Data from: Mouse gingival single cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis
Data files
Nov 30, 2023 version files 30.81 KB
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eLife_raw_data_11.14_TK.xlsx
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README.md
Abstract
Periodontitis, one of the most common non-communicable diseases, is characterized by chronic oral inflammation and uncontrolled tooth supporting alveolar bone resorption. Its underlying mechanism to initiate aberrant oral barrier immunity has yet to be delineated. Here, we report a unique fibroblast subpopulation activated to guide oral inflammation (AG fibroblasts) identified in a single-cell RNA sequencing gingival cell atlas constructed from the mouse periodontitis models. AG fibroblasts localized beneath the gingival epithelium and in the cervical periodontal ligament responded to the ligature placement and to the discrete topical application of Toll-like receptor stimulants to mouse maxillary tissue. The upregulated chemokines and ligands of AG fibroblasts linked to the putative receptors of neutrophils in the early stages of periodontitis. In the established chronic inflammation, neutrophils together with AG fibroblasts appeared to induce type 3 innate lymphoid cells (ILC3s) that were the primary source of interleukin-17 cytokines. The comparative analysis of Rag2-/- and Rag2gc-/- mice suggested that ILC3 contributed to the cervical alveolar bone resorption interfacing the gingival inflammation. We propose that the AG fibroblast–neutrophil–ILC3 axis as a previously unrecognized mechanism which could be involved in the complex interplay between oral barrier immune cells contributing to pathological inflammation in periodontitis.
README: Mouse gingival single cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis
https://doi.org/10.5061/dryad.hqbzkh1pb
The dataset contents quantitative characterization of the mouse ligature-induced periodontitis model and the newly developed mouse maxillary topical application (MTA) model, which was activated by unmethylated CpG oligonucleotide (Tlr9 ligand) and P. gingivalis LPS (Tlr2/4 ligand). The data correspond to the presented Figures in the published paper in eLife, not to those in the BioRxiv reprint.
Description of the data and file structure
The measurement raw data of periodontal defect area and microCT bone loss are provided in a single Exl file. The figure number in the Exl file corresponds to the figures in the eLife paper. Here are the description of dataset available in Dryad:
CHARACTERIZATION OF THE LIGATURE-INDUCED PERIODONTITIS IN MICE
Fig. 1-figure supplement 1B: A ligature (5.0 silk suture) was placed around the maxillary second molar (M2) of wild-type (WT) mice. Representative intra-oral photographs of the maxilla on Day 0, prior to ligature placement (healthy gingiva), and on Days 1, 3, and 7 after ligature placement. The gingival defect area was measured and normalized to the circumferential area of the maxillary first molar (M1) (n = 6). Gingival defects appeared on Day 3.
Fig. 1-figure supplement 1D: Micro-computed tomography (microCT) images of the maxilla taken from the lateral view. Alveolar bone loss was determined from the total distance between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) of the buccal or palatal bone at six sites in the ligated side (n = 6). Alveolar bone loss was apparent on Day 7.
Fig. 1-figure supplement 1E: Alveolar bone loss was assessed at the mesiobuccal cusp (M1-1) and distobuccal cusp (M1-2) of the first molar, the mesiobuccal cusp (M2-1), distobuccal cusp (M2-2), and distal cusp (M2-3) of the second molar, and the buccal cusp (M3) of the third molar by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) on the buccal or palatal side of the alveolar bone (n = 6).
THE LIGATURE-INDUCED PERIODONTITIS IN WT, Rag2-/- AND Rag2-/-Il2rg-/- MICE
Fig. 8E: Using microCT images, the alveolar bone loss was determined from the total distance between the CEJ and the ABC of the buccal bone or palatal bone at six sites in the ligated side of WT, Rag2-/- and Rag2-/-Il2rg-/- at Day 7 (n= 6 per group).
Fig. 8H: The number of tartrate-resistant acid phosphatase (TRAP) staining positive osteoclasts in a 0.01 mm2-area of the buccal and palatal bone in the cervical PDL site of WT, Rag2-/- and Rag2-/-Il2rg-/- at Day 7 (n= 6 per group).
Fig. 8I: The number of TRAP staining positive osteoclasts in a 0.01 mm2-area of the buccal and palatal bone in the apical PDL site of WT, Rag2-/- and Rag2-/-Il2rg-/- at Day 7 (n= 6 per group).
