HomeScienceOral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin...

Oral microbial extracellular DNA initiates periodontitis through gingival degradation by fibroblast-derived cathepsin K in mice

[ad_1]

Animal care

All protocols for animal experiments were reviewed and approved by the University of California Los Angeles (UCLA) Animal Research Committee (ARC# 2003-009) and followed the Public Health Service Policy for the Humane Care and Use of Laboratory Animals and the UCLA Animal Care and Use Training Manual guidelines. C57BL/6 J wild type mice (Jackson Laboratory, Bar Harbor, ME) were used in this study. Animals had free access to regular rodent diet and water ad libitum and were maintained in standard housing conditions with 12-hour-light/dark cycles in the Division of Laboratory Animal Medicine at UCLA. All animal experiments were designed, conducted, and reported following the Animal Research: Reporting of In vivo Experiments guideline (ARRIVE 2.0)59.

Human subjects

All protocols involving human subjects were reviewed and approved by the UCLA Institutional Review Board (UCLA-IRB 11-002483). The participants provided verbal informed consent to take part in the study. Saliva samples were collected from 20- to 40-year-old healthy human subjects. Subgingival plaque samples were collected from 20- to 40-year-old human subjects with clinical diagnosis of periodontitis. The detailed protocols for further sample processing are described below.

Osteoadsorptive fluorogenic substrate probes

OFS-1 and OFS-3 were synthesized and characterized as described previously10. The probes were dissolved in 0.7 mL of 0.9% sodium chloride (NaCl) in water to a concentration of 50 μM and stored at 4 °C in the dark until use.

Evaluation of gingival swelling, alveolar bone resorption and Ctsk activation in a ligature-induced mouse model of periodontitis

A silk thread was gently tied around the left maxillary second molar of 8- to 12-week-old female C57BL6/J wild type mice under general inhalation anesthesia with isoflurane (Henry Schein, Melville, NY). To characterize Ctsk activation, OFS-1 or OFS-3 was prepared and characterized as previously described15 with minor modifications to further improve yield, and 100 µl of 10 µM OFS prepared in 0.9% NaCl solution was injected through the retro-orbital venous plexus one day prior to euthanasia. At 1, 3, 5, and 7 days after the ligature placement, mice were euthanized by 100% CO2 inhalation. The maxillary gingival tissues were then digitally photographed and harvested, and the fluorescent signal was measured with the IVIS Spectrum Imaging System (IVIS Lumina II: Perkin Elmer, Waltham, MA). The gingival swelling area was measured using a Java-based image processing program (ImageJ: NIH, Bethesda, MD) and normalized to the circumferential area of the maxillary second molar. After evaluation of Ctsk using the IVIS, the maxillary bones were harvested from each mouse and fixed in 10% buffered formalin (Thermo Fisher Scientific, Waltham, MA). The fixed maxillary bones were X-rayed at an energy level of 60 kV and 166 µA, and 3D images were reconstructed (Skyscan 1275: Bruker, Billerica, MA). Alveolar bone loss was measured at the middle of the second molar from the cementoenamel junction to the alveolar bone crest. Statistical analysis was performed using two-way analysis of variance with Tukey’s multiple-comparison test to assess the difference among multiple experimental groups. P < 0.05 was considered as statistically significant.

Histological and IHC evaluation of ligature-induced periodontitis in mice

The harvested maxillae with gingival tissue at 1, 3, and 7 days after ligature placement were fixed in 10% buffered formalin (Thermo Fisher Scientific, Waltham, MA) and decalcified in 10% EDTA (Sigma-Aldrich, Saint Louis, MO) for 3 weeks. After the decalcification, samples were embedded in paraffin. Histological cross-sections were stained by hematoxylin and eosin (H&E) and evaluated on a light microscope. Adjacent paraffin sections (4 µm) were immunohistochemically stained for Ctsk (anti-cathepsin K antibody #ab19027, Abcam, Waltham, MS) at 1:1000 dilution after heat-induced epitope retrieval procedure followed by the secondary antibody application and diaminobenzidine staining and methylene blue counterstaining. Ctsk+ gingival connective tissue cells were counted in the 10 mm2 area.

For some histological sections, IHC analysis of type XIV collagen (COL14A1 Polyclonal antibody, #PA5-49916, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution was also performed following the protocol. Osteoclasts were evaluated by tartrate-resistant acid phosphatase (TRAP) staining using a commercially available kit (Acid Phosphatase TRAP kit, Sigma-Aldrich, St. Louis, MO) following the manufacturer’s protocol.

