Abstract

Background and Objective

   Periodontitis is a chronic inflammatory condition that can be
   associated with systemic diseases like diabetes and cardiovascular
   disease. This study investigates the role of AIM2, a key inflammasome
   component, in periodontitis, focusing on its involvement in
   inflammation, DNA repair, and systemic disease links.

Methods

   AIM2 expression was analyzed in saliva and gingival crevicular fluid
   (GCF) from periodontitis patients. A mouse periodontitis model and in
   vitro gingival fibroblast experiments were used to study AIM2’s role.
   Gene Set Enrichment Analysis (GSEA) and Protein-Protein Interaction
   (PPI) network analysis explored AIM2’s systemic disease associations.

Results

   AIM2 was significantly upregulated in periodontitis patients and
   models, correlating with increased IL-1β, ASC, and Caspase-1.
   Immunofluorescence revealed AIM2’s nuclear localization and
   co-localization with inflammatory markers. GSEA linked high AIM2
   expression to cardiovascular diseases, while its suppression showed
   protective effects. PPI analysis identified interactions with DNA
   repair proteins (THOC2, SETX, ATM), suggesting a role in genomic
   stability and systemic disease.

Conclusion

   AIM2 drives local inflammation in periodontitis and may connect
   periodontitis to systemic diseases via DNA repair and systemic
   inflammation. This highlights AIM2 as a potential therapeutic target
   for managing periodontitis and associated systemic risks.

Clinical Significance

   Targeting AIM2 could offer a dual therapeutic strategy to control
   periodontal inflammation and mitigate systemic disease risks, such as
   cardiovascular disorders.

   Keywords: periodontitis, AIM2, inflammasome, systemic diseases, DNA
   repair

Introduction

   Gingival tissue inflammation, alveolar bone resorption elaborated
   periodontitis is a prevalent chronic inflammatory disease caused by
   microbial infection in dental plaque and could cause periodontal loss
   significantly. Moreover, a growing body of evidence suggests that
   periodontitis transcends the oral cavity and has a great impact on the
   systemic health increasing the risk for systemic diseases, such as
   cardiovascular diseases, diabetes, and others.[38]^1–3 The host immune
   response is critical to the progression of periodontitis and represents
   a key mechanism during that progression.

   The AIM2 (Absent in Melanoma 2) inflammasome is a multiprotein complex
   assembled largely constituted by the AIM2 receptor, the adaptor protein
   ASC (apoptosis-associated speck-like protein containing a CARD) and the
   effector protein caspase-1. AIM2 recruits ASC upon recognition of
   cytosolic dsDNA (double-stranded dsDNA). ASC then facilitates the
   assembly of caspase-1. Activated caspase-1 cleaves proIL-1β into IL-1β,
   and proIL-18 to IL-18. This then triggers the cleaving of pro-IL-1β and
   pro-IL-18 into their mature versions, IL-1β and IL-18, initiating a
   robust inflammatory response. The recognition that pathogens by pattern
   recognition receptors plays a critical role in host defense and the
   pathogenesis of inflammatory diseases. AIM2 is deeply studied in the
   different inflammatory diseases such as infectious diseases, autoimmune
   diseases and cancer via its regulation of inflammation and its effect
   on disease progression.[39]^4–6

   AIM2 has now become of interest to us in oral health. They reported
   that AIM2 expression is highly increased in the patients with different
   stages of periodontitis, suggesting that AIM2 may be involved in
   pathogenesis of periodontitis.[40]^7 It appears that such upregulation
   is associated with the persistence and exacerbation of chronic
   inflammation in periodontal tissues. Carrillo-Gálvez et al also showed
   that AIM2 inflammasome activation contributes to alveolar bone
   resorption in the periodontitis by elevating the release of
   IL-1β.[41]^8 Although the local inflammatory role of AIM2 appears to be
   well established, it may also play a role in systemic diseases, such as
   cardiovascular and metabolic disorders, through systemic inflammatory
   responses. AIM2 has been linked to DNA repair and aging pathways and
   has been shown to interact with key genes like ATM and SETX which have
   a role in maintaining genomic stability as well as regulating the aging
   of the cellular.[42]^9

   They may explain the link between upregulated AIM2 and age-associated
   diseases. In addition, tightly linked with AIM2 expression are
   polymorphisms in inflammation genes, which may render subjects to
   periodontitis susceptibility or may alter the intensity and duration of
   the inflammatory response, which then will affect the disease
   progression. As an example, a study conducted by Li et al found that
   AIM2 gene polymorphisms play a major role in an individual’s
   periodontitis susceptibility.[43]^10 This indicates that AIM2 may be a
   significant factor for periodontitis.

   The literature goes on to suggest that in addition to having a role in
   local inflammation, AIM2 also participates in systemic inflammation,
   DNA repair and aging-related signaling pathways. In this way, this
   indicates that AIM2 can function as a key link between periodontitis
   and systemic diseases which may aggravate the pathological processes
   related to cardiovascular and metabolic disorders.[44]^11–13 As a
   result, AIM2 is of great significance in investigation of its
   multifaceted role, particularly in systemic diseases.

   Based on previous research, this study will extend to examine the AIM2
   expression in periodontitis models and also aims to understand AIM2′s
   specific roles in periodontal inflammation progression. Therefore, we
   hypothesize that in addition to exacerbating periodontitis via local
   inflammatory responses, AIM2 also influences systemic progression of
   the disease by modulating DNA repair and aging related pathways.

