Gli1+ Inflamm-Stem Cells Engage Extracellular Vesicles to Prime Aberrant Neutrophils for Exacerbating Periodontal Immunopathology

doi:10.21203/rs.3.rs-5036104/v1

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Gli1+ Inflamm-Stem Cells Engage Extracellular Vesicles to Prime Aberrant Neutrophils for Exacerbating Periodontal Immunopathology

https://doi.org/10.21203/rs.3.rs-5036104/v1

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Periodontitis is a prevalent and progressive detrimental disease which is characterized by chronic inflammation, the immunopathological mechanisms being not yet fully understood. Mesenchymal stem cells (MSCs) play crucial roles as immunoregulators and maintain tissue homeostasis and regeneration, but their in vivo function in immunopathology and periodontal tissue deterioration is still unclear. Here, we utilized multiple transgenic mouse models to specifically mark, ablate and modulate Gli1⁺ cells, a critical and representative subset of MSCs in the periodontium, to explore their specific role in periodontal immunopathology. We reveal that Gli1⁺ cells, upon challenging by an inflammatory microenvironment, significantly induce rapid trafficking and aberrant activation of neutrophils thus exacerbating alveolar bone resorption. Mechanistically, extracellular vesicles (EVs) released by Gli1⁺ cells act as crucial immune regulators in the periodontal tissue, mediating the recruitment and activation of neutrophils through increased generation of reactive oxygen species and trigger of the nuclear factor kappa-B signaling. Importantly, specific inhibition of EV release from Gli1⁺ cells or pharmacological therapy using GANT61 ameliorates periodontal inflammation and alveolar bone loss. Collectively, our findings identify previously unrecognized roles of Gli1⁺ cells in orchestrating infiltration and priming aberrant activation of neutrophils under inflammation, which provide pathological insights and potential therapeutic targets for periodontitis.

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Immunology/Inflammation

Periodontitis

Gli1

mesenchymal stem cells

neutrophils

extracellular vesicles

immunopathology

Periodontitis has become a major public health issue, affecting approximately 50% of the global population and predisposing individuals to multiple systemic diseases, such as cardiovascular disease, diabetes, and Alzheimer’s disease^1,2. A thorough grasp of the underlying cellular and molecular mechanisms is essential for devising effective therapeutic and preventive strategies for periodontitis and its associated conditions. During the progression of periodontitis, a variety of immune cells, such as neutrophils, macrophages, T cells, and B cells, play important roles in influencing the immune response and tissue cells by cell-cell communication and release of cytokines, thus contributing to increased inflammation and bone resorption^3–7. Notably, while neutrophils, as the first leukocytes to accumulate at the inflammation sites, are crucial for antimicrobial immunity, insufficient and excessive recruitment, as well as improper activation of neutrophils in the periodontium, are linked to inflammatory periodontal bone loss⁸. Nevertheless, the mechanisms regulating neutrophils during the progression of periodontitis are not yet fully understood. Mesenchymal stem/stromal cells (MSCs), with multipotent capabilities, are crucial for maintaining periodontal homeostasis and promoting periodontal tissue regeneration^9–11. Despite extensive in vitro studies, the in vivo characteristics and responses of periodontal MSCs in various pathophysiological conditions are far from understood. In particular, as resident stem cells, MSCs are presumed to actively influence the surrounding cells, such as endothelial cells and immune cells, thus contributing to microenvironment remodeling^12,13. Therefore, elucidating the role of periodontal MSCs in regulating neutrophils during periodontitis and the in vivo implications will provide a more comprehensive understanding of the pathogenesis of periodontitis.

Within recent years, accumulating evidence has highlighted that Gli1⁺ cells, marked by the expression of the transcription factor Gli1, constitute a significant subpopulation of MSCs across various organs and hold critical biological functions^14–18. In particular, Gli1⁺ cells have been identified to be an important subset of odontogenic and periodontogenic dental stem cells crucial for the tooth development, remodeling and regeneration^11,17,19. Our recent research has further demonstrated that Gli1⁺ cells in the periodontal ligament (PDL) are vital for both alveolar bone regeneration of avulsed tooth and orthodontic force-induced bone remodeling, indicating that they are highly responsive to the niche signals^20,21. As for the pathogenesis of periodontitis, while a recent study has revealed that the osteogenic differentiation function of Gli1⁺ MSCs is compromised with reduced contribution to periodontium homeostasis²², the role of Gli1⁺ cells in periodontitis is not fully understood, particularly regarding their interactions with other cells. To be noted, as bilayered membranous nanoparticles released by nearly all cell types, extracellular vesicles (EVs) contain a range of signaling molecules, such as proteins, lipids, and nucleic acids, and are recognized as important messengers in cell-to-cell communication²³. Although the effects of EVs from exogenous transplanted MSCs have been extensively studied, the role of EVs from endogenous Gli1⁺ cells in periodontal immunopathology remains to be elucidated.

In this study, we first generated Gli1-CreER^T2;mT/mG mice to trace Gli1⁺ cells and their descendants in ligature-induced periodontitis. We identified Gli1⁺ cells as active participants in periodontitis, with increased proximity to infiltrated neutrophils, indicating potential interactions. In addition, in vitro experiments showed that lipopolysaccharide (LPS) pre-stimulated Gli1⁺ cells could effectively recruit neutrophils as well as induce neutrophil deregulation and extracellular traps (NETs) formation. Next, to gain in vivo evidence, we specifically deleted Gli1⁺ lineage cells in Gli1-Cre transgenic mice crossed with DTA mice, confirming that endogenous Gli1⁺ cells contributed to the recruitment and activation of neutrophils during periodontitis. Mechanistically, EVs released from LPS pre-stimulated Gli1⁺ cells also had the potential to recruit and activate neutrophils, which involved increased generation of reactive oxygen species (ROS) and activation of nuclear factor kappa-B (NF-κB) signaling pathway, as confirmed by transcriptome sequencing and in vitro tests. Based on this, we then generated Gli1-creER^T2;Rab27a^fl/fl mice to conditionally inhibit EV release of Gli1⁺ cells. Results showed that the recruitment and activation of neutrophils, as well as the periodontal bone loss was inhibited, demonstrating the in vivo relevance of EV-mediated Gli1⁺ cell-neutrophil modulation. Moreover, we further showed that pharmacological inhibition of Gli1⁺ cells using GANT61 could effectively alleviate periodontal inflammation and bone loss in periodontitis. Collectively, this study reveals previously unrecognized roles of periodontal MSCs, specifically Gli1⁺ cells, in orchestrating neutrophil infiltration and activation during periodontal immunopathology. Our findings offer mechanistic insights into the complex mechanisms underlying periodontitis, presenting a novel example of stromal-immune cell interactions in inflammatory diseases and shedding light on new therapeutic strategies.

