Porphyromonas gingivalis поощряет передачу Т-клеток через канал сигнала NF-_B/CXCR3 в экспериментальном аутоиммунном энцефаломиелите.

Introduction

Multiple sclerosis (MS), a common autoimmune disease of the central nervous system (CNS), is characterized by inflammation and demyelination and is a leading cause of disability in young adults [ 1–5 ]. Experimental autoimmune encephalomyelitis (EAE), which closely resembles MS in clinical and pathological features, is widely used as an ideal animal model for MS research [ 6–9 ]. In EAE pathogenesis, Th1 cells dominate the peripheral immune response and can independently induce severe CNS inflammation [ 10–13 ]. The principal mechanism involves activated peripheral CD4⁺ T cells—particularly Th1 and Th17 subsets—crossing the blood–brain barrier (BBB) into the CNS, releasing proinflammatory cytokines, and triggering myelin and neuronal injury [ 1 , 14 , 15 ]. This homing of peripheral T cells is a complex, tightly regulated process orchestrated by chemokines and adhesion molecules. In MS and EAE, the inflammatory CNS milieu induces high expression of chemokines such as CXCL9, CXCL10 and CCL2 [ 16 , 17 ], along with adhesion molecules such as ICAM-1 and VCAM-1 [ 17 ]. These ligands bind to corresponding receptors on T cells, including CXCR3, CCR7, CCR2, LFA-1 and VLA-4 [ 18 , 19 ], thereby promoting T-cell migration and homing to sites of inflammation.

Periodontitis is a chronic inflammatory disease caused by periodontal pathogens and is one of the most prevalent chronic diseases worldwide [ 20–24 ]. Increasing evidence indicates its association with multiple systemic conditions, including MS [ 25–31 ]. Porphyromonas gingivalis ( P. gingivalis ), a keystone periodontal pathogen [ 32–34 ], is found at elevated abundance in patients with MS and individuals with periodontitis have a higher risk of developing MS [ 35 , 36 ]. Oral administration of P. gingivalis has been shown to exacerbate EAE symptoms in mice [ 37 ], although the underlying mechanisms remain unclear. Notably, Th1 cells in periodontal lesions exhibit significantly elevated CXCR3 expression [ 38 ]. Moreover, CXCR3 antagonists effectively inhibit P. gingivalis lipopolysaccharide (LPS)-induced osteoclastogenesis and bone resorption [ 39 , 40 ], underscoring the pivotal role of CXCR3 in periodontal immune responses.

Given that CXCR3–CXCL10 signaling represents an important, though not exclusive, chemokine axis involved in T-cell migration in CNS autoimmunity [ 41–45 ], periodontitis may aggravate EAE by upregulating CXCR3 expression on peripheral T cells, thereby enhancing their homing to the CNS and amplifying neuroinflammation. However, whether P. gingivalis -induced periodontitis promotes EAE progression through this mechanism has not been clearly elucidated. Therefore, in this study, we aimed to determine whether P. gingivalis -induced periodontitis exacerbates EAE progression by enhancing the homing capacity of peripheral T cells through CXCR3 upregulation, and to elucidate the underlying molecular pathways. Our findings may provide new insights into the immunopathological link between periodontitis and MS and help identify potential therapeutic targets for MS.

Materials and methods

Animals and experimental groups

Thirty-two female C57BL/6J mice (6–8 weeks; Beijing Vital River Laboratory Animal Technology Co., Ltd.) were housed under specific pathogen-free conditions at the Animal Experimental Center of Beijing Meide Kangna. Female mice were selected to maintain methodological consistency with commonly used MOG 35–55 -induced EAE protocols and comparability across experimental groups. Mice were acclimatized for a minimum of 3 days prior to experimentation. All procedures were approved by the Animal Experimental Ethics Committee of Beijing Meide Kangna and conducted in accordance with Institutional Animal Care and Use Committee guidelines. Animals were randomly assigned ( n = 8/group) into four groups: healthy control (CON), periodontitis only ( P ), EAE only (EAE) and EAE with periodontitis (EAE + P). The sample size ( n = 8/group, n = number of mice) was chosen based on prior EAE studies with comparable endpoints and variability, while minimizing animal use in accordance with ethical principles. For flow cytometry, immunofluorescence and Western blot analyzes, animals from the same cohort were randomly selected as a subset for tissue collection at the endpoint.