Materials and Methods

Study Subjects and Sample Collection

   This study involved 33 patients diagnosed with chronic periodontitis
   (CP) and 27 healthy individuals serving as the control group.
   Periodontitis was diagnosed following the 2017 classification system of
   the World Workshop on the Classification of Periodontal and Peri -
   Implant Diseases and Conditions.[45]^14 All participants provided
   informed consent, and the consent process was approved by the Ethics
   Committee of Hainan Medical University. The study adheres to the
   ethical principles outlined in the Declaration of Helsinki. No specific
   approval number was assigned to this project. For patients who meet the
   criteria, at the initial diagnosis, 500 μL of saliva was collected
   using the natural flow collection method with an EP tube respectively;
   gingival crevicular fluid was collected using the filter paper method
   with a filter paper strip of 1 mm×3 mm.

Inclusion Criteria

     * Patients aged 18 to 75 years who had not undergone periodontal
       treatment in the past six months.[46]^15
     * Periodontitis is diagnosed by probing pocket depth≥4 mm and
       clinical attachment loss≥2 mm.
     * No history of systemic diseases, including cardiovascular disease,
       diabetes, or immune system disorders.

Exclusion Criteria

     * Pregnant or lactating women.
     * Individuals who received antibiotic treatment within the last six
       months.
     * Smokers or those with a history of smoking.
     * Non-steroidal anti-inflammatory drugs (NSAIDs) or
       immunosuppressants used within 6 months.

Enzyme-Linked Immunosorbent Assay (ELISA)

   ELISA was conducted following the manufacturer’s protocol. Each sample
   was run in triplicate to ensure accuracy. Optical density (OD) readings
   were taken at 450 nm, and concentrations were calculated based on a
   standard curve.

ELISA Kits Used

     * Human Interleukin-1β (IL-1β) ELISA kit (Jianglaibio, JL13662).
     * Human IFI16 ELISA kit (Jianglaibio, JL11400).
     * Human Caspase-1 ELISA kit (BYabscience, BYHS100009).
     * Human ASC ELISA kit (BYabscience, BY-[47]EH115791).
     * Human NLRP3 ELISA kit (Jianglaibio, JL10272).
     * Human AIM2 ELISA kit (Jianglaibio, JL49566).
     * Human Gasdermin D Protein (GSDMD) ELISA Kit (BYabscience,
       BYHS501266).

Animal Model Establishment

   Eight-week-old C57BL/6 mice were used to establish the periodontitis
   animal model. All mice were obtained from the institutional animal
   facility and housed in a pathogen-free environment. The 2017
   classification system of the World Workshop on the Classification of
   Periodontal and Peri-Implant Diseases and Conditions was used to
   diagnose periodontitis.[48]^14 The Ethics Committee of Hainan Medical
   University approved the consent process. All participants gave informed
   consent. The Ethics Committee of Hainan Medical University approved all
   animal experiments. This project adhered to the ethical principles of
   Guide for the Care and Use of Laboratory Animals. Periodontitis Model
   Construction: Orthodontic wire (0.25 mm) was passed between the first
   and second molars, and a 4–0 surgical suture was tied around the wire
   on both the buccal and palatal sides. The ligature was placed mesially
   to the first molar, with the excess wire trimmed to allow penetration
   into the gingival sulcus. The ligature was checked and retied every two
   days. After three weeks, one mouse from both the control and
   experimental groups was euthanized for Micro-CT imaging, which showed
   significant alveolar bone loss in the model group, confirming
   successful periodontitis induction. The control group did not undergo
   ligation but received the same treatment otherwise.

Micro-CT Analysis

   After 4 weeks of standard housing, the experimental mice were
   euthanized via cervical dislocation under general anesthesia.
   Subsequently, the left mandibular bone samples containing the first,
   second and third mandibular molars were collected. The mandibles are
   then scanned with a high-resolution micro-CT (SkyScan1176, Bruker). The
   scanning parameters were 50 kV and 200 μA with a 9 μm resolution. The
   scanned images were reconstructed, and the bone mineral density (BMD)
   and bone volume (BV) were calculated.

Immunohistochemistry (IHC) Staining

Tissue Preparation

   Mouse gingival tissues were fixed in 4% paraformaldehyde for 24 hours,
   embedded in paraffin, and sectioned into 4-μm thickness for
   immunohistochemical (IHC) analysis.

IHC Staining

   After deparaffinization and antigen retrieval at 4°C, tissue sections
   were incubated overnight with primary antibodies to AIM2, IL-1β and
   Caspase-1 (1: 200 dilution). Detection was performed using
   3,3’-diaminobenzidine (DAB) for chromogenic visualization, and nuclei
   were counterstained with hematoxylin. Images were acquired using an
   Olympus BX51 microscope. Under microscopic examination,
   hematoxylin-stained nuclei displayed a blue coloration, while
   DAB-positive expression regions exhibited a brown-yellow signal.
   Quantitative image analysis was conducted using ImageJ software.