2.1. Gli1⁺ cell dynamics in ligature-induced periodontitis

To investigate the dynamics of Gli1⁺ cells during periodontitis, we applied ligature-induced periodontitis model to Gli1-CreER^T2;mT/mG mice (Fig. 1A). Mice without ligature insertion served as control. Both Gli1⁺ cells and their descendants (Gli1-lineage cells) were marked by green fluorescent protein (GFP) following tamoxifen injection. As early as 18 h post-ligature insertion, more Gli1⁺ cells expressed proliferating cell nuclear antigen (PCNA) compared to control which indicated that Gli1⁺ cells were highly responsive to the niche signals (Figure S1A and B). Meanwhile, the percentage of Gli1⁺ cells expressing PCNA in total Gli1⁺ cells was also higher than control group at both 3 and 7 days post-ligature insertion (Fig. 1B and C), which was consistent with more Gli1⁺ cell in the interdental regions in the periodontitis group compared to control (Figure S1C and D). In control group, approximately 60% of Gli1⁺ cells were positive for runt-related transcription factor 2 (RUNX2), an osteogenic marker, indicating that the majority of Gli1⁺ cells were functionally associated with osteogenesis under physiological conditions. However, the ratio of Gli1⁺RUNX2⁺ double-labeled cells to the total Gli1⁺ cells in the periodontitis group was significantly reduced at both 3 and 7 days post-ligature insertion (Fig. 1D and E), suggesting that periodontitis inhibited osteogenic differentiation of Gli1⁺ cell. These findings suggest that Gli1⁺ cells are active participants in periodontitis, and undergo phenotypic and functional changes in response to periodontal inflammation.

As an inflammatory condition driven by dysbiotic imbalance, periodontitis is marked by the infiltration of various immune cells. Flow cytometry results showed that CD11b⁺Ly6G⁺ neutrophils were the most abundant CD45⁺ leukocytes infiltrated in the periodontal tissue at 18 h, 3 days as well as 7 days post-ligature insertion (Figure S1E). Moreover, flow cytometry and immunofluorescence staining results showed that the interdental regions exhibited neutrophil infiltration as early as 18 h, which significantly increased at both 3 and 7 days post-ligature insertion (Figure S1F-H), indicating that in ligature-induced periodontitis model, neutrophils exhibited sustained and extensive infiltration, highlighting their prolonged involvement in the inflammatory response within periodontal tissues²⁴. Notably, we found that Gli1⁺ cells and neutrophils (marked by Ly6G) were in significantly increased proximity after periodontitis modeling (Fig. 1F and G). The spatial correlation prompted us to explore whether Gli1⁺ cells could modulate neutrophils and contribute to periodontal immunopathology.

2.2. LPS pre-stimulated Gli1⁺ cell supernatants induced neutrophil migration and activation in vitro.

To explore whether Gli1⁺ cells could modulate neutrophils in vitro, we applied flow cytometry-activated cell sorting (FACS) to isolate Gli1⁺ cells from cultured bone MSCs of Gli1-CreER^T2;mT/mG mice, and stimulated them with LPS or equal volume of PBS as control for 24 h. Then, the medium was changed into culture medium and the cell supernatants were collected after 24 h which was used to treat neutrophils (Fig. 2A). We first evaluated the impacts on the migration ability of neutrophils through transwell assay. As shown by crystal violet staining and cell counter, LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants group) significantly promoted neutrophil migration, while the effects of the normal Gli1⁺ cell supernatants (Supernatants group) were negligible compared to the control group (Fig. 2B-D). These results indicate that LPS pre-stimulated Gli1⁺ cell supernatants could effectively recruit neutrophils in vitro.

After being recruited to inflammatory sites, neutrophils exert their immune defense effects in a variety of ways, mainly including degranulation and production of NETs²⁵. Next, we examined whether Gli1⁺ cells affected the functions of neutrophils following their recruitment. Ultrastructure of neutrophils was observed by transmission electron microscopy (TEM), which showed that the cytoplasmic granules were significantly decreased while lots of autophagosomes appeared in the Stimulated supernatants group, with no obvious differences between the control and Supernatants groups (Fig. 2E-G), suggesting that LPS pre-stimulated Gli1⁺ cell supernatants could promote neutrophil degranulation and autophagy. We then evaluated the production of NETs by both scanning electron microscopy (SEM) and fluorescence staining. SEM results showed that neutrophils cultured in LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants group) produced many filaments that extended toward and interwound into a network, representing a typical structure of NETs, which was not observed in the control and normal Gli1⁺ cells supernatants groups (Fig. 2H). Moreover, immunofluorescence staining results showed that NETs-related proteins, myeloperoxidase (MPO) and citrullinated histone H3 (citH3), were clearly observed in the Stimulated supernatants group, with citH3 forming fibrous reticular structures; in contrast, these proteins were almost undetectable in the control and Supernatants groups (Fig. 2H and I). SYTOX staining also verified that neutrophils cultured in the Stimulated supernatants group exhibited more SYTOX positive cells with extended filamentary structure (Fig. 2H). These results indicated that LPS pre-stimulated Gli1⁺ cell supernatants could promote neutrophils NETs formation. Since neutrophil apoptosis is essential for inflammation resolution and its delay can cause ongoing local inflammation and tissue damage²⁶, the effects of Gli1⁺ cells on neutrophils apoptosis were detected by flow cytometry. Results showed that LPS pre-stimulated Gli1⁺ cell supernatants significantly inhibited neutrophils apoptosis, while the effects of normal Gli1⁺ cells supernatants were negligible compared to the control group (Fig. 2J and K).

Collectively, these findings suggest that LPS pre-stimulated Gli1⁺ cell supernatants could effectively induce neutrophil migration, deregulation and NETs formation, as well as inhibit neutrophil apoptosis in vitro.

2.3. Deletion of Gli1 ⁺ cells suppressed neutrophil recruitment and activation in vivo and ameliorated periodontal destruction in periodontitis.

