test (C–F) Human articular chondrocytes were incubated with IL‐1β (20 ng/mL) with or without healthy donor neutrophil EVs. At 24 hours, real‐time PCR quantification of markers of hypertrophy and/or targets of miR‐455‐3p: COL10A1 and RUNX2, ALPL, and MMP13 were monitored. Data are reported as fold change from vehicle control (dotted line) **P < 0.01 vs IL‐1β Kruskal‐Wallis test. (G and H) Human articular chondrocytes were treated with combinations of IL‐1β (20 ng/mL) and EVs (3 × 10⁷), with or without presence of a control or specific antagomir. Data are normalized to IL‐1β control. *P < 0.05 vs IL‐1β, #P < 0.05 vs IL‐1β+EV following a two‐way ANOVA multiple comparison. Each data point identifies distinct EV donors. ANOVA, analysis of variance; EC, endothelial cell; EV, extracellular vesicle; IL, interleukin; miR, microRNA; NØ, neutrophil; PCR, polymerase chain reaction; PLT, platelet.

Figure 6

Characterization of RA neutrophil EVs. EVs were obtained from RA and HD neutrophils. (A) Quantification of EV number per cell. (B) Median size distribution of HD and RA neutrophil EVs (C) Average Nanosight profiles for six EV donors per group; mean ± SD (D–F) CD66b and AnxA1 expression on EVs from HD and patients with RA neutrophils. (Left) Representative ImageStream profiles. (Right) Quantitation of positive events (mean ± SD, n = 5–6 preparations per group) *P < 0.05, **P < 0.01 Mann‐Whitney test. (G and H) Uptake of RA neutrophil EVs by human chondrocytes and modulation by anti‐AnxA1 Ab. (Left) Representative ImageStream images showing EV uptake (green) by the whole cells. (Right) Quantitative data (mean ± SD of n = 3 experiments; #P < 0.05 Kruskal‐Wallis test with Dunn's multiple comparison). (I) miR‐455‐3p content for 3 × 10⁷ EV per donor, as analyzed by RT‐PCR (n = 7–11). (J) Human articular chondrocytes were stimulated IL‐1β (20 ng/mL) with or without RA EVs (3 × 10⁷) for 24 hours. Collagen type‐X gene expression was quantified by RT‐PCR (mean ± SD of 4 replicates from two donors). Ab, antibody; EV, extracellular vesicle; HD, healthy donor; IL, interleukin; MFI, mean fluorescence intensity; miR, microRNA; RA, rheumatoid arthritis; RT‐PCR, real‐time polymerase chain reaction.