Culture of P. gingivalis

P. gingivalis (strain W83, BNCC) was cultured anaerobically on enriched sterile blood agar containing 3.7% brain heart infusion (BHI), 2% agarose, 0.05% L-cysteine, 0.0005% hemin, 0.0001% menadione and 5–7% defibrinated sheep blood. Anaerobic conditions were generated using an AnaeroPack system (MGC, Japan). After 4–5 days of incubation, a single well-isolated colony was aseptically inoculated into BHI broth (3.7% BHI, 0.05% L-cysteine, 0.0005% hemin, 0.0001% menadione) and cultured under anaerobic conditions for an additional 48 h.

Induction of EAE

EAE was induced by subcutaneous immunization with a 10 mg/mL solution of myelin oligodendrocyte glycoprotein (MOG 35–55 ) peptide (QYAOBIO, China) emulsified 1:1 (v/v) with complete Freund's adjuvant (Sigma-Aldrich, USA) containing 5 mg/mL Mycobacterium tuberculosis H37Ra [ 46 ]. A total of 100 µL of emulsion was administered at four sites along the lower back (25 µL/site) in a single immunization. On Day 0 and again within 48 h, each mouse received an intraperitoneal injection of 200 µL pertussis toxin (GlpBio, China) at 1 µg/mL. Group allocation was performed using a random number table. From the day of immunization, neurological deficits were evaluated daily using Weaver's 15-point neurological function scale [ 47 ] ( Table 1 ). Neurological scoring was independently performed by two investigators blinded to group allocation. This scale characterizes disease progression by assessing each limb individually.

Establishment of a periodontitis model

Periodontitis was induced by placing P. gingivalis –soaked 6–0 silk ligatures around the maxillary second molars 7 days before EAE induction. Mice then received daily oral inoculation with 100 µL of P. gingivalis suspension (1 × 10 9 CFU/mL) for 28 consecutive days. Successful model establishment was confirmed by consistent clinical signs of gingival inflammation and histological evidence of inflammatory cell infiltration.

Histopathology

Decalcified tooth–gingiva–alveolar bone blocks were harvested and fixed in 4% paraformaldehyde, followed by decalcification in EDTA until adequately softened. Samples were then rinsed, dehydrated through graded ethanol (70, 80, 95 and 100%), cleared in xylene and embedded in paraffin. Serial sections (3–5 μm) were cut and mounted on glass slides, baked at 60 °C, deparaffinized in xylene and rehydrated through graded ethanol to water. Sections were stained with hematoxylin and eosin (H&E), dehydrated, cleared and coverslipped. Periodontal inflammation and alveolar bone resorption were evaluated histologically.

Immunofluorescent staining

Paraffin-embedded spinal cord sections were baked at 60 °C for 1 h, deparaffinized in xylene and rehydrated using graded ethanol. Antigen retrieval was performed by boiling in Tris-ethylenediamine tetra-acetic acid buffer (pH 9.0) for 15 min. Sections were blocked with 5% goat serum (Servicebio, China) for 1 h and incubated overnight at 4 °C with rat anti-mouse CD4 (Servicebio, China) and rabbit anti-mouse CXCR3 (Servicebio, China) for CD4/CXCR3 staining or with rat anti-mouse CD4 (Servicebio, China) and rabbit anti-mouse NF-κB p65 (Servicebio, China) for CD4/p65 staining.

After phosphate-buffered saline (PBS) washes, Alexa Fluor 488 goat anti-rat IgG (Servicebio, China) and Alexa Fluor 647 goat anti-rabbit IgG (Servicebio, China) were applied for 1 h in the dark. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (~1 µg/mL; Servicebio, China) and imaged using a Leica TCS SP5 confocal microscope (63×) with optimized laser settings. Image acquisition and quantitative analyzes were performed by investigators blinded to group allocation using predefined criteria.