Next, to further investigate whether Gli1⁺ cells were able to modulate neutrophils in vivo, we generated Gli1-CreER^T2;Rosa-DTA mice (Figure S2A and B) to conditionally delete Gli1⁺ cells, and performed ligature-induced periodontitis model (Fig. 3A). Immunofluorescence results showed that deletion of Gli1⁺ cells reduced Ly6G⁺ cells infiltration in the periodontium of the interdental regions between the first and second molars in Gli1-CreER^T2;Rosa-DTA mice in contrast to their Rosa-DTA counterparts at both 3 and 7 days post-ligature insertion (Fig. 3B and C; Figure S3A and B). Flow cytometry analysis of CD11b⁺Ly6G⁺ neutrophils in the periodontal tissue around the second molar showed similar results (Fig. 3D and E; Figure S3C and D). Moreover, neutrophil NETs formation in the inflamed regions was observed by Ly6G and citH3 staining. Results indicated that citH3 was obviously increased after periodontitis modeling with close proximity to Ly6G⁺ neutrophils in the interdental periodontium, which was notably decreased following the deletion of Gli1⁺ cells (Fig. 3F and G). Taken together, these results suggest that Gli1⁺ cells could promote the recruitment and activation of neutrophils in periodontitis condition.

Given the critical role of neutrophils in periodontitis pathogenesis, we then explored whether deletion of Gli1⁺ cells could effectively ameliorate alveolar bone destruction. Micro-computed tomography (micro-CT) scanning was applied to alveolar bone at 7 and 14 days post-ligature insertion to investigate the roles of Gli1⁺ cells in alveolar bone destruction. The scanning and reconstruction images revealed that deletion of Gli1⁺ cells effectively suppressed alveolar bone destruction in both the interdental and furcation regions of the second molar, along with decreased CEJ–ABC (cemento-enamel junction-alveolar bone crest) distance on the buccal surface of the second molar and increased bone volume to total volume ratio (BV/TV) of interdental regions between the first and second molars (Fig. 3H-J and Figure S3E-G), which was consistent with less neutrophil infiltration and NETs formation. H&E staining further confirmed that deletion of Gli1⁺ cells effectively attenuated alveolar bone resorption at 7 days (Figure S3H). Staining for tartrate-resistant acid phosphatase (TRAP), a classic method to identify osteoclasts, verified that deletion of Gli1⁺ cells inhibited osteoclasts at 7 days, which was consistent with the suppression of inflammation and bone loss (Figure S3I). These results suggest that Gli1⁺ cells could remarkedly aggravate alveolar bone destruction during periodontitis.

Collectively, these data indicate that Gli1⁺ cells play a critical role in neutrophil recruitment and activation in vivo, thus contributing to periodontal immunopathology and exacerbating bone resorption.

2.4. LPS pre-stimulated Gli1 ⁺ cell supernatants induced pro-inflammatory transcriptional activation of neutrophils and increased their ROS levels.

Then, we intended to investigate the underlying mechanism. To assess whether LPS pre-stimulated Gli1⁺ cell supernatants influenced the gene expression profiles of neutrophils, we conducted RNA-sequencing (RNA-seq) analysis on neutrophils treated with these supernatants compared to control neutrophils. The analysis identified a total of 6023 differentially expressed genes (DEGs) following LPS pre-stimulated Gli1 + cell supernatants treatment (Fig. 4A), with 3057 genes upregulated and 2966 downregulated (Fig. 4B). Of the upregulated DEGs detected in Stimulated supernatants group, biological process gene ontology (GO) enrichment analysis revealed that these genes were mainly involved in immune and inflammatory processes, including the “Cellular response to lipopolysaccharide”, “Activation of innate immune responses”, “Inflammatory responses”, “Immune responses” and “Immune system processes” (Fig. 4C). In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the upregulated DEGs showed significant enrichment in inflammation-related pathways, especially “NF-kappa B signaling pathway” (Fig. 4D). Gene set enrichment analysis (GSEA) also revealed that the “NF-kappa B signaling pathway” was significantly upregulated in neutrophils cultured in the stimulated supernatants (Fig. 4E). Quantitative real-time PCR (qRT-PCR) results further verified that the expression of most NF-Kappa B signaling pathway transcripts were upregulated in stimulated supernatants-treated neutrophils (Fig. 4F). Immunofluorescence analysis also showed that stimulated supernatants treatment dramatically promoted NF-κB expression, and most NF-κB translocated from cytoplasm to nucleus (Fig. 4G), implicating the activation of NF-κB signaling pathway.

It has been reported that neutrophil activation relies on the production ROS, which are crucial for their diverse functions, including degranulation, NET formation, and bacterial phagocytosis^27,28. Therefore, we investigated whether LPS pre-stimulated Gli1⁺ cell supernatants could increase the ROS levels in neutrophils. GSEA revealed that the “Oxidative stress response” was upregulated in neutrophils cultured in the stimulated supernatants (Fig. 4H), indicating increased intracellular ROS levels. Flow cytometry analysis further confirmed that the stimulated supernatants significantly elevated neutrophil ROS levels (Fig. 4I and J), which were consistent with the immunofluorescence results (Fig. 4K).

Collectively, these data indicate that LPS pre-stimulated Gli1⁺ cell supernatants induced pro-inflammatory transcriptional profile of neutrophils, mainly involving activation of NF-κB signaling pathway, and increased ROS levels, which may mediate the regulatory effects of Gli1⁺ cells.

2.5. EV release was indispensable for LPS pre-stimulated Gli1⁺ cells to recruit and activate neutrophils.

Given that EVs are crucial for intercellular communication and play a key role in mediating the regulatory effects of MSCs²⁶, we further investigated whether EVs are involved in the recruitment and activation of neutrophils by LPS pre-stimulated Gli1⁺ cells. We isolated EVs from the conditioned culture medium through gradient centrifugation, as described in previous study²⁹. Nanoparticle tracking analysis (NTA) showed that the diameters of LPS pre-stimulated Gli1⁺ cell-EVs were ranged from 50 to 500 nm, with a peak at 137.1 nm (Figure S4A). TEM observation further revealed that LPS pre-stimulated Gli1⁺ cell-EVs had typical cup-shaped morphology with bilayered membranes (Figure S4B). For “Stimulated supernatants-EVs”, EVs were removed from LPS pre-stimulated Gli1⁺ cells supernatants. For “EVs”, EVs were extracted from LPS pre-stimulated Gli1⁺ supernatants and added into alpha minimum essential medium (α-MEM) containing 20% EV-free FBS. As to the effects of EVs on the recruitment of neutrophils, crystal violet staining in transwell assay showed that removing EVs from the LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants-EVs group) inhibited neutrophil migration to the lower chamber compared to the LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants group). In contrast, the addition of EVs (EVs group) significantly enhanced neutrophil migration compared to the control group (Fig. 5A and B). The cell counter results were consistent with those obtained from crystal violet staining (Fig. 5C). These results suggest that EVs released from LPS pre-stimulated Gli1⁺ cells have the potential to recruit neutrophils in vitro.

