Delivery of miR-146a to Ly6Chigh Monocytes Inhibits Pathogenic Bone Erosion in Inflammatory Arthritis

Abstract

Rationale: Monocytes play critical roles in the pathogenesis of arthritis by contributing to the inflammatory response and bone erosion. Among genes involved in regulating monocyte functions, miR-146a negatively regulates the inflammatory response and osteoclast differentiation of monocytes. It is also the only miRNA reported to differentially regulate the cytokine response of the two classical Ly6C^high and non-classical Ly6C^low monocyte subsets upon bacterial challenge. Although miR-146a is overexpressed in many tissues of arthritic patients, its specific role in monocyte subsets under arthritic conditions remains to be explored. Methods: We analyzed the monocyte subsets during collagen-induced arthritis (CIA) development by flow cytometry. We quantified the expression of miR-146a in classical and non-classical monocytes sorted from healthy and CIA mice, as well as patients with rheumatoid arthritis (RA). We monitored arthritis features in miR-146a^-/- mice and assessed in vivo the therapeutic potential of miR-146a mimics delivery to Ly6C^high monocytes. We performed transcriptomic and pathway enrichment analyses on both monocyte subsets sorted from wild type and miR-146a^-/- mice. Results: We showed that the expression of miR-146a is reduced in the Ly6C^high subset of CIA mice and in the analogous monocyte subset (CD14⁺CD16^-) in humans with RA as compared with healthy controls. The ablation of miR-146a in mice worsened arthritis severity, increased osteoclast differentiation in vitro and bone erosion in vivo. In vivo delivery of miR-146a to Ly6C^high monocytes, and not to Ly6C^low monocytes, rescues bone erosion in miR-146a^-/- arthritic mice and reduces osteoclast differentiation and pathogenic bone erosion in CIA joints of miR-146a^+/+ mice, with no effect on inflammation. Silencing of the non-canonical NF-κB family member RelB in miR-146a^-/- Ly6C^high monocytes uncovers a role for miR-146a as a key regulator of the differentiation of Ly6C^high, and not Ly6C^low, monocytes into osteoclasts under arthritic conditions. Conclusion: Our results show that classical monocytes play a critical role in arthritis bone erosion. They demonstrate the theranostics potential of manipulating miR-146a expression in Ly6C^high monocytes to prevent joint destruction while sparing inflammation in arthritis.

Keywords: arthritis; bone erosion; miR-146; monocyte subsets; osteoclast.

Figure 1

Inflammatory Ly6C^high monocytes are amplified in arthritis. DBA/1 mice were immunized with bovine type II collagen and boosted on day 21 to induce collagen-induced arthritis (CIA). Non-immunized (NI) DBA/1 mice were used as controls. (A) Mice were sacrificed 40 days after immunization and frequencies of Ly6C^high (grey symbols) and Ly6C^low (black symbols) monocytes quantified in the blood. (B-C) Temporal changes in Ly6C^high and Ly6C^low monocyte subsets were quantified in peripheral blood (B) and spleen (C), compared to day 0 of arthritis. Arthritis severity was assessed by measuring the hind-paw swelling for each mouse along the course of the disease (grey triangles). (D) At day 50 post-immunization, leukocytes were isolated from ankle joints of CIA (grey bars) and NI (dark bars) mice. Representative dot plots for F4/80^high macrophages and Ly6C^high monocytes are shown (gated on CD45⁺ Ly6G^- CD11b⁺ cells). Bar graphs show flow cytometric enumeration of Ly6C^high monocytes (Mo Ly6C^high), macrophages (MΦ) and neutrophils. (E) Activation status of Ly6C^high monocytes from ankle joints was assessed by membrane staining of CD80 and MHCII and TNF-α intracellular cytokine staining. The gates were set using isotype control antibodies. The frequency at each time point represents the mean ± SEM of 3-8 mice/group. Differences between groups were compared using two-way ANOVA (A), two-way repeated measures ANOVA (B, C), and non-parametric Mann-Whitney test (D). ** p<0.01, *** p<0.001.