Fig. 8-figure supplement 1A: Alveolar bone loss was assessed at six sites by measuring the distance from the CEJ to the ABC on the buccal or palatal side of the alveolar bone of wild-type (WT), Rag2-/-, and Rag2-/-Il2rg-/- mice on Day 7 following ligature placement (n = 6).
Fig. 8-figure supplement 1B: Bone volume/total volume (BV/TV), bone surface, trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the buccal side of alveolar bone of the second molar were measured on Day 7 (n = 6).
Fig. 8-figure supplement 1D: The gingival defect area was measured and normalized to the circumferential area of M1 (n = 5).
Sharing/Access information
Links to other publicly accessible locations of the data: Single-cell RNA sequencing data are deposited to NIH Gene Expression Omnibus (GSE228635).
Methods
Evaluation of gingival defect and alveolar bone resorption in a ligature-induced mouse model of periodontitis
A silk thread was gently tied around the left maxillary second molar of 8- to 12-week-old female WT, Rag2-/-, and Rag2-/-Il2rg-/- mice under general inhalation anesthesia with isoflurane (Henry Schein, Melville, NY, USA) following the published protocol [21] (PMCID: PMC3707981 DOI: 10.1016/j.jim.2013.05.002).
WT mice were randomly chosen and euthanized by 100% CO2 inhalation on Days 1, 3, and 7 after ligature placement (n=6 per time point). WT mice without ligature placement were used as Day 0 pre-periodontitis control (n=6). The ligature-induced mouse model using WT mice was replicated at least 2 times in our laboratory.
Rag2-/- and Rag2-/-Il2rg-/- mice were euthanized on Day 7 (n=6) and a separate set of WT mice were used as a control group and euthanized on Day 7 (n=6) in this experiment.
The palatal tissue was digitally photographed, and maxillae were harvested and fixed in 10% buffered formalin (Thermo Fisher Scientific, Waltham, MA, USA). The gingival defect area was measured from digital photographs, using the ImageJ Java-based image-processing program (NIH, Bethesda, MD, USA), and normalized to the circumferential area of the maxillary first molar. Fixed maxillae were subjected to microCT imaging at an energy level of 60 kV and 166 µA, and 3D images were reconstructed from microCT scans (Skyscan 1275: Bruker, Billerica, MA, USA). Alveolar bone loss was assessed at three sites (mesiobuccal cusp, distobuccal cusp, and distal cusp) of the first molar, two sites (mesiobuccal cusp and distobuccal cusp) of the second molar, and one site (buccal cusp) of the third molar by measuring the distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) on the buccal and palatal side of the alveolar bone. Total bone loss was calculated from the six-site total CEJ–ABC distance. The bone volume/total volume (BV/TV) ratio, bone surface, trabecular number (Tb.N), and trabecular thickness (Tb.Th) in the buccal side of alveolar bone of the second molar were determined using the proprietary analysis program (CTan: Bruker, Billerica, MA, USA).
Evaluation of gingival effect in the maxillary topical application (MTA) model
We have developed a method to apply chemical therapeutic agents topically to the mouse maxillary tissue [24, 78]. We used the maxillary topical application (MTA) model to apply oral microbial components [8] (PMCID: PMC9474870 DOI: 10.1038/s42003-022-03896-7). The present study used the MTA model using 1 µg/ml unmethylated CpG oligonucleotide (CpG ODN: InvivoGen, San Diego, CA) or P. gingivalis lipopolysaccharide (LPS: InvivoGen). First, a custom-made oral appliance was fabricated using clear dental resin, covering the maxillary/palatal tissue between the molars. Mice were anesthetized and placed on a supine position. CpG ODN (3 µl) or LPS (3 µl) was pipetted over the maxillary tissue and the oral appliance was placed to hold the solution with a bite block for 1 hr in an anesthetization chamber. Mice were then transferred to the operation table and the oral appliance and bite block were removed. In general, there was no remaining solution in the mouth. Mice were returned to the cage in the vivarium. On Day 4 of the MTA, mice were euthanized by 100% CO2 inhalation and the maxillary tissue was harvested and fixed with 10% buffered formalin for histological evaluation.