Synovial macrophage diversity and activation of M-CSF signaling in post-traumatic osteoarthritis

Reviewer #1 (Public Review):

Summary:
The authors recently reported a scRNA-seq-based study focused on synovial fibroblasts using a mouse model of post-traumatic OA (Ref. 21). In the present manuscript, they reanalyzed the scRNA-seq data to investigate the diversity and roles of macrophages. In addition to their original scRNA-seq data (Ref. 21), they utilized the deposited data of other OA or RA models (Ref. 25-27) and compared cell types in the synovium. The authors extracted the macrophage/monocyte group, compared differentially expressed genes (DEGs) between OA and RA synovium, and analyzed macrophage subsets, including trajectory analysis. They further estimated the crosstalk between stromal and immune cells via M-CSF signaling, and transcription factors for monocyte differentiation.

Strengths:
The descriptions are comprehensive, based on the scRNA-seq data including the original and other independent studies.

Weaknesses:
Meanwhile, methods of sample preparation must be different, for example, the extent and location of excised synovium. The comparison with other studies is meaningful and informative; however, caution should be exercised regarding the potentially significant impact of methodological differences on the analysis results.

The various data obtained from these technologies are comprehensive and useful; however, they are just estimates. Without confirmation by experiments, it is impossible to determine how much of it can be believed. This issue is not limited to this paper.

Most of all signaling pathways and molecules described in the latter part of this study are previously known.

Reviewer #2 (Public Review):

Summary:
The manuscript by Knights et al set out to identify the specific immune cells and their contribution to the development of osteoarthritis. They performed a comprehensive analysis of scRNA-seq and flow cytometry using different stages of the PTOA model and sought to identify specific synovial macrophages in OA. Computational analysis revealed that M-CSF signaling in synovium plays an important role in stromal-immune crosstalk in OA. They also found that four transcription factors including Pu.1, Cebp-alpha, Cebp-beta, and Jun regulate the differentiation of monocytes into pro-inflammatory synovial macrophages in OA.

Strengths:
The main strength of this study is the profiling of immune cells which will be a valuable resource for better understanding the pathogenesis of OA. The work is technically sound, and the level of analysis of gene expression, clustering, cell-cell communication, and dynamic changes in gene modules over time is state-of-the-art.

The reviewer appreciates that the authors uncovered the transcriptional network that regulates the differentiation of synovial macrophages in OA. In addition, the identification of M-CSF signaling as a major crosstalk axis in OA development is also intriguing.

Weaknesses:
Although the scRNA-seq analysis of immune cells in OA is quite convincing, the data has been rather descriptive and superficial at this stage. The authors did not show the in vivo significance of their findings in OA development.