Figure 2

Expression of miR-146a by Ly6C^high monocytes is decreased in arthritic individuals. Monocyte subsets were FACS sorted from mouse and human samples, isolated from either healthy or arthritic individuals. Relative miR-146a expression was quantified using RT-qPCR in (A) Ly6C^high (black circles) or Ly6C^low (white circles) monocytes isolated from blood of healthy mice, (B) CD14⁺ (black squares) and CD16⁺ (white squares) monocytes isolated from blood samples collected from healthy donors, (C) Ly6C^high monocytes isolated from healthy (NI, black circles) mice and collagen-induced arthritic (CIA, black triangles) mice, (D) CD14⁺ monocytes isolated from the blood of either healthy donors (CT, black squares) or patients with rheumatoid arthritis (RA, black triangles). (E) Relative miR-146a expression was quantified using RT-qPCR in CD14⁺ monocytes isolated from the peripheral blood (PBRA, black triangles) or synovial fluid (RASF, black diamond) of RA patients. Data are presented as mean ± SEM of 3-8 biological replicates corresponding to either individual (human) or 20-25 pooled (mice) samples. Two-tailed Wilcoxon test (A-B, E) and Mann-Whitney test (C-D) were used. ** p<0.01, ns: not significant.

Figure 3

MiR-146a deficiency increases osteoclast differentiation and inflammatory arthritis-induced bone erosion. miR-146a^-/- (KO) mice or wild type (WT) littermates were injected i.p. on day 0 and 1 with K/BxN serum to induce serum-transfer arthritis (STA). Control groups were injected with PBS. Mice were sacrificed 11 days after the first injection (n=4-10 mice/group). (A) Arthritis severity was monitored. Significant differences to the wild-type group are marked. Data are representative of four independent experiments. (B) Knee sections were processed for TRAP and hematoxylin staining. Representative sections are shown (5x magnification). The number of TRAP positive osteoclast cells was counted at pannus site. (C) Micro-computed tomography analysis was performed at day 10 for 3-dimensional reconstruction of the anklebones and quantification of trabecular micro-architecture parameters of proximal femur bone, including trabecular thickness (Tb.Th, mm) and bone volume (BV/TV, %). (D) Temporal changes in osteoclast differentiation capacity of splenic and bone marrow precursors isolated from arthritic miR-146a KO or WT mice were monitored at the indicated time points. The number of TRAP-positive multinucleated osteoclasts was quantified using automated fluorescence imaging (n=20 areas/well, n=4 mice/group). (E) Osteoclast precursors from STA or PBS miR-146a^-/- (KO) mice were cultivated in osteoclast differentiation medium for 7 days. Number of TRAP-positive multinucleated osteoclasts was quantified using automated fluorescence imaging (n=20 areas/well, n=4 mice/group). Osteoclast differentiation potential of miR-146a deficient progenitors isolated from arthritic or healthy mice after 9 days of culture (n=4). Data are presented as mean ± SEM and representative of four independent experiments. Differences between groups were compared using two-way repeated measures ANOVA (A), Mann-Whitney test (B and E), or two-way ANOVA (C, D) * p<0.05, ** p<0.01, *** p<0.001.

Figure 4

In vivo delivery of miR-146a mimics to Ly6C^high monocytes interferes with pathogenic bone erosion during inflammatory arthritis in wild type and knockout mice. (A-D) The DMAPAP/DOPE cationic liposome was formulated with either miR-146a mimics (miR-146a) or non-targeting miRNA mimics (miR-CT) and injected (0.5mg/kg) either only once (A), twice (B), or weekly over a 3-weeks period (C-D) from day 23. Mice were sacrificed 24 hours after the last injection. DBA/1 mice were immunized with bovine type II collagen and boosted on day 21. (A) Fold change of miR-146a expression in splenic Ly6C^high and Ly6C^low monocyte subsets isolated from pooled mice injected with miR-146a mimic-containing lipoplexes compared to miR-CT mimic-injected animals. (B) Expression level of miR-146a in splenic Ly6C^high monocytes following treatment with either miR-146a mimics- or miR-CT mimics-containing lipoplexes. (C-D) At euthanasia, knee (C) and ankle (D) joints were collected and processed for histopathological measurements. (C) Tissue sections were processed for TRAP and hematoxylin staining. Representative sections are shown (20x magnification). The number of TRAP positive osteoclast precursor cells (OCP) and osteoclasts (OC) was counted at the sites of bone erosion. (D) Tissue sections were stained with Safranin O/Fast green for evaluation of bone erosion. Representative sections are shown (20x magnification) and erosion score plotted. (E) MiR-146a^-/- (KO) mice were injected i.p. on day 0 and 1 with K/BxN serum to induce arthritis. Mice were injected with miR-146a- or miR-CT-containing lipoplexes at day 4, 7 and 9. Micro-computed tomography analysis was performed for 3-dimensional reconstruction of knee joints and quantification of trabecular micro-architecture of proximal femur bone, including the following parameters: bone specific surface (BS/BV, mm-1) and trabecular thickness (Tb.Th, mm). Data are presented as mean ± SEM of 1-5 technical replicates of 4-5 pooled biological samples (A-B) or of 5-10 individual mice (C-E). Data refer to one representative experiment out of 2. * p<0.05, ** p<0.01, ns: not significant versus controls using Mann-Whitney (A-D), or Kruskal-Wallis (E) tests.

