DOI: 10.5061/dryad.hqbzkh1pb
title: Data from: Mouse gingival single cell transcriptomic atlas identified a novel fibroblast subpopulation activated to guide oral barrier immunity in periodontitis

description: 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.
description: <strong>Evaluation of gingival defect and alveolar bone
 resorption in a ligature-induced mouse model of
 periodontitis</strong> A silk thread was gently
 tied around the left maxillary second molar of 8- to 12-week-old female
 WT, <em>Rag2</em><sup>-/-</sup>, and
 <em>Rag2<sup>-/-</sup>Il2rg<sup>-/-</sup>
 </em>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% CO<sub>2</sub>
 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.
 <em>Rag2</em><sup>-/-</sup> and
 <em>Rag2<sup>-/-</sup>Il2rg<sup>-/-</sup>
 </em>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). <strong>Evaluation
 of gingival effect in the maxillary topical application (MTA)
 model</strong> 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 <em>P. gingivalis</em> 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.
description: # Mouse gingival single cell transcriptomic atlas identified a novel
 fibroblast subpopulation activated to guide oral barrier immunity in
 periodontitis 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). *
 .