Cell Culture and Transfection

   Gingival fibroblasts(GFs) were isolated from healthy gingival tissues
   of volunteers and cultured in Dulbecco’s Modified Eagle Medium
   supplemented by 10% fetal bovine serum (FBS). Cells were maintained at
   37°C in a 5% CO[2] atmosphere. For experiments, cells were seeded in
   6-well plates and transfected at 80–90% confluency. Overexpression of
   AIM2: Gingival fibroblasts were transfected with pcDNA3.4-AIM2-3xFlag
   plasmid using Lipofectamine 6000 (Invitrogen), with empty vector (Vec)
   as the control. Cells were collected 48 hours after transfection for
   subsequent mRNA and protein analysis. Knockdown of AIM2: Small
   interfering RNA (siRNA) was used to specifically knock down AIM2
   expression, with Lipofectamine 6000 (Invitrogen) used for transfection.
   Non-targeting siRNA (NC) was used as a control. Cells were collected
   for analysis 48 hours after transfection.

Quantitative Real-Time PCR (qRT-PCR)

   RNA was extracted using the EastepR Super Total RNA Extraction Kit from
   Promega (Beijing), followed by cDNA synthesis using the HifairR III
   First Strand cDNA Synthesis Kit from Yeasen Biotech (Shanghai). SYBR
   Green Master Mix (Yeasen, Shanghai) was used for quantitative PCR on a
   Roche LightCyclerR 96 platform. The 2-DDCt technique was used to
   determine the gene expression levels of AIM2, IL-1β and Caspase-1 using
   the GAPDH internal control.

Western Blot Analysis

   BCA Protein Quantification Kits (Beyotime Biotech Inc) were used to
   quantify the proteins extracted with RIPA buffer. SDS-PAGE was used to
   separate samples (40 ug), which were then transferred onto PVDF
   membranes from Millipore. After blocking with 5% milk fat for 2 h the
   membranes were incubated at 4degC overnight with primary antibodies.
   These included AIM2 (Bioss bs5986R 1:1000), ASC (Bioss, bs-6741R,
   1:1000), IL-1β(Bioss, bs-6319R, 1:1000), Cleaved Caspase-1 (Affinity
   Biosciences, AF4005, 1:1000), Caspase-1 (Bioss, bs-10442R, 1:1000), and
   β-actin (Proteintech 20536-1AP, 1:5000). Secondary antibodies
   conjugated with HRP were applied to anti-rabbit or anti-mouse secondary
   antibodies (Jackson 111-035-013, 1:10,000). Thermo Scientific’s ECL
   detection system was used to visualize the protein bands. ImageJ
   software was then used to analyze them.

Immunofluorescence Staining

   The cells were fixed with 4% paraformaldehyde and permeabilized by 0.5%
   Triton X - 100 over 20 minutes. QuickBlock™ solution (Beyotime Biotech
   Inc) was applied for 1 hour to block nonspecific binding. Primary
   antibodies targeting AIM2, IL-1β, ASC, and Caspase-1 (1:200 dilution)
   were incubated at 4°C overnight. Images were captured with a confocal
   microscopy using Alexa Fluor 594 or 488 conjugated secondary antibodies
   (1:500). Nuclei were counterstained in DAPI. Quantification was
   performed using ImageJ software.

Bioinformatics Analysis

   RNA sequencing was performed on AIM2 overexpression and knockdown
   groups to identify differentially expressed genes (DEGs). GSEA and PPI
   network analysis were conducted using the STRING database and
   visualized with Cytoscape software to identify AIM2-regulated key
   proteins and pathways.

Statistical Analysis

   Data were analyzed using GraphPad Prism 9.0 software. All quantitative
   data were expressed as mean ± SEM. Statistical differences between
   groups were analyzed using Student’s t-test or Mann-Whitney U-test,
   with P < 0.05 deemed significant.

Results

Expression of AIM2, IFI16, NLRP3, IL-1β, ASC, and GSDMD in Saliva and
Gingival Crevicular Fluid of Periodontitis Patients

   In saliva and gingival Crevicular Fluid (GCF), 33 chronic periodontitis
   patients (CP) and 27 healthy controls were compared to determine the
   expression levels of AIM2, IFI16 and GSDMD. All molecules were
   significantly more abundant in the CP group compared with controls
   ([49]Figure 1).

Figure 1.

   [50]Figure 1
   [51]Open in a new tab

   Expression of AIM2, IFI16, NLRP3, IL-1β, ASC, and GSDMD in Saliva and
   Gingival Crevicular Fluid (GCF) of Periodontitis Patients. This figure
   compares the concentrations of inflammatory and pyroptosis-related
   molecules (AIM2, IFI16, NLRP3, and IL-1β) in the saliva (A) and GCF (B)
   of periodontitis patients (CP) and healthy controls (Control). Results
   show significantly higher expression levels of these molecules in
   periodontitis patients, with statistical significance denoted by
   asterisks (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). The
   findings suggest that these molecules play a key role in the
   inflammatory response associated with periodontitis.

Expression of AIM2, IL-1β, ASC, and Caspase-1 in a Periodontitis Animal Model

   In our periodontitis mouse model, micro-CT analysis ([52]Figure 2A)
   revealed significant periodontal bone loss in the periodontitis group
   compared to controls, confirming the successful model establishment.
   Quantitative results in [53]Figures 2B and [54]C indicated a
   significant reduction in bone density and bone volume in the
   periodontitis group (P < 0.001).

Figure 2.

