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Rac-Null Leukocytes Are Associated with Increased Inflammation-Mediated Alveolar Bone Loss

Published:November 22, 2013DOI:https://doi.org/10.1016/j.ajpath.2013.10.018
      Periodontitis is characterized by altered host-biofilm interactions that result in irreversible inflammation-mediated alveolar bone loss. Genetic and epigenetic factors that predispose to ineffective control of biofilm composition and maintenance of tissue homeostasis are not fully understood. We elucidated how leukocytes affect the course of periodontitis in Rac-null mice. Mouse models of acute gingivitis and periodontitis were used to assess the early inflammatory response and patterns of chronicity leading to loss of alveolar bone due to inflammation in Rac-null mice. Leukocyte margination was differentially impaired in these mice during attachment in conditional Rac1-null (granulocyte/monocyte lineage) mice and during rolling and attachment in Rac2-null (all blood cells) mice. Inflammatory responses to subgingival ligatures, assessed by changes in peripheral blood differential leukocyte numbers, were altered in Rac-null compared with wild-type mice. In response to persistent subgingival ligature-mediated challenge, Rac-null mice had increased loss of alveolar bone with patterns of resorption characteristic of aggressive forms of periodontitis. These findings were partially explained by higher osteoclastic coverage of the bone-periodontal ligament interface in Rac-null compared with wild-type mice. In conclusion, this study demonstrates that leukocyte defects, such as decreased endothelial margination and tissue recruitment, are rate-limiting steps in the periodontal inflammatory process that lead to more aggressive forms of periodontitis.
      Periodontits is characterized by progressive inflammation-induced alveolar bone loss (ABL) as a result of altered host-biofilm interactions in the gingival crevice. It is generally accepted that periodontal pathogens are required but not sufficient to induce loss of clinical attachment; many host and environmental factors dramatically modify the expression of disease.
      • Offenbacher S.
      Periodontal diseases: pathogenesis.
      Chronic periodontal inflammation is characterized by a complex myelo-lymphoid cell infiltrate and is associated with irreversible loss of alveolar bone. Host-derived factors, including chemokines, cytokines, and matrix-degrading enzymes (metalloproteases), contribute to tissue destruction and disease progression.
      • Page R.C.
      • Kornman K.S.
      The pathogenesis of human periodontitis: an introduction.
      Mounting evidence supports the major role of host responses modulated through genetics as being the major determinants of the outcomes of the classic chronic inflammatory condition we know as periodontitis.
      • Bartold P.M.
      • Van Dyke T.E.
      Periodontitis: a host-mediated disruption of microbial homeostasis: unlearning learned concepts.
      Rac1 and Rac2 GTPases regulate actin cytoskeleton, which plays essential roles in directional leukocyte migration, phagocytosis, and superoxide (O2) production by the NADPH oxidase during neutrophil responses to chemoattractants.
      • Glogauer M.
      • Marchal C.C.
      • Zhu F.
      • Worku A.
      • Clausen B.E.
      • Foerster I.
      • Marks P.
      • Downey G.P.
      • Dinauer M.
      • Kwiatkowski D.J.
      Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.
      Rac1 is ubiquitously expressed, whereas Rac2 is exclusively present in cells of hematopoietic origin. Differential activation of Rac1 and Rac2 in response to different concentrations of chemoattractant partially explains how neutrophils kill invading bacteria while limiting oxidative damage to adjacent healthy tissue.
      • Zhang H.
      • Sun C.
      • Glogauer M.
      • Bokoch G.M.
      Human neutrophils coordinate chemotaxis by differential activation of Rac1 and Rac2.
      In neutrophils, Rac1 seems to be the major regulator of migration and Rac2 of O2 production.
      • Glogauer M.
      • Marchal C.C.
      • Zhu F.
      • Worku A.
      • Clausen B.E.
      • Foerster I.
      • Marks P.
      • Downey G.P.
      • Dinauer M.
      • Kwiatkowski D.J.
      Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.
      Rac1 seems to play a critical role in both functions during osteoclast differentiation, although in macrophages it may be primarily responsible for regulating cell morphology and membrane ruffling and may not be required for migration.
      • Wang Y.
      • Lebowitz D.
      • Sun C.
      • Thang H.
      • Grynpas M.D.
      • Glogauer M.
      Identifying the relative contributions of Rac1 and Rac2 to osteoclastogenesis.
      • Wells C.M.
      • Walmsley M.
      • Ooi S.
      • Tybulewicz V.
      • Ridley A.J.
      Rac1-deficient macrophages exhibit defects in cell spreading and membrane ruffling but not migration.
      The protein product of RAC2 regulates several processes central to immune and inflammatory responses, including proliferation of primary T cells and differentiation toward the Th1 subtype, dendritic cell migration, neutrophil NADPH oxidase activity, and B-cell maturation.
      • Kim C.
      • Dinauer M.C.
      Rac2 is an essential regulator of neutrophil nicotinamide adenine dinucleotide phosphate oxidase activation in response to specific signaling pathways.
      • Li B.
      • Yu H.
      • Zheng W.
      • Voll R.
      • Na S.
      • Roberts A.W.
      • Williams D.A.
      • Davis R.J.
      • Ghosh S.
      • Flavell R.A.
      Role of the guanosine triphosphatase Rac2 in T helper 1 cell differentiation.
      • Benvenuti F.
      • Hugues S.
      • Walmsley M.
      • Ruf S.
      • Fetler L.
      • Popoff M.
      • Tybulewicz V.L.J.
      • Amigorena S.
      Requirement of Rac1 and Rac2 expression by mature dendritic cells for T cell priming.
      • Croker B.A.
      • Tarlinton D.M.
      • Cluse L.A.
      • Tuxen A.J.
      • Light A.
      • Yang F.-C.
      • Williams D.A.
      • Roberts A.W.
      The Rac2 guanosine triphosphatase regulates B lymphocyte antigen receptor responses and chemotaxis and is required for establishment of B-1a and marginal zone B lymphocytes.
      The role of autoimmunity and chronic inflammatory diseases as selective pressure during the course of human evolutionary history remains to be evaluated. Therefore, the maintenance of susceptibility alleles for autoimmune diseases may also be regarded as a possible by-product of adaptation to pathogen exposure. Nonetheless, recent observations indicate that RAC2 may be involved in host-pathogen genetic conflicts; consequently, the gene may represent a target of natural selection and may harbor variants that affect the susceptibility to infectious diseases or other immunologic phenotypes in humans.
      • Sironi M.
      • Guerini F.R.
      • Agliardi C.
      • Biasin M.
      • Cagliani R.
      • Fumagalli M.
      • Caputo D.
      • Cassinotti A.
      • Ardizzone S.
      • Zanzottera M.
      • Bolognesi E.
      • Riva S.
      • Kanari Y.
      • Miyazawa M.
      • Clerici M.
      An evolutionary analysis of RAC2 identifies haplotypes associated with human autoimmune diseases.
      The current hypothesis is that under identical subgingival challenge, Rac1- and Rac2-null phagocytes (neutrophils and macrophages) are associated with differing levels of increased alveolar bone resorption through altered inflammatory responses. The present study aimed to investigate the roles of leukocyte Rac1 and Rac2 in the onset and progression of periodontitis.