We then examined whether EVs activated neutrophils following their recruitment. Flow cytometry results showed that removing EVs from LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants-EVs group) significantly inhibited neutrophils ROS production. In contrast, the addition of EVs (EVs group) remarkedly promoted ROS production compared to the control group. Immunofluorescence results were consistent with those of flow cytometry (Fig. 5D-F). These results indicate that EVs released from LPS pre-stimulated Gli1⁺ cells markedly elevate the ROS levels in neutrophils. Moreover, we treated LPS-stimulated Gli1⁺ cells with GW4869 to inhibit EV secretion, which significantly reduced neutrophils ROS production compared to the Stimulated supernatants group (Figure S5), further confirming that EVs play an important role in promoting neutrophils ROS production. To further assess neutrophil activation, we examined the activation of NF-κB signaling pathway and the formation of NETs. qRT-PCR analysis revealed that when EVs were removed from the LPS pre-stimulated Gli1⁺ cell supernatants (Stimulated supernatants-EVs group), the expression levels of Nfkb1 and Nfkb2 in the NF-κB signaling pathway were reduced. In contrast, the EV-treated group displayed higher expression levels compared to the control group (Fig. 5G). These findings were corroborated by immunofluorescence staining, which demonstrated that EVs not only promoted NF-κB expression but also facilitated its translocation from the cytoplasm to the nucleus (Fig. 5H). Moreover, immunofluorescence staining indicated that removing EVs from LPS pre-stimulated Gli1 + cell supernatants (Stimulated supernatants-EVs group) significantly inhibited NETs formation, whereas adding EVs (EVs group) promoted NETs formation compared to the control (Fig. 5I and J). These results suggest that EVs from LPS pre-stimulated Gli1⁺ cells can activate the pro-inflammatory NF-κB signaling pathway and induce NETs formation in neutrophils.

Collectively, these findings indicate that EVs released from LPS pre-stimulated Gli1⁺ cells could effectively recruit neutrophils and promote ROS production to activate neutrophils in vitro.

2.6. Genetic inhibition of EV release suppressed Gli1 ⁺ cell-mediated recruitment and activation of neutrophils in vivo and ameliorated periodontal destruction in periodontitis.

To further investigate the effects of Gli1⁺ cells-released EVs on neutrophils in vivo, we generated Gli1-CreER^T2;Rab27a^fl/fl mice (Figure S6A) to conditionally delete Rab27a gene, a key regulator of vesicular fusion and trafficking, from Gli1⁺ cells to inhibit EV release, and performed ligature-induced periodontitis model (Fig. 6A). First, the neutrophil infiltration level in the inflamed regions was measured. Immunofluorescence staining results showed that inhibition of EV release in Gli1⁺ cells reduced the infiltration of Ly6G⁺ cells in the periodontium of the interdental regions between the first and second molars in Gli1-CreER^T2;Rab27a^fl/fl mice compared to their Rab27a^fl/fl counterparts, at both 3 and 7 days post-ligature insertion (Fig. 6B and C; Figure S6B and C). Flow cytometry results similarly showed reduction of neutrophil infiltration in the inflamed regions around the second molar in the Gli1-CreER^T2;Rab27a^fl/fl mice compared to their Rab27a^fl/fl counterparts at both 3 and 7 days (Fig. 6D and E; Figure S6D and E). Then, we evaluated the functional status of neutrophils by assessing the formation of NETs. Immunofluorescence results showed that inhibition of EV release in Gli1⁺ cells of Gli1-CreER^T2;Rab27a^fl/fl mice resulted in significantly fewer NETs compared to Rab27a^fl/fl mice (Fig. 6F and G). These data suggest that EVs released from Gli1⁺ cells can recruit neutrophils and enhance their NETosis in periodontitis. Further, the role of Gli1⁺ cells-released EVs on pathological bone resorption of periodontitis was evaluated by micro-CT scanning and histological analysis. Micro-CT analysis and H&E staining showed that inhibition of EV release from Gli1⁺ cells effectively alleviate alveolar bone destruction in both the interdental and furcation regions of the second molar, along with decreased CEJ-ABC distance on the buccal surface of the second molar and increased BV/TV of interdental regions between the first and second molars (Fig. 6H-J; Figure S6F). These data suggest that EVs released from Gli1⁺ cells induce alveolar bone destruction in periodontitis.

Collectively, these findings indicate that genetic inhibition of EV release reduces Gli1⁺ cell-mediated neutrophil recruitment and activation in vivo, and alleviates periodontal destruction in periodontitis.

2.7. Administration of Gli1 inhibitor GANT61 suppressed neutrophil recruitment and activation and alleviated periodontal destruction in periodontitis.

Based on the above results, we explored the potential of Gli1⁺ cells as a therapeutic target for periodontitis and evaluated the therapeutic efficacy of GANT61, a small molecule inhibitor of Gli transcription factors within the Hedgehog signaling pathway^30,31. In particular, GANT61 was injected every other day for three times following periodontal ligation (Fig. 7A). Immunofluorescence staining results revealed a significant reduction in both neutrophil infiltration and NETs formation in the GANT61-treated group (Fig. 7B-D). Moreover, micro-CT analysis showed considerable alveolar bone destruction in control mice, while those treated with GANT61 exhibited significantly less bone loss (Fig. 7E-G). These findings collectively suggest that GANT61 has the potential to effectively alleviate Gli1⁺ cell-mediated neutrophil recruitment and activation in vivo, and alleviate periodontal inflammation and destruction, providing novel insights into the clinical management of periodontitis.

Periodontitis is a common chronic inflammation disease that occurs in the periodontal tissues and often leads to alveolar bone resorption³². Various immune cells are involved in the progression of periodontitis; in particular, excessive infiltration and activation of neutrophils have been shown to exacerbate the immunopathology of periodontitis⁸. In addition to immune cells, stromal cells within the periodontal tissues also play a critical role in the development of periodontitis. Periodontal MSCs, as an important component of periodontal tissues, not only contribute to the growth, development, and regeneration of periodontal tissues under normal conditions^9,11,19, but may also interact with other cells in the microenvironment³³. Previous studies on the function of periodontal MSCs have primarily focused on in vitro experiments^34–36. It remains unclear whether and how periodontal MSCs regulate the immune response in periodontitis. In this study, we utilized multiple transgenic mouse models to specifically mark, ablate and modulate the function of Gli1⁺ cells to explore their specific role in periodontitis. Our findings reveal that Gli1⁺ cells significantly exacerbate periodontal immunopathology and alveolar bone resorption (Fig. 8). The specific mechanism involves the secretion of EVs by Gli1⁺ cells, which recruit and activate neutrophils (Fig. 8). Specific ablation of Gli1⁺ cells or pharmacological therapy using GANT61 significantly ameliorates periodontal immunopathology and alveolar bone resorption (Fig. 8). This research provides compelling evidence of active neutrophil regulation by Gli1⁺ cells, offering new insights into the immunopathological mechanisms of periodontitis and paving the way for targeted therapies.