Flow cytometry

Flow cytometry was performed to quantify CD4 + CXCR3 + T cells in murine peripheral blood and splenic lymphocytes. Cells were stained for viability using the Zombie NIR™ Fixable Viability Kit (BioLegend, USA) for 15 min in the dark, washed with PBS and then incubated for 20 min in the dark with Brilliant Violet 421™ anti-mouse CD4 (BioLegend, USA) and APC anti-mouse CXCR3 (BioLegend, USA). For in vivo measurements, cells were analyzed directly without in vitro culture; for in vitro assays, splenic lymphocytes were first incubated with P. gingivalis for 6 h at 37 °C and then processed using the same staining protocol. Data were acquired on a Cytek NL-CLC3000 flow cytometer (Cytek, USA), with > 10,000 CD4 + events collected for each sample. Gating included FSC-A versus FSC-H to exclude doublets, FSC-A versus SSC-A to remove debris and Zombie NIR–negative gates to select live cells. CXCR3 expression was then analyzed within the CD4 + T-cell population.

Reference-based mRNA-seq

Transcriptomic data were obtained from our previously published dataset (NCBI BioProject accession: PRJNA1233427), which includes mRNA-seq of CD4⁺ T cells isolated from the spleens of EAE and EAE + P mice at day 17 post-immunization ( n = 4 per group).

For the present study, raw sequencing reads were downloaded and reprocessed. Quality control was performed to remove low-quality reads, adapter contamination and poly- N sequences. Clean reads were aligned to the Mus musculus reference genome (mm10) using the STAR aligner. Polymerase chain reaction (PCR) duplicates were marked using Picard, and single nucleotide polymorphisms were called using the Genome Analysis Toolkit. Gene expression levels were quantified using HTSeq-count. Differential expression analysis was performed using DESeq2 (when biological replicates were available) or DEGseq (when applicable).

Gene ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was conducted using the GOseq R package, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using KOBAS. Protein–protein interaction networks were predicted via the STRING database and visualized using Cytoscape.

All original sequencing data are publicly available in the NCBI Sequence Read Archive under BioProject PRJNA1233427.

Cell culture

To examine the effects and mechanisms of P. gingivalis on peripheral CD4 + CXCR3 + T cells, mouse splenic lymphocytes were divided into three experimental groups: (1) Control group (CON): untreated splenic lymphocytes; (2) P. gingivalis group ( P ): splenic lymphocytes stimulated with P. gingivalis (1 × 10^8 CFU/mL) for 6 h; and (3) Nuclear factor kappa B ( NF-κB) inhibitor + P. gingivalis group (NF-κB inhibitor + P): splenic lymphocytes pretreated with the NF-κB inhibitor pyrrolidine dithiocarbamate (25 μM; Ambeed, USA) for 30 min, followed by P. gingivalis stimulation (1 × 10^8 CFU/mL) for 6 h. This experiment was designed as a stimulus–inhibition assay to determine whether NF-κB inhibition attenuated the response induced by P. gingivalis under stimulated conditions. After treatment, CD4 + T cells were isolated by negative selection using a CD4 + T-Cell Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer's instructions.

Quantitative reverse-transcription PCR (qRT–PCR)

CD4 + T cells were isolated from each group for qRT‒PCR analysis of Cxcr3 , Rela and Nfkbia mRNA expression. Total RNA was extracted using RNAiso Plus reagent (Takara Bio, Japan), and RNA concentration and purity were assessed using a spectrophotometer. cDNA was synthesized using the RT Easy™ II Kit (FOREGENE, China), following the manufacturer's instructions. qRT‒PCR was performed on a SLAN-96S Real-Time PCR system (Shanghai Hongshi, China) using a 10 μL reaction mixture containing SYBR qPCR SuperMix Plus (Sichuan Lanyun, China) and gene-specific primers (Chengdu Qingke Biotechnology Co., China).

The amplification protocol included an initial denaturation at 95 °C for 2 min, followed by 40 amplification cycles and a final melt curve analysis. Primer sequences are listed in Table 2 . Gene expression was calculated using the 2^-ΔΔCt method.

Western blotting

Splenic T cells were lysed using an ultrasonic homogenizer at 30–35 kHz and 50 μA for 2 min with continuous stirring to ensure complete disruption. Total protein concentration was measured using a Bicinchoninic Acid Protein Assay Kit (Solarbio, China). Proteins were precipitated with acetone, dissolved, boiled for 5 min and stored at −70 °C until use.

Next, proteins were separated by molecular weight on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, then transferred onto polyvinylidene fluoride membranes (Merck Millipore, USA) using a Trans-Blot chamber (Bio-Rad, USA). Membranes were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween-20 (TBST) for 1.5 h at 37 °C. Following this, they were incubated for 2 h at 37 °C with primary antibodies diluted in TBST containing 5% bovine serum albumin.

