Neural EGFL like 1 as a potential pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug

Abstract

Arthritis, an inflammatory condition that causes pain and cartilage destruction in joints, affects over 54.4 million people in the US alone. Here, for the first time, we demonstrated the emerging role of neural EGFL like 1 (NELL-1) in arthritis pathogenesis by showing that Nell-1-haploinsufficient (Nell-1^+/6R) mice had accelerated and aggravated osteoarthritis (OA) progression with elevated inflammatory markers in both spontaneous primary OA and chemical-induced secondary OA models. In the chemical-induced OA model, intra-articular injection of interleukin (IL)1β induced more severe inflammation and cartilage degradation in the knee joints of Nell-1^+/6R mice than in wildtype animals. Mechanistically, in addition to its pro-chondrogenic potency, NELL-1 also effectively suppressed the expression of inflammatory cytokines and their downstream cartilage catabolic enzymes by upregulating runt-related transcription factor (RUNX)1 in mouse and human articular cartilage chondrocytes. Notably, NELL-1 significantly reduced IL1β-stimulated inflammation and damage to articular cartilage in vivo. In particular, NELL-1 administration markedly reduced the symptoms of antalgic gait observed in IL1β-challenged Nell-1^+/6R mice. Therefore, NELL-1 is a promising pro-chondrogenic, anti-inflammatory dual-functional disease-modifying osteoarthritis drug (DMOAD) candidate for preventing and suppressing arthritis-related cartilage damage.

Keywords: Cartilage damage; Disease-modifying osteoarthritis drug (DMOAD); Inflammation; Neural EGFL like 1 (NELL-1); Osteoarthritis; Runt-related transcription factor 1 (RUNX1).

Fig. 1.. Schematic depicting the intra-articular injection animal model.

‘Control’ group: 6 μl PBS per injection for 14 days; ‘NELL-1’ group: 6 μl PBS per injection for 7 days followed by 2 μg recombinant human NELL-1 in 6 μl PBS per injection for the next 7 days; ‘IL1β’ group: 100 ng recombinant human IL1β in 6 μl PBS per injection for 14 days; and ‘IL1β + NELL-1’ group: 100 ng IL1β in 6 μl PBS per injection for 7 days to trigger inflammation, and 100 ng IL1β + 2 μg NELL-1 in 6 μl PBS per injection for the next 7 days. All injections were performed twice daily.

Fig. 2.. Characterization of WT and Nell-1-haploinsufficient (Nell-1^+/6R) mouse knee joints at 3 and 18 months of age.

Representative photos of 3- (A), and 18-month- (B) old female WT and Nell-1^+/6R mouse knee joints. H&E staining was performed for histological analysis, while safranin O was used to stain proteoglycans. Expression of anabolic marker type II collagen (Collagen, type II), catabolic marker Mmp13, as well as proinflammatory markers interleukin (Il)1β and Il6 was evaluated by IF staining. DAPI was used for nuclear counterstaining. HC: uncalcified hyaline zone of articular cartilage; CC: calcified zone of articular cartilage. Solid arrows indicate the erosion in HC; open triangles indicate severe loss of HC. Bar = 500 μm. Relative RNA expression in the tibial cartilage is presented in Supplementary Fig. 5.

Fig. 3.. Characterization of mouse knee joints after 14 days of intra-articular injections.

Representative photos of 2.5-month-old female WT (A) and Nell-1^+/6R (B) mouse knee joints after 14 days of intra-articular injections (3 months old at the end of treatment). The injection schematic of each group is presented in Fig. 1. H&E staining was performed for histological analysis, while safranin O was used to stain proteoglycans. Expression of Collagen, type II, Mmp13, Il1β, and Il6 was evaluated by IF staining. DAPI was used for nuclear counterstaining. HC: uncalcified hyaline zone of articularx cartilage; CC: calcified zone of articular cartilage. Relative RNA expression in the tibial cartilage is presented in Supplementary Fig. 7, while gait scores are summarized in Supplementary Fig. 8. Corresponding videos of Nell-1^+/6R mice (B) are provided in Supplementary Videos 1–7. Bar = 500 μm.

Fig. 3.. Characterization of mouse knee joints after 14 days of intra-articular injections.

Representative photos of 2.5-month-old female WT (A) and Nell-1^+/6R (B) mouse knee joints after 14 days of intra-articular injections (3 months old at the end of treatment). The injection schematic of each group is presented in Fig. 1. H&E staining was performed for histological analysis, while safranin O was used to stain proteoglycans. Expression of Collagen, type II, Mmp13, Il1β, and Il6 was evaluated by IF staining. DAPI was used for nuclear counterstaining. HC: uncalcified hyaline zone of articularx cartilage; CC: calcified zone of articular cartilage. Relative RNA expression in the tibial cartilage is presented in Supplementary Fig. 7, while gait scores are summarized in Supplementary Fig. 8. Corresponding videos of Nell-1^+/6R mice (B) are provided in Supplementary Videos 1–7. Bar = 500 μm.

Fig. 4.. Effects of NELL-1 on gene expression in primary mouse articular chondrocytes (mARCs).