In the early stages of periodontitis, neutrophils are the first immune cells to arrive at the site of infection³⁷. They perform immune defense functions through mechanisms such as phagocytosis, degranulation, and the production of NETs³⁸. However, while effectively eliminating pathogens, neutrophils also release large amounts of ROS and enzymatic substances, which can damage periodontal support structures such as alveolar bone and PDL, leading to tissue destruction^39,40. The effective regulation of neutrophil is crucial to prevent excessive damage to periodontal tissues and control disease progression. Nevertheless, the mechanisms of neutrophil recruitment and activation during periodontitis are not fully understood. Specifically, how neutrophils are attracted and activated by signaling molecules within periodontal tissues, and how they interact with other immune or tissue cells, are key areas of ongoing research. Understanding these mechanisms more thoroughly could lead to more effective strategies for controlling the progression of periodontitis. Of note, while most current research focus on the impact of immune responses on periodontal tissues, the role of periodontal tissues themselves, especially periodontal stromal cells, in regulating immune cell function remains underexplored. This study highlights the critical role of periodontal Gli1⁺ cells in periodontitis, showing that these cells exacerbate inflammation-induced bone resorption by recruiting and activating neutrophils. This discovery not only enhances our understanding of the interaction between periodontal MSCs and the immune system, but also offers new potential therapeutic targets for periodontitis.

Gli1⁺ cells play a crucial role in the growth, development, and homeostasis of periodontal tissues, representing an important subpopulation of periodontal MSCs^11,19. Traditional research have mainly focused on the role of Gli1⁺ cells in physiological condition^19,41. While a recent study has revealed that the osteogenic differentiation function of Gli1⁺ cells is compromised in periodontitis⁴², the role of Gli1⁺ cells, especially how they may influence periodontal immunopathological processes, remains unclear. In this study, we generated Gli1-CreER^T2;mT/mG mice to trace Gli1⁺ cells and their descendants, and discovered that in physiological condition, more than half of periodontal Gli1⁺ cells expressed the osteogenic marker RUNX2, indicating that the majority of Gli1⁺ cells within periodontal tissues predominantly contributing to osteogenesis which is consistent with previous research. However, in the context of periodontitis, we observed notable increase in the proliferative activity of Gli1⁺ cells, accompanied by a significant reduction in their osteogenic differentiation potential. This suggests that Gli1⁺ cells are active participants of periodontitis, and undergo phenotypic and functional changes in response to periodontal inflammation, a phenomenon that necessitates further detailed investigation. Additionally, Gli1⁺ cells were found in close proximity to neutrophils, indicating potential interactions. By specific deletion of Gli1⁺ lineage cells in Gli1-CreER^T2;Rosa-DTA transgenic mice, we found that under periodontal inflammatory conditions, Gli1⁺ cells exhibited strong immunoregulatory functions by mobilizing and activating neutrophils, which led to further periodontal immune dysregulation and exacerbated the destruction of periodontal bone tissue. Our study reveals the phenotypic changes and previously unrecognized roles of Gli1⁺ cells in periodontal immune responses and alveolar destruction, elucidating their immunoregulatory functions and enhancing our understanding of the periodontitis pathological mechanisms. Since periodontitis involves various cell types, it remains to be explored whether and how Gli1⁺ cells might influence other immune cells. Moreover, the question of whether other periodontal MSCs also exhibit immunopathological regulatory functions requires further in-depth investigation.

MSCs regulate immune cells through various mechanisms, including the secretion of cytokines, growth factors, chemokines, metabolites, and EVs^43,44. Despite the extensive research on the effects of EVs from exogenously transplanted MSCs^45–47, whether and how endogenous Gli1⁺ cell-released EVs contribute to periodontal immunopathology remains to be elucidated. Our research found that EVs secreted by LPS pre-stimulated Gli1⁺ cells could significantly recruit and activate neutrophils in vitro, which indicates that EVs released by Gli1⁺ cells are an important regulator of neutrophils. More importantly, we generate Gli1-creER^T2;Rab27a^fl/fl mice to conditionally inhibit EV released by Gli1⁺ cells which showed reduced neutrophil recruitment and activation, thereby alleviating bone resorption in periodontitis. This discovery reveals that EVs secreted by Gli1⁺ cells are important regulatory factors of periodontal immunity, which helps to further understand the molecular mechanism of Gli1⁺ cell-mediated bone resorption in periodontitis, and provides a new direction for the treatment of periodontitis. The specific factors within EVs that mediate the above effects, and the involved downstream pathways remain to be further elucidated. Moreover, we discovered that without EVs, the LPS pre-stimulated Gli1⁺ cell supernatants could also partially promote the migration and activation of neutrophils. This suggests that, aside from EVs, there are other substances that may mediate the regulatory effects of Gli1⁺ cells on neutrophils, which requires further investigation.

GANT61 functions by specifically targeting and inhibiting Gli transcription factors within the Hedgehog signaling pathway, which are essential for the activity of Gli1⁺ cells^30,31. Our research found that GANT61 could effectively alleviate periodontal immunopathology, indicating GANT61 as a potential therapeutic drug for periodontitis. A significant limitation of the current application of GANT61 is lack of cell specificity. This non-specificity may affect non-target cells, which can lead to unintended side effects and reduced therapeutic efficacy. To address this issue, future research should focus on developing more targeted strategies to precisely regulate Gli1⁺ cells, which has the potential to improve therapeutic efficacy while minimizing adverse effects, thereby contributing to safer and more effective treatments for periodontitis.

In summary, our findings reveal that endogenous Gli1⁺ cells play a crucial role in the pathology of periodontitis by recruitment and activation of neutrophils, which is mediated through releasing EVs. Additionally, we have identified GANT61 as a potential therapeutic drug for periodontitis. This study uncovers a previously unrecognized pathogenic mechanism in periodontitis and highlights Gli1⁺ cells as a promising therapeutic target. It provides a novel example of stromal-immune cell interactions in inflammatory diseases and offers new insights for the clinical management of such conditions.

