Animals and experimental groups

Thirty-six C57BL/6 wild-type, aged 10–12 weeks, were used. The mice were maintained in a specific pathogen-free environment, 12:12 h light/dark cycle at 24 ± 0.5 °C and 40–70% relative humidity. The Institutional Animal Care and Use Committee of the Forsyth Institute reviewed and approved the experimental protocols. Mice were divided into five groups of 7 to 8 mice per group, including mice with no ligature (baseline) and mice used on day 1, 10, 20, and 30 post-ligature placements, as shown in Fig. 1A. The ligatures were placed using a 7-0 silk suture subgingivally around the maxillary right and left second molars with the knot positioned at the palatal side, as previously reported [23]. Ligature placement was performed under general anesthesia using a ketamine/xylazine cocktail (87.5 mg of ketamine per kg of body weight and 12.5 mg of xylazine per kg of body weight) via intraperitoneal injection. The procedure was performed using fine microsurgical instruments, a microscope, a cold-light source system, and animal-holding support to allow the maximum mouth opening without causing mechanical damage to the oral mucosa of the anesthetized animal.

Fig. 1 Alveolar bone resorption and mRNA expression levels of RANKL and OPG in periodontal tissues of mice with ligature-induced experimental periodontal disease. A In vivo experimental design (n = 36, 7–8 mice per group). The baseline group was left untreated, and four groups had ligatures around maxillary right and left second molars for different timepoints corresponding to 1,10, 20, and 30 days and then sacrificed. Black arrow: placement of ligatures. Red arrows: sacrifice. B Left: representative images of the left maxillae from each experimental group (buccal view). Right: quantification of the area between the alveolar bone crest level and the cementoenamel junction of the three maxillary molars, using Fiji software (ImageJ). C Levels of mRNA expression of proteins involved in periodontal tissue metabolism RANKL and OPG. The right and left gingival tissue of each mouse were pooled together and represented one sample. Baseline: mice without ligature placement. Lig. D1, D10, D20, and D30: mice with ligature placed for different timepoints corresponding to 1,10, 20, and 30 days, respectively. (n = 36, 7–8/group, Mean ± SEM, ANOVA, *p < 0.05, ****p < 0.0001) Full size image

Assessment of alveolar bone loss and periodontal inflammatory process

To confirm the alveolar bone loss as a direct assessment of experimental periodontal disease, we measured the loss of bone in defleshed mouse jaws dissected immediately after the animals were euthanized. After removing ligatures and gingival tissue from the sacrificed animals, the maxillae were defleshed by dermestid beetles for 4–5 days. Then, the samples were cleaned with 5% hydrogen peroxide for 8 h and washed thoroughly with water. The samples were stained with methylene blue (1% in water) for 10 s to clearly distinguish between the bone and the teeth before the morphometric analysis of the bone loss. Then, the samples were mounted and photographed at × 10 magnification using an inverted microscope (Axiovert 200, Zeiss, Thornwood, NY, USA) and AxioVision 4.8 software. The area between the alveolar bone crest level and the cementoenamel junction of the three maxillary molars was measured using ImageJ and calculated in micrometers.

For further confirmation of bone metabolism and the resorptive process, the gingival tissues were collected around all maxillary molars and fixed in RNAlater (Life Technologies, Carlsbad, CA, USA). Then, the samples were homogenized, and total RNA was isolated through the RNeasy® Mini kit (QIAGEN, Germantown, MD, USA) according to the manufacturer's instructions. The purity was tested using a NanoDrop ND-1000 spectrophotometer (absorbance ratio at 260 and 280 nm, ThermoFisher Scientific, Waltham, MA, USA). One µg of RNA was reverse-transcribed to cDNA through High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, ThermoFisher, Waltham, MA, USA) using RT random primers, dNTP mix, RT buffer, nuclease-free water, ribonuclease (RNase) inhibitor, and reverse transcriptase for quantitative PCR (Q-PCR) analysis using StepOnePlus Real-Time PCR system (Applied Biosystems, ThermoFisher, Waltham, MA, USA). The detection assay was done using the TaqMan™ Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA), 50 ng cDNA of gingival tissue, and TaqMan Gene Expression Assays of RANKL and OPG (Additional file 1: Table S1). Data were analyzed using the 2−ΔΔCT method, and the expression of each target gene was then calculated as a fold-change relative to the controls using β-actin as an endogenous control.