The primary antibody used were as follows: rabbit anti-mouse CXCR3 (1:1000), rabbit anti-mouse NF-κB p65 (1:1000), phospho-NF-κB p65 (1:1000), phospho-IκBα (1:1000), IκBα (1:1000) and tubulin (1:5000) as a loading control.

After washing with TBST, the membranes were incubated with the secondary antibody, biotinylated goat anti-rabbit IgG (Jackson ImmunoResearch, USA), for 1 h at 37 °C. This was followed by treatment with 0.1 µg/mL horseradish peroxidase-conjugated streptavidin (Jackson ImmunoResearch, USA) to enhance the signal. Protein bands were detected using enhanced chemiluminescence reagents (Amersham Healthcare, USA) and visualized with a Vilber Lourmat TFX-35 WL transilluminator. Band intensities were quantified using ImageJ software (NIH, USA) and normalized to tubulin to control for protein loading variability.

Cell transwell migration experiment

To investigate the effects of P. gingivalis on the migratory capacity of peripheral T cells in mice, splenic T cells were divided into three experimental groups: (1) Control group (CON): untreated splenic lymphocytes; (2) P. gingivalis group ( P ): splenic lymphocytes stimulated with P. gingivalis (1 × 10^8 CFU/mL) for 6 h; and (3) CXCR3 inhibitor + P. gingivalis group (CXCR3inhibitor + P): splenic lymphocytes pretreated with the CXCR3 inhibitor AMG 487 (20 μM; final concentration as indicated) for 30 min, followed by P. gingivalis (1 × 10^8 CFU/mL) stimulation for 6 h.This experiment was designed as a stimulus–inhibition assay to determine whether CXCR3 inhibition attenuated P. gingivalis -induced T-cell migration under stimulated conditions.

After stimulation, CD4⁺ T cells were isolated from the treated splenic lymphocyte suspensions by negative selection (Miltenyi Biotec, Germany) according to the manufacturer's instructions, and the purified T cells were used for the Transwell migration assay. Purified T cells from each group were resuspended in serum-free medium and adjusted to 1 × 10 6 cells/mL. For the migration assay, the lower chamber of the Transwell insert (5.0 μm pore size; Corning, USA) was filled with medium containing the chemokine CXCL10 (100 ng/mL). A total of 100 μL of the cell suspension was seeded into the upper chamber, and the inserts were incubated at 37 °C with 5% CO₂ for 4 h.

After 4 h, the inserts were removed, the medium was aspirated and the upper surface of the membrane was gently wiped with a PBS-moistened cotton swab to remove non-migrated cells. The inserts were subsequently transferred into a 24-well plate containing 1 mL of 4% paraformaldehyde and fixed for 15 min at room temperature. After discarding the fixative, membranes were washed twice with PBS, stained with 1 mL of crystal violet solution for 10 min and rinsed thrice again with PBS. Migrated cells were then photographed and quantified under a microscope.

Elisa

The gingipain concentration in gingival tissue homogenates was measured using a commercial Elisa kit (Saipeisen, China), according to the manufacturer's instructions. Briefly, gingival tissues were homogenized in cold PBS and centrifuged to collect supernatants. Samples and standards were added to pre-coated plates and incubated with HRP-conjugated detection antibody. After washing, tetramethylbenzidine (TMB) substrate was applied and the reaction was stopped with stop solution. Absorbance was read at 450 nm using a Thermo microplate reader (Thermo Fisher Scientific, USA).

Statistical analysis

Statistical analyzes were performed using GraphPad Prism version 9.0 (GraphPad Software, USA). Data are expressed as mean ± standard deviation. Longitudinal neurological scores ( Figure 1B ) were analyzed using two-way repeated-measures ANOVA with Bonferroni's multiple comparisons test, with time and experimental group as factors. Other comparisons among groups were conducted using one-way analysis of variance followed by Bonferroni's post hoc test. The Bonferroni correction was applied to all pairwise comparisons within each individual dataset. A p -value < 0.05 was considered statistically significant. For key outcomes, effect sizes and 95% confidence intervals (CIs) are reported to aid interpretation.