Figure 5

MiR-146a is a regulator of osteoclastogenesis in Ly6C^high monocytes. (A) Total RNA was extracted from FACS sorted Ly6C^high or Ly6C^low blood monocytes isolated from either miR-146a^-/- (KO) or wild type (WT) mice (pool of 40 mice/group). After reverse transcription, transcriptomic analysis was performed using mouse GeneChip arrays (Affymetrix). Hierarchical cluster analysis was performed. (B) Venn diagram illustrates the intersection between genes differentially expressed between Ly6C^high monocytes of miR-146a^-/- (KO) and wild type (WT) mice, genes of the Osteoclast differentiation Pathway SuperPath, and genes differentially expressed between WT Ly6C^high and Ly6C^low monocytes. (C) STRING analysis generated a confidence view. Thick lines represent strong associations. (D-E) DBA/1 mice were immunized with bovine type II collagen on day 0 and 21, and analyses were performed on day 28. Relative RelB (D) and Traf6 (E) mRNA expression in Ly6C^high or Ly6C^low splenic monocytes isolated from arthritic mice. The DMAPAP/DOPE cationic liposome was formulated with either miR-146a mimics (0.5mg/kg) (miR-146a, light grey bars) or non-targeting miRNA mimics (miR-CT, black and white bars) and injected twice on day 23 and 27 (pool of 5 mice/group). Mice were sacrificed 24 hours after the last injection and Ly6C^high monocytes sorted from pooled spleens (n=2-3 pools of 5 mice/group). Data are presented as mean ± SEM of 3 experimental replicates. ** p<0.01 using Kruskal-Wallis test (D-E).

Figure 6

miR-146a controls osteoclast progenitor differentiation through RelB. (A) Primary bone marrow precursors were differentiated by culturing with M-CSF (15 ng/ml) and RANKL (40 ng/ml). Fold change of expression levels of miR-146a, RelB and Trap gene were quantified during osteoclastogenesis. (B) Osteoclast precursors from miR-146a^-/- (KO) mice or wild type littermates (WT) were cultivated in osteoclast differentiation medium for 7 days. Number of TRAP-positive multinucleated osteoclasts was quantified using automated fluorescence imaging (n=20 areas/well, n=4 mice/group). Representative pictures from four independent experiments are shown (10x magnification). (C) Functional assay was performed on an Osteo Assay Surface plate. Relative resorption area was quantified using image J software (n=6 areas/well, two experimental replicates of 4 mice/group pooled). (D) Expression levels of miR-146a were quantified in bone marrow precursors from miR-146a^-/- (KO) mice or wild type littermates (WT). (E) miR-146a^-/- (KO) mice or wild type (WT) littermates were injected i.p. on day 0 and 1 with K/BxN serum to induce serum-transfer arthritis (STA). The DMAPAP/DOPE cationic liposome was formulated with either CTRL siRNA (si-CT, dark grey bars) or si-RNA targeting RelB gene (si-RelB, light Grey bars) and injected (0.5mg/kg) twice from day 4 and 7. Mice were sacrificed on day 10. Osteoclast precursors from STA miR-146a^-/- (KO) mice injected with siRNA CTRL (si-CT) or siRNA RelB (si-RelB) and were cultivated in osteoclast differentiation medium for 7 days. Relative RelB and Trap mRNA expression was quantified using RTqPCR. (F) Number of TRAP-positive multinucleated osteoclasts was quantified using automated fluorescence imaging (n=20 areas/well, n=4 mice/group). Data are presented as mean ± SEM, (A) present data from 6 mice/group. Differences between groups were compared using Mann-Whitney test (A, B, C and D). E and F present as mean 2 data from 5 mice/group, no statistical analysis can be carried out. * p<0.05, ** p<0.01, *** p<0.001, ordinary one-way ANOVA t-test.