Author Response
We thank all reviewers for constructive critiques. We plan to perform new experiments and revise our manuscript accordingly. The text and Figures are currently undergoing the revision process. Below highlights our revision plan.
eLife assessment
The findings of this article provide valuable information on the changes of cell clusters induced by chronic periodontitis. The observation of a new fibroblast subpopulation, which was named as AG fibroblasts, was quite interesting, but needs further evidence. The strength of evidence presented is incomplete.
RESPONSE: We discovered a new subpopulation of gingival fibroblasts, named AG fibroblasts, using non-biased single cell RNA sequencing (scRNA-seq) of mouse gingival samples undergoing the development of ligature-induced periodontitis. AG fibroblasts exhibited a unique gene expression profile: [1] constitutive expression of type XIV collagen; and [2] ligature-induced upregulation of chemokines such as CXCL12. As a biomedical data science experiment, we validated the scRNA-seq observation using immunohistochemical experiment, which showed the presence of type XIV collagen-positive and CXCL12-positive gingival fibroblasts localized immediately under the gingival epithelium and the coronal region of periodontal ligament.
We agree that the functional/pathological role of AG fibroblasts must be further explored. We have hypothesized that AG fibroblasts initially sense the pathological stress including oral microbial stimuli and secrete inflammatory signals through chemokine expression. To address this hypothesis, in this revision, we plan to analyze a separate scRNA-seq data for AG fibroblast gene expression profile derived from mouse gingival tissues that have been stimulated by Toll-Like Receptor 9 (TLR9) ligand (unmethylated CpG oligonucleotide) and TLR2/4 ligand (LPS). This approach mimics the initial pathological stress applied to gingival tissue. The new insight of AG fibroblasts will be presented in the revision.
Reviewer #1 (Public Review):
In this article, the authors found a distinct fibroblast subpopulation named AG fibroblasts, which are capable of regulating myeloid cells, T cells and ILCs, and proposed that AG fibroblasts function as a previously unrecognized surveillant to orchestrate chronic gingival inflammation in periodontitis. Generally speaking, this article is innovative and interesting, however, there are some problems that need to be addressed to improve the quality of the manuscript.
RESPONSE: We appreciate this comment. As suggested, we further investigated the surveillant function of AG fibroblasts by reanalyzing the scRNA-seq data for stress sensing receptors such as Toll-Like Receptors (TLR). Therefore, we analyzed AG fibroblast gene expression profile when the putative ligands to TLR2/4 and TLR9 are applied to mouse gingival tissue instead of ligature placement. We believe that this first step analysis should warrant to dissect further the function of AG fibroblasts in the future.
Results:
- It is recommended to add HE staining and immunohistochemistry staining to observe the inflammation, tissue damage, and repair status from 0 to 7 days, so that readers can understand cell phenotype changes corresponding to the periodontitis stage. The observation index can include inflammation and vascular related indicators.
RESPONSE: As recommended, representative histological figures will be included. We will further perform new immunohistochemistry experiment of mouse gingival tissue (D0, D1, D4, D7). We plan to highlight the infiltration of CD45+ immune cells. We also plan to highlight the progressive degeneration of gingival collagen fiber by picrosirius red staining.
- Figure 1A-1D can be placed in the supplementary figure.
RESPONSE: Combining the new data above, Figure 1 will be revised as suggested.
- I suggest the authors to put the detection of the existence of AG fibroblasts before exploring its relationship with other types of cells.
- The layout of the picture should be closely related to the topic of the article. It is recommended to readjust the layout of the picture. Figure 1 should be the detection of AG cells and their proportion changes from 0 to 7 days. In other figures, the authors can separately describe the proportion changes of myeloid cells, T cells and ILCs, and explored the association between AG fibroblasts and these cell types.
RESPONSE: As suggested, the presentation order of Figures and text will be revised to bring the information about AG fibroblasts first. The chemokine-receptor analysis is moved below.
Methods:
It is recommended to separately list the statistical methods section. The statistical method used in the article should be one-way ANOVA.
RESPONSE: A separate statistical method section is created. As pointed out, we used one-way ANOVA with post-hoc Tukey test (when multiple groups were compared).
Reviewer #2 (Public Review):
This study proposed the AG fibroblast-neutrophil-ILC3 axis as a mechanism contributing to pathological inflammation in periodontitis. However, the immune response in the vivo is very complex. It is difficult to determine which is the cause and which is the result. This study explores the relevant issue from one dimension, which is of great significance for a deeper understanding of the pathogenesis of periodontitis. It should be fully discussed.
RESPONSE: We agree with this comment. We expanded the current understanding of oral immune signal communication in Discussion and highlight how AG fibroblast may fit to it.
- Many host cells participate in immune responses, such as gingival epithelial cells. AG fibroblast is not the only cell involved in the immune response, and the weight of its role needs to be clarified. So the expression in the conclusion should be appropriate.
RESPONSE: Following this critique, we revised INTRODUCTION, DISCUSSION and CONCLUSION, to highlight how AG fibroblasts function within a comprehensive immune response network.
- This study cannot directly answer the issue of the relationship between periodontitis and systemic diseases.
RESPONSE: We agree with this critique. We either deleted or de-emphasized the relationship between periodontitis and systemic diseases throughout the text.