Evaluation of gene expression of Il1b, Il6, Il17a, and Tnfsf11 (RANKL) in a ligature-induced mouse model of periodontitis

At 1, 3, and 7 days after ligature placement, total RNA was extracted from the harvested ligature side or non-ligature side of the maxillary gingival tissues with the RNeasy Mini Kit (QIAGEN, Germantown, MD) and quantified with a Thermo Scientific NanoDrop 1000 ultraviolet-visible spectrophotometer (NanoDrop Technologies, Wilmington, DE). After treatment with DNase I (Thermo Fisher Scientific), cDNA was synthesized from 1 µg of total RNA using Super Script III reverse transcriptase (Super Script VILO: Thermo Fisher Scientific).

Taqman-based qRT-PCR was performed using commercially available primer/probe mixes as follows, Il1b (Mm00434228_m1, Thermo Fisher Scientific), Il6 (Mm00446190_m1, Thermo Fisher Scientific), Il17a (Mm00439618_m1, Thermo Fisher Scientific) and Tnfsf11 (RANKL) (Mm00441908_m1, Thermo Fisher Scientific) in combination with a mouse Gapdh internal control mix (Mm99999915_g1, Thermo Fisher Scientific). Target gene expression was quantitatively analyzed using the ΔΔCT method. Statistical analysis was performed using Student’s t test to assess the difference between the ligature side group and the non-ligature side group at each time point. P < 0.05 was considered as statistically significant.

Examination of the function of Ctsk in the initial stage of periodontitis

Following ligature placement, mineral oil (Sigma-Aldrich) alone or supplemented with odanacatib (Selleckchem, Houston, TX) at a dose of 90 µg/100 µl was orally administered. Human studies reported a long elimination half-live of odanacatib, which was estimated to be 3 to 4 days60,61. In this study, odanacatib was administered once before the ligature placement. The gingival swelling area was measured, and the maxillary bones were scanned by microCT at 7 days after ligature placement as described above. Alveolar bone resorption was measured at the middle of the second molar from the cementoenamel junction to the alveolar bone crest. The average of bone area/total area in the alveolar bone on the buccal and palatal side of the second molar was measured from the apex of the root to the cementoenamel junction.

Harvested maxillae were decalcified and prepared for histological sections. The paraffin sections were stained with picrosirius red (PolyScience, Niles, IL). The collagen fiber structure of the gingival connective tissue and periodontal ligament was evaluated using confocal laser scanning microscopy (SP8: Leica Microsystems, Wetzlar, Germany). Picrosirius red was visualized via excitation with a 20 mW DPSS 561 nm and emission collection at 635-685 nm bandwidth. The connective tissue area was measured (ImageJ) and normalized to the area between tooth surface and the surface of the alveolar bone. Statistical analysis was performed using Student’s t test to assess the difference between the experimental groups. P < 0.05 was considered as statistically significant.

Evaluation of steady state gene expression profiles in a ligature-induced mouse model of periodontitis by scRNA-seq

The naïve untreated maxillary gingival tissue (designated as Day 0) and the gingival tissues after 1 day of ligature placement (designated as Day 1) were harvested and subjected to gingival cell dissociation as following.

Collagenase II treatment

The tissues were cut into ~1 mm2 pieces and placed immediately into 20 ml digestion buffer containing 1 mg/ml collagenase II (Life Technologies, Grand Island, NY), 10 units/ml DNase I (Sigma-Aldrich) and 1% bovine serum albumin (BSA; Sigma-Aldrich) in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies). The chopped tissues were incubated in the digestion buffer for 20 minutes at 37 °C on a 150 rpm shaker. The tissues were then passed through a 70 µm cell strainer, pelleted at 1500 rpm for 10 minutes at 4 °C before being resuspended in phosphate-buffered saline (PBS; Life Technologies) that was supplemented with 0.04% BSA (Cell suspension A) and counted to generate “Cell suspension A”.

Trypsin treatment

The parts of the tissues that did not pass through the 70 µm cell strainer after collagenase II treatment, were subjected to additional incubation in 10 ml of 0.25% trypsin (Life Technologies) and 10 units/ml DNase I for 30 minutes at 37 °C on a 150 rpm shaker. Trypsin was neutralized with 10 ml of fetal bovine serum (FBS; Life Technologies), and the tissues were passed through a 70 µm cell strainer, which was washed with 10 ml DMEM. The collected cells were then pelleted at 1500 rpm for 10 minutes at 4 °C, resuspended in PBS that was supplemented with 0.04% BSA and counted to generate “Cell suspension B”.