      Materials and Methods

      Mice and Study Design

      Rac1–conditional null (granulocyte/monocyte lineage),
      • Glogauer M.
      • Marchal C.C.
      • Zhu F.
      • Worku A.
      • Clausen B.E.
      • Foerster I.
      • Marks P.
      • Downey G.P.
      • Dinauer M.
      • Kwiatkowski D.J.
      Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.
      Rac2-null, and wild-type (WT) mice aged 8 to 10 weeks were used in accordance with the Guide for the Humane Use and Care of Laboratory Animals, and all the experiments were approved by the University of Toronto Animal Care Committee. Leukocyte–endothelial cell interactions were assessed using a model of acute gingivitis and gingival intravital microscopy. A split-mouth design was used to evaluate ABL by inducing periodontitis for 21 days on the maxillary left side, with the right side serving as the control. Gingival leukocyte infiltration, ABL, and osteoclastic coverage of the bone-PDL interface were assessed by immunofluorescence, bone morphometric and micro–computed tomography (micro-CT) analyses, and tartrate-resistant acid phosphatase (TRAP) stains.

      Complete Blood Cell Count

      Total and differential circulating leukocyte numbers were measured using a Hemavet machine (Drew Scientific, Waterbury, CT). Baseline leukocyte numbers were assessed in age- and sex-matched mice of all genotypes. Furthermore, leukocyte counts were measured during the course of ligature-induced ABL. For this purpose, 20 μL of anticoagulated peripheral blood (diluted 10:1 in 0.5 mol/L EDTA at pH 8) was collected by saphenous serial phlebotomy at baseline (day −1), 24 hours, and 7, 14, and 21 days after ligature placement. Sham mice that underwent the same procedure as ligatured mice except for the placement of a ligature were used as controls for leukocyte number counts.

      Gingival Intravital Microscopy

      Acute gingival inflammation was induced in mice of all genotypes by injecting tumor necrosis factor α (TNF-α) (Roche Diagnostics, Laval, QC, Canada) in mandibular labial gingiva. Four- to six-week-old male mice weighing 20 to 25 g received 10 μL of 1-μg/mL TNF-α intragingivally on the labial side between the mandibular incisors under brief isoflurane anesthesia. Anesthetized mice were rapidly placed in the supine position under a stereomicroscope at ×2.5 magnification. The lower lip was gently retracted using the 0.5-mL tuberculin syringe loaded with the TNF-α aliquot to be delivered by holding it parallel to the lower incisors. TNF-α was injected into the alveolar mucosa on the midline. Mice recovered spontaneously from anesthesia and were returned to a cage until preparation for microscopic visualization 2 hours later. TNF-α stimulates the expression of IL-8 by endothelial cells, of adhesion molecules by leukocytes, and of endothelial cells through granule release and transcriptional up-regulation. A 50-μL mixture of 6 mg/kg of fluorescein isothiocyanate–dextran (Mw 150 kDa; Sigma-Aldrich, St. Louis, MO) for vasculature visualization and 0.15 mg/kg of rhodamine 6G (Molecular Probes, Invitrogen, Carlsbad, CA) for leukocyte labeling in PBS was injected i.v. via one of the two side tail veins before microscopic visualization. Rhodamine 6G stains circulating leukocytes by mainly binding to mitochondria. After i.v. injection, mice were transferred onto a glass support plate and set on the inverted microscope with appropriate body temperature monitoring and maintenance during the entire experiment. Additional light was used for identification of postcapillary venules 20 to 40 μm in diameter for assessment of leukocyte–endothelial cell interactions (rolling and attachment) in visible light. One hundred micrometers length of vessel was visualized, and cells moving along the vessel wall were considered rolling, whereas those that remained stationary for the 30-second observation period were considered attached.

      Ligature-Induced ABL

      Mice were anesthetized with a mixture of 100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine and were placed in supine under a stereomicroscope. One 9-0 silk suture was placed in the gingival sulcus of the maxillary left second molar (M2) and was tied on the palatal side using a surgeon's knot (day 0). The knot was positioned in the distopalatal angle of M2. Blood samples for complete blood cell counts were collected at baseline (day −1), 1 day after ligature placement (day 1), and at 1 week (day 7), 2 weeks (day 14), and 3 weeks (day 21). On day 21, mice were euthanized, and the presence of ligature was verified before further processing. Subsets of mice of all genotypes were used for histologic analysis. For another subset, maxillae were skinned, defleshed in a colony of dermestid beetles at Royal Ontario Museum, and freeze-fumigated for 7 days (−20°C). Dry skulls were used for assessment of ABL by morphometry and micro-CT.

      Micro-CT Analysis of Bone Loss

      Each dry skull was scanned at a resolution of 7 μm in all three spatial dimensions using a desktop SkyScan1172 micro-CT scanner (Bruker-microCT, Kontich, Belgium) and was analyzed using CT-Analyzer software version 1.6.1 (Toronto Centre for Phenogenomics, Toronto, ON, Canada). The frontal plane of the specimens was set parallel to the X-ray beam axis. Using ImageJ64 software version 1.47 (NIH, Bethesda, MD) from Wright Cell Imaging Facility, stacks of reconstructed scans were gaussian low-pass filtered, and the plane connecting the tips of the distopalatal cusps of the right and left M2s were positioned horizontally before acquiring the orthogonal view for each specimen. Using the coronal and sagittal views, mesiobuccal, distobuccal, mesiopalatal, and distopalatal sites of M2s were identified. The interproximal ABL was measured as perpendicular from the mid-cemento-enamel junction (CEJ) to a plane tangential to buccal and palatal alveolar bone crests on the transaxial view. Furcation involvement was measured from the buccal CEJ to the lowest bone level along the palatal root. The buccal infrabony defect was approximated by measuring a rectangular surface from the buccal CEJ to the bone in the sagittal plane.

      Morphometry

      Dry skulls were stained with methylene blue (1% in water), and images of the buccal aspects of the right (healthy) and left M2s were taken at ×5 magnification using a video camera mounted on a stereomicroscope. Horizontal bone loss was measured from the CEJ to the alveolar bone crest at the midbuccal, mesial, and distal roots on the buccal aspect of the M2 on the ligature and contralateral control side using ImageJ64 software. Masson trichrome–stained coronal sections in the M2 area were used to identify inflammatory cell infiltrates and to measure the loss of attachment in response to chronic subgingival challenge as a result of ABL. TRAP staining for bone-resorbing osteoclasts was used to quantify osteoclast coverage of the bone-PDL interface 21 days after the induction of periodontitis.

      Immunofluorescence

      Gingival infiltration of neutrophils 21 days after ligature placement was assessed by immunofluorescence using antibodies against CD45 common leukocyte antigen and Ly6G neutrophil-specific surface antigen. Paraffin-embedded coronal sections in the maxillary M2 area were dewaxed and incubated with Alexa Flour 488–anti-Ly6G alone or mixed with PE–Cy7–anti-CD45 (eBioscience, San Diego, CA) for 1 hour at room temperature in the dark. Nuclear staining was performed using Hoechst DNA as previously described.
      • Ilvesaro J.
      • Tavi P.
      • Tuukkanen J.
      Connexin-mimetic peptide Gap 27 decreases osteoclastic activity.
      Phagocytosis by neutrophils was assessed on cells recruited i.p. by injecting 1 mL of 0.2-mg/mL zymosan A in the lower right quadrant of the abdomen. Cells were collected by peritoneal lavage performed 2 hours later and were counted using a hemocytometer. Neutrophils were incubated with opsonized Alexa Fluor 488–labeled zymosan (Life Technologies, Grand Island, NY) at a ratio of 1:10 for 30 minutes at 37°C in the dark. Leukocytes were then stained with rhodamine 6G, cytospinned, and assessed for phagocytosis using fluorescence microscopy.

