The alveolar bone and gingival tissue surrounding the maxillary second molar of C57BL/6J mice presented healthy connective tissue supporting the dentition with minimal CD45+ immune cell infiltration and well-developed collagen architecture (Figure 1A, Figure 1—figure supplement 1A). The placement of a ligature around the maxillary left second molar (Abe and Hajishengallis, 2013) induced a localized and small gingival connective tissue degradation and infiltration of CD45+ immune cells near the cervical area (Figure 1B). The noticeable gingival defect and more prominent though still localized CD45+ immune cell infiltration developed after day 3 following ligature placement (Figure 1C, Figure 1—figure supplement 1A and B). On day 7, the gingival connective tissue was largely degraded, and chronic inflammation was developed (Figure 1D, Figure 1—figure supplement 1A and B). Three-dimensional (3D) reconstruction of micro-computed tomography (microCT) images (Figure 1—figure supplement 1C) further revealed a reduction in alveolar bone height starting from day 3, which progressively increased on day 7 (Figure 1—figure supplement 1D and E). Overall, the observed pattern of periodontal tissue degradation was consistent with that reported in previous studies (Tamura et al., 2021; Marchesan et al., 2018).

Left-side palatal gingiva tissue was harvested from mice on day 0 (i.e., healthy gingiva without ligature placement) and on days 1, 4, and 7 after ligature placement, and gingival cells were dissociated for single-cell RNA sequencing (scRNA-seq) (Kondo et al., 2022; Okawa et al., 2022a). On days 0 and 1, the major cell types identified included epithelial cells expressing cadherin 1 (Cdh1) (Groeger and Meyle, 2019) and type XVII collagen (Col17a1) (Kamaguchi et al., 2018), fibroblasts expressing type I collagen (Col1a1) (Takahashi et al., 2019) and lumican (Lum) (Lallier et al., 2005), B cells expressing membrane spanning 4 domains A1 (Ms4a1) (Bruno et al., 2010) and cluster of differentiation 79A (Cd79a) (Sparger et al., 2018), T cells expressing epsilon subunit of T cell receptor complex (Cd3e) (Alcover et al., 2018) and cluster of differentiation 5 (Cd5) (Fujihashi et al., 1989), and myeloid cells expressing lysozyme 2 (Lyz2) (Cross et al., 1988) and integrin subunit alpha M (Itgam) (Okubo et al., 2016; Figure 1E and Figure 1—figure supplement 2). On days 4 and 7, an additional endothelial cell fraction expressing selectin P (Selp) (Gotsch et al., 1994) and selectin E (Sele) (Komatsu et al., 2012) emerged (Figure 1E and Figure 1—figure supplement 2), suggesting increased inflammatory neovascularization with the progression of periodontal inflammation. The proportion of B cells was increased on day 1, and the proportion of myeloid cells increased progressively from days 4–7 (Figure 1F). In addition, the proportion of fibroblasts was increased on day 7 (Figure 1F).

We previously identified two distinct subpopulations of gingival fibroblasts, differentiated by expression of type XIV collagen (Col14a1) (Kondo et al., 2022), and these were also detected in our current scRNA-seq data from days 0–7 (Figure 2A). Gene Ontology (GO) enrichment analysis of the Col14a1-expressing fibroblast subpopulation revealed expression of major gene clusters related to immune regulation, including ‘Regulation of leukocyte migration’ (Figure 2B). This immune regulatory phenotype appears to be unique to Col14a1-expressing fibroblasts, which are therefore referred to as ‘fibroblasts activated to guide leukocyte migration’ or AG fibroblasts. The other subpopulation of Col1a1-expressing fibroblasts appeared to keep typical fibroblastic features and is thus referred to as KT fibroblasts (Figure 2C). GO analysis suggested that KT fibroblasts primarily maintained connective tissue remodeling but did not express an immune regulatory phenotype. An additional fibroblast subpopulation expressing smooth muscle actin alpha 2 (Acta2) was detected on day 7, and these were identified as myofibroblasts (MF) (Sasaki et al., 2020; Figure 2A). We found that the proportions of KT and AG fibroblasts were equal on day 0 and on days 1 and 4 after ligature placement (Figure 2E and F). However, on day 7, the proportion of AG fibroblasts decreased, and the MF fraction emerged (Figure 2E and F).