Results

P. gingivalis -induced periodontitis aggravates the clinical signs of EAE in mice

The periodontitis mice model was successfully established by P. gingivalis infection and ligation, as confirmed by inflammatory cell infiltration and alveolar bone resorption on H&E staining (Fig. S1). In addition, gingipain levels in gingival tissue homogenates were quantified by Elisa and showed no statistically significant difference between the P and EAE+P groups, indicating comparable P. gingivalis burden across the two periodontitis conditions (Fig. S2). Weaver's neurological function scale was used to assess clinical signs and confirm successful model establishment in EAE mice. Neurological scores were recorded daily from day 0 to day 21 post-immunization to evaluate the effect of periodontitis on disease progression. Both EAE and EAE+P mice developed neurological deficits by day 7, and peak scores occurred on day 17. Compared with the EAE group, EAE+P mice displayed significantly higher neurological scores from day 15 onward and this difference persisted until day 21 ( p < 0.0001). This indicates that P. gingivalis -induced periodontitis exacerbated clinical symptoms during both the peak and recovery phases of EAE ( Figure 1 ).

P. gingivalis -induced periodontitis increases CD4⁺ T-cell infiltration in the spinal cord of EAE mice

Immunofluorescence staining was used to assess CD4⁺ T-cell infiltration in the spinal cords of mice. At day 21 post-immunization, corresponding to the recovery phase of EAE, the EAE+P group exhibited a significantly higher number of infiltrating CD4⁺ T cells in the spinal cord than the EAE group ( p < 0.0001). These results indicate that P. gingivalis -induced periodontitis promotes the homing of peripheral CD4⁺ T cells to the CNS during EAE recovery ( Figure 2 ).

Transcriptomic data analysis reveals Cxcr3 upregulation and NF-κB pathway activation in splenic T cells of EAE mice with P. gingivalis -induced periodontitis

To investigate how P. gingivalis -induced periodontitis modulates systemic T-cell responses in EAE, we reanalyzed transcriptome data from our previously published RNA-seq dataset (NCBI BioProject: PRJNA1233427). This dataset profiled splenic CD4⁺ T cells from EAE and EAE+P mice at the peak of disease (17 dpi).

The reanalysis identified 5,335 DEGs, including 2,297 upregulated and 3,038 downregulated genes in EAE+P relative to EAE. Among these, Cxcr3 was significantly upregulated in EAE+P, along with several inflammation- and chemokine-related genes such as Cxcl10 , Cxcl9 , Cd4 , Nfkb1 and Nfkb2 ( p < 0.01, p < 0.001).

KEGG pathway enrichment revealed that these DEGs were predominantly involved in NF-κB signaling, cell adhesion molecules and T-cell receptor signaling. GO analysis further showed enrichment in biological processes associated with immune regulation, T-cell activation and lymphocyte chemotaxis.

Taken together, these findings suggest that P. gingivalis -induced periodontitis enhances Cxcr3 expression in splenic T cells through NF-κB pathway activation. This potentially contributes to the exacerbated systemic immune activation observed in EAE ( Figure 3 ).

P. gingivalis -induced periodontitis increased the proportion of CD4 + CXCR3 + T cells in the peripheral blood of EAE mice

Given the critical role of CD4⁺CXCR3⁺ T cells in T-cell trafficking during EAE, we assessed their frequency in peripheral blood by flow cytometry to investigate whether P. gingivalis -induced periodontitis modulates peripheral immune cell migration. The proportion of CD4⁺CXCR3⁺ T cells was significantly elevated in the P , EAE and EAE+P groups compared with the CON group ( p < 0.01, p < 0.001, p < 0.0001). Notably, the EAE+P group exhibited a significantly higher frequency of these cells than either the EAE or P groups alone ( p < 0.05, p < 0.001). No significant difference was observed between the P and EAE groups ( Figure 4 ). These findings indicate that both P. gingivalis -induced periodontitis and EAE independently increase the proportion of peripheral CD4⁺CXCR3⁺ T cells, and their combination exerts an additive effect during EAE.