   [55]Figure 2
   [56]Open in a new tab

   Expression of AIM2, IL-1β, ASC, and Caspase-1 in a Periodontitis Animal
   Model (A) Micro-CT images compare the periodontal bone structure in
   normal control mice (Ctrl) and periodontitis model mice (PD), showing
   significant bone loss in the PD group. (B) Quantitative analysis
   reveals significantly lower bone mineral density (BMD) in the
   periodontitis group compared to controls (***P < 0.001). (C) Bone
   volume (BV) is also significantly reduced in the periodontitis group
   (***P < 0.001). (D) Immunohistochemistry demonstrates significantly
   higher expression of AIM2, IL-1β, ASC, and Caspase-1 in periodontal
   tissues of periodontitis mice, with representative images at low (2×)
   and high (10×) magnification. The blue box indicates the enlarged area,
   and red arrows highlight regions of high expression in inflamed
   tissues. Quantitative data are presented as mean ± SEM (***P < 0.001).
   These results underscore the structural and molecular changes
   associated with periodontitis, emphasizing the role of inflammatory and
   pyroptosis-related markers in disease progression.

   Immunohistochemical analysis ([57]Figure 2D) demonstrated a marked
   upregulation of AIM2 in the periodontal tissues of periodontitis mice,
   especially in areas of active inflammation (indicated by the red
   arrow). Similarly, IL-1β, ASC, and Caspase-1 expression levels were
   significantly higher in periodontitis mice, showing a strong
   co-localization with AIM2 in inflamed regions.

   Histological and quantitative analyses further verified that compared
   with the control group, the expression of AIM2, IL-1β, ASC, and
   Caspase-1 was significantly upregulated in the periodontitis model.

Effects of AIM2 Overexpression and Knockdown on IL-1β, ASC, and Caspase-1
Expression in Gingival Fibroblasts

   To further investigate the role of AIM2 in gingival fibroblasts (GFs),
   we analyzed the effects of AIM2 overexpression (OE-AIM2) and knockdown
   (siAIM2) on the expression of inflammatory factors IL-1β, ASC, and
   Caspase-1.

   [58]Figure 3 A shows the mRNA expression levels of IL-1β, ASC, and
   Caspase-1 in AIM2 overexpression (OE-AIM2) and control (Vec) cells. The
   results indicate that AIM2 overexpression significantly upregulated the
   mRNA levels of IL-1β, ASC, and Caspase-1, demonstrating AIM2’s active
   role in promoting the expression of these inflammatory factors.

Figure 3.

   [59]Figure 3
   [60]Open in a new tab

   Effects of AIM2 Overexpression and Knockdown on IL-1β, ASC, and
   Caspase-1 Expression in Gingival Fibroblasts This figure demonstrates
   the regulatory role of AIM2 in the expression of key inflammatory
   mediators. (A) qRT-PCR results show that AIM2 overexpression (OE-A)
   significantly upregulates mRNA levels of IL-1β, ASC, and Caspase-1,
   while the control group (Vec) shows no effect. (**P < 0.01 vs the Vec
   group, ***P < 0.001 vs the Vec group) (B) Western blot analysis
   confirms increased protein levels of AIM2, IL-1β, ASC, and
   Cleaved-Caspase-1 upon AIM2 overexpression. (C) Quantitative analysis
   reveals a higher Cleaved-Caspase-1/pro-Caspase-1 ratio, indicating
   enhanced inflammation. (D) Conversely, AIM2 knockdown (siAIM2)
   significantly reduces mRNA levels of IL-1β, ASC, and Caspase-1 compared
   to the control (SI-NC) (*P < 0.05 vs the SI-NC group, **P < 0.01 vs the
   SI-NC group). (E) Western blot shows decreased protein levels of these
   mediators, particularly Cleaved-Caspase-1, upon AIM2 knockdown. (F)
   Quantitative analysis confirms a reduced
   Cleaved-Caspase-1/pro-Caspase-1 ratio, suggesting diminished
   inflammation. Together, these findings highlight AIM2 as a key
   regulator of inflammatory responses in gingival fibroblasts.

   At the protein level, Western blot analysis in [61]Figures 3B and [62]C
   further validated this phenomenon. AIM2 overexpression not only
   increased the protein expression of AIM2 itself but also significantly
   elevated the levels of IL-1β, ASC, and Caspase-1, particularly the
   active form of Caspase-1 (Cleaved-Caspase-1). In its precursor form
   (pro-Caspase-1), Caspase-1 is inactive, but AIM2 overexpression
   promoted its cleavage into the active form, which is crucial for
   amplifying the inflammatory response. The increase in
   Cleaved-Caspase-1, along with the elevated ratio of Cleaved-Caspase-1
   to pro-Caspase-1, indicates a significant increase in inflammasome
   activation, potentially enhancing the intensity of the inflammatory
   response.

   Conversely, [63]Figure 3D displays the mRNA expression levels of IL-1β,
   ASC, and Caspase-1 in AIM2 knockdown (siAIM2) and control (NC) cells.
   After AIM2 knockdown, the mRNA levels of these inflammatory factors
   were significantly reduced, further supporting AIM2’s critical role in
   regulating the expression of these factors.

   Western blot results in [64]Figures 3E and [65]F further illustrate the
   impact of AIM2 knockdown on protein expression. Knockdown of AIM2 led
   to a significant reduction in the protein levels of IL-1β, ASC,
   Caspase-1, and Cleaved-Caspase-1, indicating that AIM2 downregulation
   inhibited Caspase-1 activation, reduced the generation of active
   Caspase-1, and potentially weakened the inflammatory response.

Immunofluorescence Double Staining Reveals AIM2 Localization and Its
Relationship with Inflammatory Factors

   Immunofluorescence double staining was performed to explore AIM2
   localization in gingival fibroblasts and its relationship with the
   inflammatory factors IL-1β, ASC, and Caspase-1. Confocal microscopy
   allowed the visualization of the distribution and co-localization of
   these molecules within the cells.