To further characterize AG fibroblasts and their ‘Regulation of leukocyte migration’ phenotype, we analyzed expression of CC motif chemokine ligands (CCLs) and CXC motif chemokine ligands (CXCLs) within fibroblast subpopulations in our scRNA-seq dataset (Cekici et al., 2014). Results show that AG fibroblasts activated expression of Ccl8, Ccl11, Ccl19, Cxcl1, Cxcl10, and Cxcl12 immediately after ligature placement on days 1 and 4 (Figure 3A). Chemokine expression levels then decreased on day 7, suggesting that AG fibroblasts might initiate early leukocyte migration into gingival tissue. Given that activation of Toll-like receptors (TLR) is known to increase chemokine expression, we further assessed expression of TLRs in fibroblast subpopulations. We found that AG fibroblasts displayed temporal activation of Tlr2, Tlr3, and Tlr4 expression on days 1 and 4 (Figure 3B). Similarly, the TLR downstream genes Myd88, Irak1, Map3k7, and RelA were also expressed on days 1 and 4 (Figure 3B). These data suggest that AG fibroblasts may guide the establishment of an early inflammatory environment within the gingiva and thereby promote periodontitis pathogenesis.

To validate the presence of AG fibroblasts, day 1 gingiva and periodontal tissue histological sections were subjected to immunohistochemistry with antibodies against COL14A1 and CXCL12 (Figure 3C). We detected COL14A1- and CXCL12-positive AG fibroblasts localized near gingival epithelial cells in the connective tissue papillae and free gingiva, as well as in the cervical zone of the periodontal ligament (PDL) space (Figure 3C). We note that this localization pattern of AG fibroblasts appears to be highly suitable for early immune surveillance during periodontitis pathogenesis. Thus, we have hypothesized that AG fibroblasts initially sense the pathological stress including oral microbial stimuli through TLRs and secrete inflammatory signals through chemokine expression.

We previously reported that the microbial composition of the mouse ligature did not mirror the human oral microbial composition (Kondo et al., 2022). To mitigate this critical discrepancy in the mouse periodontitis model, we developed the MTA model. The MTA model applied oral microbial biofilm directly to the maxillary gingiva and held under a custom-made oral appliance (Figure 4A) for 1 hr. We previously reported that human oral microbial biofilm, but not planktonic microbes, induced initial gingival tissue degradation in the MTA model, suggesting that extracellular components of human oral biofilm could play an important role in the initiation of periodontitis (Kondo et al., 2022). In this study, we used the MTA model without the placement of a ligature to further characterize the behavior of AG fibroblasts. This study discretely applied ligands of TLR9 and TLR2/4: unmethylated CpG ODN and P. gingivalis LPS, respectively. Four days after the topical application, mouse maxillary tissue was harvested for histological analysis and for scRNA-seq (Figure 4B; Kondo et al., 2022). CpG ODN induced the expression of cathepsin K (Ctsk) in the PDL and gingival connective tissue (Figure 4C) validating the previous report (Kondo et al., 2022). Furthermore, there were signs of localized Ctsk+ osteoclastic activities on the surface of alveolar bone (Figure 4C).

The scRNA-seq gingival cell composition of the MTA of CpG ODN and LPS indicated the early periodontitis pattern (Figure 4E) consistent with day 1 scRNA-seq of the ligature-induced periodontitis (Figure 1F). The MTA of CpG ODN and LPS identified AG fibroblasts along with KT fibroblasts, whereas the MTA of LPS induced the additional MF (Figure 4E). Further analysis demonstrated the expression of CCL and CXCL chemokines by AG fibroblasts activated by the MTA of CpG ODN and LPS (Figure 4F). The scRNA-seq of MTA of CpG ODN did not capture the transcriptional activation of Tlr9; however, the downstream effector genes associated with TLR9 such as Myd99, Irak1, and RelA were found to be upregulated in AG fibroblasts (Figure 4G). The MTA of LPS appeared to upregulate Tlr2 and Tlr4 gene transcription of AG fibroblasts and of MF albeit at lesser degrees (Figure 4G). These results indicated that AG fibroblasts were activated by CpG ODN and LPS through the MTA model. As such, the extracellular substances of human oral biofilm such as microbial extracellular DNA and LPS might be an important trigger of gingival inflammation.

The infiltration of proinflammatory neutrophils into the gingiva has been extensively characterized in the mouse model of ligature-induced periodontitis (Hajishengallis, 2020; Silva et al., 2019). Here, we found that macrophages expressing cluster of differentiation 86 (Cd86) (Lam et al., 2014) and integrin subunit alpha X (Itgax) (Agarbati et al., 2021) were predominant on day 0 in the myeloid cell fraction from healthy gingiva (Figure 5A and B, Figure 5—figure supplement 1). In contrast, the proportion of neutrophils expressing CXC motif chemokine receptor (Cxcr2) (Hashim et al., 2021) and G0/G1 switch gene 2 (G0s2) (Zhang et al., 2017) increased after ligature placement and during periodontitis development from days 1–7 (Figure 5A and B, Figure 5—figure supplement 1), suggesting continuous neutrophil infiltration into the early and established gingival lesion. Moreover, after ligature placement, gingival neutrophils upregulated the expression of triggering receptor expressed on myeloid cells 1 (Trem1), indicating that these cells are activated and participating in the amplification of inflammatory signals (Bouchon et al., 2000; Dopheide et al., 2013; Figure 5C). Strikingly, the Trem1-expressing activated neutrophils also show upregulation of matrix metalloproteinase 9 (Mmp9; Figure 5C)—a protein associated with extracellular matrix degeneration within gingival tissue (Corotti et al., 2009). We further detected expression of tumor necrosis factor (Tnf) and transforming growth factor beta 1 (Tgfb1) in both macrophages and neutrophils on day 0 and after ligature placement (Figure 5D). Collectively, these myeloid cell behaviors are consistent with those reported in prior studies on periodontitis development, thus validating our scRNA-seq data.

Myeloid cells are also known to stimulate other immune cells through the expression of CCL and CXCL chemokines, many of which are associated with periodontitis development (Souto et al., 2014). Our scRNA-seq analysis revealed that macrophages expressed Ccl2, Ccl9, Cxcl4, and Cxcl6, and neutrophils expressed Ccl3, Ccl4, Ccl6, Cxcl2, and Cxcl3 throughout periodontitis development (Figure 5E and F). These data suggest that chemokines and cytokines produced by macrophages and neutrophils in inflamed tissue may amplify and polarize the immune response toward chronic gingival inflammation.

The interaction between chemokine ligands and their receptors has been extensively studied. Here, to evaluate the interaction between chemokine ligands strongly expressed by AG fibroblasts and chemokine receptors in innate immune cells, we matched AG fibroblast-expressed chemokines to expression of their putative receptors in myeloid cells. Results show that AG fibroblasts may regulate macrophages via the expression Ccl8 and Ccl11, which encode chemokines that can interact with CC chemokine receptors (CCRs) encoded by Ccr2 and Ccr5 in macrophages. Similarly, gene expression signatures suggest that AG fibroblast-mediated neutrophil regulation may occur through CCL8–CCR1, CXCL1–CXCR2, and CXCL12–CXCR4 interactions (Figure 6A). Notably, all chemokine–receptor pairs are expressed throughout periodontitis development, although expression of factors mediating the interaction between AG fibroblasts and macrophages was decreased on day 7.