Cell suspension A and Cell suspension B were combined into one tube and cell viability was determined. For scRNA-seq, gingival cells were treated with the Chromium single-cell 3’ solution (10X Genomics, San Francisco, CA) using microfluidic partitioning to capture single cells and prepared the barcoded, next-generation sequencing cDNA library (10X Genomics, San Francisco, CA). The Cell Ranger software was used to align reads, generate feature-barcode matrix and perform clustering. The Cell Ranger output of scRNA-seq data was then analyzed using an R toolkit for single-cell genomics (Seurat, https://satijalab.org/seurat/). Cells with fewer than 800 detected genes were discarded. The signature gene was used to identify: B cells (Cd19), T cells (Cd3e), Myeloid cells (Lyz2), epithelial cells (Krt5) and fibroblasts (Col1a1). The expression of Ctsk was determined in each cell cluster. The myeloid cluster and fibroblast cluster was further subclustered for designated analyses.

Evaluation of Ctsk activation by topical application of cultured oral biofilm or planktonic bacteria

Saliva samples from 20- to 40-year-old healthy human subjects were collected and diluted to 25% with PBS. The diluted saliva was centrifuged at 2600 × g for 10 minutes to pellet large debris and eukaryotic cells. Prior to seeding of the oral biofilm, 100 µl of the diluted saliva was grown in 1 ml of SHI medium62 for 17–18 hours under anaerobic conditions (10% CO2, 10% H2, and 80% N2). This overnight grown oral microbial community was pelleted and washed with PBS. For biofilm seeding, cells were diluted cells to an optical density at 600 nm of 0.1 into 100% SHI medium supplemented with 5 mM CaCl2. Further, 1 ml of this diluted oral community was seeded onto oral appliances which were custom-made of clear dental resin (GC America, Alsip, IL) and incubated under anaerobic conditions at 37 °C for 5 days. Cultured oral community was treated with 10 U/ml DNase I and resuspended in PBS to prepare a planktonic bacteria solution free of EPSs including eDNA.

The palates were covered by an oral appliance with cultured oral biofilm, or three µl of planktonic bacteria solution (3 × 107 CFU) were topically applied to the palate and the palates were covered by an oral appliance. After 1 hour of covering the palate, the oral appliances were removed. One hundred µl of 10 µM OFS solution was injected through the retro-orbital venous plexus one day prior to euthanasia. At 4 days after the topical application of cultured oral biofilm or planktonic bacteria, the OFS fluorescent signal was measured. Statistical analysis was performed using two-way analysis of variance with Tukey’s multiple-comparison test to assess the difference among multiple experimental groups. P < 0.05 was considered as statistically significant.

Evaluation of Ctsk activation by topical application of CpG DNA or LPS

Three µl of 1 µg/ml of CpG ODN (InvivoGen, San Diego, CA), 1 µg/ml of LPS from P. gingivalis (InvivoGen) or 1 µg/ml of control ODN (InvivoGen) were topically applied to the palate as described above, and 100 µl of 10 µM OFS solution was injected through the retro-orbital venous plexus one day prior to euthanasia. At 4 days after the topical application of CpG ODN, P. gingivalis LPS or control ODN, the OFS fluorescent signal was measured. Statistical analysis was performed using two-way analysis of variance with Tukey’s multiple-comparison test to assess the difference among multiple experimental groups. P < 0.05 was considered as statistically significant. In addition, the harvested maxillas were subjected to immunohistochemial staining for Ctsk and stained with HE as described above.

Evaluation of steady state gene expression profiles in mouse gingival tissues with topical application of CpG DNA or LPS by scRNA-seq

At 4 days after the palatal topical application of CpG ODN or P. gingivalis LPS, the maxillary gingival tissues were harvested from freshly isolated mouse maxillas. Single cells were dissociated from the maxillary gingival tissues, and scRNA-seq was performed as described above.

Evaluation of gene expression of TLR9 in gingival fibroblasts and skin fibroblasts

Primary gingival fibroblasts or skin fibroblasts from 8- to 12-week-old female wild type mice were cultured using an explant method as previously reported63. The cells were cultured in DMEM with 10% FBS and 100 U penicillin/0.1 mg/ml streptomycin (Life Technologies) at 37 °C, 5% CO2 in a humidified incubator.

Total RNA was extracted from the gingival fibroblasts or skin fibroblasts, and cDNA was synthesized as described above. Taqman-based RT-qPCR was performed using a commercially available primer/probe mix for Tlr9 (Mm00446193_m1, Thermo Fisher Scientific). Statistical analysis was performed using Student’s t test to assess the difference between the experimental groups. P < 0.05 was considered as statistically significant.

Evaluation of induction of Ctsk secretion or production of Ctsk protein in gingival fibroblasts by CpG DNA

Primary mouse gingival fibroblasts were cultured in DMEM supplemented with 10% FBS and 100 U penicillin/0.1 mg/ml streptomycin in the presence of 0, 0.1, 1 or 10 µg/ml of CpG ODN at 37 °C, 5% CO2 in a humidified incubator for 24 hours.