      Results

      Rac-Null Leukocytes Exhibit Dysfunctional Interactions with Gingival Microvascular Endothelium in Acute Gingival Inflammation

      Intravital microscopy revealed a leukocyte rolling rate of 3933 ± 151 cells/106 circulating leukocytes per minute in WT mice 2 hours after TNF-α–induced acute gingivitis (means ± SD). Rac1-null mice had a comparable rolling rate, whereas in Rac2-null mice it was found to be significantly lower compared with WT controls (means ± SD: 3502 ± 121 and 1760 ± 40 cells/106 circulating leukocytes per minute, respectively). The number of leukocytes (means ± SD) attached to gingival microvascular endothelium was 756 ± 38 cells/106 circulating leukocytes per minute in WT mice, significantly higher in Rac1-null mice (1208 ± 60 cells/106 circulating leukocytes per minute), and significantly lower in Rac2-null mice (400 ± 40 cells/106 circulating leukocytes per minute) (Figure 1A).
      Figure thumbnail gr1
      Figure 1A: Rac-null mice have impaired leukocyte-endothelium interactions. Gingival intravital microscopy in acute gingivitis revealed a reduced leukocyte rolling rate along microvascular endothelium in Rac2-null mice compared with WT and Rac1-null mice. The number of attached leukocytes to endothelium during the observation period was significantly higher in Rac1-null mice and significantly lower in Rac2-null mice. Numbers have been normalized to circulating total leukocyte numbers. Data are shown as means ± SD; five mice per group. ∗P < 0.05, one-way analysis of variance. B: Rac1-null leukocytes may fail to retract uropods and project lamellipodia in vivo. Four snapshot images were taken during intravital video microscopic analysis at 1-second intervals for a Rac1-null, Rac2-null, and WT mouse. Normal attached leukocytes (WT) were able to retract the uropod and project a lamellipodia to migrate through the endothelial wall within the observation period. Rac1-null attached leukocytes were more prone to detach from endothelium before projection of lamellipodia and seemed to fail to retract the uropod. Rac2-null attached leukocytes behaved similar to normal leukocytes despite a lower rolling rate along the endothelium. The white arrows denote leukocyte attached to endothelium; red arrows, direction of blood flow; solid blue arrows, leading edge/lamellipodia; dashed blue arrows, trailing edge/uropod. Insets show magnified leukocyte attached to endothelium during the observation period.
      To investigate a potential mechanism for this finding of altered margination/transmigration, randomly chosen attached leukocytes were observed in intravital microscopy video recordings. Leukocytes of Rac1-null mice seemed to be less efficient in forming and maintaining the lamellipodia necessary for transmigration, whereas leukocytes of Rac2-null mice, although capable of forming lamellipodia, had a lower rolling rate compared with Rac1-null mice and leukocytes of WT mice (Figure 1B).
      Peripheral blood white blood cell counts revealed significantly higher leukocyte numbers in the blood of Rac2-null mice compared with WT and Rac1-null mice. Neutrophil and monocyte numbers were increased in Rac2-null compared with WT and Rac1-null mice. Mice of all genotypes had similar lymphocyte numbers. All Rac2-null mice had higher numbers of total leukocytes and neutrophils than all WT mice tested and higher numbers of monocytes than most WT mice. Red blood cell parameters showed no significant differences among strains, although erythrocytes of Rac1-null mice were smaller in volume with a higher hemoglobin concentration (Table 1).
      Table 1Baseline Complete Blood Counts
      Complete blood cell countWTRac1-nullRac2-null
      Leukocytes (k/μL)7.43 ± 2.18.49 ± 2.5412.82 ± 2.1
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      PMNs (k/μL)1.26 ± 0.691.44 ± 0.853.77 ± 1.5
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      Monocytes (k/μL)0.48 ± 0.410.41 ± 0.370.86 ± 0.53
      P < 0.05, Rac2-null versus Rac1-null.
      Lymphocytes (k/μL)5.61 ± 1.636.49 ± 1.797.8 ± 1.91
      P < 0.05, Rac2-null versus WT.
      Eosinophils (k/μL)0.05 ± 0.040.12 ± 0.150.39 ± 0.44
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      Basophils (k/μL)0.02 ± 0.020.03 ± 0.050.02 ± 0.01
      RBCs (mil/μL)8.85 ± 1.469.27 ± 1.359.71 ± 1.15
      Hematocrit (%)46.75 ± 8.2142.14 ± 7.4853.54 ± 5.25
      P < 0.05, Rac2-null versus Rac1-null.
      MCV (fL)52.74 ± 2.0145.48 ± 5.13
      P < 0.05, Rac1-null versus WT.
      55.26 ± 2.27
      P < 0.05, Rac2-null versus WT.
      Hemoglobin (g/dL)12.47 ± 2.6412.91 ± 214.19 ± 2.31
      MCH (pg)13.87 ± 0.913.94 ± 0.6914.57 ± 1.26
      MCHC (g/dL)25.8 ± 2.4631 ± 3.57
      P < 0.05, Rac2-null versus Rac1-null.
      P < 0.05, Rac1-null versus WT.
      26.37 ± 2.37
      Platelets (k/μL)533 ± 105605 ± 186687 ± 108
      P < 0.05, Rac2-null versus WT.
      Data are given as means ± SD. The Turkey-Kramer test was used to compare phenotypes among strains.
      MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PMN, polymorphonuclear neutrophil; RBC, red blood cell.
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      P < 0.05, Rac1-null versus WT.

      Neutrophils of Rac-Null Mice Exhibit Altered Responses in Chronic Ligature-Induced Periodontitis