Animals

All animal experiments in this study were conducted with the approval of Institutional Animal Care and Use Committee of the Fourth Military Medical University. C57BL/6J (#000664), Gli1-CreER^T2 (#007913), ROSA26-mT/mG (#007676), and ROSA26-eGFP-DTA mice (#006331) were purchased from the Jackson Laboratory. Rab27a-flox mice (#T066643) were purchased from GemPharmatech. Gli1-CreER^T2 mice were crossed with ROSA26-mT/mG mice, ROSA26-eGFP-DTA mice, Rab27a^fl/fl to generate Gli1CreER^T2;mT/mG mice, Gli1-CreER^T2;Rosa-DTA mice, Gli1-CreER^T2;Rab27a^fl/fl mice, respectively. All mice were maintained under specific pathogen free conditions (24°C, 12 h light/dark cycles and 50% humidity), and fed with food and water at libitum. Genotype identification was conducted using PCR following established protocols from published studies^48,49. In brief, mouse tail tissue was lysed in tail lysis buffer (Vigen Biotec, USA) overnight at 55°C, followed by heating at 85°C for 1 h. The supernatant was then collected for DNA amplification and gel electrophoresis. The relevant primers were shown in the Table S1. Tamoxifen (MCE, USA) was injected intraperitoneally at 100 µg/g for 4 consecutive days to induce Cre-mediated DNA recombination, and the ligature-induced periodontitis model was performed after 7 days.

Establishment of the Ligature-induced Periodontitis Model

The maxillary second molars of 6-8-week-old mice were ligatured with 5 − 0 silk sutures according to published research²⁴. In brief, silk sutures were wrapped around the neck of the tooth with knots tying on the buccal side. For the therapeutic application of GANT61, 40 mg/kg GANT61 (MCE, USA) or equal volume of corn oil was intraperitoneally injected into the mice every other day for three times following periodontal ligation.

Immunofluorescence Staining

Immunofluorescent staining was performed according to previously published methods²⁹. Briefly, the mice maxillary samples were separated, fixed in 4% paraformaldehyde (PFA) (Sigma, USA) at 4°C for 8 h and were decalcified in 17% ethylene diamine tetraacetic acid (EDTA) (Sigma-Aldrich, USA) at 4°C for 21 days, with the decalcified solution changed every other day. Samples were then washed thoroughly with PBS to remove the PFA, dehydrated in 30% sucrose solution overnight, embedded in optimal cutting temperature (OCT) compound (Leica, Germany) and sliced into 10 µm thick sections. For tissue samples, the sections were permeabilized in 0.5% Triton (Sigma-Aldrich, USA) at room temperature for 10 mins, blocked with goat serum at room temperature for 30 min, incubated with primary antibodies overnight at 4°C, incubated with secondary antibodies at 37°C for 1 h, and sealed with a mounting medium containing 4,’-6-diamidino-2-phenylindole (DAPI) (Abcam, USA). For cell samples, cells were seeded in round coverslip, fixed with 4% PFA for 5 min, and then underwent the same procedure as mentioned above. Images were captured using confocal scanning laser microscopes (FV3000, Olympus, Japan and LSM900, ZEISS, German) and analyzed using the ImageJ software. The SYTOX dye (Alphabio, China) was used to detect cellular nucleic acids. The antibodies used were as follows: anti-PCNA antibody (13110T, Cell Signaling Technology, USA, diluted 1:100), anti-RUNX2 antibody (12556, Cell Signaling Technology, USA, diluted 1:200), anti-Ly6G antibody (sc-53515, Santa Cruz, USA, diluted 1: 100), anti-Histone H3 (citrulline R2 + R8 + R17) (ab5103, Abcam, USA, diluted 1:300), anti-MPO (GB-15224, Servicebio, China, diluted 1:200), anti-NF-κB (sc-109, Santa Cruz, USA, diluted 1:100), Alexa Fluor 647-conjugated Goat anti-Rabbit secondary antibody (4414, Cell Signaling Technology, USA, diluted 1:200), Alexa Fluor 594-conjugated Donkey anti-Rat secondary antibody (A21209, Invitrogen, USA, diluted 1:200), Alexa Fluor 594-conjugated Donkey anti-Rabbit secondary antibody (A21207, Invitrogen, USA, diluted 1:200), Alexa Fluor 488-conjugated Donkey anti-Rabbit secondary antibody (A21206, Invitrogen, USA, diluted 1:200), and Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, USA, diluted 1:200).

Flow Cytometry Analysis

Mice were sacrificed and the periodontal tissue around the upper second molar was collected. After being washed by cold PBS, the tissue was digested with 1 mg/mL collagenase I (Gibco, USA) for 30 min at 37°C. The supernatants were collected and filtered through a 100 µm cell strainer, centrifuged at 500 g for 5 min, and resuspended with 100 µL PBS to obtain single-cell suspension. Then, the cells were stained with CD45 Monoclonal Antibody (30-F11), PE (12-0451-82, Invitrogen, USA, 0.125 µg/test), CD11b Monoclonal Antibody (M1/70), Brilliant Violet™ 421 (404-0112-80, Invitrogen, USA, 0.125 µg/test) and Ly6G Monoclonal Antibody (1A8-Ly6g), APC (17-9668-82, Invitrogen, USA, 0.5 µg/test) for 30 min on ice in the dark. The cells were washed twice and PI (SL7091, Coolaber, China, diluted 1:100) was added 5 min before the detection. Then samples were detected on a flow cytometer (Cytomics FC 500, Beckman-Coulter, USA). The results were analyzed using FlowJo_V10 software.

Micro-CT Analysis

At the endpoints of each experiment, the mice were sacrificed and the maxillae were dissected and fixed with 4% PFA solution overnight at 4℃. The fixed maxillae were scanned using micro-CT (Siemens Inveon, Germany and PerkinElmer, USA) with a resolution of 18 µm, a voltage of 50 kV and a current of 80 µA. Micro-CT data was reconstructed and analyzed by VGStudio MAX software (Volume Graphics, Germany). The CEJ-ABC distance on the buccal surface of the second molar and the BV/TV in the interdental regions between first and second molars were analyzed.