   AIM2 was primarily localized in the nuclear region, especially in AIM2
   overexpression (OE-A) cells where the nuclear signal was significantly
   enhanced. Conversely, AIM2 knockdown (siA) cells exhibited a marked
   reduction in AIM2 expression. DAPI staining highlighted the cell
   nuclei, and merged images ([66]Figure 4A) demonstrated AIM2’s
   co-localization with the nuclei. Quantitative analysis confirmed a
   significant increase in AIM2’s nuclear localization following
   overexpression.

Figure 4.

   [67]Figure 4
   [68]Open in a new tab

   Immunofluorescence Double Staining Reveals AIM2 Localization and Its
   Relationship with Inflammatory Factors This figure illustrates the
   localization of AIM2 and its regulatory effects on key inflammatory
   factors in gingival fibroblasts. (A) Immunofluorescence results (400x
   magnification) show the distribution of AIM2 in gingival fibroblasts.
   AIM2 is primarily localized in the nucleus in the overexpression group
   (OE-A), with significantly enhanced signals (*P < 0.05 vs the VEC
   group), while its expression is notably reduced in the knockdown group
   (siA) (*P < 0.05 vs the SI-NC group). DAPI stains the nuclei, and the
   merged images show co-localization of AIM2 with the nucleus.
   Quantitative analysis indicates that AIM2 overexpression significantly
   increases its nuclear localization. (B) Immunofluorescence results for
   IL-1β show that IL-1β expression is significantly increased in the
   overexpression group and mainly localized in the nucleus (*P < 0.05 vs
   the VEC group). AIM2 knockdown significantly reduces IL-1β expression,
   which is primarily localized in the cytoplasm (**P < 0.01 vs the SI-NC
   group). In the control groups (Vec and si-NC), IL-1β is also primarily
   expressed in the cytoplasm, suggesting that AIM2 may regulate IL-1β
   expression to influence the inflammatory response. (C)
   Immunofluorescence results for ASC show a punctate distribution within
   the cells, suggesting the formation of inflammasome aggregates. AIM2
   overexpression significantly increases ASC expression, particularly
   around the nucleus (*P < 0.05 vs the VEC group), while AIM2 knockdown
   reduces the formation of these aggregates (*:P < 0.05 vs the SI-NC
   group). In the control groups, ASC is also primarily expressed in the
   cytoplasm. (D) Immunofluorescence results for Caspase-1 show a punctate
   aggregate distribution within the cells, and the number of aggregates
   is significantly increased in the AIM2 overexpression group,
   concentrating near the nucleus (**P < 0.01 vs the VEC group). AIM2
   knockdown significantly reduces Caspase-1 expression and the formation
   of aggregates (*P < 0.05 vs the SI-NC group). This suggests that AIM2
   not only promotes Caspase-1 expression but may also regulate its
   activation, further amplifying the inflammatory cascade.

   The analysis of IL-1β levels indicated a substantial increase in IL-1β
   expression in the overexpression group, predominantly localized within
   the nucleus. In contrast, knockdown of AIM2 led to a significant
   reduction in IL-1β expression, which was primarily found in the
   cytoplasm ([69]Figure 4B). Both control groups exhibited a similar
   cytoplasmic distribution, suggesting that AIM2 may regulate IL-1β
   expression, influencing the inflammatory response.

   ASC exhibited a punctate distribution within the cells, indicative of
   potential inflammasome formation. AIM2 overexpression markedly
   increased ASC expression and enhanced its aggregation around the
   nucleus, while AIM2 knockdown significantly reduced these aggregates.
   In control cells, ASC was primarily expressed in the cytoplasm
   ([70]Figure 4C), supporting AIM2’s role in promoting inflammasome
   assembly.

   Caspase-1 showed a similar punctate distribution pattern, with the
   number of aggregates increasing significantly in AIM2 overexpression
   cells, particularly near the nucleus ([71]Figure 4D). This finding
   suggests that AIM2 not only elevates Caspase-1 expression but also
   regulates its activation, further amplifying the inflammatory response.
   In contrast, AIM2 knockdown significantly decreased both Caspase-1
   expression and the formation of aggregates.

RNA Seq Uencing Reveals Key Pathways and Molecular Mechanisms Regulated by
AIM2

   RNA-seq analysis of AIM2-overexpressing (OE-AIM2) and AIM2-knockdown
   (siAIM2) gingival fibroblasts, followed by GO and KEGG enrichment
   analyses, revealed distinct functional roles for AIM2. Overexpression
   of AIM2 primarily influenced biological processes and pathways related
   to immune response, inflammation, and gene regulation, including
   “antimicrobial peptide production”, “regulation of inflammatory
   response”, “inflammasome assembly”, and “NF-κB signaling ([72]Figure 5A
   and [73]C)”. In contrast, AIM2 knockdown significantly enriched
   processes and pathways associated with “cell cycle regulation”,
   “apoptosis”, “genome stability maintenance”, “DNA repair”, and
   “autophagy”. These findings highlight AIM2’s critical involvement in
   immune-inflammatory responses and cellular homeostasis, suggesting its
   potential role in diseases linked to immune dysregulation and genomic
   instability ([74]Figure 5B–D).

Figure 5.