      Silk ligatures were placed around the maxillary M2 unilaterally, and circulating leukocytes were measured in WT, Rac1-null, and Rac2-null mice 1 day before and 1, 7, 14, and 21 days after (chronic phase) placement compared with sham mice that received no ligature. To assess the impact of Rac1 and Rac2 on peripheral blood cells during a localized chronic inflammation, complete blood cell counts were performed at each time point. The inflammatory response in WT mice was characterized by a significant increase in circulating monocytes and lymphocytes 24 hours after induction and an almost complete return to baseline levels for neutrophils. Rac1-null mice responded with similar increases in circulating lymphocyte and neutrophil numbers but a significant decrease in circulating monocytes. Rac2-null mice responded with a significant increase in circulating neutrophils 1 day after induction, which persisted above baseline levels until day 21 (Table 2). Circulating lymphocyte numbers remained relatively steady throughout the induction (Table 3).
      Table 2Post-Induction Complete Blood Cell Counts (Acute Phase)
      Complete blood cell count24 hours
      WTRac1-nullRac2-null
      ShamLigatureShamLigatureShamLigature
      Leukocytes (k/μL)8.01 ± 0.879.41 ± 3.238.41 ± 2.6211.04 ± 3.7414.1 ± 1.8415.89 ± 2.83
      P < 0.05, Rac2-null versus WT.
      PMNs (k/μL)1.79 ± 0.632.45 ± 1.061.53 ± 0.833.18 ± 1.893.69 ± 1.356.4 ± 1.21
      P < 0.05, Rac2-null versus WT.
      P < 0.05, ligature versus sham.
      P < 0.05, Rac2-null vs Rac1-null.
      Monocytes (k/μL)0.26 ± 0.150.48 ± 0.290.24 ± 0.120.79 ± 0.690.82 ± 0.381.41 ± 1.18
      Lymphocytes (k/μL)5.87 ± 0.766.34 ± 26.61 ± 1.836.88 ± 1.289.23 ± 1.197.95 ± 1.16
      Eosinophils (k/μL)0.07 ± 0.050.1 ± 0.110.03 ± 0.020.17 ± 0.30.4 ± 0.090.12 ± 0.04
      P < 0.05, ligature versus sham.
      Basophils (k/μL)0.01 ± 0.010.03 ± 0.060.01 ± 0.010.05 ± 0.10.19 ± 0.040.02 ± 0.01
      RBCs (mil/μL)8.8 ± 1.289.23 ± 0.379.49 ± 0.679.66 ± 0.518.00 ± 1.0710.16 ± 0.22
      P < 0.05, Rac2-null versus WT.
      P < 0.05, ligature versus sham.
      Hematocrit (%)47.9 ± 8.1346.74 ± 1.8345.5 ± 3.2844.86 ± 2.9947.52 ± 6.5753.86 ± 0.45
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null vs Rac1-null.
      MCV (fL)53.12 ± 2.1550.66 ± 1.0347.98 ± 2.6746.42 ± 0.97
      P < 0.05, Rac1-null versus WT.
      56.68 ± 0.6953.32 ± 1.17
      P < 0.05, Rac2-null versus WT.
      P < 0.05, ligature versus sham.
      Hemoglobin (g/dL)11.98 ± 1.8912.86 ± 0.8413.14 ± 1.5414.2 ± 0.9111.76 ± 2.4514.6 ± 0.97
      P < 0.05, Rac2-null versus WT.
      MCH (pg)13.32 ± 0.4013.9 ± 0.8713.84 ± 1.1214.68 ± 0.52
      P < 0.05, ligature versus sham.
      14.04 ± 1.1314.42 ± 1.01
      P < 0.05, Rac2-null versus WT.
      P < 0.05, Rac2-null vs Rac1-null.
      MCHC (g/dL)25.14 ± 1.6027.36 ± 1.1228.84 ± 1.7731.62 ± 0.74
      P < 0.05, Rac2-null vs Rac1-null.
      P < 0.05, Rac1-null versus WT.
      24.82 ± 2.1927.02 ± 1.32
      Platelets (k/μL)596 ± 43566 ± 165703 ± 107695 ± 202636 ± 138764 ± 214
      Data are given as means ± SD. The Student's t-test was used for all pairs (ie, treatment and sham). The Turkey-Kramer test was used to compare phenotypes among strains.
      MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PMN, polymorphonuclear neutrophil; RBC, red blood cell.
      P < 0.05, Rac2-null versus WT.
      P < 0.05, ligature versus sham.
      P < 0.05, Rac2-null vs Rac1-null.
      § P < 0.05, Rac1-null versus WT.
      Table 3Post-Induction Complete Blood Cell Counts (Chronic Phase)
      Complete blood cell countWTRac1-nullRac2-null
      ShamLigatureShamLigatureShamLigature
      7 days
      Leukocytes (k/μL)10 ± 1.096.52 ± 2.05
      P < 0.05, ligature versus sham.
      8.53 ± 1.497.56 ± 2.6614.4 ± 1.289.86 ± 0.98
      P < 0.05, ligature versus sham.
      PMNs (k/μL)2.08 ± 0.691.88 ± 0.631.42 ± 0.451.83 ± 1.253.73 ± 0.752.97 ± 0.94
      Monocytes (k/μL)0.41 ± 0.210.23 ± 0.090.39 ± 0.230.3 ± 0.210.38 ± 0.150.5 ± 0.29
      Lymphocytes (k/μL)7.38 ± 1.184.3 ± 1.64
      P < 0.05, ligature versus sham.
      6.49 ± 1.765.37 ± 1.399.66 ± 1.006.26 ± 0.56
      P < 0.05, ligature versus sham.
      Eosinophils (k/μL)0.13 ± 0.070.08 ± 0.120.19 ± 0.220.05 ± 0.10.45 ± 0.090.11 ± 0.11
      P < 0.05, ligature versus sham.
      Basophils (k/μL)0.03 ± 0.020.04 ± 0.060.03 ± 0.050.01 ± 0.010.17 ± 0.110.02 ± 0.03
      P < 0.05, ligature versus sham.
      RBCs (mil/μL)9.26 ± 1.049.65 ± 0.359.74 ± 1.419.74 ± 0.468.82 ± 0.99.55 ± 0.66
      Hematocrit (%)47.44 ± 5.4549.02 ± 2.1840.6 ± 6.8744.12 ± 3.7549.42 ± 5.3950 ± 4.57
      MCV (fL)51.18 ± 0.6250.8 ± 1.647.72 ± 2.5246.7 ± 3.13
      P < 0.05, Rac1-null versus WT.
      55.72 ± 1.6752.34 ± 2.05
      P < 0.05, ligature versus sham.
      Hemoglobin (g/dL)13.38 ± 1.9713.72 ± 0.5212.68 ± 2.5013.6 ± 0.813.42 ± 1.3513.9 ± 1.03
      MCH (pg)14.4 ± 0.7814.26 ± 0.4114.82 ± 0.7114.38 ± 0.315.18 ± 0.3514.6 ± 0.48
      MCHC (g/dL)28.14 ± 1.5128.1 ± 0.2331.24 ± 2.9830.92 ± 2.57
      P < 0.05, Rac1-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      27.2 ± 1.3927.92 ± 0.68
      Platelets (k/μL)616 ± 142590 ± 185669 ± 199602 ± 136487 ± 123626 ± 190
      14 days
      Leukocytes (k/μL)7.77 ± 1.824.86 ± 1.98
      P < 0.05, ligature versus sham.
      6.76 ± 2.378.48 ± 2.7210.88 ± 6.1413.08 ± 4.03
      P < 0.05, ligature versus sham.
      PMNs (k/μL)1.62 ± 1.001.32 ± 0.751.50 ± 0.412.49 ± 1.353.21 ± 1.74.08 ± 1.21
      Monocytes (k/μL)0.44 ± 0.220.18 ± 0.050.17 ± 0.100.24 ± 0.110.27 ± 0.170.31 ± 0.19
      Lymphocytes (k/μL)5.47 ± 1.343.23 ± 1.15
      P < 0.05, ligature versus sham.
      4.95 ± 1.885.73 ± 2.647.19 ± 4.158.38 ± 2.67
      P < 0.05, ligature versus sham.