Histological Analysis

The isolated maxillae were fixed in 4% PFA solution overnight at 4℃ and decalcified in 17% EDTA (Sigma-Aldrich, USA) at 4°C for 21 days, with the decalcified solution changed every other day. The decalcified maxillae were rinsed with running water thoroughly, dehydrated using graded ethanol, embedded in paraffin and sliced into 4 µm thick sections. The sections were deparaffinized, and underwent H&E staining (Leica, Germany) and TRAP staining (Sigma–Aldrich, USA), according to the manufacturer’s instructions. Images were acquired using a Leica microscope.

Sorting and Culture of Gli1 ⁺ Cells

4-week-old Gli1-CreER^T2;mT/mG mice received intraperitoneal injections of tamoxifen for four consecutive days to induce the expression of GFP in Gli1⁺ cells. Seven days later, the mice were sacrificed, and the femurs and tibias were dissected and washed with ice-cold sterile PBS. Then, the bone marrow was flushed with α-MEM (Invitrogen, USA) containing 20% fetal bovine serum (FBS) (Tianhang, China), 2 mm L-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA), with the remaining bones being cut into small pieces. The cell suspension was centrifuged at 500 g for 5 min, and then the cell pellet and bone fragments were seeded in cell culture flasks (Thermo, USA) at 37°C with 5% CO₂. Cell medium was changed every three days and cell passaging was initiated when the cells reached approximately 80%-90% confluence. The third passage of cells was used for FACS of GFP positive cells. Specifically, cells were digested with 0.25% trypsin (MP Biomedicals, USA), followed by collection through centrifugation at 500 g for 5 min. After resuspension in PBS containing 2% FBS at a density of 1 × 10⁶ cells/mL, GFP-positive cells were subsequently sorted using a flow cytometer (BD, USA)²¹. The sorted cells were washed by sterile PBS for twice and then cultured in α-MEM (Invitrogen, USA) containing 20% FBS (Tianhang, China), 2 mm L-glutamine (Invitrogen, USA), and 1% penicillin/streptomycin (Invitrogen, USA) at 37°C with 5% CO₂.

Extraction and Culture of Neutrophils

Mice was sacrificed, and the femurs and tibias were dissected. Bone marrow was flushed out with ice-cold sterile PBS using syringe, followed by erythrocyte lysis in RBC lysis buffer (Yuanye Bio-Technology, China). 3 mL Histopaque-1119 (Sigma, USA) was added to the bottom of the 15 mL centrifugation tube, with 3 mL Histopaque-1077 (Sigma, USA) in the middle, and the cell suspension on the top. After centrifugation at 800 g for 30 min, neutrophils at the interface of the Histopaque-1119 and Histopaque-1077 layers were collected and washed by ice-cold sterile PBS for twice. Then, the neutrophils were cultured in Roswell Park Memorial Institute (RPMI) 1640 (Gibco, USA) supplemented with 10% FBS (Tianhang, China) and 1% penicillin/streptomycin (Invitrogen, USA) or resuspended in Hanks balanced salt solution (HBSS).

Collection and Treatment of Cell Supernatants

Gli1⁺ cells were stimulated with LPS (Sigma, USA, 2 µg/mL) or equal volume of PBS as control for 24 h. Then, the medium was changed into α-MEM (Invitrogen, USA) containing 20% FBS (Tianhang, China) and the cell supernatants were collected after 24 h which was used to treat neutrophils as “Stimulated supernatants” or “Supernatants”.

Collection and Treatment of EVs Derived from LPS Pre-stimulated Gli1 ⁺ Cells

Gli1⁺ cell was stimulated with LPS (Sigma, USA, 2 µg/mL) or equal volume of PBS as control for 24 h, and the medium was changed into α-MEM (Invitrogen, USA) containing 20% EV-free FBS (Tianhang, China). For “EVs” group, cell supernatants were collected after 24 h and the EVs were isolated by gradient centrifugation. Briefly, cell supernatants were centrifuged at 800 g for 10 min to remove cells, 2000 g for 10 min to remove cell debris, and 16000 g for 30 min to pellet EVs²⁹, which were characterized by NTA and TEM, and then used to treat neutrophils. For “Stimulated supernatants-EVs” group, the stimulated supernatants were collected and depleted of EVs by ultracentrifugation²⁹. For “GW4869” group, Gli1⁺ cell was co-stimulated with LPS (Sigma, USA, 2 µg/mL) and GW4869 (MCE, USA, 10 µM) for 24 h. Then, the medium was changed into α-MEM (Invitrogen, USA) containing 20% EV-free FBS (Tianhang, China) and the cell supernatants were collected after 24 h.

Transwell Migration Assay

Different cell supernatants or α-MEM (Invitrogen, USA) were added to the lower chamber. Then, the neutrophils were resuspended in 100 µL RPMI 1640 (Gibco, USA) and seeded to the upper chamber of a transwell plate (5 × 10⁵/well). After 30 min, the medium of the lower chamber was collected and the number of cells that had migrated into the lower chamber was determined by cell counter. In addition, the transwell membrane's lower surface was fixed with 4% PFA for 15 min and then stained with 0.1% crystal violet (Servicebio, China) for 30 min. Migrated cells were observed and captured under a microscope in randomly selected visual fields.

NTA

To evaluate the size distribution of EVs, NTA was conducted as the protocol described in a previous study²¹. In brief, EVs were resuspended in PBS at a final concentration of 6.6 × 10⁷ particles/mL and then analyzed using a PMX ZetaView instrument (Particle Metrix, Germany).

SEM

First, neutrophils were seeded in round coverslip, then they were fixed, dehydrated, and dried to eliminate moisture. After drying, they were coated with a thin conductive layer, typically gold or platinum. The prepared samples were then mounted on SEM stubs and placed in a vacuum chamber. Finally, the samples were examined under a scanning electron microscope (ThermoFisher Quattro S, ThermoFisher, Czech Republic).

TEM

For cell samples, neutrophils were initially fixed in a 2.5% glutaraldehyde solution to preserve their cellular structures. This was followed by post-fixation with 1–2% osmium tetroxide to further stabilize lipids and proteins. Dehydration was carried out through a graded series of ethanol solutions, starting from 30% and progressing to 100%, to gradually remove water from the samples. After dehydration, the samples were infiltrated with a resin. The infiltration was performed by gradually increasing the concentration of resin in ethanol until only pure resin remained. The samples were then embedded in fresh resin and polymerized, usually by heating at 60°C for 24–48 h. Once the resin blocks were hardened, ultrathin sections (approximately 60–90 nm thick) were cut. These sections were collected on copper grids and contrasted with heavy metals, such as uranyl acetate and lead citrate, to enhance the electron density and improve image contrast. Finally, the prepared grids were examined under a transmission electron microscope (HT7800, HITACHI, Japan). For EV samples, EVs were first resuspended in 2% PFA, then dropped onto 200-mesh copper grids. After removing excess liquid with filter paper, the samples were stained with uranyl acetate (Sigma-Aldrich, USA) for 2 min at room temperature. Following a distilled water rinse and drying process, images were captured using a transmission electron microscope (HT7800, HITACHI, Japan).