   [75]Figure 5
   [76]Open in a new tab

   RNA Sequencing Reveals Key Biological Pathways and Molecular Mechanisms
   Regulated by AIM2 (A) GO enrichment analysis of gingival fibroblasts
   after AIM2 overexpression (OE-AIM2). Results show that AIM2
   overexpression significantly influences biological processes related to
   immune response, inflammation, and gene regulation, with “antimicrobial
   peptide production”, “regulation of inflammatory response”, and “DNA
   repair” being the top enriched GO terms. (B) GO enrichment analysis of
   gingival fibroblasts after AIM2 knockdown (siAIM2). The significantly
   enriched biological processes include “cell cycle regulation”,
   “apoptotic process”, and “maintenance of genome stability”, suggesting
   AIM2’s role in these key cellular functions. (C) KEGG pathway
   enrichment analysis of gingival fibroblasts after AIM2 overexpression.
   Cells with AIM2 overexpression are significantly enriched in pathways
   such as “inflammasome assembly”, “NF-κB signaling pathway”, and
   “apoptosis”, suggesting that AIM2 may promote inflammation and cell
   death through these pathways. (D) KEGG pathway enrichment analysis of
   gingival fibroblasts after AIM2 knockdown. Cells with AIM2 knockdown
   are significantly enriched in pathways such as “cell cycle regulation”,
   “DNA repair”, and “autophagy”, indicating that AIM2 may suppress these
   pathways.

GSEA Enrichment and Protein-Protein Interaction Network Reveal Potential
Mechanisms Regulated by AIM2

   The Disease Ontology (DO) enrichment analysis revealed that AIM2
   overexpression was significantly associated with disease categories
   such as “multiple endocrine neoplasia type 1”, “polycystic ovary
   syndrome”, “infertility”, and “autoimmune diseases ([77]Figure 6A)”,
   while AIM2 knockdown enriched categories included “hereditary tumor
   syndromes”, “mitochondrial dysfunction”, and “neurodegenerative
   diseases ([78]Figure 6B)”. Gene set enrichment analysis (GSEA) further
   demonstrated that AIM2 overexpression was linked to periodontitis and
   systemic diseases, including cardiovascular (eg, acute myocardial
   infarction, hypertension), metabolic (eg, diabetes), neurological (eg,
   Alzheimer’s disease), and respiratory diseases, whereas AIM2 knockdown
   showed reduced enrichment for these conditions ([79]Figure 6A and
   [80]B). PPI network analysis identified key molecular interactions:
   under AIM2 overexpression, networks involving THOC2-PNN/SETX, SETX-ATM,
   PNN-RBM25, and ATM-WRN/GEN1 were prominent, highlighting roles in RNA
   metabolism, DNA repair, and transcriptional regulation ([81]Figure 6C).
   In contrast, AIM2 knockdown weakened interactions, particularly in
   mitochondrial function (ACO2-PCK2/SLC25A11), cell proliferation
   (KRAS-APC/ALDH1A1), cell division (CENPE-RANBP2/SASS6), and RNA
   processing (THOC2-SRSF6/EIF5B) ([82]Figure 6D). These findings
   underscore AIM2’s broad influence on disease-related pathways and
   molecular networks, connecting it to immune, metabolic, and
   neurological disorders.

Figure 6.

   [83]Figure 6
   [84]Open in a new tab

   GSEA Enrichment and Protein-Protein Interaction Network Reveal
   Potential Mechanisms Regulated by AIM2 This figure shows the molecular
   mechanisms regulated by AIM2 based on GSEA enrichment analysis and PPI
   network analysis. (A and B) display the top 30 significantly enriched
   Disease Ontology (DO) terms and their corresponding Normalized
   Enrichment Score (NES) and Q values after AIM2 overexpression and
   knockdown. AIM2 overexpression is associated with various systemic
   diseases, including periodontitis and cardiovascular diseases,
   suggesting AIM2 may serve as a key link between periodontitis and
   systemic conditions. In contrast, AIM2 knockdown significantly reduces
   the enrichment of disease-related terms, indicating potential
   protective effects. (C and D) show the PPI networks for AIM2
   overexpression (C) and knockdown (D), identifying key protein
   interactions altered by changes in AIM2 expression. In the
   overexpression network (C), significant interactions
   include:THOC2-PNN/THOC2-SETX: involved in RNA metabolism and
   transcription regulation. SETX-ATM: related to DNA damage response and
   genome stability. PNN-RBM25: involved in RNA splicing and
   post-transcriptional regulation. ATM-WRN/GEN1: involved in DNA repair
   pathways. In the knockdown network (D), core interactions are
   significantly reduced, highlighting AIM2’s crucial role in gene
   regulatory networks. Key interactions include:ACO2-PCK2/SLC25A11:
   associated with mitochondrial function and metabolic pathways.
   KRAS-APC/ALDH1A1: involved in tumor suppression, cell proliferation,
   and metabolism. CENPE-RANBP2/SASS6: primarily involved in regulating
   cell division and mitosis. THOC2-SRSF6/EIF5B: related to RNA
   post-transcriptional processing and protein synthesis.