Isolation of adult mouse brain cells

Isolation of cells from the adult mouse brain was done according to a previously published protocol [24]. Briefly, mice were euthanized and transcardially perfused with cold PBS (10 mL/min). Then, each fresh brain was transferred into a 50 ml Falcon tube containing 10 mL cold 1 × HBSS (R&D Systems, Inc., Canada) and minced with a sterile No.15 scalpel blade on ice. The tissue samples were spun for 5 min at 400 × g at 4 °C, and the supernatant was aspirated. 10 mL of HBSS supplemented with 85 units dispase II (Sigma Aldrich, catalog number: D4693-1G) + 0.25 units DNaseI (Zymo Research, catalog number: E1010) + 2.5 units (CDU) collagenase Type I (Sigma Aldrich, catalog number: C0130) + 1 μg Nα-Tosyl-L-lysinechloromethyl ketone hydrochloride (Sigma Aldrich, catalog number: T7254) was added to the minced whole-brain tissue. The tubes were incubated in a water bath at 37 °C for 1 h. Then, the digested brain tissue with the enzyme cocktail was transferred into a 15 mL Dounce homogenizer (Sigma-Aldrich) and dissociated on ice using the large clearance pestle. The homogenized brain cell suspension was transferred to a 50 mL tube containing 5 mL 10% Fetal Bovine Serum (FBS) through the 70 µm cell strainer (Miltenyi Biotec). The cells were filtered by repeatedly washing the cell strainer with 5 mL 10% FBS. The obtained cell suspension was centrifuged for 5 min at 400 × g at 4 °C; the supernatant was discarded. Then, the cells were resuspended in 16 mL of 35% Percoll (Sigma-Aldrich, St. Louis, MO, USA). The sample was split equally into two 15 mL tubes, overlayed with 5 mL 1 × HBSS, and rested on ice for 5 min. The samples were spun at 45 min at 4 °C and 800 × g without a break to get the different density gradient layers. Finally, the supernatant, including the myelin layer, was discarded. The pelleted mixed brain cells were resuspended and prepared for flow cytometric analyses without further passaging to prevent any phenotypic changes.

Flow cytometry and T-SNE analysis

Brain cells were washed with PBS and stained with the zombie UV™ Fixable Viability Kit (Biolegend, San Diego, CA, USA) for 30 min in the dark. The extracellular staining was performed in PBS containing 5% FBS, using the following antibodies: anti-CD45 (30-F11, Biolegend), anti-CD11b (M1/70, Biolegend), anti-CD68 (FA-11, Biolegend), anti-MHCII (M5/114.15.2, Biolegend), and anti-CX3CR1(SA011F11, Biolegend) for 30 min at 4◦C in the dark (Additional file 1: Table S2). Cells were analyzed on Attune™ NxT acoustic focusing cytometer (Invitrogen) using a sequential gating strategy according to the FSC/SSC and SSC/SSC parameters, live/dead staining, and CD45/CD11b markers. The multiparametric flow cytometry data analysis was performed using FlowJo software (CA, USA). The data were analyzed using dimensionality reduction with the t-Distributed Stochastic Neighbor Embedding (tSNE) algorithm. The tSNE algorithm computes two new derived parameters from a user-defined selection of cytometric parameters. The following workflow was used to compare samples effectively: (1) data clean up by applying manual gates to exclude doublets, debris, and dead cells from each sample. (2) Use the down-sample algorithm on the CD11b+ CD45low gated populations of each sample to 20,000 events to significantly reduce calculation time. (3) Concatenate all samples (3 samples per experimental group) to generate a single two-dimensional data space created by tSNE. (4) Dimensionally reduce (create tSNE parameters) on the concatenated file using default settings in FlowJo, iterations 1000, perplexity 30, and learning rate (eta) 521. (5) Analysis of differential expression of microglial cell markers in different mice groups.