      Eosinophils (k/μL)0.19 ± 0.30.09 ± 0.150.1 ± 0.130.02 ± 0.010.18 ± 0.160.22 ± 0.22
      Basophils (k/μL)0.07 ± 0.120.03 ± 0.040.03 ± 0.040.01 ± 0.010.04 ± 0.210.09 ± 0.11
      RBCs (mil/μL)9.06 ± 1.039.03 ± 0.599.40 ± 0.579.41 ± 1.079.54 ± 0.349.59 ± 0.57
      Hematocrit (%)47.02 ± 5.8746.58 ± 1.7645.46 ± 1.2943.5 ± 1.6952.66 ± 2.0350.1 ± 10.45
      P < 0.05, Rac2-null versus WT.
      MCV (fL)52.08 ± 3.1451.64 ± 1.7846.72 ± 2.1848.4 ± 2.7
      P < 0.05, Rac1-null versus WT.
      55.16 ± 0.6352.42 ± 1.89
      P < 0.05, ligature versus sham.
      P < 0.05, Rac2-null versus WT.
      Hemoglobin (g/dL)11.92 ± 3.2212.9 ± 0.6513.98 ± 1.0813.6 ± 1.2714.52 ± 0.8514.2 ± 0.8
      MCH (pg)14.28 ± 1.3114.3 ± 0.2314.18 ± 0.4414.76 ± 0.615.18 ± 0.4514.8 ± 0.26
      MCHC (g/dL)27.42 ± 1.8227.68 ± 0.5530.76 ± 2.2731.22 ± 2.0727.56 ± 1.0328.3 ± 1.09
      Platelets (k/μL)693 ± 217531 ± 169625 ± 169773 ± 186693 ± 94688 ± 177
      21 days
      Leukocytes (k/μL)7.39 ± 3.019.08 ± 1.439.34 ± 1.189.26 ± 1.5312.96 ± 1.4513.4 ± 1.19
      P < 0.05, Rac2-null versus Rac1-null.
      P < 0.05, Rac2-null versus WT.
      PMNs (k/μL)1.05 ± 0.952.06 ± 0.701.57 ± 0.782.43 ± 1.072.83 ± 0.323.75 ± 0.51
      P < 0.05, ligature versus sham.
      P < 0.05, Rac2-null versus WT.
      Monocytes (k/μL)0.27 ± 0.110.41 ± 0.220.35 ± 0.180.25 ± 0.070.39 ± 0.180.35 ± 0.09
      Lymphocytes (k/μL)5.74 ± 2.926.33 ± 1.727.27 ± 0.346.56 ± 2.059.46 ± 1.909.22 ± 1.06
      P < 0.05, ligature versus sham.
      Eosinophils (k/μL)0.36 ± 0.30.17 ± 0.240.16 ± 0.180.01 ± 0.000.21 ± 0.230.06 ± 0.04
      Basophils (k/μL)0.11 ± 0.130.026 ± 0.0420.03 ± 0.030.03 ± 0.030.07 ± 0.040.05 ± 0.07
      RBCs (mil/μL)8.14 ± 3.028.69 ± 1.849.62 ± 1.019.67 ± 0.478.82 ± 1.169.33 ± 0.44
      Hematocrit (%)37.1 ± 12.145.16 ± 2.0444.12 ± 2.0447.16 ± 2.0448.88 ± 7.5549 ± 1.86
      MCV (fL)51.38 ± 4.651.78 ± 2.0950.82 ± 3.0248.8 ± 1.51
      P < 0.05, Rac1-null versus WT.
      53.54 ± 2.5952.58 ± 1.46
      P < 0.05, Rac2-null versus WT.
      Hemoglobin (g/dL)9.41 ± 3.7111.82 ± 2.8212.35 ± 1.4013.7 ± 0.7713.5 ± 1.4613.4 ± 0.45
      MCH (pg)14.42 ± 9.5714.08 ± 7.6414.2 ± 1.1014.22 ± 0.314.92 ± 0.5414.42 ± 0.36
      MCHC (g/dL)27.42 ± 3.1426.16 ± 1.4427.98 ± 1.5029.2 ± 0.87
      P < 0.05, Rac1-null versus WT.
      27.62 ± 1.3527.44 ± 0.79
      Platelets (k/μL)388 ± 163531 ± 201474 ± 182829 ± 176
      P < 0.05, ligature versus sham.
      P < 0.05, Rac2-null versus Rac1-null.
      627 ± 174753 ± 77
      Data are given as means ± SD. The Student's t-test was used for all pairs (ie, treatment and sham). The Turkey-Kramer test was used to compare phenotypes among strains.
      MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PMN, polymorphonuclear neutrophil; RBC, red blood cell.
      P < 0.05, ligature versus sham.
      P < 0.05, Rac1-null versus WT.
      P < 0.05, Rac2-null versus Rac1-null.
      § P < 0.05, Rac2-null versus WT.
      Neutrophils were present in high numbers around silk filaments, pocket epithelium, and connective tissue in WT compared with Rac-null mice 21 days after induction of periodontitis. Most neutrophils appeared to localize around blood vessels in gingival connective tissue and PDL (Figure 2A). Although neutrophilic infiltrate around the ligature seemed less pronounced in Rac-null mice, other cells types were found in high numbers around the silk filaments, particularly in Rac2-null mice, possibly monocytes, lymphocytes, or epithelial cells. Functionally, neutrophils of Rac2-null mice exhibited impaired phagocytosis of zymosan particles (phagocytic efficiency: WT, 80%; Rac1-null, 64%; and Rac2-null, 12%) (Figure 2B).
      Figure thumbnail gr2
      Figure 2Rac-null neutrophils have impaired migratory and phagocytic functions. A: Immunofluorescence for neutrophil-specific antigen Ly6G was used to identify neutrophil infiltrates around ligature (L; circled by a dashed black line, ×200 in phase contrast) silk filaments (SF), inside pocket epithelium (PE), and connective tissue of periodontal ligament (PDL) 21 days after induction of periodontitis. Significant numbers of neutrophils were detected in WT mice, surrounding SF (black arrows), inside gingival tissues (white arrows), and adjacent to cementum (CE) on tooth roots (top left panels). Rac1-null and Rac2-null mice had fewer neutrophils inside the tissue and surrounding silk filaments compared with WT mice. Neutrophils of Rac-null mice were seen predominantly intravascularly (white arrows) or in the perivascular compartment (bottom panels). Anti-CD45 (common leukocyte antigen), anti-Ly6G (neutrophil-specific antigen) immunofluorescence combined with nuclear polymorphic morphology confirmed the specificity of neutrophil identification. Neutrophil size was found at approximately one-tenth the size of an SF (top right panels). B: Neutrophils were recruited into the peritoneal cavity by zymosan injection (in vivo phagocytosed particles), collected, and incubated ex vivo with fluorescent-labeled zymosan particles at a ratio of 1:10. Neutrophils of WT and Rac1-null mice were more efficient in uptake of zymosan particles (ex vivo phagocytosed particles) compared with Rac2-null neutrophils. Most labeled particles were extracellular in Rac2-null mice (top panels). Phagocytic efficiency was measured as the number of intracellular fluorescent-labeled zymosan particles divided by the total number of zymosan particles per field (10 fields in ×100 magnification per genotype). WT, Rac1-null, and Rac2-null neutrophils had phagocytic efficiency of 80%, 64%, and 12%, respectively. Nuclear staining (Hoechst) shows significant cellular infiltrate around SF in all genotypes. FITC, fluorescein isothiocyanate.

      Rac-Null Leukocytes Are Associated with Increased ABL

      Assessment of ABL by morphometry and micro-CT analysis revealed significant differences in Rac-null mice compared with WT controls. On day 21 after molar ligature placement, the means ± SD CEJ to alveolar bone crest distances on the buccal aspects of ligated M2s were 98 ± 1.1 μm, 196 ± 15 μm, and 185 ± 20 μm in WT, Rac1-null, and Rac2-null mice, respectively. These differences were statistically significant compared with WT controls and contralateral healthy molars (Figure 3).
      Figure thumbnail gr3
      Figure 3Rac-null leukocytes are associated with increased ABL in experimental periodontitis. Molar ligatures were placed around maxillary M2s on day 0, and mice were sacrificed on day 21; their skulls were defleshed and assessed for ABL by morphometry. A: ABL was measured as the distance from the CEJ to the alveolar bone crest (ABC) on the facial aspect of ligated molars (the average of three measurements at the mesial, middle, and distal line angles) and compared with contralateral nonligated molars. In WT mice, the means ± SD CEJ-ABC distances on control and ligated molars were 54 ± 1 and 128 ± 1 μm, respectively. In Rac1- and Rac2-null mice, the same distances were 50 ± 1.1 and 116 ± 9 μm and 60 ± 1 and 190 ± 17 μm, respectively. B: Statistical analysis of the amount of bone loss between genotypes showed significant bone loss in Rac2-null mice (see ). Data are shown as means ± SD; five mice per group. P < 0.05, one-way analysis of variance.
      Micro-CT analysis was used to assess site-specific ABL in ligature-induced periodontitis. Intragroup comparisons between proximal, buccal infrabony, and furcation (F) ABL showed significant F involvement in all groups compared with proximal ABL (means ± SD: WT: proximal, 0.169 ± 0.054 mm; F, 0.548 ± 0.073 mm; Rac1-null: proximal, 0.21 ± 0.045 mm; F, 0.657 ± 0.03 mm; and Rac2-null: proximal, 0.253 ± 0.036 mm; F, 0.744 ± 0.128 mm). Intergroup comparisons showed significant proximal ABL in all the groups compared with baseline. Rac1- and Rac2-null had significantly higher buccal infrabony ABL (4.39 ± 0.367 and 4.335 ± 0.665 mm, respectively) compared with WT (1.589 ± 0.539 mm) (n = 5 WT, n = 4 Rac1, and n = 4 Rac2) (Figure 4). These findings suggest a more aggressive vertical type of bone loss in Rac-null mice consistent with rapid onset and progression.
      Figure thumbnail gr4
      Figure 4Rac-null leukocytes are associated with more severe forms of attachment loss around molars in periodontitis. A: Site-specific ABL was measured by micro-CT analysis. Mesial and distal sites for proximal bone loss measurement were chosen at contact points between the first-second and second-third molars (left top and middle panels). Bone loss was measured as perpendicular from the mid-CEJ to a plane tangential to the buccal and palatal alveolar crests (right top panels). Furcation involvement was measured from the buccal CEJ to the lowest level of alveolar bone along the palatal root of the M2 at the furcation level (right bottom panels). B: Statistical analysis of site-specific bone loss showed significant furcation involvement in all groups compared with proximal bone loss. There was significant proximal bone loss in all groups compared with the healthy side. Rac1- and Rac2-null mice had significantly higher buccal infrabony bone loss. P < 0.05 versus control sides, one-way analysis of variance. P < 0.05, Rac-null versus WT mice, one-way analysis of variance. C: Rac2-deficient leukocytes are associated with more severe attachment loss. CAL and connective tissue attachment (CTA) were measured on coronal sections of ligated M2s 21 days after induction. Masson trichrome stain was used for morphometric analysis of attachment loss. CAL was measured from the CEJ to the base of the periodontal pocket and connective tissue attachment from the base of the pocket to the alveolar crest. Statistical analysis revealed a similar CAL in all strains in response to ligature (L; black arrows) but was significantly wider CTA in Rac2-null mice. P < 0.05, one-way analysis of variance. AB, alveolar bone; B, bone; C, control; c, cementum; D, dentin; JE, junctional epithelium; Lig, ligature; OE, oral epithelium; PDL, periodontal ligament; Prox, proximal. Data are shown as means ± SD; five mice per group.
      Significant clinical attachment loss (CAL) was found in all mice using histomorphometry on Masson trichrome–stained coronal sections in the M2 area. Although the CAL was found to be similar among strains, there was significantly wider connective tissue attachment in Rac2-null mice, partially explaining the higher bone loss in these mice (means ± SD: CAL: WT, 94.08 ± 5.01 μm; Rac1-null, 86.26 ± 3.81 μm; and Rac2-null, 84.24 ± 10.82 μm; and connective tissue attachment: nonligated, 89.6 ± 0.37 μm; WT, 93.65 ± 17.26 μm; Rac1-null, 98.68 ± 24.52 μm; and Rac2-null, 106.4 ± 6.83 μm) (Figure 4C).

      Rac-Null Mice Have Increased Osteoclastic Coverage of the Bone-Ligament Interface in Induced Periodontitis

      Osteoclast coverage at the bone-periodontal ligament interface was assessed by TRAP and TRAP-nuclear staining from root tip to alveolar crest. TRAP staining of coronal sections of ligated molars and surrounding bone showed higher osteoclastic coverage in Rac-null mice compared with WT mice. Means ± SD osteoclastic coverage around molars with periodontitis was 0.37 ± 0.05 mm, 0.49 ± 0.06 mm, and 0.45 ± 0.1 mm in WT, Rac1-null, and Rac2-null mice, respectively, significantly higher than that around healthy molars (WT, 0.21 ± 0.08 mm; Rac1-null, 0.22 ± 0.08 mm; and Rac2-null, 0.19 ± 0.04 mm) (Figure 5A). Osteoclasts from Rac2-null mice have significantly fewer nuclei compared with WT and Rac1-null osteoclasts, with a means ± SD ratio of nuclei to micrometer of TRAP-stained bone of 0.11 ± 0.014 mm, 0.11 ± 0.013, and 0.077 ± 0.007 in WT, Rac1-null, and Rac2-null mice, respectively (Figure 5B).
      Figure thumbnail gr5
      Figure 5Rac-null mice have increased osteoclastic coverage of the bone-periodontal ligament (PDL) interface in experimental periodontitis. Coronal sections of ligated M2s and surrounding structures were stained for TRAP to assess osteoclastic coverage on day 21 after induction of periodontitis. A: Increased osteoclastic activity was observed around the molars (solid arrows) with ligatures compared with the contralateral molars (dashed arrows, physiologic) in all genotypes. Rac1- and Rac2-null mice had higher osteoclastic coverage of the bone-PDL interface compared with WT mice. TRAP stain was quantified relative to length of bone along the bone-PDL interface from the tooth apex to the alveolar crest. Alveolar bone around diseased molars showed a significant twofold increase in osteoclastic coverage compared with healthy molars. Statistical analysis revealed significantly greater osteoclastic coverage in Rac2-null mice compared with WT mice (2.4-fold increase). B: Coronal sections stained for TRAP and nuclei (Hoechst) were used to quantify osteoclast maturity. Numbers of nuclei were counted per length of TRAP-stained bone and compared between groups. Rac2-null osteoclasts have a significantly lower number of nuclei compared with WT and Rac1-null osteoclasts. OE, oral epithelium; JE, junctional epithelium; D, dentin; PDL, periodontal ligament; AB, alveolar bone; P, pulp. P < 0.05, control (C) versus ligature (L) per genotype, t-test. P < 0.05, one-way analysis of variance for L groups. Data are shown as means ± SD; five mice per genotype.

      Discussion

      The present study demonstrated that neutrophil/monocyte-altered migratory function is associated with more severe ABL in response to a persistent subgingival challenge. This phenotype was characterized by increased osteoclastic coverage at the alveolar bone to the periodontal ligament interface. We further showed that the more severe bone loss in Rac2-null mice compared with Rac1-null and WT mice is not associated with osteoclast size as determined by number of nuclei per TRAP-stained bone-ligament interface length. These findings may be, in part, explained by the impaired neutrophil margination and extravasation in gingiva in response to proinflammatory stimulation as observed by intravital microscopy, which may lead to more rapid activation of adaptive immunity and bone resorption.
      Rac-null leukocytes have been reported to exhibit altered responses to shear stresses due to blood flow through impairment of pseudopod formation. Rac1- and Rac2-null leukocytes had an ability to form pseudopods in response to platelet-activating factor but did not respond to shear stress in vitro. This resulted in induced cell adhesion and microvascular stasis.
      • Makino A.
      • Glogauer M.
      • Bokoch G.M.
      • Chien S.
      • Schmid-Schönbein G.W.
      Control of neutrophil pseudopods by fluid shear: role of Rho family GTPases.
      This may, in part, explain the observation of increased endothelial attachment of Rac1-null leukocytes in acute gingivitis compared with WT leukocytes and reduced rolling and attachment of Rac2-null leukocytes. This indicates that the absence of Rac1 in leukocytes results in altered intravascular margination in the late stages (postrolling), whereas the absence of Rac2 results in altered intravascular margination in the early stages (rolling). Despite leukocytosis in the absence of inflammation and prominent release of neutrophils into the circulation in response to proinflammatory induction, Rac2-null leukocytes had a decreased attachment rate, which may be explained by the observed reduced rolling rate. Genetic mutations in human RAC2 have been found to cause severe phagocytic immunodeficiency characterized by severe infections in infancy.
      • Ambruso D.R.
      Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.
      • Kurkchubasche A.G.
      • Panepinto J.A.
      • Tracy Jr., T.F.
      • Thurman G.W.
      • Ambruso D.R.
      Clinical features of a human Rac2 mutation: a complex neutrophil dysfunction disease.
      This phenotype was reproduced in Rac2-null mice and resembles human leukocyte adhesion deficiency, being termed leukocyte adhesion deficiency type IV.
      • Pai S.-Y.
      • Kim C.
      • Williams D.A.
      Rac GTPases in human diseases.
      Furthermore, Rac-null leukocytes may have impaired transmigration of the endothelial wall because vascular cell adhesion molecule 1–mediated Rac signaling is thought to control endothelial cell-cell interactions and leukocyte transmigration.
      • Alevriadou B.R.
      CAMs and Rho small GTPases: gatekeepers for leukocyte transendothelial migration: focus on “VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration.”.
      • van Wetering S.
      • van den Berk N.
      • van Buul J.D.
      • Mul F.P.J.
      • Lommerse I.
      • Mous R.
      • Klooster ten J.-P.
      • Zwaginga J.-J.
      • Hordijk P.L.
      VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration.
      These functional requirements for adequate neutrophil function are critical for maintaining nonpathogenic subgingival biofilms. A study in germfree and conventional rats has confirmed that microorganisms are necessary for the occurrence of periodontal inflammation in response to silk ligatures placed around molars.
      • Rovin S.
      • Costich E.R.
      • Gordon H.A.
      The influence of bacteria and irritation in the initiation of periodontal disease in germfree and conventional rats.
      One explanation for pathogenic subgingival biofilm formation in this model could be the fact that silk is plaque retentive. Diseases characterized by neutropenia or by impairment of leukocyte adhesion, chemotaxis, phagocytosis (Chédiak–Higashi syndrome), and cathepsin C production (Papillon–Lefèvre syndrome) are associated with severe forms of periodontitis.
      • Schenkein H.A.
      Host responses in maintaining periodontal health and determining periodontal disease.
      • Meyle J.
      Leukocyte adhesion deficiency and prepubertal periodontitis.
      An interesting finding of the present study was the altered inflammatory response to the localized subgingival biofilm in Rac-null mice within 24 hours after induction. WT mice responded with a prominent monocyte increase in the circulation as expected in a physiologic inflammatory response. These monocytes would clear apoptotic neutrophils in the tissue, limit local damage, and decide the fate of the inflammatory response based on the ability to control the proinflammatory stimulus, ie, subgingival ligature and plaque. In Rac1-null mice, despite similar neutrophil release into the circulation to WT, peripheral blood monocyte numbers have progressively decreased. In Rac2-null mice, although a massive neutrophil pool was released into the circulation within 24 hours after ligature placement, monocytes have also decreased into the circulation, similar to Rac1-null mice. Note that inflammation around a single tooth can elicit a significant circulatory response. However, in our model the ligature was placed around second molars, therefore inducing periodontitis at adjacent surfaces of neighboring molars in addition to the M2. A total of 10 sites of 96, or 10.4% of sites per mouse, were affected. In addition, these findings suggest altered neutrophil-monocyte cross talk early in the inflammatory response for Rac-null leukocytes, possibly due to early neutrophil-altered recruitment and function in gingival tissues. Other studies have reported an altered inflammatory response in Rac-null mice and humans with a RAC2 inhibitory mutation, including chemotaxis and recruitment at sites of inflammation.
      • Glogauer M.
      • Marchal C.C.
      • Zhu F.
      • Worku A.
      • Clausen B.E.
      • Foerster I.
      • Marks P.
      • Downey G.P.
      • Dinauer M.
      • Kwiatkowski D.J.
      Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.
      • Ambruso D.R.
      Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation.
      • Kurkchubasche A.G.
      • Panepinto J.A.
      • Tracy Jr., T.F.
      • Thurman G.W.
      • Ambruso D.R.
      Clinical features of a human Rac2 mutation: a complex neutrophil dysfunction disease.
      • Roberts A.W.
      • Kim C.
      • Zhen L.
      • Lowe J.B.
      • Kapur R.
      • Petryniak B.
      • Spaetti A.
      • Pollock J.D.
      • Borneo J.B.
      • Bradford G.B.
      • Atkinson S.J.
      • Dinauer M.C.
      • Williams D.A.
      Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense.
      • Williams D.A.
      • Tao W.
      • Yang F.
      • Kim C.
      • Gu Y.
      • Mansfield P.
      • Levine J.E.
      • Petryniak B.
      • Derrow C.W.
      • Harris C.
      • Jia B.
      • Zheng Y.
      • Ambruso D.R.
      • Lowe J.B.
      • Atkinson S.J.
      • Dinauer M.C.
      • Boxer L.
      Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.
      This can be partially explained by a defect in tail retraction during cell migration.
      • Gardiner E.M.
      • Pestonjamasp K.N.
      • Bohl B.P.
      • Chamberlain C.
      • Hahn K.M.
      • Bokoch G.M.
      Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis.
      The increased and rapid ABL in Rac-null mice may be, in part, explained by a more sudden onset of adaptive immunity due to the altered innate immune response to proinflammatory stimulation. Although Rac-null mice seemed to have fewer neutrophils around silk filaments, in the junctional epithelium and connective tissue, they showed a mononuclear cellular infiltrate around silk filaments similar to WT mice. This is consistent with the current hypothesis that the onset of ABL and CAL occurs in established periodontal lesions characterized by a predominant mononuclear cell infiltrate (plasma cells and T lymphocytes).
      • Page R.C.
      • Offenbacher S.
      • Schroeder H.E.
      • Seymour G.J.
      • Kornman K.S.
      Advances in the pathogenesis of periodontitis: summary of developments, clinical implications and future directions.
      • Page R.C.
      • Schroeder H.E.
      Pathogenesis of inflammatory periodontal disease: a summary of current work.
      It is established that inflammatory macrophages, B and T lymphocytes, and activated fibroblasts can generate pro-osteoclastic environments in periodontium mainly by synthesizing prostaglandin E2, IL-1β, TNF-α, IL-17, receptor activator of nuclear factor kappa-B ligand, and matrix metalloproteinases.
      • Graves D.T.
      • Li J.
      • Cochran D.L.
      Inflammation and uncoupling as mechanisms of periodontal bone loss.
      • Graves D.
      Cytokines that promote periodontal tissue destruction.
      • Graves D.
      • Cochran D.
      The contribution of interleukin-1 and tumor necrosis factor to periodontal tissue destruction.
      Therefore, the observation of increased osteoclastic coverage of bone-ligament interface may, in part, be explained by a prominent adaptive immune response in Rac-null mice. Another explanation for these finding may be a direct influence of Rac on osteoclast maturation and function. It is currently believed that Rac is involved in the fusion of osteoclast precursors, membrane ruffling, and actin ring formation required for resorptive activity.
      • Razzouk S.
      • Lieberherr M.
      • Cournot G.
      Rac-GTPase, osteoclast cytoskeleton and bone resorption.
      • Goldberg S.R.
      • Georgiou J.
      • Glogauer M.
      • Grynpas M.D.
      A 3D scanning confocal imaging method measures pit volume and captures the role of Rac in osteoclast function.
      Rac-null mice, particularly Rac1-null mice, were found to have higher bone mineral density compared with WT controls, with decreased intratrabecular separation and increased trabecular volume and number.
      • Kawano T.
      • Troiano N.
      • Adams D.J.
      • Wu J.J.
      • Sun B.-H.
      • Insogna K.
      The anabolic response to parathyroid hormone is augmented in Rac2 knockout mice.
      • Magalhaes J.K.R.S.
      • Grynpas M.D.
      • Willett T.L.
      • Glogauer M.
      Deleting Rac1 improves vertebral bone quality and resistance to fracture in a murine ovariectomy model.
      Therefore, the high bone mineral density before the onset of ABL in the current model does not explain the more rapid bone loss in Rac2-null mice. On the contrary, a high bone mineral density would reduce the rate of ABL. Although the role of low bone mineral density as an independent risk factor for periodontitis in humans is still a subject of debate, most studies of this relationship suggest that osteopenia/osteoporosis may be an independent risk factor for periodontitis.
      • Reddy M.S.
      • Morgan S.L.
      Decreased bone mineral density and periodontal management.
      • Brennan-Calanan R.M.
      • Genco R.J.
      • Wilding G.E.
      • Hovey K.M.
      • Trevisan M.
      • Wactawski-Wende J.
      Osteoporosis and oral infection: independent risk factors for oral bone loss.
      • Geurs N.C.
      • Lewis C.E.
      • Jeffcoat M.K.
      Osteoporosis and periodontal disease progression.
      The present study is consistent with the new paradigm that the host immune response to persistent subgingival microbial challenge is critical to the resolution of inflammation and the restoration of periodontal tissue homeostasis.
      • Van Dyke T.E.
      The management of inflammation in periodontal disease.
      • Kantarci A.
      • Hasturk H.
      • Van Dyke T.E.
      Host-mediated resolution of inflammation in periodontal diseases.
      • Preshaw P.M.
      Host response modulation in periodontics.
      • Scott A.E.
      • Milward M.
      • Linden G.J.
      • Matthews J.B.
      • Carlile M.J.
      • Lundy F.T.
      • Naeeni M.A.
      • Lorraine Martin S.
      • Walker B.
      • Kinane D.
      • Brock G.R.
      • Chapple I.L.C.
      Mapping biological to clinical phenotypes during the development (21 days) and resolution (21 days) of experimental gingivitis.
      Disruption and reduction of subgingival plaque and calculus may not be sufficient to arrest disease progression in the long term, although immediate maintainable results are obvious. Modulation of host immune responses to subgingival pathogenic biofilms may prevent recurrence of disease or slow progression due to unresolved periodontal low-grade inflammation. Alteration in leukocyte RAC expression and function is one example of a potential host genetic predisposing factor. A recent study of RAC2 intraspecific genetic diversity analyzed two RAC2 regions and found three major haplotypes in humans, one of which was associated with autoimmune diseases, such as multiple sclerosis and Crohn disease.
      • Sironi M.
      • Guerini F.R.
      • Agliardi C.
      • Biasin M.
      • Cagliani R.
      • Fumagalli M.
      • Caputo D.
      • Cassinotti A.
      • Ardizzone S.
      • Zanzottera M.
      • Bolognesi E.
      • Riva S.
      • Kanari Y.
      • Miyazawa M.
      • Clerici M.
      An evolutionary analysis of RAC2 identifies haplotypes associated with human autoimmune diseases.
      One study reported up-regulation of RAC2 in periodontitis-affected tissues by whole-transcriptome gene expression analysis.
      • Abe D.
      • Kubota T.
      • Morozumi T.
      • Shimizu T.
      • Nakasone N.
      • Itagaki M.
      • Yoshie H.
      Altered gene expression in leukocyte transendothelial migration and cell communication pathways in periodontitis-affected gingival tissues.
      These findings support a potential role for genetic and epigenetic changes in RAC2 transcription in higher susceptibility to inflammation-mediated ABL in humans.
      In conclusion, the present study brings new light to the host genetic/epigenetic immune predisposition to rapid and severe bone resorption in response to persistent subgingival challenge. Impairment of leukocyte margination and transmigration into gingival tissues during the acute phase of inflammation affects the course of local inflammation to exogenous stimuli and more rapid resorption of tooth-supporting bone. These findings may aid in identifying critical immune factors involved in host-biofilm interactions to maintain homeostasis and control pro-osteolytic environments.

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