Discussion

   This study analyzed AIM2 expression in the saliva and gingival
   crevicular fluid of periodontitis patients, using a mouse model and
   functional experiments in gingival fibroblasts to reveal AIM2’s role in
   periodontitis and systemic diseases. The results indicate that AIM2
   expression is significantly increased in both periodontitis patients
   and mouse model. In addition, AIM2 facilitates expression of
   inflammatory media, such as IL-1β, ASC and Caspase-1 to amplify
   inflammatory response.[85]^16^,[86]^17 AIM2 clearly plays a central
   role in periodontitis, and hinted at a possible crucial link between
   periodontal disease and systemic disorders,[87]^18 while the function
   and the mechanism of AIM2 in periodontal inflammation is less validated
   than that of NLRP3.[88]^19 They signal through common pathway[s],
   although whether the mechanisms are the same remain[s] to be
   verified. Moreover, IFI16, another member of this family acting as an
   IFN-inducible protein family, has identical function to AIM2 in sensing
   intracellular pathogenic DNA to trigger inflammasomes.[89]^20 Because
   of AIM2’s specific role in periodontitis remains to be established, we
   investigated its expression and its association with the IL-1β, ASC and
   Caspase-1 inflammatory mediators important to the disease.

   IL-1β, ASC, and Caspase-1 are essential to the inflammatory
   response.[90]^21 The maturation and secretion of IL-1β (a potent
   pro-inflammatory cytokine) is a process that largely requires
   inflammasome activation and, on the whole, is mediated by
   Caspase-1.[91]^22 As an adaptor protein, ASC bridges inflammasome
   sensors AIM2 or NLRP3 to Caspase-1 to form the active inflammasome
   complex and initiates the inflammatory cascade.[92]^23 Caspase-1 is the
   effector protein which cleaves pro-IL-1β and other precursors to
   generate the active cytokine.[93]^24 To better understand AIM2’s role
   in inflammatory mechanisms of periodontitis, a study of co-expression
   patterns of AIM2, IL-1β, ASC and Caspase-1 was conducted. In our study,
   we have confirmed through histological and quantitative analysis, the
   co-expression of AIM2 with IL-1β, ASC and Caspase-1. This finding
   suggests that AIM2 is of central importance in modulating inflammatory
   response and may participate in periodontal tissue pathological damage
   through regulating the expression and activation of the above key
   inflammatory factors. These results are in agreement with recent
   studies and provide further confirmation of the role of AIM2 in
   regulating the inflammatory response in periodontitis.[94]^25 The AIM2
   ([95]Figure 4) mainly localizes in the nuclear region by
   immunofluorescence double staining. Further, in over expression
   conditions, AIM2 localizes with inflammatory proteins such as ASC,
   Caspase 1 and IL 1β around the nucleus confirming its role in the
   inflammatory response. By the virtue of this localization, AIM2 could
   regulate the expression and the activation of inflammatory factors by
   its nuclear or perinuclear signaling, thereby influencing the pathology
   of periodontal disease. Finally, upon aiming to purify AIM2 during our
   research of its role in periodontitis, the high expression of AIM2 mRNA
   levels along with its interaction with the other key factors of
   inflammation point to a direct part of AIM2 in the initiation and
   amplification of the inflammatory response. Our results further cement
   AIM2 as a major regulator of inflammatory responses in periodontitis,
   and offer theoretical basis for AIM2 as a potential drug target.
   Further studies must be performed in other populations of patients to
   better define the role of AIM2 as well as examine its possible role as
   a therapeutic target.