Expression of Il1β (A-A2), Il6 (B-B2), Tnfβ (C-C2), Mmp13 (D-D2), Adamts5 (E-E2), Ptgs2 (F-F2), Col2α1 (G-G2), and Nell-1 (H-H2) was quantified by real-time PCR, and the data were normalized to the respective levels of WT-mARCs (A-H and A1-H1) or Nell-1^+/6R-mARCs (A2-H2) before treatment (dashed lines). Mean + SD of three independent experiments performed in duplicate are shown. One-way ANOVA and two-sample t-test analyses were performed. *, P < 0.05, a suggestive difference; **, P < 0.005, a statistically significant difference.

Fig. 4.. Effects of NELL-1 on gene expression in primary mouse articular chondrocytes (mARCs).

Expression of Il1β (A-A2), Il6 (B-B2), Tnfβ (C-C2), Mmp13 (D-D2), Adamts5 (E-E2), Ptgs2 (F-F2), Col2α1 (G-G2), and Nell-1 (H-H2) was quantified by real-time PCR, and the data were normalized to the respective levels of WT-mARCs (A-H and A1-H1) or Nell-1^+/6R-mARCs (A2-H2) before treatment (dashed lines). Mean + SD of three independent experiments performed in duplicate are shown. One-way ANOVA and two-sample t-test analyses were performed. *, P < 0.05, a suggestive difference; **, P < 0.005, a statistically significant difference.

Fig. 5.. Effects of NELL-1 on gene expression in primary human articular chondrocytes (hARCs).

Expression of IL1β (A-A3), IL6 (B-B3), TNFα (C-C3), MMP13 (D-D3), ADAMTS5 (E-E3), PTGS2 (F-F3), COL2α1 (G-G3), and NELL-1 (H-H3) was quantified by real-time PCR, and the data were normalized to the respective levels of pathological normal/health (NM)-hARCs (A-H and A1-H1), osteoarthritis (OA)-hARCs (A2-H2), or rheumatoid arthritis (RA)-hARCs (A3-H3) before treatment (dashed lines). Mean + SD of three independent experiments performed in duplicate are shown. One-way ANOVA and two-sample t-test analyses were performed. *, P < 0.05, a suggestive difference; **, P < 0.005, a statistically significant difference.

Fig. 5.. Effects of NELL-1 on gene expression in primary human articular chondrocytes (hARCs).

Expression of IL1β (A-A3), IL6 (B-B3), TNFα (C-C3), MMP13 (D-D3), ADAMTS5 (E-E3), PTGS2 (F-F3), COL2α1 (G-G3), and NELL-1 (H-H3) was quantified by real-time PCR, and the data were normalized to the respective levels of pathological normal/health (NM)-hARCs (A-H and A1-H1), osteoarthritis (OA)-hARCs (A2-H2), or rheumatoid arthritis (RA)-hARCs (A3-H3) before treatment (dashed lines). Mean + SD of three independent experiments performed in duplicate are shown. One-way ANOVA and two-sample t-test analyses were performed. *, P < 0.05, a suggestive difference; **, P < 0.005, a statistically significant difference.

Fig. 6.. Effects of Nfatc1- and Runx1-KD on NELL-1’s anti-inflammatory potency.

Stable scramble, Nfatc1-, and Runx1-KD ATDC5 clones were established (Supplementary Fig. 13). Expression of proinflammatory genes Il1β (A-A3), Il6 (B-B3), and Tnfα (C-C3) was quantified by real-time PCR, and the data were normalized to the respective levels of ATDC5 (A-C), ATDC5 (scramble) (A1-C1), ATDC5 (Nfatc1-KD) (A2-C2), or ATDC5 (Runx1-KD) (A3-C3) cells before treatment (dashed lines). Mean + SD of three independent experiments performed in duplicate are shown. One-way ANOVA and two-sample t-test analyses were performed. N.D.: not detectable. *, P < 0.05, a suggestive difference; **, P < 0.005, a statistically significant difference.

Fig. 7.. Expression of Runx1 in mouse knees with intra-articular NELL-1 administration.

Using IF, Runx1 expression was observed in 2.5-month-old female WT (A) and Nell-1^+/6R (B) mouse knee joints after 14 days of intra-articular injections (3 months old at the end of treatment). DAPI was used for nuclear counterstaining. HC: uncalcified hyaline zone of articular cartilage; CC: calcified zone of articular cartilage. Bar = 500 μm.

Fig. 8.. Schematic depicting NELL-1’s effects in articular cartilage.

(A) Focal wear and tear of HC with early chondrocyte clustering became evident in the tibial plateau cartilage of 3-month-old Nell-1^+/6R mice, while severe loss of HC was observed in the knees of 18-month-old Nell-1^+/6R mice. (B) Our previous studies revealed that the NELL-1 → NFATc1 → RUNX3 → IHH cascade in chondrocytes is responsible for NELL-1’s pro-chondrogenic bioactivities. Here, we demonstrate that RUNX1, instead of NFATc1, is essential for NELL-1 to exhibit its anti-inflammatory properties in chondrocytes.