Apoptosis Assay

Neutrophils were cultured in different medium for 8 h, followed by apoptosis detection using the PE Annexin V Apoptosis Detection Kit I (BD Biosciences, 559763). Briefly, the cells were washed with cold PBS and resuspended in 100 µL of 1× binding buffer. Subsequently, 5 µL of PE Annexin V and 5 µL of 7-amino-Actinomycin D (7-AAD) solution were added to the cell suspension, which was then incubated at room temperature for 30 min. After incubation, an additional 400 µL of binding buffer was added. The samples were analyzed using a flow cytometer

(Cytomics FC 500, Beckman-Coulter, USA) within 1 h. The results were analyzed using FlowJo_V10 software. The apoptotic rate of neutrophils was calculated as the combined percentage of Annexin V⁺/7-AAD⁻ and Annexin V⁺/7-AAD⁺ cells.

Oxidative Stress Evaluation

Cell ROS production was detected with the ROS assay kit (Beyotime Biotechnology, China). Briefly, neutrophils receiving different treatments were stained with DCFH-DA (10 µМ) for 20 min at 37°C, and the fluorescence was examined by flow cytometry (Cytomics FC 500, Beckman-Coulter, USA) and fluorescence microscopy (Olympus, Japan).

RNA Sequencing

Neutrophils were cultured in LPS pre-stimulated Gli1⁺ cell supernatants or control medium for 8 h and then washed with PBS for 3 times. Total RNA was extracted using the MIsZOL Reagent (Mishushengwu, China) according to the manufacturer’s protocol. RNA purity and quantification were evaluated using the NanoDrop 2000 spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Then the libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit according to the manufacturer’s instructions. The transcriptome sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The libraries were sequenced on a llumina Novaseq 6000 platform and 150 bp paired-end reads were generated. Raw reads of fastq format were firstly processed using fastp and the low quality reads were removed to obtain the clean reads. The clean reads were mapped to the reference genome using HISAT2. FPKM of each gene was calculated and the read counts of each gene were obtained by HTSeq-count. PCA analysis were performed using R (v 3.2.0) to evaluate the biological duplication of samples. Differential expression analysis was performed using the DESeq2. Q value < 0.05 and foldchange > 2 or foldchange < 0.5 was set as the threshold for DEGs. Volcano mapping and Hierarchical clustering diagram was drawn using R (v 3.2.0) according to DGEs. Based on the hypergeometric distribution, GO and KEGG pathway enrichment analysis of upregulated genes in stimulated supernatants group were performed to screen the significant enriched term using R (v 3.2.0), respectively. GSEA was performed using GSEA software.

qRT-PCR

Total RNAs were extracted using MIsZOL Reagent (Mishushengwu, China) following the manufacturer’s instructions and purified through phenol-chloroform extraction. Complementary DNA (cDNA) was prepared by TaqMan Reverse Transcription Reagents (Takara) as previously described⁵⁰. The primer sequences can be found in Table S2. Genes were amplified and analyzed by qRT PCR using Bio-Rad SYBR Green Premix Ex Taq (Takara) in a Roche LightCycler 96 system.

Statistical Analysis

Data were expressed as the mean ± standard deviation (SD). GraphPad Prism 8.0 was used for statistical analysis. Two groups were compared by two-tailed Student’s t test, and multiple groups were compared by one-way ANOVA with Turkey's or Dunnet's post-hoc test. P < 0.05 (*, P < 0.05; **, P < 0.01; ***, P < 0.001) were considered to have a statistical difference.

Conflicts of Interest

The authors declare no conflicts of interest.

Author contributions

X.Y.C., C.X.Z. and H.G. contributed equally to the experimental performing, data collection and analysis, and manuscript drafting. S.Y.F., X.Y.H, and J.C. contributed to experimental performing and data collection. J.X.L., Y.R.G., A.Q.L., J.N.L. and X.H.Z. contributed to data acquisition and analysis. C.M., H.W., F.F., P.P. and H.K.X. contributed to contributed to data analysis and interpretation. B.D.S., K.X. and Y.J. contributed to the project conception, experimental design and supervision. All authors have read and approved the current version of the manuscript.

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (81930025, 82100969, 82370949, 82371020, 82401201, 82100992), the Young Science and Technology Rising Star Project of Shaanxi Province (2023KJXX-027 and 2024ZC-KJXX-122), the Key Research and Development Program of Shaanxi Province (2023-YBSF-489), the China Postdoctoral Science Foundation (BX20230485), and the "Rapid Response" Research projects (2023KXKT017 and 2023KXKT090).

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You are reading this latest preprint version

Gli1+ Inflamm-Stem Cells Engage Extracellular Vesicles to Prime Aberrant Neutrophils for Exacerbating Periodontal Immunopathology

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1. Gli1⁺ cell dynamics in ligature-induced periodontitis

2.2. LPS pre-stimulated Gli1⁺ cell supernatants induced neutrophil migration and activation in vitro.

2.5. EV release was indispensable for LPS pre-stimulated Gli1⁺ cells to recruit and activate neutrophils.

3. Discussion

4. Experimental Section

Declarations

Conflicts of Interest

Author contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

Gli1+ Inflamm-Stem Cells Engage Extracellular Vesicles to Prime Aberrant Neutrophils for Exacerbating Periodontal Immunopathology

Status:

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1. Gli1+ cell dynamics in ligature-induced periodontitis

2.2. LPS pre-stimulated Gli1+ cell supernatants induced neutrophil migration and activation in vitro.

2.5. EV release was indispensable for LPS pre-stimulated Gli1+ cells to recruit and activate neutrophils.

3. Discussion

4. Experimental Section

Declarations

Conflicts of Interest

Author contributions

Acknowledgments

References

Additional Declarations

Supplementary Files

Status:

Version 1

2.1. Gli1⁺ cell dynamics in ligature-induced periodontitis

2.2. LPS pre-stimulated Gli1⁺ cell supernatants induced neutrophil migration and activation in vitro.

2.5. EV release was indispensable for LPS pre-stimulated Gli1⁺ cells to recruit and activate neutrophils.