P. gingivalis -induced periodontitis increased the proportion of CD4 + CXCR3 + T cells in the splenic lymphocytes of EAE mice

Given the spleen's central role in T-cell activation and chemokine receptor regulation, we next quantified the frequency of CD4⁺CXCR3⁺ T cells within splenic lymphocytes by flow cytometry. The EAE + P group exhibited a significantly higher proportion of CD4⁺CXCR3⁺ T cells compared to the CON, P and EAE groups ( p < 0.0001, p < 0.05, p < 0.01). Furthermore, both the P and EAE groups showed significant increases in this population relative to the CON group ( p < 0.001), while no statistically significant difference was observed between the P and EAE groups ( Figure 5 ). These findings indicate that EAE alone enhances the frequency of splenic CD4⁺CXCR3⁺ T cells, an effect that is further potentiated by concurrent P. gingivalis -induced periodontitis.

P. gingivalis -induced periodontitis increases infiltration of CD4 + CXCR3 + T cells in the spinal cord of EAE mice

Immunofluorescence staining was performed to assess CD4⁺CXCR3⁺ T-cell infiltration in the spinal cord. At day 21 post-immunization, EAE+P showed a significantly higher number of infiltrating CD4⁺CXCR3⁺ T cells than EAE ( p < 0.0001) ( Figure 6 ).

P. gingivalis -induced periodontitis enhances NF-κB activation (p65 nuclear translocation) in CD4⁺ T cells infiltrating the spinal cord of EAE mice

NF-κB p65 nuclear translocation in spinal cord–infiltrating CD4⁺ T cells was analyzed by immunofluorescence to assess NF-κB activation in CNS-infiltrating T cells in vivo. The proportion of CD4⁺ T cells showing nuclear p65 localization was notably increased in the EAE and EAE+P groups compared to the CON and P groups ( p < 0.0001). Notably, p65 nuclear translocation in spinal cord–infiltrating CD4⁺ T cells was significantly higher in the EAE+P group than in the EAE group ( p < 0.0001) ( Figure 7 ). These results show that P. gingivalis -induced periodontitis is associated with enhanced NF-κB activation (p65 nuclear translocation) in spinal cord–infiltrating CD4⁺ T cells during the disease state of EAE mice.

P. gingivalis enhances the chemotactic ability of peripheral T cells by upregulating CXCR3

To assess whether P. gingivalis alters the migratory capacity of splenic T cells, a Transwell migration assay was performed. Compared to the CON group, the P group showed a significant increase in the number of migrated T cells following CXCL10 stimulation. In contrast, the CXCR3inhibitor+P group showed markedly reduced T-cell migration compared with the P group ( p < 0.0001) ( Figure 8 ).

P. gingivalis increases the proportion of CD4⁺CXCR3⁺T cells in the spleen via an NF-κB pathway

To investigate whether P. gingivalis affects the distribution of CXCR3⁺ T-cell subsets in the spleen, we quantified CD4⁺CXCR3⁺T cells in splenic lymphocytes via flow cytometry. The proportion of CD4⁺CXCR3⁺ T cells was significantly increased in the P group compared with that in the CON group, indicating that P. gingivalis promotes the expansion of CXCR3-expressing CD4⁺ T cells in the spleen.

In the NF-κB inhibitor+P group, the proportion of CD4⁺CXCR3⁺ T cells was significantly reduced compared with that in the P group, indicating that the expansion of this subset is at least partially dependent on NF-κB signaling ( p < 0.0001) ( Figure 9 ).

P. gingivalis activates the NF-κB pathway to enhance CXCR3 expression in T cells

To further elucidate how P. gingivalis regulates CXCR3 expression on T-cell surfaces, we conducted in vitro T-cell culture experiments. Western blot analysis of mouse splenic T cells revealed significantly increased phosphorylation of p65 and IκBα, along with enhanced IκBα degradation, in the P group compared with that in the CON group. Notably, total p65 protein levels remained unchanged across all experimental groups, confirming that the observed increases in p-p65 and nuclear p65 reflect enhanced phosphorylation and nuclear translocation of p65 rather than changes in total p65 expression. These results indicate that P. gingivalis stimulation activated the NF-κB signaling pathway.

To verify the critical role of NF-κB signaling in P. gingivalis -induced CXCR3 upregulation, an NF-κB inhibitor was added prior to the co-culture of P. gingivalis with T cells. Western blot analysis confirmed that inhibition of NF-κB signaling significantly suppressed p65 and IκBα phosphorylation and prevented IκBα degradation, effectively blocking NF-κB activation. Concomitantly, P. gingivalis -induced CXCR3 protein and mRNA expression was significantly downregulated, and the proportion of CD4 + CXCR3 + T cells was also decreased ( p < 0.0001) ( Figure 10 ).