   Furthermore, by using GSEA enrichment analysis, we extended our
   association analysis between AIM2 and systemic diseases within
   periodontitis. It was found that the overexpression of AIM2 is linked
   to cardiovascular diseases (acute myocardial infarction, hypertension
   and cerebral artery diseases) and metabolic diseases (such as
   diabetes), a well as neurological diseases (such as Alzheimer’s disease
   and cerebral ischemia).[96]^26–28 This raises the possibility that AIM2
   functions as a correlation between periodontal disease and
   cardiovascular disease through enhancing systemic inflammatory
   response, which makes contribution to the pathological correlation
   between these two conditions. Previous studies have shown a
   bidirectional relationship between cardiovascular disease and
   periodontitis. AIM2 could be of importance in this regard. On the other
   hand, the depletion of AIM2 substantially decreased enrichment to
   related gene sets, especially to gene sets in pathways for
   cardiovascular diseases. This indicates that AIM2 knockdown will be
   protective, slowing down disease progression. Furthermore, this finding
   confirms the bidirectional regulatory role of AIM2 in systemic
   diseases, and overexpression may exacerbate pathological processes, but
   knockdown may be protective.[97]^29^,[98]^30 Along with its function in
   the inflammatory response, AIM2 is associated with DNA damage repair.
   Secondly, PPI network analysis identified significant interactions
   between AIM2 and several proteins associated with RNA metabolism,
   transcriptional regulation and DNA repair (ie THOC2, SETX, ATM and WRN)
   upon over expression of AIM2. This suggests that AIM2 may participate
   in the progression of inflammation related diseases by affecting the
   stability of genome, possibly by these key pathways. SETX and ATM are
   mainly reported to be involved in DNA repair and genome stability, and
   these processes can affect the development of chronic diseases such as
   heart disease, Alzheimer’s and diabetes, indirectly. In many long lived
   bat species, telomere length did not significantly shorten with age and
   there are indications that DNA repair genes like ATM and SETX are
   important for telomere dynamics. Accordingly, healthy aging may be
   maintained by ATM and SETX to preserve genome stability and ultimately,
   age related diseases such as Alzheimer’s disease and cardiovascular
   disease.[99]^9 The work demonstrates that, through the ATM/p53
   signaling pathway, WRN plays an important role in inhibiting oxidative
   stress induced apoptosis in human lens epithelial cells. DNA
   methylation has been found to downregulate WRN expression. This
   suggests that WRN and ATM not only have the function of DNA repair but
   are involved in other reactions to oxidative stress and are associated
   with age related disease including Alzheimer and cardiovascular
   disease.[100]^31 The roles of the MRN complex and WRN protein in the
   ATM pathway are critical for DNA double stranded break repair by their
   activation. This may lead to a new understanding of other diseases,
   since DNA damage and repair are closely associated with the onset of
   aging, cancer, and neurodegenerative diseases (ie,
   Alzheimer’s).[101]^32 Experiments using AIM2 knockdown revealed
   significant changes in gene axes expression of metabolic functions,
   cell proliferation and cell division. Firstly, the association of the
   ACO2-PCK2/SLC25A11 axis with mitochondrial function and metabolic
   regulation is close. The key genes that are involved in tricarboxylic
   acid cycle and energy metabolism include ACO2 (acetyl-CoA synthase 2)
   and SLC25A11 (mitochondrial malate transporter). Mitochondrial
   dysfunction usually leads to more severe neurodegenerative diseases
   like Alzheimer’s as studies show.[102]^33 Gluconeogenesis is a vastly
   important metabolic reaction and PCK2 (phosphoenolpyruvate
   carboxykinase 2) is a key enzyme in that reaction and regulates
   cellular response to glucose metabolism.[103]^34 Secondly, KRAS is
   necessary for cell proliferation and suppresses tumor progression in
   part through the control of the APCCDH1 E3 ligase and the deacetylase
   ALDH1A1. While under the PubMed and Google Scholar databases, KRAS is a
   known proto-oncogene that had been mutated in several types of cancer
   including pancreatic cancer and lung cancer.[104]^35 APC (adenomatous
   polyposis coli) is a negative regulator of such Wnt signaling pathway
   and APC loss or mutation is a common event in the pathogenesis of
   colorectal cancer. ALDH1A1 (aldehyde dehydrogenase 1 family member A1)
   is involved in vitamin A metabolism and involved also in stem cell
   function and oxidative stress response.[105]^36 In the end, the
   CENPE-RANBP2/SASS6 axis has an important role in cell division and
   mitosis. RANBP2 (RAN binding protein 2) is involved in nuclear pore
   complex function, SASS6 (sperm associated antigen 6), functioning in
   centriole duplication,[106]^37^,[107]^38 and CENPE (centromere protein
   E) is essential for microtubule attachment to the chromosomes during
   mitosis.

   In summary, AIM2’s regulatory role extends beyond inflammation,
   affecting metabolic regulation, cell proliferation, and division. These
   findings further emphasize AIM2’s bidirectional regulatory role in
   systemic diseases: it may exacerbate pathological processes through
   overexpression, but knockdown may provide protective effects. AIM2’s
   role in metabolism, proliferation, and cell cycle regulation,
   especially in the context of chronic diseases like cardiovascular
   disease and Alzheimer’s disease, is crucial and warrants further
   investigation.

Limitations

   Future research should address several limitations, despite the fact
   that this study revealed AIM2’s potential role in periodontitis.
   Firstly, this study primarily used gingival fibroblasts as a model.
   Although these cells play a crucial role in periodontitis, periodontal
   tissue also includes other cell types, such as osteoblasts and immune
   cells, whose roles in the inflammatory response were not thoroughly
   investigated. Future studies should extend to other cell types to
   comprehensively uncover AIM2’s mechanisms of action.

   Additionally, we did not conduct detailed functional experiments in
   this study to validate the direct effects of AIM2 on cell senescence or
   inflammation. By supplementing functional experiments, especially in
   vivo validation using AIM2 knockout or overexpression animal models, a
   better understanding of AIM2’s specific role in periodontitis and
   systemic diseases could be achieved.

   In terms of bioinformatics analysis, we primarily relied on data from
   this study and did not integrate data from public databases for deeper
   exploration. Future research could enhance the generalizability and
   robustness of the findings by integrating data from public databases.
   Such supplementary studies would provide stronger evidence for AIM2 as
   a potential therapeutic target.

Conclusion and Outlook

   As an important component of the inflammasome, AIM2 recognizes
   intracellular double-stranded DNA and activates an inflammatory
   response, playing a key role in the pathogenesis of periodontitis. Our
   study demonstrates that AIM2 is significantly elevated in the saliva
   and gingival crevicular fluid of patients with periodontitis,
   suggesting that it may exacerbate periodontal tissue damage by
   triggering the production of inflammatory factors. Additionally, the
   high expression of AIM2 may connect periodontitis with systemic
   diseases, such as cardiovascular disease, through systemic
   inflammation.

   Moreover, AIM2 is closely related to DNA repair and aging. When
   recognizing DNA damage, AIM2 activates the inflammatory response to
   eliminate damaged cells and maintain tissue homeostasis. However,
   prolonged inflammation may lead to genome instability, thereby
   accelerating the progression of aging-related diseases. Notably, AIM2’s
   interactions with DNA repair proteins such as ATM and SETX suggest its
   potential influence on DNA repair and aging.

   In conclusion, AIM2’s role in periodontitis extends beyond inflammation
   regulation and may also influence systemic diseases by impacting DNA
   repair and aging processes. This study provides new research directions
   to develop AIM2 into a potential therapeutic target for the future.

Acknowledgments