Culture supernatant or cell solution lysed with RIPA Lysis and Extraction buffer (VWR, Radnor, PA, USA) supplemented with a protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The Ctsk protein concentration in the culture supernatant or lysed cell solution was determined by a colorimetric method (OD at 450 nm) using a Ctsk ELISA kit (MyBioSource, San Diego, CA, USA). Statistical analysis was performed using one-way analysis of variance with Bonferroni’s multiple-comparison test to assess the difference only of pairs relative to the control group. P < 0.05 was considered as statistically significant.

Extraction of iDNA or eDNA from a human saliva-derived oral microbial community

eDNA and iDNA were extracted from human saliva-derived biofilms. After biofilm growth in 100% SHI medium supplemented with 5 mM CaCl2 under anaerobic conditions at 37 °C for 5 days, planktonic cells were removed by gently aspirating the medium and carefully washing once with 500 µl PBS. Following the wash, 250 µl of PBS was added in each well, and microbial cells were harvested by scraping and pipetting with a sterile pipette tip. The bacterial cells were then transferred to an Eppendorf tube and pelleted at 3250 × g for 15 minutes at 4 °C for DNA isolation. The supernatant was filtered through 0.22 µm syringe filters to exclude bacterial cells. While the bacterial pellet was used for iDNA isolation, the cell- free supernatant was further processed for eDNA isolation.

iDNA extraction

The bacterial pellet was used for iDNA extraction using the Epicentre MasterPure DNA extraction and Purification Kit (Lucigen, Middleton, WI) according to the manufacturer’s instructions64.

eDNA extraction

eDNA was extracted from the cell-free supernatants of the microbial biofilm according to a previously described protocol65. Briefly, the cell-free supernatants (containing eDNA) were mixed with 2 volumes of absolute ethanol and a 1/10th volume of sodium acetate (3 M, pH 5.2) containing 1 mM EDTA. After overnight precipitation at −80 °C, eDNA was pelleted by centrifugation at 13,000 rpm at 4 °C for 20 minutes followed by a wash with ice-cold 70% ethanol. The eDNA was then air dried, dissolved in sterile deionized water and quantified using NanoDrop.

Evaluation of Ctsk activation by topical application of eDNA or iDNA

Three µl of 1 µg/ml solution of eDNA or iDNA were topically applied to the palate as described above, and 100 µl of 10 µM OFS solution was injected through the retro-orbital venous plexus one day prior to euthanasia. At 4 days after the topical application of eDNA or iDNA, the OFS fluorescent signal was measured. Statistical analysis was performed using two-way analysis of variance with Tukey’s multiple-comparison test to assess the difference among multiple experimental groups. P < 0.05 was considered as statistically significant.

Evaluation of eDNA and iDNA of mouse oral biofilm harvested from recovered ligatures

The ligatures were recovered from the mouse model of periodontitis 1 day (n = 4) and 7 days (n = 4) after placement. iDNA and eDNA samples were prepared separately as above and subjected to 16 S rRNA sequencing of the V4 region (Laragen, Inc, Culver City, CA). Demultiplexed sequences were imported into Qiime 2 (v2020.11). Low quality sequences containing bases with Phred quality values <20 were trimmed and denoised using the DADA2 package66. The amplicon sequence variants generated after the denoising were taxonomically assigned by comparison to the HOMD database.

SYTOX Green/Orange staining of the subgingival plaque from a periodontitis patient and the recovered ligature

Subgingival plaque was collected from a periodontitis patient and stained with SYTOX Green (Thermo Fisher Scientific). The eDNA scaffold in the plaque was evaluated via fluorescence microscopy (Zeiss Axio Imager M2 with Zen 2.5 pro software). Sytox green was visualized using epifluorescence through a ×100/1.4 Plan Achromat objective at 450–490 nm bandwidth excitation and 500–550 nm bandwidth emission filters. The recovered ligature from the mouse periodontitis model was stained with SYTOX Orange and was visualized similarly but with 538–562 nm bandwidth excitation and 570–640 nm bandwidth emission filter settings.

Statistics and reproducibility

For statistical analysis, Student’s t test or one-way analysis of variance with Tukey’s multiple-comparison test or with Bonferroni correction was performed. A significant difference was defined by p < 0.05. The sample size and number of replicates ware reported in the legends of figures. The data were showed as mean and standard deviation.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.


[ad_2]

Source link

RELATED ARTICLES

LEAVE A REPLY

Please enter your comment!
Please enter your name here

Most Popular

Recent Comments

%d bloggers like this: