Antimicrobial-Induced Oral Dysbiosis Exacerbates Naturally Occurring Alveolar Bone Loss

Abstract

Periodontitis-mediated alveolar bone loss is caused by dysbiotic shifts in the commensal oral microbiota that upregulate proinflammatory osteoimmune responses. Study purpose was to determine whether antimicrobial-induced disruption of the commensal microbiota has deleterious effects on alveolar bone. We administered an antibiotic cocktail, minocycline, or vehicle-control to sex-matched C57BL/6T mice from age 6- to 12-weeks. Antibiotic cocktail and minocycline had catabolic effects on alveolar bone in specific-pathogen-free mice. We then administered minocycline or vehicle-control to male mice reared under specific-pathogen-free and germ-free conditions, and we subjected minocycline-treated specific-pathogen-free mice to chlorhexidine oral antiseptic rinses. Alveolar bone loss was greater in vehicle-treated specific-pathogen-free vs. germ-free mice, demonstrating that the commensal microbiota drives naturally occurring alveolar bone loss. Minocycline- vs. vehicle-treated germ-free mice had similar alveolar bone loss outcomes, implying that antimicrobial-driven alveolar bone loss is microbiota dependent. Minocycline induced phylum-level shifts in the oral bacteriome and exacerbated naturally occurring alveolar bone loss in specific-pathogen-free mice. Chlorhexidine further disrupted the oral bacteriome and worsened alveolar bone loss in minocycline-treated specific-pathogen-free mice, validating that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone. Minocycline enhanced osteoclast size and interface with alveolar bone in specific-pathogen-free mice. Neutrophils and plasmacytoid dendritic cells were upregulated in cervical lymph nodes of minocycline-treated specific-pathogen-free mice. Paralleling the upregulated proinflammatory innate immune cells, minocycline therapy increased T_H1 and T_H17 cells that have known pro-osteoclastic actions in alveolar bone. This report reveals that antimicrobial perturbation of the commensal microbiota induces a proinflammatory oral dysbiotic state that exacerbates naturally occurring alveolar bone loss.

INTRODUCTION:

The indigenous commensal oral microbiota critically regulates osteoimmune processes and alveolar bone homeostasis in periodontal health and disease^1,2. Longitudinal and cross-sectional studies in periodontally healthy patients receiving regular care have shown that naturally occurring alveolar bone loss occurs at a rate of 0.03 to 0.07 mm per year^3-5. The outcomes from these clinical investigations suggest that the indigenous oral microbiota causes alveolar bone loss in periodontal health. Gnotobiotic animal studies comparing germ-free (GF) mice to mice harboring a commensal microbiota have confirmed that the indigenous oral microbiota drives naturally occurring alveolar bone loss^6-8. Irie, Novince, and Darveau (2014) carried out the first known osteoimmunology study investigating mechanisms mediating the commensal oral microbiota’s catabolic effects on alveolar bone⁸. The authors showed that the burden of indigenous oral microbiota induces periodontal immune response effects, which support osteoclast-mediated bone resorption in the healthy periodontium⁸.

Periodontal disease-driven inflammatory alveolar bone destruction is caused by a polymicrobial disruption of the homeostatic relationship between the host and oral microbiota^1,2,9,10. Periodontitis-induced dysbiotic shifts in the oral microbiota upregulate proinflammatory immune response mechanisms that enhance osteoclastogenesis and drive inflammatory alveolar bone osteolysis^1,2,9. Hajishengallis et al. (2011) delineated that naturally occurring alveolar bone loss is exacerbated by perio-pathogenic bacteria-induced shifts in the commensal oral microbiota⁷. The seminal report demonstrated that Porphyromonas gingivalis triggers changes in the composition and abundance of the oral microbiota, which drives inflammatory periodontal bone loss⁷.

Systemic antibiotic administration^11,12 and chlorhexidine oral antiseptic rinses^13,14 cause shifts in the healthy human oral microbiome. However, it is unknown whether antimicrobial-induced perturbations in the healthy commensal microbiota influence the periodontal osteoimmune response and alveolar bone homeostasis. Considering that periodontitis-driven alveolar bone loss is caused by a polymicrobial disruption of host homeostasis^1,2,9,10, the current study’s purpose was to discern whether antimicrobial perturbation of the commensal oral microbiota has deleterious effects on alveolar bone.

MATERIALS & METHODS:

Mice:

Five-week-old male murine-pathogen-free C57BL/6T mice were purchased from Taconic Biosciences (Rensselaer, NY, USA) and housed under specific-pathogen-free (SPF) conditions at Medical University of South Carolina (MUSC). Room temperature and humidity were maintained within the advised ranges per the eighth edition of the Guide for the Care and Use of Laboratory Animals, animals were maintained on a 12 hour: 12 hour light:dark schedule, and received an autoclaved NIH-31 M diet. SPF mice were provided one week to acclimate to the SPF vivarium before initiating treatment. Male SPF mice were administered vehicle-control, minocycline hydrochloride (100 mg/L), or antibiotic cocktail (vancomycin [500 mg/L], imipenem/cilastatin [500 mg/L], and neomycin [1 g/L]) in drinking water from age 6- to 12-weeks. In two separate experiments, male SPF mice were subjected to the following treatments from age 6- to 12-weeks: (1) vehicle-control administration through the drinking water, minocycline hydrochloride (100 mg/L) administration through the drinking water, or minocycline hydrochloride (100 mg/L) administration through the drinking water with twice-daily 0.12% chlorhexidine gluconate oral rinses. (2) vehicle-control administration through the drinking water, or vehicle-control administration through the drinking water with twice-daily 0.12% chlorhexidine gluconate oral rinses. The 0.12% chlorhexidine gluconate oral rinses were performed by taking up 150 μL chlorhexidine gluconate solution into a LASIK Expanded Spear sponge (#581709; Beaver Visitec, Waltham, MA, USA), which was inserted into the murine oral cavity for 30 seconds. Chlorhexidine gluconate oral rinses were performed every 12 hours, and animals were euthanized 12 hours following the final oral rinse.

Female C57BL/6T SPF mice were administered vehicle-control or minocycline hydrochloride (100 mg/L) from age 6- to 12-weeks. Experimental groups were euthanized at age 12-weeks and age 18-weeks to assess immediate and sustained antibiotic treatment effects.

Germ-free (GF) C57BL/6T mice were acquired from Taconic Biosciences, and bred and maintained in sterile isolators at MUSC Gnotobiotic Animal Core. Room temperature and humidity were maintained within the advised ranges per the Guide for the Care and Use of Laboratory Animals (8th ed., National Academies Press, Washington, DC; 2011); animals were maintained on a 12 hour: 12 hour light:dark schedule, and received a sterilized Teklad 8656 diet (Harlan Laboratories, Inc.Indianapolis, IN, USA). The gnotobiotic facility at MUSC is surveyed for serology, parasites, aerobic/anaerobic bacteria and fungi by direct testing of colony animals. Serological, Helicobacter and pinworm evaluation is performed by Charles River Laboratory (CRL) and covers the agents identified in their Assessment Profile and Infectious Disease PCR profile. Additional quality control testing is performed in-house and is designed to detect fastidious microbes, fungi, anaerobes, aerobes and microaerophiles. Male GF mice were administered vehicle-control or minocycline hydrochloride (100 mg/L) in drinking water from age 6- to 12-weeks. Two to three mice were housed per cage for all studies. Antibiotic drinking water solutions were prepared, sterile filtered, and refreshed every other day. Treatment groups were euthanized at age 12-weeks. Germ-free status of animals was validated by performing 16S qRT-PCR analysis on fecal pellets isolated immediately prior to sacrifice (Fig. S1)

All work with mice was approved by the MUSC Institutional Animal Care and Use Committee and reported in accordance with the ARRIVE 2.0 guidelines. All work with mice was performed in accordance with the US National Research Council's Guide for the Care and Use of Laboratory Animals, the US Public Health Service's Policy on Humane Care and Use of Laboratory Animals, and Guide for the Care and Use of Laboratory Animals.

Antimicrobial treatment was initiated at age 6-weeks since this is the C57BL/6 murine developmental age when the immune system is considered mature^15,16 and alveolar bone formation is complete^17,18. Alveolar bone formation depends on the eruption of the teeth^19,20, a developmental process which is principally complete by age 5-weeks in mice^17,18,20,21. The experimental antibiotic cocktail of vancomycin, imipenem/cilastatin, and neomycin was employed to deplete the indigenous commensal microbiota²². Minocycline hydrochloride was strategically chosen based on the intention to administer a broad-spectrum antibiotic monotherapy commonly prescribed to healthy adolescents and young adults for extended periods. Roughly 1/3 of adolescents and young adults are prescribed systemic antibiotics at antimicrobial doses for acne therapy, and greater than 40% of these people receive minocycline therapy^23-25. The mean duration of systemic antibiotic therapy in adolescents and young adults has been reported to range from 4 - 11 months^23-26. The 100 mg/L minocycline hydrochloride drinking water concentration supported administering a 25 mg/kg murine daily dose, which is considered equivalent to a 2.0 mg/kg clinical daily dose^27-29. The 0.12% chlorhexidine gluconate oral antiseptic rinses were performed in minocycline-treated SPF mice to further perturb the oral microbiota.

qRT-PCR 16S rDNA Analysis:

Maxillary and mandibular gingiva and colon contents were harvested from each animal at sacrifice, flash-frozen, and stored at −80°C. Genomic DNA was extracted from gingival isolates and colon contents using the DNEasy Powersoil Pro Kit (Qiagen, Hilden, Germany). Total DNA was quantified via NanoDrop 1000 (Thermo Scientific, Waltham, MA, USA). Phylum level analysis was performed via qRT-PCR amplification of 16S rDNA target genes. A 20 μL PCR reaction was carried out using 10 μL of SYBR Green Fast Master Mix (Applied Biosystems, Foster City, CA, USA), 6.4 μL of forward/reverse primers (800 nM/μL) (Integrated DNA Technology, Coralville, IA, USA), and 3.6 μL of sample DNA (5 ng/μL)^30,31. A 30-cycle PCR reaction protocol was performed on the StepOnePlus System (Applied Biosystems); cycle number 25 was used as the cutoff for non-specific amplification of the Universal 16S gene^31,32. Initial denaturing step at 95°C for 5 minutes; 30 cycles of 95°C for 15 seconds, 61.5°C for 15 seconds, 72°C for 20 seconds; final elongation step of 72°C for 5 minutes^30,31. Samples were run in triplicate. Oral bacterial load was determined by normalizing the Universal 16S gene to a bacterial DNA standard (ZymoBIOMICS, Zymo Research, Irvine, CA), as previously described^31,33. Relative quantification of Universal 16S rDNA was carried out by the 2^−ΔC_T method³⁴. Phylum level outcomes are reported relative to the Universal 16S gene, as described previously³¹. Relative quantification of phylum-level rDNA was performed via the comparative C_T method (2^−ΔΔCT)³⁵. Integrated DNA Technologies forward (F) / reverse (R) primer sequences included:

Universal 16S³⁰: F=5’-AAACTCAAAKGAATTGACGG-3’; R=5’-CTCACRRCACGAGCTGAC-3’.

Proteobacteria³⁰: F=5’-TCGTCAGCTCGTGTYGTGA-3’; R=5’-CGTAAGGGCCATGATG-3’.

Actinobacteria³⁰: F=5’-TACGGCCGCAAGGCTA-3’; R=5’-TCRTCCCCACCTTCCTCCG-3’.

Bacteroidetes³⁰: F=5’-CRAACAGGATTAGATACCCT-3’; R=5’-GGTAAGGTTCCTCGCGTAT-3’.

Firmicutes³⁰: F=5’-TGAAACTYAAAGGAATTGACG-3’; R=5’-ACCATGCACCACCTGTC-3’.

Bacterial phyla examined (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes) in fecal specimens are the predominant bacterial phyla in the murine gut bacteriome^30,36. Bacterial phyla evaluated in gingival specimens (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes) are the predominant phyla in the murine oral bacteriome^37,38.

Micro-CT:

Maxillae, mandibles, and femurs were fixed in 10% neutral-buffered-formalin for twenty-four hours at room temperature and thereafter stored in 70% ethanol at 4°C. Maxilla specimens (Fig. 1) and femur specimens (Fig. 6) were micro-CT imaged via the Bruker Skyscan 1176 scanner (Skyscan Co, Belgium) with a 0.5 mm thick aluminum filter; X-ray tube potential (50 kVp); X-ray intensity (497 μA); Integration time (65 ms); Rotation step (0.3°); Isotropic voxel size (9 μm³). Three-dimensional images were reconstructed and a fixed threshold of 350 Hounsfield Units were used to discriminate mineralized tissue. Maxilla specimens (Fig. 2) and mandible specimens (Fig. 3) were scanned with the Scanco Medical μCT 40 scanner (Scanco Medical; Wangen-Brüttisellen, Switzerland): X-ray tube potential (70 kVp); X-ray intensity (114 μA); Integration time (200 ms); Isotropic voxel size (10 μm³). Three-dimensional images were reconstructed and a fixed threshold of 1250 Hounsfield units was used for mineralized tissue analysis. Reconstructed images were consistently oriented prior to analyzing with the AnalyzePro Analysis software (AnalyzeDirect, Seattle, WA, USA)^31,39. Data are reported in accordance with standardized nomenclature⁴⁰.

Figure 1. Systemic Antibiotic Therapy has Detrimental Effects on Alveolar Bone Homeostasis.

(a) Male C57BL/6T specific-pathogen-free (SPF) mice were administered vehicle (VEH), antibiotic cocktail (ABX), or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks; specimens were isolated for analyses. (b) 16S rDNA analysis in gingiva specimens evaluating oral bacterial load (n=4-5/gp). Bacterial load was determined by normalizing the Universal 16S gene to a bacterial DNA standard; relative quantification was performed by the 2^−ΔC_T method, (c-g) Alveolar bone loss was evaluated at the maxillary first molar by measuring the CEJ-ABC linear distance at the mesiobuccal, mid-lingual, and distobuccal line angles. The average alveolar bone loss was determined by averaging the CEJ-ABC distance at the mesiobuccal, mid-lingual, and distobuccal line angles. (c) Representative images of CEJ to ABC linear measurements (*green lines) at the mesiobuccal, mid-lingual, and distobuccal line angles; adjustments consistently applied across images from each group. Linear alveolar bone loss outcomes: (d) mesiobuccal, (e) mid-lingual, (f) distobuccal, (g) average (n=4-5/gp). One-way ANOVA with Holm-Sidak test; data reported as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001.

Figure 6. Antibiotic-Induced Gut Dysbiosis Impact on Osteoimmune Processes at Non-Oral Skeletal Sites.

Male C57BL/6T specific-pathogen-free mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks; specimens were isolated for analyses. 16S rDNA analysis in colon contents evaluating (a) gut bacterial load and (b) gut bacterial phyla communities (n=5-6/gp). Bacterial load was determined by normalizing the Universal 16S gene to a bacterial DNA standard; relative quantification was performed by the 2^−ΔC_T method. Phylum outcomes were determined by normalizing the phyla genes to the Universal 16S gene; relative quantification was performed via the 2^−ΔΔCT method. (c-e) Micro-CT analysis of cortical bone at the femur mid-diaphysis (n=5-6/gp); (c) Representative micro-CT images (adjustments consistently applied across images from each group); (d) quantitative analysis of cortical thickness (Ct.Th) and (e) cortical bone mineral density (Ct.BMD). (f-i) Histomorphometric analysis of TRAP+ osteoclast cellular endpoints in the proximal tibia; TRAP+ cells lining bone with ≥ 3 nuclei were designated as osteoclasts (n=4/gp). (f) Representative images of TRAP+ osteoclast cells lining bone in the proximal tibia; adjustments consistently applied across images from each group. (g) N.Oc/B.Pm = osteoclast number per bone perimeter. (h) Oc. Ar/Oc = average osteoclast area. (i) Oc.Pm/B.Pm = osteoclast perimeter per bone perimeter. (j,k) nCounter gene expression analysis was performed in femoral long bone marrow (LBM) to assess critical osteoclast signaling factors (n=4/gp): (j) Rankl and Opg mRNA; (K) Rankl:Opg ratio. Absolute quantification of mRNA was performed; data normalized to the geometric means of spiked-in positive controls, negative controls, and built-in internal control genes. (l-n) Flow cytometric analysis of CD4⁺ T-helper cell subsets in femoral LBM (n=4/gp). Quantitative analysis of (l) CD3⁺CD4⁺CD183⁺T-bet⁺ T_H1 cells, (m) CD3⁺CD4⁺RORγt⁺AHR⁻ T_H17 cells, (n) CD3⁺CD4⁺CD25⁺FoxP3⁺ T_REG cells; reported as % CD3⁺CD4⁺ cells. (o-q) Immunofluorescent analysis of osteoblasts in the secondary spongiosa of proximal tibia; DAPI+OCN+ cells lining bone were designated as osteoblasts (n=6/gp.) (o) Representative images of DAPI+OCN+ osteoblasts in proximal tibia sections (blue, DAPI; green, OCN – FITC). Arrows indicate dual-labeled osteoblasts. (p) N.Ob/B.Pm = osteoblast number per bone perimeter. (q) Ob.Pm/B.Pm = osteoblast perimeter per bone perimeter. Unpaired t-test; data are reported as mean ± SEM; *p<0.05 vs. VEH, **p<0.01 vs. VEH.

Figure 2. Antimicrobial-Induced Oral Dysbiosis Exacerbates Naturally Occurring Alveolar Bone Loss.

(a) Male C57BL/6T (specific-pathogen-free) SPF and germ-free (GF) mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Minocycline-treated SPF mice also received chlorhexidine oral antiseptic rinses (MINO & CHX) from age 6- to 12-weeks to further perturb the oral microbiota. Animals were euthanized at age 12-weeks; specimens were isolated for analyses. 16S rDNA analysis in gingiva specimens evaluating (b) oral bacterial load and (c) oral bacterial phyla communities (n=6/gp). Bacterial load was determined by normalizing the Universal 16S gene to a bacterial DNA standard; relative quantification was performed by the 2^−ΔC_T method. Phylum outcomes were determined by normalizing the phyla genes to the Universal 16S gene; relative quantification was performed via the 2^−ΔΔCT method. (d-h) Alveolar bone loss was evaluated at the maxillary first molar by measuring the CEJ-ABC linear distance at the mesiobuccal, mid-lingual, and distobuccal line angles. The average alveolar bone loss was determined by averaging the CEJ-ABC distance at the mesiobuccal, mid-lingual, distobuccal line angles. (d) Representative images of CEJ to ABC linear measurements (*blue lines) at the mesiobuccal, mid-lingual, and distobuccal line angles; adjustments consistently applied across images from each group. Alveolar bone loss outcomes: (e) mesiobuccal, (f) mid-lingual, (g) distobuccal, (h) average (n=6/gp). One-way ANOVA with Tukey test; data reported as mean ± SEM; *p<0.05, **p<0.01, ***p<0.001.

Figure 3. Antibiotic Disruption of the Commensal Microbiota Reduces Cortical Thickness and Enhances Osteoclastogenesis in Alveolar Bone.

Male C57BL/6T specific-pathogen-free mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks; specimens were isolated for analyses. (a-c) Micro-CT analysis of alveolar bone cortical thickness and bone mineral density in the mandible (n=6/gp) (a) Representative micro-CT images of buccal cortical bone (*perpendicular red lines) and lingual cortical bone (*perpendicular blue lines) in the mandibular first molar furcation. (b) Quantitative alveolar bone cortical thickness (Ct.Th) outcomes at the buccal and lingual cortical plates in the furcation of the mandibular first molar. (c) Quantitative cortical bone mineral density (Ct.BMD) findings in the alveolar bone of the mandibular first molar. (d-g) Histomorphometric analysis of TRAP+ osteoclast cellular endpoints in the alveolar bone of the maxillary first molar furcation; TRAP+ cells lining bone with ≥ 3 nuclei were designated as osteoclasts (n=4-6/gp). (d) Representative images of TRAP+ osteoclast cells lining bone; adjustments consistently applied across images from each group. (e) N.Oc/B.Pm = osteoclast number per bone perimeter. (f) Oc. Ar/Oc = average osteoclast area. (g) Oc.Pm/B.Pm = osteoclast perimeter per bone perimeter. (h,i) qRT-PCR gene expression analysis was performed in mandible bone marrow (MBM) to assess critical osteoclast signaling factors (n=5-6/gp): (h) Rankl and Opg mRNA; (i) Rankl:Opg ratio. Relative quantification of mRNA was performed via the 2^−ΔΔCT method; Gapdh was utilized as an internal control gene. (j-l) Immunofluorescent analysis of osteoblasts in the maxillary first molar furcation; DAPI+OCN+ cells lining bone were designated as osteoblasts (n=6/gp.) (j) Representative images of DAPI+OCN+ osteoblasts in the maxillary first molar furcation (blue, DAPI; green, OCN – FITC). Arrows indicate dual-labeled osteoblasts. (k) N.Ob/B.Pm = osteoblast number per bone perimeter. (l) Ob.Pm/B.Pm = osteoblast perimeter per bone perimeter. Unpaired t-test; data are reported as mean ± SEM; *p<0.05 vs. VEH, **p<0.01 vs. VEH, ***p<0.001 vs. VEH.

Alveolar bone loss was evaluated at the maxillary first molar by measuring the linear distance from the cementoenamel junction (CEJ) to the alveolar bone crest (ABC). The measurement began at the CEJ, the anatomical site where the enamel meets the cementum, and ended at the ABC, the anatomical site where the cortical plates merge with the alveolar bone proper. The coronal height of contour was determined at three anatomical sites of interest, which included the mid-lingual, mesiobuccal, and distobuccal line angles. The coronal height of contour served as the midpoint for carrying out CEJ-ABC linear measurements at each anatomical site of interest. Initial measurement was performed at the mid-point, and measurements were then carried out 10 and 20pm distal and mesial to the midpoint (5 measurements per line angle).

Alveolar bone cortical thickness was assessed in the bifurcation of the mandibular first molar. Separate analyses were carried out for the buccal and lingual cortical plates. The ROI was centered at the midpoint between the mesial and distal roots. Cortical thickness was assessed in a 200 μm region of interest, which included evaluating a 100 μm region mesial to the midpoint and a 100 μm region distal to the midpoint. Buccal cortical plate measurements were performed via drawing a perpendicular line from the endocortical surface to the periosteal surface of the buccal cortical plate. Lingual cortical plate measurements were performed via drawing a perpendicular line from the endocortical surface to the periosteal surface of the lingual cortical plate.

Alveolar bone mineral density was evaluated at the mandibular first molar. First, the ROI was defined by morphing a cylinder at the mesial root of the mandibular first molar, which included the buccal cortical plate, lingual cortical plate, and the alveolar bone proper. Next, the root was virtually removed / extracted from the alveolus by segmenting at the periodontal ligament space. Analyze 12.0 Bone Microarchitecture Analysis software (AnalyzeDirect) was then used to measure the bone mineral density in a region extending from the alveolar bone crest to 400 μm apically.

Femur cortical thickness and bone mineral density were assessed using Analyze 12.0 Bone Microarchitecture Analysis software (AnalyzeDirect). Transverse CT slices were analyzed in a 500 μm segment of the femur mid-diaphysis.

Histomorphometry:

Maxillae and tibiae were fixed in 10% neutral-buffered-formalin for twenty-four hours at room temperature, stored in 70% ethanol at 4°C, decalcified in 14% ethylenediaminetetraacetic acid for 21 days, and then processed for paraffin histology. Sagittal sections were cut through the maxillary first molar and frontal sections were cut through the proximal tibia. Tartrate-resistant acid phosphatase (TRAP) stain^8,31 was performed for histomorphometric analysis of osteoclasts in the maxillary first molar furcation alveolar bone and proximal tibia. TRAP+ multinucleated (≥ 3 nuclei) cells lining bone were considered osteoclasts. Images were acquired at 200X via the Nikon Eclipse TS1000 microscope (Nikon, Melville, NY, USA). Images were stitched using Adobe Photoshop (Adobe, San Jose, CA, USA) and scored via ImageJ software (ImageJ 1.52a, NIH, Bethesda, MD, USA). Blinded histomorphometric analysis of TRAP+ osteoclast cellular endpoints was performed by two independent investigators. Data are reported in accordance with standardized nomenclature⁴¹.

Immunofluorescence:

Osteoblast immunofluorescence analysis was performed in paraffin-embedded sagittal sections of the maxillary first molar and frontal sections of proximal tibia. Samples were deparaffinized with xylenes, rehydrated with graded ethanols, and then briefly washed. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at 100°C for 2 minutes. Samples were cooled to room temperature and washed in deionized water. Specimens were permeabilized in 1x PBS containing 0.2% Triton X-100 and blocked in 10% goat serum for one hour at room temperature. Specimens were then incubated with 1:100 dilution of anti-osteocalcin (OCN) polyclonal antibody (Millipore Sigma, Burlington, MA, USA) overnight at 4°C. Sections were washed in 1X PBS and then incubated with a 1:2000 FITC-goat anti-rabbit (Santa Cruz Biotechnology, Dallas, TX, USA) for one hour at room temperature (protected from light). Samples were washed in 1X PBS and mounted via ProLong Diamond Antifade Mountant with DAPI (Life Technologies, Carlsbad, CA, USA). Images were acquired at 200X with a Keyence BZ-X810 fluorescence microscope. Images were stitched and overlaid with the Keyence BZ-X Analysis Software (Keyence, Osaka, Japan). Analysis of OCN+DAPI+ osteoblastic cells lining bone were evaluated in the maxillary first molar furcation alveolar bone. In the proximal tibia, OCN+DAPI+ osteoblastic cells lining trabecular bone were analyzed. Analysis was limited to the secondary spongiosa, beginning 250 μm distal to the growth plate and extended distally 1000 μm (50 μm from endocortical surfaces). Images were scored using ImageJ software version 1.52a.

qRT-PCR mRNA Analysis:

Mandible bone marrow and mandibular gingiva were isolated from each animal at sacrifice. Mandible bone marrow was flushed with TRIzol (Invitrogen, Carlsbad, CA, USA) and stored at −80°C. Mandibular bone marrow and gingival RNA extraction was performed using the TRIzol method, as previously reported^31,39. Total RNA was quantified via NanoDrop 1000 (Thermo Scientific). cDNA was synthesized from RNA using Taqman Random Hexamers and Reverse Transcription Reagents (Applied Biosystems), according to the manufacturer’s protocol^31,39. cDNA was amplified via TaqMan Fast Advanced Master Mix and TaqMan primer-probes on the StepOnePlus System (Applied Biosystems), per the manufacturer’s instructions^31,39. A 20 μL PCR reaction was performed using 10 μL of Taqman Fast Advanced Master Mix (2x), 1 μL of primer-probe (20x), 2 μL of sample cDNA (10x), and 7 μL of nuclease-free water. Thermocycler protocol was 50°C for 2 minutes, 95°C for 2 minutes, followed by 40 cycles of 95°C for 1 second, 60°C for 20 seconds. Relative quantification of mRNA was performed via the comparative C_T method (2^−ΔΔCT)³⁵; Gapdh was utilized as an internal control gene. TaqMan primer-probe sequences included: Tnfsf11 (Rankl) = Mm00441908_m1; Tnfrsf11b (Opg) = Mm00435451_m1; Il12 = Mm00434174_m1; Il6 = Mm00446190_m1; Gapdh = Mm99999915_g1.

nCounter Gene Expression.

Femur bone marrow was isolated from each animal at sacrifice. Femur marrow was flushed with TRIzol (Invitrogen); RNA extracted following the manufacturer’s protocol^31,39. Total RNA was quantified by NanoDrop 1000 for gene expression analysis by nCounter Mouse PanCancer Panel (NanoString Technologies, Seattle, WA, USA). The nCounter gene expression platform is a multiplexed probe detection system, that relies on a probe library constructed with two sequence-specific probes for each gene of interest^42,43. According to the manufacturer protocol, RNA specimens were hybridized and products were analyzed on the nCounter preparation station. Data analysis was performed using nSolver v2.6 analysis software (NanoString Technologies). Absolute quantification of mRNA reported with data normalized to the geometric means of spiked-in positive controls, negative controls, and built-in internal control genes, as described previously^31,39.

Flow Cytometry:

Live Cell Analysis:

Cervical lymph node cells were isolated, washed, counted, and resuspended at 100,000 cells in 50 μL of flow cytometry buffer. Cells were treated with FcR-block (Miltenyi Biotec, Bergisch Gladbach, Germany) and labeled for surface markers via antibodies, per the manufacturer’s instructions^31,39. Dead cells were excluded from analysis by labeling with propidium iodide viability dye (Miltenyi Biotec), per the manufacturer’s instructions^31,39. Neutrophils: anti-CD11b-APC (Miltenyi Biotec; clone REA592), anti-Ly6C-FITC (Miltenyi Biotec; clone REA796), anti-Ly6G-VioBlue (Miltenyi Biotec; clone 1A8). M1-macrophages: anti-CD11b-APC (Miltenyi Biotec; clone REA592), anti-MHC II-FITC (Miltenyi Biotec; clone REA528), anti-CD206-PE (Miltenyi Biotec; clone MR6F3), anti-CD64-APC-Vio770 (Miltenyi Biotec; clone REA286). Plasmacytoid dendritic cells: anti-CD11c-PE-Vio770 (Miltenyi Biotec; clone REA754), anti-MHC II-FITC (Miltenyi Biotec; clone REA528), anti-B220-VioBlue (Miltenyi Biotec; clone REA755). A minimum of 5,000 gated cells were analyzed per specimen. Data was acquired by the MACSQuant System (Miltenyi Biotec) and analyses were performed via FlowJo 11.0 software (TreeStar, Ashland, OR, USA), as previously reported^31,39.

Transcription Factor Analysis:

Cervical lymph node cells, mandible bone marrow cells, and femur bone marrow cells were isolated, washed, counted, and resuspended at 100,000 cells in 50 μL of flow cytometry buffer. Cells were treated with FcR-block (Miltenyi Biotec) and labeled for surface markers via antibodies, per the manufacturer’s instructions^31,39. Dead cells were excluded from analysis by labeling with eFluor 780 viability dye (eBioscience, Santa Clara, CA, USA), per the manufacturer’s instructions^31,39. Intracellular stains for transcription factors were carried out after fixing-permeabilizing per the fixation-permeabilization buffer manufacturer’s protocol (eBioscience), as previously reported^31,39. T_H1 cells: anti-CD3-PE-Vio770 (Miltenyi Biotec; clone REA641), anti-CD4-FITC (Miltenyi Biotec; clone REA604), anti-CD183-PE (Miltenyi Biotec; clone CXCR3-173), anti-T-bet-APC (Miltenyi Biotec; clone REA102). T_H17 cells: anti-CD3-APC-Vio770 (Miltenyi Biotec; clone REA641), anti-CD4-FITC (Miltenyi Biotec; clone REA604), anti-RORγt-APC (Miltenyi Biotec; clone REA278), anti-AHR-PE-Vio770 (eBioscience; clone 4MEJJ). T_REG cells: anti-CD3-APC-Vio770 (Miltenyi Biotec; clone REA641), anti-CD4-FITC (Miltenyi Biotec; clone REA604), anti-CD25-PE-Vio770 (Miltenyi Biotec; clone 7D4), anti-FoxP3-PE (Miltenyi Biotec; clone REA788). A minimum of 5,000 gated cells were analyzed per specimen. Data was acquired by the MACSQuant System (Miltenyi Biotec) and analyses were performed via FlowJo 11.0 software (TreeStar), as previously reported^31,39.

Statistical Analysis:

One-way ANOVA, with Holm-Sidak post-hoc test, was used to assess changes in linear alveolar bone loss and the oral microbiome of male SPF mice treated with vehicle, minocycline, or antibiotic cocktail (Fig. 1). One-way ANOVA, with Tukey post-hoc test, was employed to evaluate alterations in linear alveolar bone loss and the oral microbiome of male GF and SPF mice treated with vehicle, minocycline, or minocycline & chlorhexidine (Fig. 2). Unpaired t-tests were performed to assess alterations in the oral microbiome and alveolar bone loss in male SPF mice subjected to vehicle or vehicle & chlorhexidine (Fig. S2). Unpaired t-tests were carried out to assess differences in mandible, femur, osteoclastogenesis, osteoblastogenesis, innate immune cells, adaptive immune cells, and inflammatory markers in gingiva of male SPF mice subjected to vehicle vs. minocycline treatment (Figs. 3-6, S3). Unpaired t-tests were used to assess changes in linear alveolar bone loss and the oral microbiome of female SPF mice treated with vehicle or minocycline, and euthanized at age 12-weeks. Unpaired t-tests were performed to assess changes in linear alveolar bone loss and the oral microbiome of female SPF mice treated with vehicle or minocycline, and euthanized at age 18-weeks (Fig. 7, S4). Data was analyzed using GraphPad Prism 9.0 (GraphPad, La Jolla, CA, USA). Data are presented as mean ± standard error of the mean (SEM). Significance is denoted as *p<0.05, **p<0.01, ***p<0.001. Power analysis was carried out with the Biostatistical Unit of the MUSC Bioinformatics Core.

Figure 7. Antibiotic Therapy has Lasting Detrimental Effects on the Oral Microbiome and Alveolar Bone Homeostasis.

(a) Female C57BL/6T specific-pathogen-free (SPF) mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks and age 18-weeks; specimens were isolated for analyses. 16S rDNA analysis in gingiva specimens evaluating oral bacterial phyla communities in (b) 12-week-old female SPF mice (n=6/gp) and (c) 18-week-old female SPF mice (n=6/gp). Phylum outcomes were determined by normalizing the phyla genes to the Universal 16S gene; relative quantification was performed via the 2^−ΔΔCT method. Alveolar bone loss was evaluated at the maxillary first molar by measuring the CEJ-ABC linear distance at the mesiobuccal, mid- lingual, and distobuccal line angles (d-h) 12-week-old female SPF mice (n=6/gp) and (i-m) 18-week-old female SPF mice (n=6/gp). The average alveolar bone loss was determined by averaging the CEJ-ABC distance at the mesiobuccal, mid-lingual, distobuccal line angles. (d,i) Representative images of CEJ to ABC linear measurements (*purple lines) at the mesiobuccal, mid-lingual, and distobuccal line angles; adjustments consistently applied across images from each group. Alveolar bone loss outcomes: (e,j) mesiobuccal, (f,k) mid- lingual, (g,l) distobuccal, (h,m) average. Unpaired t-test; data are reported as mean ± SEM; *p<0.05 vs. VEH, **p<0.01 vs. VEH.

RESULTS

Systemic Antibiotic Therapy has Detrimental Effects on Alveolar Bone Homeostasis.

Male SPF mice were subjected to systemic treatment with vehicle-control, an experimental antibiotic cocktail, or minocycline at a clinically relevant dose from age 6- to 12-weeks. Animals were euthanized at age 12-weeks to determine whether systemic antibiotics could impact alveolar bone homeostasis (Fig. 1a). 16S rDNA analysis in gingival specimens showed that the oral bacterial load was reduced by systemic antibiotic therapy (Fig. 1b). Compared to vehicle-control, oral bacterial load was decreased in SPF mice subjected to treatment with antibiotic cocktail (mean diff. = 30.21; P < 0.0001) and minocycline (mean diff. = 13.15; P = 0.0034). Antibiotic cocktail suppressed the oral bacterial load more substantially than minocycline treatment (mean diff. = 17.06; P = 0.0007).

Micro-CT analysis evaluating the linear distance from the cementoenamel junction (CEJ) to alveolar bone crest (ABC) was carried out at the maxillary first molar to investigate differences in alveolar bone loss (Fig. 1c-g). CEJ to ABC measurements were performed at the mesiobuccal (Fig. 1c,d), mid-lingual (Fig. 1c,e), and distobuccal (Fig. 1c,f) line angles of the maxillary first molar, and these values were used to determine the average alveolar bone loss at the maxillary first molar (Fig. 1c,g). Antibiotic cocktail vs. vehicle-control treated SPF mice had greater average alveolar bone loss (mean diff. = 28.21; P = 0.0004) (Fig. 1g), which was attributed to increased alveolar bone loss at the mesiobuccal (mean diff. = 38.07; P = 0.0099) (Fig. 1d), mid-lingual (mean diff. = 21.04; P = 0.0467) (Fig. 1e), and distobuccal (mean diff. = 25.53; P = 0.0406) (Fig. 1f) line angles of the maxillary first molar. Minocycline vs. vehicle-control treated SPF mice also had greater average alveolar bone loss (mean diff. = 59.04; P < 0.0001) (Fig. 1g), which was due to increased alveolar bone loss at the mesiobuccal (mean diff. = 65.56; P = 0.0003) (Fig. 1d), mid-lingual (mean diff. = 46.06; P = 0.0014) (Fig. 1e), and distobuccal (mean diff. = 65.49; P = 0.0003) (Fig. 1f) line angles of the maxillary first molar. Minocycline caused more profound average alveolar bone loss than antibiotic cocktail (mean diff. = 30.82; P = 0.0002) (Fig. 1c,g), which was attributed to more severe alveolar bone loss at the mesiobuccal (mean diff. = 27.49; P = 0.0215) (Fig. 1d), mid-lingual (mean diff. = 25.02; P = 0.0328) (Fig. 1e), and distobuccal (mean diff. = 39.97; P = 0.0054) (Fig. 1f) line angles of the maxillary first molar. Based on the findings that minocycline therapy caused more profound alveolar bone loss than antibiotic cocktail treatment, subsequent antibiotic treatment studies were centered on investigating minocycline treatment effects.

Antimicrobial-Induced Oral Dysbiosis Exacerbates Naturally Occurring Alveolar Bone Loss.

Vehicle-control or minocycline therapy was then administered to male mice reared under both SPF and GF conditions, from age 6- to 12-weeks. In addition, we subjected minocycline-treated male SPF mice to twice daily chlorhexidine oral antiseptic rinses from age 6- to 12-weeks (Fig. 2a). The rationale for administering minocycline to mice reared under GF conditions was to discern whether minocycline effects on alveolar bone are dependent on the microbiota. The basis for performing the chlorhexidine oral antiseptic rinses was to further disrupt the perturbed oral microbiota in minocycline-treated SPF mice, as a means to validate that antimicrobial-induced oral dysbiosis has deleterious effects on alveolar bone.

Systemic minocycline vs. vehicle-control treatment reduced the bacterial load (mean diff. = 14.02; 95% C.I. = 0.5938 to 27.45; P = 0.0402 (Fig. 2b) and suppressed Proteobacteria (mean diff. = 0.6752; C.I. = 0.2557 to 1.095; P = 0.0022) and Firmicutes (mean diff. = 0.5724; C.I. = 0.3269 to 0.8180; P < 0.0001) (Fig. 2c) in the oral bacteriome of 12-week-old SPF mice. Chlorhexidine oral antiseptic rinses induced further perturbations in the oral bacteriome of minocycline-treated SPF mice, which included suppression of Actinobacteria (mean diff. = 0.4244; C.I. = 0.01632 to 0.8325; P = 0.0410) (Fig. 2c). These data support that chlorhexidine rinses further perturb the microbiota composition in minocycline-treated SPF mice.

Average alveolar bone loss (mean diff. = 36.12; C.I. = 28.00 to 44.23; P < 0.0001) (Fig. 2h) was greater in vehicle-treated SPF vs. vehicle-treated GF mice, due to increased alveolar bone loss at the mesiobuccal (mean diff. = 35.67; C.I. = 23.58 to 47.76; P < 0.0001) (Fig. 2e), mid-lingual (mean diff. = 27.55; C.I. = 17.53 to 37.58; P < 0.0001) (Fig. 2f), and distobuccal (mean diff. = 45.10; C.I. = 27.43 to 62.78; P < 0.0001) (Fig. 2g) line angles of the maxillary first molar. These findings validate outcomes from prior investigations comparing SPF vs. GF mice, and underscores that the commensal microbiota drives naturally occurring alveolar bone loss^6-8. Alveolar bone loss outcomes were similar in minocycline-treated GF mice vs. vehicle-treated GF mice (Fig. 2d-h), which implies that antimicrobial-induced alveolar bone loss is microbiota dependent.

Minocycline-treated SPF mice vs. vehicle-treated SPF mice had greater average alveolar bone loss (mean diff. = 38.79; C.I. = 46.91 to 30.67; P < 0.0001) (Fig. 2h), which was attributed to increased alveolar bone loss at the mesiobuccal (mean diff. = 62.29; C.I. = 74.38 to 50.20; P < 0.0001) (Fig. 2e), mid-lingual (mean diff. = 15.79; C.I. = 25.81 to 5.758; P = 0.0009) (Fig. 2f), and distobuccal (mean diff. = 38.28; C.I. = 55.95 to 20.60; P < 0.0001) (Fig. 2g) line angles of the maxillary first molar. Chlorhexidine oral rinse enhanced the average alveolar bone loss in minocycline-treated SPF mice (mean diff. = 32.85; C.I. = 40.96 to 24.73; P < 0.0001) (Fig. 2h), due to causing more severe alveolar bone loss at the mesiobuccal (mean diff. = 13.63; C.I. = 25.72 to 1.538; P = 0.0216) (Fig. 2e), mid-lingual (mean diff. = 55.88; C.I. = 65.91 to 45.85; P < 0.0001) (Fig. 2f), and distobuccal (mean diff. = 29.03; C.I. = 46.70 to 11.35; P = 0.0005) (Fig. 2g) line angles of the maxillary first molar. Study findings showing that chlorhexidine oral antiseptic rinses induced further shifts in the oral bacteriome (Fig. 2c) and enhanced the alveolar bone loss phenotype in minocycline-treated SPF mice (Fig. 2d-h), imply that antimicrobial-induced oral dysbiosis exacerbates naturally occurring alveolar bone loss.

Subsequent studies were carried out in which we subjected male SPF mice to vehicle treatment or vehicle treatment with twice daily chlorhexidine oral antiseptic rinses from age 6- to 12-weeks. Studies were performed to determine the impact of chlorhexidine oral antiseptic rinse on the healthy, stable commensal oral microbiota and alveolar bone homeostasis. Chlorhexidine suppressed the oral bacterial load in vehicle-treated SPF mice (Fig. S2b) which was attributed to the reduced presence of Actinobacteria and Firmicutes (Fig. S2c). Chlorhexidine rinses attenuated the average alveolar bone loss in vehicle-treated SPF mice (Fig. S2h), which was attributed to significantly less alveolar bone loss at the mid-lingual line angle (Fig. S2f) and a trending decrease in alveolar bone loss at the distobuccal line angle (P < 0.07) (Fig. S2g) of the maxillary first molar. Outcomes from chlorhexidine rinses in vehicle-treated SPF mice imply that local antiseptic depletion of the stable commensal oral microbiota protects against naturally occurring alveolar bone loss.

Antibiotic Disruption of the Commensal Microbiota Reduces Cortical Thickness and Enhances Osteoclastogenesis in Alveolar Bone.

Linear alveolar bone loss has been attributed to changes in alveolar bone thickness and bone mineral density^44-46. Therefore, micro-CT was employed to evaluate these parameters in the alveolar bone at the mandibular first molar bifurcation of 12-week-old minocycline- vs. vehicle-treated male SPF mice (Fig. 3a-c). Minocycline treatment decreased the cortical thickness (Fig. 3a,b), but did not alter the cortical bone mineral density of the alveolar bone (Fig. 3c).

Prior research has shown that periodontal bone loss is driven by enhanced osteoclastogenesis^47,48. Therefore, alterations in bone lining osteoclastic cells (Fig. 3d-g) and differences in critical osteoclast signaling factors (Fig. 3h,i) were evaluated in the alveolar bone of 12-week-old minocycline- vs. vehicle-treated male SPF mice. Histomorphometric analysis of TRAP+ osteoclastic cells discerned that minocycline treatment increased the osteoclast cell size (Fig. 3d,f) and interface with alveolar bone at the maxillary first molar (Fig. 3d,g). qRT-PCR studies delineated that minocycline treatment decreased the expression of receptor activator of nuclear factor-κB ligand (Rankl) (Fig. 4h) and osteoprotegerin (Opg) (Fig. 3h) in the mandible alveolar bone marrow of male SPF mice. However, there were no differences in the Rankl:Opg ratio (Fig. 3i). Considering the RANKL:OPG ratio determines the availability of free RANKL to bind the RANK receptor^47,48, it does not appear that the pro-osteoclastic phenotype found in minocycline-treated male SPF mice is due to alterations in RANKL signaling.

Figure 4. Antibiotic Perturbation of the Commensal Microbiota Induces a Proinflammatory Innate Immune Response in Cervical Lymph Nodes.

Male C57BL/6T specific-pathogen-free mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks; cells were isolated from cervical lymph nodes (CLNs) for flow cytometric analyses (n=4/gp). (a) Representative dot plots and (b) quantitative analysis of CD11b⁺Ly6C⁻Ly6G⁺ neutrophils; reported as % CD11b⁺ cells. (c) Representative dot plots and (d) quantitative analysis of CD11b⁺MHC II⁺CD206⁻CD64⁺M1-macrophages; reported as % CD11b⁺ cells. (e) Representative dot plots and (f) quantitative analysis of CD11c⁺B220⁺MHC II^lo plasmacytoid dendritic cells; reported as % CD11c⁺ cells. Unpaired t-test; data are reported as mean ± SEM; *p<0.05 vs. VEH, **p<0.01 vs. VEH.

Prior studies have shown that suppressed osteoblastogenesis contributes to periodontal bone loss^49-52. Therefore, alterations were assessed in bone-lining osteoblastic cells in alveolar bone of 12-week-old minocycline vs. vehicle-treated male SPF mice (Fig. 3j-l). In situ immunofluorescence analysis of osteocalcin-positive (*marker for mature, bone-forming osteoblastic cells) bone-lining cells revealed that minocycline treatment did not alter osteoblast cell numbers (Fig. 3j,k) or interface with the alveolar bone (Fig. 3j,l) at the maxillary first molar. This implies that minocycline-induced alveolar bone loss is due to enhanced osteoclastogenesis.

Antibiotic Perturbation of the Commensal Microbiota Induces a Dysbiotic Proinflammatory Immune Response State in Cervical Lymph Nodes and Alveolar Bone Marrow.

Dysbiotic shifts in the oral microbiota stimulate the periodontal innate immune response^1,9,17. Innate immune cells influence osteoclast actions and alveolar bone homeostasis through the secretion of local signaling factors and antigen presentation processes^53,54. Therefore, alterations in innate immune response mechanisms were evaluated in the oral draining cervical lymph nodes of 12-week-old minocycline- vs. vehicle-treated male SPF mice (Fig. 4).

Oral draining cervical lymph node cells were isolated for flow cytometric analysis. Neutrophils, M1-macrophages, and plasmacytoid dendritic cells were targeted based on the intent to evaluate proinflammatory innate immune cells. Neutrophils, considered the initial line of the periodontal innate immune defense^2,55, were substantially upregulated in the cervical lymph nodes of minocycline- vs. vehicle-treated SPF mice (Fig. 4a,b). Minocycline treatment induced a trend towards increased M1-macrophages (P < 0.1) (Fig. 4c,d) and significantly increased the frequency of plasmacytoid dendritic cells (Fig. 4e,f) in the cervical lymph nodes. Whereas M1-macrophages and plasmacytoid dendritic cells are professional antigen-presenting cells^53,54, neutrophils can also present antigens that induce T-cell mediated immunity^56,57. These cells sample microbial antigens in mucosal barrier tissues and then migrate to draining lymph nodes where they present these antigens to direct T-cell mediated immunity^53,54,58,59.

In light of the increased frequency of antigen-presenting cells found in the cervical lymph nodes (Fig.4), alterations in CD4⁺ T-helper cell subsets were evaluated in 12-week-old minocycline- vs. vehicle-treated male SPF mice (Fig.5). T_H1, T_H17, and T_REG cells were targeted, since these cell populations have been shown to regulate osteoclast actions and alveolar bone homeostasis^2,9. Recognizing that antigen presentation in the lymph nodes drives the differentiation of T-helper cells that migrate to the periodontium to mount an immune defense^53,54, flow cytometric analysis was carried out in both the cervical lymph nodes and mandible alveolar bone marrow. T_H1 and T_H17 cells were increased more than 3X fold in the cervical lymph nodes (Fig. 5a-d) and roughly 2X fold in the mandible alveolar bone marrow (Fig. 5g-j) of minocycline- vs. vehicle-treated SPF mice. Minocycline treatment did not alter the frequency of T_REG cells in the cervical lymph nodes (Fig. 5e,f) or mandible alveolar bone marrow (Fig. 5k,l).

Figure 5. Antibiotic Disruption of the Commensal Microbiota Upregulates a Proinflammatory Helper T-cell Profile in Cervical Lymph Nodes and Alveolar Bone.

Male C57BL/6T specific-pathogen-free mice were administered vehicle (VEH) or minocycline (MINO) from age 6- to 12-weeks. Animals were euthanized at age 12-weeks; cells were isolated from cervical lymph nodes (CLNs) and mandible bone marrow (MBM) for flow cytometric analyses. (a-f) Flow cytometric analysis of CD4⁺ T-helper cell subsets in CLNs (n=4/gp). (a) Representative dot plots and (b) quantitative analysis of CD3⁺CD4⁺CD183⁺T-bet⁺ T_H1 cells; reported as % CD3⁺CD4⁺ cells. (c) Representative dot plots and (d) quantitative analysis of CD3⁺CD4⁺RORγt⁺AHR⁻ T_H17 cells; reported as % CD3⁺CD4⁺ cells. (e) Representative dot plots and (f) quantitative analysis of CD3⁺CD4⁺CD25⁺FoxP3⁺ T_REG cells; reported as % CD3⁺CD4⁺ cells. (g-l) Flow cytometric analysis of CD4⁺ T-helper cell subsets in MBM (n=4/gp). (g) Representative dot plots and (h) quantitative analysis of CD3⁺CD4⁺CD183⁺T-bet⁺ T_H1 cells; reported as % CD3⁺CD4⁺ cells. (i) Representative dot plots and (j) quantitative analysis of CD3⁺CD4⁺RORγt⁺AHR⁻ T_H17 cells; reported as % CD3⁺CD4⁺ cells. (k) Representative dot plots and (l) quantitative analysis of CD3⁺CD4⁺CD25⁺FoxP3⁺ T_REG cells; reported as % CD3⁺CD4⁺ cells. Unpaired t-test; data are reported as mean ± SEM; *p<0.05 vs. VEH, **p<0.01 vs. VEH, ***p<0.001 vs. VEH.

Appreciating that minocycline treatment induced a pro-inflammatory immune response state in oral-draining cervical lymph nodes and the mandible bone marrow, gene expression studies were carried out to assess pro-inflammatory mediators in mandibular gingiva⁶⁰. Tumor necrosis factor (Tnf) (Fig. S3a) and interleukin 1 beta (Il1b) (Fig. S3b) were similar in vehicle- vs. minocycline-treated male SPF mice. However, minocycline treatment induced a trending 3.5-fold increase in interleukin 6 (Il6) (P < 0.08) (Fig. S3c) and a trending 2.5 fold increase in interleukin 12 (Il12) (P < 0.07) (Fig. S3d).

Antibiotic-Induced Gut Dysbiosis Impact on Osteoimmune Processes at Non-Oral Skeletal Sites.

Systemic minocycline administration has been shown to cause disturbances across the human oral and gut microbiome communities^12,61. As a means to delineate whether antibiotic-induced changes in the gut microbiota could be contributing to the pro-inflammatory osteolytic phenotype found in the alveolar bone of minocycline-treated male SPF mice, we investigated minocycline effects on the gut microbiome and osteoimmune processes at non-oral skeletal sites. Administering systemic minocycline vs. vehicle-control from age 6- to 12-weeks caused a trend towards reduced bacterial load (P < 0.1) (Fig. 6a) and significantly suppressed Actinobacteria and Proteobacteria (Fig. 6b) in the gut bacteriome of 12-week-old male SPF mice. Minocycline-induced perturbations in the gut microbiome were not associated with changes in cortical thickness (Fig. 6c,d) or bone mineral density (Fig. 6c,e) in the femur. Histomorphometric analysis of TRAP+ osteoclastic cells in tibia showed that minocycline therapy did not influence osteoclast cell numbers (Fig. 6f,g), cell size (Fig. 6f,h), or interface with bone (Fig. 6f,i) at non-oral skeletal sites. Moreover, nCounter gene expression studies in femur bone marrow delineated that Rankl and Opg expression (Fig. 6j) were similar and there were no differences in the Rankl:Opg ratio (Fig. 6k) of minocycline- vs. vehicle-treated male SPF mice. Flow cytometric analysis revealed that minocycline treatment blunted the frequency of T_H17 cells (Fig. 6m) and T_REG cells (Fig. 6n) in the femur marrow, which suggests that minocycline has immunosuppressive actions at non-oral skeletal sites. Analysis of OCN+ osteoblastic cells in the proximal tibia demonstrated that minocycline therapy did not alter osteoblast cell numbers (Fig.6o,p) or interface with bone (Fig. 6o,q).

Antibiotic Therapy has Lasting Detrimental Effects on the Oral Microbiome and Alveolar Bone Homeostasis.

To discern whether minocycline effects on the oral microbiome and alveolar bone homeostasis were sex dependent, female SPF mice were subjected to systemic minocycline treatment from age 6- to 12-weeks. Experimental animals were euthanized at age 12-weeks to assess immediate minocycline treatment effects (Fig. 7a, S4a). A separate group of experimental animals were euthanized at age 18-weeks, 6 weeks after the cessation of antibiotic therapy, to evaluate the resiliency of the oral microbiome and sustained effects on alveolar bone homeostasis (Fig. 7a, S4a).

Minocycline treatment did not alter the bacterial load in female SPF mice at age 12-weeks (Fig. S4b) or age 18-weeks (Fig. S4c), however phylum level shifts were present in the oral bacteriome. Proteobacteria and Actinobacteria were upregulated in minocycline- vs. vehicle-treated female SPF mice at age 12-weeks (Fig. 7b). At age 18-weeks, six weeks following the termination of antibiotic therapy, Actinobacteria became more prominent, Firmicutes was trending downwards (P < 0.06), and Proteobacteria was suppressed in minocycline-treated female SPF mice (Fig. 7c).

Consistent with outcomes in male SPF mice (Fig. 2), average alveolar bone loss was greater in minocycline vs. vehicle treated female SPF mice at age 12-weeks (Fig. 7h), which was attributed to increased alveolar bone loss at the mesiobuccal (Fig. 7e) and mid-lingual (Fig. 7f) line angles of the maxillary first molar. Six weeks following the termination of antibiotic therapy, the reduced alveolar bone phenotype persisted, with an increase in average alveolar bone loss (Fig. 7m) due to increased bone loss at the mesiobuccal (Fig. 7j), mid-lingual (Fig. 7k), distobuccal (Fig. 7l) line angles of the maxillary first molar. These data show that systemic minocycline therapy induces lasting dysbiotic shifts in the murine indigenous oral microbiota and has sustained detrimental effects on alveolar bone homeostasis.

DISCUSSION

Periodontitis-induced dysbiotic shifts in the commensal oral microbiota drive a proinflammatory hyperimmune response that enhances osteoclastogenesis and inflammatory alveolar bone destruction^1,2,9. Investigations performed in gnotobiotic animal models have delineated that the indigenous oral microbiota stimulates periodontal immune response mechanisms that drive naturally occurring alveolar bone loss^6-8. The current report discerns that antimicrobial perturbation of the commensal oral microbiota dysregulates osteoimmune response mechanisms in the local alveolar bone complex, which exacerbates naturally occurring alveolar bone loss.

Clinical research suggests that shifts in the composition, not the biomass, of the oral bacteriome are responsible for the proinflammatory hyperimmune response state that causes alveolar bone destruction^62,63. Administering an antibiotic cocktail and minocycline at a clinically relevant dose to male SPF mice caused dysbiotic shifts in the murine indigenous oral bacteriome that accelerated naturally occurring alveolar bone loss. We speculate that minocycline caused more severe alveolar bone loss than the antibiotic cocktail due to more harmful, disruptive shifts in the composition of the oral microbiome. The innovative execution of the murine minocycline treatment model in male GF mice delineated that antimicrobial-induced alveolar bone loss is dependent on the microbiota. Minocycline induced-changes in the gut microbiome did not alter the osteoclast phenotype or bone morphology at non-oral skeletal sites, implying that minocycline-induced dysbiotic shifts in oral microbiota modulate local osteoimmune responses that enhance osteoclastogenesis and drive inflammatory bone loss in alveolar bone. Chlorhexidine oral antiseptic rinses further perturbed the antibiotic-induced oral dysbiosis and worsened alveolar bone loss in minocycline-treated male SPF mice, validating the premise that antimicrobial-induced dysbiotic shifts in the oral bacteriome have deleterious effects on alveolar bone. Chlorhexidine rinses in vehicle-treated male SPF mice induced shifts in the health, stable oral bacteriome that protected against naturally occurring alveolar bone loss. Notably, this highlights that oral antiseptic rinses can cause dysbiotic shifts (disease promoting) vs. eubiotic shifts (health promoting) in the oral microbiome which are dependent on the initial symbiotic relationship between the host and microbiota.

Minocycline treatment had similar detrimental effects on alveolar bone homeostasis in female and male SPF mice, however, changes in the oral microbiome differed. While minocycline treatment in male SPF mice caused decreases in Proteobacteria and Firmicutes, in female mice, minocycline treatment upregulated the expression of Actinobacteria and Proteobacteria at age 12-weeks. Six weeks following the termination of minocycline therapy, Proteobacteria became suppressed and Firmicutes was trending downwards, while Actinobacteria became more prominent in female SPF mice at age 18-weeks.

The healthy murine oral microbiome has just recently begun to be characterized^37,38,64, and ongoing research is necessary to discern how changes in the composition of the indigenous oral microbiota influence alveolar bone homeostasis. Furthermore, sex-differences in the healthy murine oral microbiome are currently unclear. The current report shows that antimicrobial-induced phyla-level shifts in the oral bacteriome of male C57BL/6 SPF mice can have dysbiotic vs. eubiotic effects on alveolar bone homeostasis that are dependent on the initial symbiotic relationship between the host and microbiota. Clinical research has shown that sex-specific differences in the oral microbiome of caries-active patients⁶⁵ and periodontitis patients⁶⁶ which have been attributed in part to differences in sex steroid hormones. This implies that minocycline’s detrimental effects on alveolar bone homeostasis found in male and female SPF mice can be caused by sex-specific dysbiotic shifts in the oral microbiome. The abundance of the bacterial phyla assessed in this report have been shown make-up greater than 90% of the murine oral bacteriome³⁸. The authors acknowledge the possibility that stability or changes of lower abundance bacterial phyla may play role in antimicrobial-induced alveolar bone loss. This highlights the need for future microbiome investigations that comprehensively investigate antimicrobial induced changes across bacterial phyla and lower-level taxa communities.

Osteoimmunology studies delineated that antibiotic perturbation of the commensal microbiota causes a dysbiotic proinflammatory immune response state in oral draining lymphoid tissues, which we speculate drives the pro-osteoclastic phenotype found in alveolar bone of minocycline-treated SPF mice. Minocycline treatment upregulated the frequency of neutrophils and plasmacytoid dendritic cells in the oral draining cervical lymph nodes. Plasmacytoid dendritic cells survey the periodontal barrier tissues for microbial antigens, and then migrate to the cervical lymph nodes where they present antigens to CD4⁺ helper T-cells. Under proinflammatory dysbiotic states, these innate immune cells drive the differentiation of T_H1 and T_H17 cells^53,54. Neutrophils also sample microbial antigens in barrier mucosal tissues and then migrate to draining lymph nodes, where they can induce the differentiation of T_H1 and T_H17 cells^56-59. Paralleling the increased frequency of proinflammatory innate immune cells found within cervical lymph nodes, T_H1 and T_H17 cells were profoundly upregulated in the cervical lymph nodes and mandible alveolar bone marrow of minocycline-treated SPF mice. Recognizing that T_H1 and T_H17 cells have pro-osteoclastic actions that drive inflammatory alveolar bone destruction^67-71, the increased presence of T_H1 and T_H17 cells likely contributes to the enhanced osteoclast phenotype and exacerbated alveolar bone loss found in minocycline-treated SPF mice.

Dysbiotic shifts in the oral microbiota stimulate the innate immune response in barrier gingival tissues, which upregulate pro-inflammatory mediators that direct CD4⁺ helper T-cell mediated immunity^1,9,17,72. Within the periodontal tissues IL6 has been shown to drive T_H17 cell responses^73,74 and Il12 has been shown to induce T_H1 cell responses^74,75. This implies that the detected upregulation of Il6 and Il12 in the mandibular gingiva of minocycline- vs. vehicle-treated SPF mice promoted the enhance T_H1 / T_H17 cell phenotype in the oral-draining cervical lymph nodes and mandibular bone marrow.

Study outcomes did not link minocycline-induced changes in the gut microbiome to alterations in osteoclastogenesis or bone morphology at non-oral skeletal sites. Interestingly, we found that minocycline decreased the frequency of both T_H17 and T_REG cells in femoral long bone marrow. Prior work has demonstrated that antibiotic perturbation of the gut microbiota impairs hematopoiesis and depletes T_REG cells in long bone marrow^76,77. Further, antibiotics have recently been shown to have immunosuppressive actions on T_H17 cells, which occur independent of their antimicrobial activity⁷⁸. The balance of pro-osteoclastic T_H17 cells / anti-osteoclastic T_REG cells has been purported to regulate osteoclastogenesis^79,80. In light of reports that probiotic modification of the gut microbiota impacts bone loss at non-oral skeletal sites through dysregulating the T_H17 / T_REG cell balance^81,82, we speculate that minocycline effects similarly suppressing T_H17 and T_REG cells contributed to the lack of alterations in osteoclasts and bone morphology found in long bones of minocycline-treated SPF mice.

Minocycline has been reported to have biological actions that extend beyond its antimicrobial properties. Experimental investigations have shown that minocycline exerts anti-apoptotic activity in the central nervous system, which has been linked to anti-inflammatory effects targeting microglia, lymphocytes, macrophages, dendritic cells, and neutrophils^83,84. Minocycline’s immunosuppressive effects have primarily been elucidated by treating immune cells in vitro, which is why minocycline has been reported to have anti-inflammatory actions independent of its antimicrobial properties^83,84. Thus, we are not ruling out that minocycline has biological actions that extend beyond its antimicrobial properties. However, the lack of differences in alveolar bone loss found in vehicle- vs. minocycline-treated GF mice, imply that minocycline’s catabolic effects on alveolar bone are dependent on its antimicrobial properties.

Systemic minocycline therapy has been shown to prevent bone loss in metabolic bone disease models, including ovariectomy-induced osteopenia⁸⁵ and diabetes-induced ostepenia⁸⁶. However, minocycline’s effects on normal bone metabolism are unclear. The concept that minocycline therapy can disrupt normal bone metabolism is indirectly supported by prior work showing that other tetracycline class drugs impair physiologic bone metabolism and skeletal homeostasis⁸⁷. Simmons et al. (1983) investigated the impact of a one-year course of systemic tetracycline therapy on mandibular bone in mature female rhesus monkeys (50 mg/kg/day). Tetracycline administration disrupted bone remodeling and dysregulated the calcium to inorganic phosphorus ratio in the mandible⁸⁷. Study outcomes reported herein provide initial evidence that systemic minocycline therapy can disrupt physiologic bone metabolism and have detrimental effects on alveolar bone homeostasis.

Prior investigations have extensively investigated systemic antibiotic therapy as an adjunctive treatment for periodontal disease. The use of systemic antibiotics as an adjunct to non-surgical periodontal therapy is largely empirically based. Notably, recent studies have called into the question the efficacy of systemic antibiotics for the treatment of periodontitis^88,89. The current report studied the impact of systemic antibiotic therapy on the healthy periodontium. Outcomes reported herein demonstrate that administering systemic antibiotics in periodontal health can induce an oral dysbiotic state that exacerbates naturally occurring alveolar bone loss. Minocycline and other tetracycline drugs are commonly administered for extended durations as a systemic treatment for acne in adolescents and young adults^23-25. Taken together, this highlights the need for future clinical research evaluating the effects of systemic antibiotics on the human oral microbiome and alveolar bone homeostasis in the healthy periodontium.

Nonstandard Abbreviations:

(SPF)

Specific-pathogen-free

(GF)

Germ-free

(ABX)

Antibiotic Cocktail

(VEH)

Vehicle-control

(MINO)

Minocycline

(CHX)

Chlorhexidine

(OCN)

Osteocalcin

(CEJ-ABC)

cementoenamel junction-alveolar bone crest

(TRAP)

Tartrate-Resistant Acid Phosphatase

(RANKL)

Receptor Activator of nuclear factor kappa-β ligand

(OPG)

Osteoprotegerin