Intercellular communication may occur via other ligand–receptor interactions, which induce downstream target gene expression in recipient cells. Here, we performed NicheNet analysis to identify potential interactions between gingival fibroblasts and myeloid cells, revealing a trend toward increasing ligand–target interactions between fibroblasts and myeloid cells from day 1 to day 7 (Figure 6B). On day 1, ligand expression was present primarily in AG fibroblasts, whereas on day 7, MFs also exhibited prominent ligand expression (Figure 6C). Analysis of target gene expression revealed that both macrophages and neutrophils expressed receptors capable of interacting with AG fibroblast ligands on day 1. In contrast, on day 7, receptor expression was almost exclusively present in neutrophils, suggesting that these cells are primarily targeted by MFs and AG fibroblasts at later stages of periodontitis development (Figure 6D).

The chemokine–receptor interaction was also suggested between AG fibroblasts and myeloid cells in the MTA of CpG DON (Figure 6E). However, in the MTA of LPS, macrophage lacked the detectable expression of chemokine receptor and the AG fibroblastic chemokine interaction appeared to be limited to neutrophils (Figure 6E).

Macrophage-colony stimulating factor (M-CSF), encoded by Csf1, and receptor activator of nuclear factor kappa-Β ligand (RANKL), encoded by Tnfsf11, are known to play critical roles in osteoclast induction (Teitelbaum, 2000). We found that in the ligature-induced periodontitis model, the expression of M-CSF was detected in various cell types including fibroblasts (Figure 7A), whereas the RNAKL expression was more restricted to fibroblasts and T cells (Figure 7B). AG fibroblasts consistently expressed M-CSF from day 0 to day 7 and neutrophils started to express M-CSF on days 4 and 7 (Figure 7C). The expression of RNAKL was detected in AG fibroblasts but not in myeloid cells (Figure 7D). T cells and type 3 innate lymphoid cells (ILC3; see below) were also shown to express M-CSF and RNAKL (Figure 7C and D).

The MTA of CpG ODN and LPS suggested the AG fibroblasts were one of the predominant cellular sources of M-CSF (Figure 7E). By contrast, the expression of RNAKL was only detected in AG fibroblasts of the MTA of CpG ODN and LPS did not seem to induce RANKL expression in the MTA model (Figure 7F). The osteoclastic activity found in the MTA of CpG ODN (Figure 4D) might be induced by M-CSF and RANKL derived from AG fibroblasts.

Our scRNA-seq dataset found that the day 0 healthy gingiva exclusively contained Th cells expressing Cd4 (Mahanonda et al., 2018). However, after ligature placement, Tc cells expressing Cd8 (Mahanonda et al., 2018) emerged, and on day 7, Cd4^-Zbtb16⁺ ILCs and Cd4⁺Foxp3⁺ Treg cells (Wei et al., 2021) were also detected (Figure 8A and B). The ILCs expressed Nfil3, a basic leucine zipper (bZIP) transcription factor required for ILC development (Eberl et al., 2015; Figure 8C). However, to our surprise, expression of Rorc, Il17a, and Il17f was detected in ILCs (Figure 8C), indicating that this gingival subpopulation is predominantly composed of type 3 ILCs (ILC3s). ILC3s and Th17 cells share similar regulatory functions. However, the role of ILC3s in the development of periodontitis has not been fully deciphered.

We thus examined the role of ILC3s in periodontitis pathogenesis by measuring ligature-induced gingival defects and alveolar bone resorption in Rag2^-/- mice, which lack functional B, Th, and Tc cells, and Rag2–IL-2 receptor common gamma (Il2rg) double-knockout mice, lacking all lymphocytes including ILCs. After ligature placement, we found that alveolar bone loss was decreased in Rag2^-/- mice and nearly eliminated in Rag2^-/-Il2rg^-/- mice (Figure 8D). MicroCT image analysis indicated a better perseveration of alveolar bone structure in Rag2^-/-Il2rg^-/- mice, relative to the other groups (Figure 8E, Figure 8—figure supplement 1A and B). However, gingival defects developed similarly in wild-type (WT), Rag2^-/-, and Rag2^-/-Il2rg^-/- mice (Figure 8—figure supplement 1C and D).

Histologically, osteoclastic resorption lacunae were observed on the alveolar bone surface at the cervical PDL and tooth apex PDL zones in WT mice (Figure 8F and G). In addition, we detected a significant decrease in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in the cervical PDL zone of Rag2^-/-Il2rg^-/- mice (Figure 8H) and in the apical PDL zone of both Rag2^-/- and Rag2^-/-Il2rg^-/- mice (Figure 8I). Collectively, these data suggest that ILC3s, not Th17 cells, are responsible for cervical alveolar bone resorption in the mouse periodontitis model, a pathological phenotype consistent with human disease (Figure 8—figure supplement 2A and B).

Based on our present data, we hypothesize that ILC3s within the gingival tissue play a pathological role in cervical alveolar bone resorption in our mouse model. We therefore aimed to identify the cells that promote ILC3 development in mice. Similar to Th17 cells, ILC3 development was shown to be triggered by IL-6 and IL-23a (Bielecki et al., 2021; Powell et al., 2015). A survey of our scRNA-seq data identified epithelial cells, fibroblasts, and myeloid cells as the source of Il6 in gingival tissue (Figure 9A). On days 1 and 4 following ligature placement, AG fibroblasts primarily expressed Il6, whereas neutrophils became the predominant source on day 7. We further found that gingival epithelial cells comprise at least four different subpopulations, plus an additional group displaying an epithelial–mesenchymal transition (EMT) phenotype on day 7; Il6 was expressed by several of these epithelial subpopulations, including the EMT subgroup (Figure 9A, Figure 9—figure supplement 1A–D). Il23a was also detected AG fibroblasts and myeloid cells (Figure 9B), with expression present in all epithelial cell subsets at various points in periodontitis development (Figure 9B, Figure 9—figure supplement 1D).

Lastly, we evaluated potential interactions between chemokine ligands expressed by AG fibroblasts and neutrophils and chemokine receptors in innate immune cells. Our data suggest the presence of a chemokine–receptor association between ILC3s and both AG fibroblasts (Figure 9C) and neutrophils (Figure 9D), although interactions with other innate immune cells are also possible. NicheNet analysis further identified potential ligand–target gene associations between lymphocytes, including ILC3s, and both fibroblasts (Figure 9E) and myeloid cells (Figure 9F). Ligand expression was more prominent in AG fibroblasts than in other fibroblast subpopulations (Figure 9G) and elevated in neutrophils relative to macrophages (Figure 9H). Additionally, target gene expression was detected in ILC3s, although it was not specific to these cells (Figure 9I and J). Thus, in total, our data suggest a regulatory role for a newly identified AG fibroblast subpopulation in the gingiva, which appears to orchestrate chronic gingival inflammation, at least in the early stages, and to promote alveolar bone resorption via stimulation of neutrophils and ILC3s.

All protocols for animal experiments were reviewed and approved by the University of California Los Angeles (UCLA) Animal Research Committee (ARC# 2003-009) and followed the Public Health Service Policy for the Humane Care and Use of Laboratory Animals and the UCLA Animal Care and Use Training Manual guidelines. C57BL/6J WT (Strain # 000664; RRID:IMSR_JAX:000664), Rag2^-/- (Strain # 008449; RRID:IMSR_JAX:008449), and Rag2^-/-Il2rg^-/- (Strain # 014593; RRID:IMSR_JAX:014593) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals had free access to regular rodent diet and water and were maintained in standard housing conditions with 12 hr light/dark cycles in the Division of Laboratory Animal Medicine at UCLA. All animal experiments were conducted according to the guideline of the Animal Research; Reporting of In Vivo Experiments (ARRIVE: Essential 10).