Altogether, these findings demonstrate that P. gingivalis promotes CXCR3 expression on T cells by activating the NF-κB signaling pathway, thereby enhancing the homing capacity of peripheral T cells in EAE mice.

Discussion

Accumulating evidence suggests that periodontitis is associated with peripheral immune system activation and can influence T-cell compartments [ 48 , 49 ]. Aberrant peripheral T-cell activation and subsequent homing to the central nervous system (CNS) play pivotal roles in the pathogenesis of both multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). In line with this, we previously demonstrated that P. gingivalis -induced periodontitis exacerbates EAE by promoting peripheral Th1 differentiation via ZAP70/NF-κB signaling, accompanied by reduced claudin-5 expression and increased blood–brain barrier (BBB) permeability [ 48 ]. Consistent with these findings, our current study shows that concurrent P. gingivalis -induced periodontitis significantly worsens the clinical symptoms of EAE during both the peak and recovery phases, as assessed by neurological scoring. Interestingly, the divergence between the EAE and EAE+P groups became more evident during the recovery phase rather than at disease onset. This pattern may suggest that P. gingivalis -induced periodontitis does not primarily alter initial EAE induction, but may contribute to sustained peripheral immune activation or delayed resolution of neuroinflammation during later disease stages. Further studies are required to clarify the mechanisms underlying this prolonged inflammatory response. However, the precise mechanisms through which periodontitis modulates peripheral T-cell chemotaxis and CNS homing require further elucidation and more direct evidence.

Extensive evidence indicates that T-cell trafficking across the BBB into the CNS in MS and EAE is a multifactorial process orchestrated by multiple chemokine and adhesion pathways. Within this network, the chemokine receptor CXCR3 and its ligands (CXCL9, CXCL10 and CXCL11) play critical roles by enhancing peripheral T-cell adhesion, facilitating BBB transmigration and promoting CNS infiltration, thereby amplifying local inflammatory responses and neuronal damage [ 19,39,40,49–51 ]. CXCR3 represents an important, though not exclusive, homing axis and its relative contribution may vary depending on T-cell subsets, the inflammatory microenvironment and disease stage. Elevated CXCR3 expression has been detected in both the peripheral blood and cerebrospinal fluid of patients with MS, accompanied by increased levels of its ligands (CXCL9/CXCL10/CXCL11) within the CNS [ 51 ]. In EAE models, genetic ablation or pharmacological blockade of CXCR3 markedly reduced Th1 cell infiltration into the CNS and alleviated demyelination [ 49 , 52 ], supporting the involvement of CXCR3 in T-cell homing/infiltration to the CNS and in disease progression. Furthermore, CXCR3 is highly expressed in Th1 cells within inflamed tissues, including periodontitis lesions [ 16 ]. Notably, systemic administration of CXCR3 antagonists significantly attenuates LPS-induced bone loss, highlighting CXCR3 as a promising therapeutic target [ 38 , 53 ].

In this study, reanalysis of our previously generated transcriptomic data from splenic T cells revealed that P. gingivalis infection significantly upregulated Cxcr3 gene expression in EAE mice. Consistent with these findings, we observed increased proportions of CD4⁺CXCR3⁺ T cells in both peripheral blood and splenic lymphocytes. Collectively, these data confirm that concomitant periodontitis increases CXCR3 expression in peripheral T cells during EAE. Functionally, our in vitro experiments demonstrated that P. gingivalis exposure increased the frequency of CXCR3⁺CD4⁺ T cells, elevated CXCR3 protein and mRNA expression and enhanced T-cell chemotaxis, whereas CXCR3 blockade effectively attenuated this migratory response. Overall, these data suggest that P. gingivalis enhances CD4⁺ T-cell chemotaxis primarily via CXCR3 upregulation. At the tissue level, we observed increased CD4⁺ T-cell infiltration in the spinal cord of EAE mice with P. gingivalis -induced periodontitis, and more specifically, robust infiltration of CD4⁺CXCR3⁺ T cells into the spinal cord was also detected, collectively supporting a mechanistic scheme in which P. gingivalis -induced periodontitis drives a CXCR3-skewed peripheral T-cell phenotype with heightened migratory potential and facilitates their CNS homing during EAE.

