CILP from Cartilage Intermediate Zone Inhibits Hyaline Cartilage Fibrosis and Chondrocyte Ferroptosis via Keap1-Nrf2 Axis in Early Osteoarthritis Exercise Therapy

Reviewer #1 (Public review):

Summary:

In this study, the authors examined the function of CLIP in exercise-mediate inhibition of osteoarthritis using an ACL transection rat model. The authors rely on rigorous experimental design and methods to demonstrate that CLIP is downregulated in osteoarthritic cartilage tissue and that CLIP expression can be rescued by moderate treadmill exercise. They further show that activation of Nrf2 signaling occurs through CLIP inhibition of Keap1-Nrf2. The results are novel as they suggest a new role for CLIP in OA pathogenesis. The following points need to be addressed in order to bring additional clarity to this work.

Strengths:

This is an interesting study that addresses an important global health issue. The significance is high and the work is novel and mechanistic.

Weaknesses:

A major concern is that a direct link between exercise and CLIP-mediated inhibition of ferroptosis via Keap1-Nrf2 pathway is not supported by the provided data. The ferroptosis studies were performed in vitro, whereas the effect of exercise was demonstrated in an OA animal model. Therefore, the data suggest a potential correlation between CLIP-Keap1-Nrf2 and exercise. This must be described as a limitation in the discussion section. Consequently, the title of the manuscript needs to better reflect the interpretation of these data.

Figure 1: Radiomics data are not described in the text. OARSI scoring of damaged and undamaged sections is not presented in the figure.

Figure 2: Data presentation is very dense in this figure. It is recommended that Figure 2 be split into two figures. Also, the histology and IHC images in Figure 2A are of poor resolution. These data do not sufficiently demonstrate early OA pathology. Clearer images to substantiate the authors' statement need to be provided.

Figure 3: The superficial zone appears to be misrepresented; it should include only the top 2-3 layers of flat chondrocyte cells.

Figure 4: This Figure should be listed as supplementary data. CTS is not spelled out in the legend. Also, a rationale for using low, medium, and high CTS needs to be provided.

Figure 5: Please describe positive and negative controls. Please elaborate on the findings of the yeast hybrid experiment in the results. Please expand KD-02 experimental condition in the legend and results.

Figure 6: Please move Figure S2 into the main Figures and describe the results in section 2.9 which describes ferroptosis.

In the results section, it is recommended that the authors describe all panels of the figures appropriately in sequential order. The authors are advised to provide publication-quality figures and, in some cases, to split figure panels into new figures as well as to ensure that the fonts and data are legible. Finally, the use of non-conventional abbreviations (such as G3 for passage-3 chondrocytes, CG for the control condition, and OE for overexpression) may confuse the readership, and describing each abbreviation when used for the first time is required.

Reviewer #2 (Public review):

Summary:

Recent studies indicate a beneficial role for moderate-intensity exercise in early osteoarthritis (OA). This manuscript by Jia et al. investigates the role of cartilage intermediate layer protein (CILP) and moderate exercise in maintaining hyaline cartilage integrity following anterior cruciate ligament transection (ACLt) in rats. Single-cell RNA-sequencing of OA and OA+ exercise knee joints from rats at 4 weeks post-ACLt revealed the upregulation of CILP and a higher Col2/Col1 ratio in OA knee chondrocytes from ACLt rats that exercised on a treadmill. CILP was downregulated in the damaged portions, compared to healthy regions of knee cartilage of patients undergoing total knee arthroplasty. In the rat ACLt model, CILP is downregulated in the OA cartilage but not in OA + exercise cartilage. Using CLIP1 over-expression and knockdown in passage 3 cultures of primary rat chondrocytes, the authors demonstrate that the loss of CILP is associated with higher ROS, lipid peroxidation, and iron content in chondrocytes whereas its overexpression is protective against these changes. CILP binds to Keap1, and its overexpression disrupts Keap1/Nrf2 interaction and attenuates Nrf2 ubiquitination. The authors conclude that exercise protects the articular cartilage intermediate zone and the associated upregulation of CILP facilitates Keap1-Nrf2 interaction to prevent chondrocyte ferroptosis and hyaline cartilage fibrosis.

Strengths:

The study is interesting, and the experiments are conducted well. The methodology is well-described. The data presented strongly support the downregulation of CILP in human OA cartilage and its potential role in regulating Keap1/Nrf2 interaction and chondrocyte ferroptosis.

Weaknesses:

The data do not support a role for CILP in exercise-mediated inhibition of hyaline cartilage fibrosis in early OA. The reason for selecting CILP from the ScRNA-seq for further analysis is not clear. The manuscript is put together sloppily. The abstract, introduction, and results were written confusingly, and hard to follow. Some of the figures were confusing as well. Still, the study is interesting.