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) following the published protocol (Abe and Hajishengallis, 2013; PMCID: PMC3707981; DOI: https://doi.org/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 two 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). The gingival defect area was measured from digital photographs using the ImageJ Java-based image-processing program (NIH, Bethesda, MD) 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). 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).

We have developed a method to apply chemical therapeutic agents topically to the mouse maxillary tissue (Okawa et al., 2022a; Okawa et al., 2022b). We used the MTA model to apply oral microbial components (Kondo et al., 2022) (PMCID: PMC9474870; DOI: 10.1038/s42003-022-03896-7). This 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.

Fixed maxillae were decalcified in 10% EDTA (Sigma-Aldrich) for 3 wk and then embedded in paraffin. Histological cross-sections were stained with hematoxylin and eosin (HE) and evaluated on a light microscope. Adjacent paraffin sections (4 µm) were subjected to a heat-induced epitope retrieval procedure and then immunohistochemically stained with polyclonal antibodies to CD45 (#PA5-11671, Thermo Fisher Scientific), COL14A1 (#PA5-49916, Thermo Fisher Scientific), CXCL12 (#PA5-30603, Thermo Fisher Scientific), or Ctsk (PA5-14270, Thermo Fisher Scientific) at the suggested dilution, followed by secondary antibody application, diaminobenzidine staining, and methylene blue counterstaining.

Using maxillary cross-sections of WT, Rag2^-/-, and Rag2^-/-Il2rg^-/- mice, osteoclasts were evaluated by TRAP staining using a commercially available kit (Acid Phosphatase TRAP kit, Sigma-Aldrich), according to the manufacturer’s instructions. TRAP-positive cells were counted in the 0.01 mm² area.

On days 1, 4, and 7 after ligature placement, and on day 4 of the MTA of CpG ODN or LPS, mice were euthanized by 100% CO₂ inhalation. Maxillary gingival tissues (n = 4 per group) were harvested from freshly isolated mouse maxillae.

Cell Ranger was used to align reads, generate feature–barcode matrices, and perform clustering and gene expression analyses on the scRNA-seq data, and the output from this program was analyzed using the R-program Seurat (https://satijalab.org/seurat/). Cells with <2400 genes detected or >1% mitochondrial gene expression were filtered out as low-quality cells. Individual gene counts for each cell were divided by the total gene counts for that cell and multiplied by a scale factor of 10,000; natural-log transformation was then applied to the counts. The FindVariableFeatures function was used to select 2000 variable genes with default parameters, and the ScaleData function was used to scale and center the counts in the dataset. Principal component analysis and Uniform Manifold Approximation and Projection dimensional reduction were performed on variably expressed genes. The cluster markers were found using the FindAllMarkers function, and cell types were manually annotated based on the cluster markers. Cell types were assigned based on expression of cell marker genes, and gene expression within different cell types was displayed using dot plots and violin plots.

Annotation and visualization of GO terms were performed by Metascape (http://metascape.org/gp/index.html#/main/step1). The top 100 differentially expressed genes in each population were input and filtered with the term ‘immune.’ Filtered genes were then input, and only ‘biological process’ gene sets were retrieved from the GO database.

NicheNet (v.1.0.0, https://github.com/saeyslab/nichenetr; Browaeys et al., 2020; Saeys Lab, 2023) was used to predict interactions between cell types. In brief, the integrated Seurat object containing each cell subpopulation was input into the NicheNet Seurat wrapper. Sender cells and receiver cells were determined, and interactions between active ligands expressed by sender cells and target receptors expressed by receiver cells were predicted based on information in signaling and ligand–receptor databases.

Statistical analysis to assess differences among multiple experimental groups was performed using one-way ANOVA with Tukey’s multiple-comparison test; p<0.05 was considered to be statistically significant.