Isolation and heat-inactivation of mouse microbiome associated with experimental periodontal disease

We isolated and collected the microbial species from the ligatures around the maxillary molars to characterize the direct impact of the mouse microbiome associated with PD on mouse microglial cells. To standardize the multiplicity of infection (MOI) of bacteria within and between individual experiments and avoid the live organisms exhibiting a rapid doubling time, which can quickly lead to culture overgrowth, medium exhaustion, and subsequent microglial death, we used heat-inactivated bacteria to activate microglial cells [25]. The ligatures were recovered from euthanized animals and were gently washed with PBS to remove food residue and other debris. Subsequently, the ligatures were placed in Eppendorf tubes containing Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Billings, MT, USA) without any supplements. The bacteria were dispersed by vortexing for 2 min at 3000 rpm, followed by removing ligatures from the tubes as previously described [26]. Then, the number of bacteria in the suspension was determined spectrophotometrically. Finally, heat-inactivation of the total microbiome was prepared by heating the culture media for 30 min at 65 °C in a shaking water bath. The preparation was checked for sterility by plating on agar media. Heat-inactivated bacteria were stored at − 80 °C until use.

Identification and culture of Klebsiella variicola

In separate experiments, using 16S rRNA gene sequencing, we identified the microbial composition of the ligatures and the brains of 5xFAD mice, a transgenic mouse model of AD, after 4 weeks of ligature-induced experimental periodontitis. We identified individual species that grew in the live cultures. Klebsiella variicola was the major species detected in direct cultures of both ligatures and brain specimens in 5xFAD mice, confirmed by direct brain cultures and 16S rRNA sequencing. We used K. variicola in subsequent studies to explore its impact on microglial cells as a periodontal bacteria in an AD-like mouse model. To prepare K. variicola for co-culture experiments with microglial cells, K. variicola was plated on Trypticase™ Soy Agar (TSA II™) with sheep blood plates aerobically at 37 °C. After 2–3 days of incubation, colonies were picked with a sterile metal loop and were grown in liquid Tryptic Soy Broth medium (TSB) aerobically at 37 °C for 1 day. Then, the number of bacteria in the suspension was determined spectrophotometrically. Finally, heat-inactivation of cells was prepared by heating bacteria-containing culture media for 30 min at 65 °C in a shaking water bath. The preparation was checked for sterility by plating. Heat-inactivated K. variicola was stored at − 80 °C until use.

Microglial cell culture

The mouse microglial cell line BV2 (generated from primary mouse microglia transfected with a v-raf/v-myc oncogene carrying retrovirus (J2)) [27] was purchased from Biological Bank and Cell Factory (BBCF, Italy, www.iclc.it). BV2 cells were cultured in RPMI 1640 medium (Gibco), supplemented with 1% penicillin–streptomycin (Gibco) and 10% fetal bovine serum (FBS, ATLANTA biologicals). Cells were maintained at 37 °C in a 5% CO 2 humidified atmosphere. Every 2 days, microglial cells were washed and dislodged from the surface of the flask by gentle pipetting with phosphate-buffered solution twice and transferred into a 50 mL tube. After spinning the culture medium containing cells, the supernatant was discarded, and the cell pellet was resuspended in 5 mL of culture medium. Then, 2 × 106 cells were plated into new 75 cm2 flasks containing 15 mL prewarmed culture medium and placed in the incubator. Cells were passaged 2–3 times. All experiments were typically done on passage number 7. When cells reached 60–70% confluency, they were transferred into 24-well plates (1 × 105 cells/well). q-PCR and flow cytometry were used to measure the expression level of specific genes involved in microglial activation and the phagocytic capacity of Aβ by microglial cells, respectively.