We next investigated the molecular pathway underlying CXCR3 upregulation in peripheral T cells in EAE mice with P. gingivalis -induced periodontitis. Re-analysis of our previously generated transcriptomic sequencing revealed a pronounced elevation of Cxcr3 expression in peripheral T cells from P. gingivalis -infected EAE mice, together with upregulation of NF-κB-associated genes. Enrichment analysis further identified NF-κB signaling as a key pathway activated by P. gingivalis infection.

Previous studies have established that NF-κB signaling regulates CXCR3-mediated T-cell migration into the CNS [ 50 ], and shared genetic profiles between periodontitis and MS are predominantly enriched in the NF-κB pathway [ 54 , 55 ]. Moreover, P. gingivalis LPS is known to activate NF-κB signaling in oral epithelial cells and periodontal ligament stem cells. Consistently, our study demonstrated that P. gingivalis stimulation significantly enhanced p65 and IκBα phosphorylation and promoted IκBα degradation in splenic T cells, thereby activating the NF-κB pathway. Correspondingly, we observed increased frequencies of CXCR3⁺CD4⁺ T cells, elevated CXCR3 protein and mRNA expression and enhanced chemotaxis. Importantly, pharmacological inhibition of NF-κB signaling markedly reduced the phosphorylation and degradation processes, resulting in significantly reduced CXCR3⁺CD4⁺ T-cell frequency and CXCR3 expression. These results demonstrate that P. gingivalis upregulates CXCR3 expression on T cells through activation of the NF-κB signaling pathway. Consistent with these ex vivo findings, we observed enhanced NF-κB activation in vivo, as evidenced by increased p65 nuclear translocation in CD4⁺ T cells infiltrating the spinal cord of EAE mice with P. gingivalis -induced periodontitis.

Taken together, our results support the notion that P. gingivalis -induced periodontitis is associated with increased CXCR3 expression on T cells in the context of NF-κB pathway activation, consistent with enhanced T-cell migratory capacity and CNS infiltration and with more severe EAE pathology. This NF-κB–CXCR3 axis may therefore represent a plausible pathway linking peripheral T-cell activation to increased CNS homing potential during EAE, and its upregulation coincides with worse clinical disease severity.

Although we provide supporting in vivo and in vitro evidence, the mechanistic inference is based primarily on in vitro perturbation assays, and inhibitor effects under basal conditions were not independently assessed. In addition, only female mice were studied, limiting the assessment of potential sex-dependent effects. Future studies employing in vivo functional blockade or genetic approaches in both sexes are warranted to strengthen causal inference and generalizability. The specific P. gingivalis virulence determinants responsible for the observed effects also warrant further investigation. Despite these limitations, this work provides a useful foundation for subsequent studies exploring the oral–CNS immune axis in autoimmune neuroinflammation.

Conclusion

In summary, our findings show that P. gingivalis -induced periodontitis exacerbates EAE and support a potential NF-κB/CXCR3-mediated mechanism involving NF-κB pathway activation, increased CXCR3 expression on peripheral T cells and enhanced T-cell migratory capacity. Increased infiltration of CD4⁺CXCR3⁺ T cells into the CNS was associated with more severe neurological deficits. These findings suggest that the NF-κB/CXCR3 axis may represent a potential mechanistic link between periodontitis and neuroinflammation, providing new insights into the immunopathological connection between oral infection and MS progression. Targeting this pathway may represent a promising adjunctive therapeutic strategy for MS patients, particularly those with concomitant periodontal disease.

Supplementary Material

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Supplementary Material

FigS1.tif

Supplementary Material

Acknowledgements

The authors thank Dr. Yang Wang (Beijing Institute of Otolaryngology, Beijing, China) for their technical assistance.

All authors contributed to manuscript revision, read and approved the submitted version.

Funding Statement

This work was supported by the National Natural Science Foundation of China (82370955) to JL.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethics statement

The animal experiment was reviewed and approved by the Ethics Committee of Beijing MDKN Biotechnology Co., Ltd. (Approval No: MDKN-2021-073).

Supplemental Material

Supplemental data for this article can be accessed at https://doi.org/10.1080/20002297.2026.2736905 .

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Supplementary Materials

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Supplementary Material