Gene expression by microglial cells using q-PCR

To assess the effect of the whole PD-associated microbiome or K. variicola on the expression level of specific genes involved in microglial activation, microglial cells were seeded into 24-well plates and incubated in RPMI supplemented with 2% FBS and 1% penicillin–streptomycin for 24 h. The bacterial suspension (whole PD-associated microbiome from the ligature or K. variicola) was added to the microglial cells at different multiplicities of infection (MOI). After 24 h of co-incubation, total cellular RNA was isolated using the RNeasy® Mini kit (QIAGEN, Germantown, MD, USA) according to the manufacturer's instructions. The purity of RNA was tested using a NanoDrop ND-1000 spectrophotometer (ratio of absorbance at 260 and 280 nm, Thermo Scientific, Waltham, MA, USA). The detection assay was done using the TaqMan™ Fast Advanced Master Mix (ThermoFisher Scientific, Waltham, MA, USA), 5 ng cDNA of microglial cells, and TaqMan Gene Expression Assays (Additional file 1: Table S1). Data were analyzed using the 2−ΔΔCT method, and the expression of each target gene was then calculated as a fold-change relative to the controls using β-actin as an endogenous control.

Amyloid β phagocytosis by microglial cells

We used flow cytometry to assess the effect of the whole PD-associated microbiome or K. variicola on the phagocytosis of Aβ by microglial cells. Microglial cells were seeded into 24-well plates and incubated in RPMI supplemented with 2% FBS and 1% penicillin–streptomycin for 24 h. Bacteria were added at different multiplicities of infection (MOI). After 24 h of co-incubation, 1 µg/mL HiLyte Fluor 488-conjugated Aβ42 (Anaspec, Fremont, USA) was added to the cultures for 2 h. Microglial cells were harvested and washed three times with PBS containing 5% fetal bovine serum (FBS). The final cell suspension was spun down at 200 × g for 5 min; the supernatant was eliminated, and the cells were resuspended in 200 µL of PBS + 5% FBS. Phagocytosis of Aβ42 was measured as the percentage of β-amyloid+ microglial cells and analyzed on an Attune™ NxT acoustic focusing cytometer (Invitrogen). Data analysis was done using the FlowJo software.

TLR-mediated activation of microglial cells

We used two strategies to test the role of TLRs in microglial response to K. variicola. After cells were seeded into 24-well plates and incubated in RPMI supplemented with 2% FBS and 1% penicillin–streptomycin for 24 h, first, we targeted TLR2. Microglial cells were pre-treated for 1 h with either TLR2 antagonist (T2.5) at a concentration of 10 µg/mL (InvivoGen) or TLR2 agonist, lipoteichoic acid (LTA-SA) at a concentration of 10 µg/mL (InvivoGen). Then, we targeted TLR9, which is downstream of TLR2. We pre-treated the microglial for 1 h with TLR9 antagonist (ODN2088) at a concentration of 1 µM (InvivoGen) before introducing K. variicola. In parallel, we used the TLR9 agonist, CpG oligonucleotide (ODN1826), at a concentration of 1 µM (InvivoGen). These conditions are shown in Additional file 1: Table S3.

Statistical analysis

All statistical analyses for bone morphometry, relative gene expression, and flow cytometry were performed using GraphPad Prism Software version 9.2.0 (GraphPad Software, La Jolla, California, USA). The normality of data distribution was determined using the Shapiro–Wilk test. Outliers were determined using the ROUT method with Q = 1% and were excluded. An unpaired t test was used to compare two experimental groups. Analysis of variance (ANOVA) followed by multiple comparison Tukey post-hoc tests was applied for three or more groups. Values are expressed as mean ± standard error of the mean. P values of < 0.05 were considered statistically significant.