Down-regulation of expression of osteoblast and osteocyte markers in periodontal tissues associated with the spontaneous alveolar bone loss of interleukin-10 knockout mice

The aim of this study was to unravel the mechanisms by which interleukin (IL)-10, a potent pleiotropic cytokine, modulates alveolar bone homeostasis in C57BL/6 wild-type (WT) and IL-10 knockout (IL-10 KO) mice, evaluated at 8, 24, and 48 wk of age. Interleukin-10 KO mice presented significant alveolar bone loss when compared with WT mice, and this was not associated with changes in leukocyte counts or bacterial load. The levels of expression of messenger RNA (mRNA) for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, transforming growth factor-beta (TGF-beta), receptor activator of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), and matrix metalloproteinase 13 (MMP13) were similar between both strains, whereas a significant decrease of tissue inhibitor of metalloproteinase 1 (TIMP1) mRNA expression was found at 48 wk in IL-10 KO mice. The osteoblast markers core binding factor alpha1 (CBFA1) and type I collagen (COL-I) were expressed at similar levels in both strains, whereas the levels of alkaline phosphatase (ALP) and osteocalcin (OCN), and those of the osteocyte markers phosphate-regulating gene endopeptidases (PHEX) and dentin matrix protein 1 (DMP1) were significantly lower in IL-10 KO mice. Our results demonstrate that the alveolar bone loss in the absence of IL-10 was associated with a reduced expression of osteoblast and osteocyte markers, an effect independent of microbial, inflammatory or bone-resorptive pathways.FAPESPCNP

CCR5 Delta 32 (rs333) polymorphism is associated with decreased risk of chronic and aggressive periodontitis: a case control analysis based in disease resistance and susceptibility phenotypes

Chronic and aggressive periodontitis are infectious diseases characterized by the irreversible destruction of periodontal tissues, which is mediated by the host inflammatory immune response triggered by periodontal infection. The chemokine receptor CCR5 play an important role in disease pathogenesis, contributing to pro inflammatory response and osteoclastogenesis. CCR5 Delta 32 (rs333) is a loss-of-function mutation in the CCR5 gene, which can potentially modulate the host response and, consequently periodontitis outcome. Thus, we investigated the effect of the CCR5 Delta 32 mutation over the risk to suffer periodontitis in a cohort of Brazilian patients (total N = 699), representative of disease susceptibility (chronic periodontitis, N = 197; and aggressive periodontitis, N-= 91) or resistance (chronic gingivitis, N = 193) phenotypes, and healthy subjects (N = 218). Additionally, we assayed the influence of CCR5 Delta 32 in the expression of the biomarkers TNFa, IL-10, IL-6, IFN-y and T-bet, and key periodontal pathogens P. gingivalis, T. forsythia, and T. denticola. In the association analysis of resistant versus susceptible subjects, CCR5 Delta 32 mutant allele-carriers proved significantly protected against chronic (OR 0.49; 95% CI 0.29-0.83; p-value 0.01) and aggressive (OR 0.46; 95% CI 0.22-0.94; p-value 0.03) periodontitis. Further, heterozygous subjects exhibited significantly decreased expression of TNFa in periodontal tissues, pointing to a functional effect of the mutation in periodontal tissues during the progression of the disease. Conversely, no significant changes were observed in the presence or quantity of the periodontal pathogens P. gingivalis, T. forsythia, and T. denticola in the subgingival biofilm that could be attributable to the mutant genotype.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) 2014/03276-0 2014/17886-

Functional Local Renin-Angiotensin System in Human and Rat Periodontal Tissue

<div><p>The initiation or progression of periodontitis might involve a local renin-angiotensin system (RAS) in periodontal tissue. The aim of this study was to further characterize the local RAS in human and rat periodontal tissues between healthy and periodontally-affected tissue. Components of the RAS were investigated using <i>in vitro</i>, <i>ex vivo</i> and <i>in vivo</i> experiments involving both human and Wistar rat periodontium. Although not upregulated when challenged with <i>P</i>. <i>gingivalis</i>-lipopolysaccharide, human gingival and periodontal ligament fibroblasts expressed RAS components. Likewise, healthy and inflamed human gingiva expressed RAS components, some of which were shown to be functional, yet no differences in expression were found between healthy and diseased gingiva. However, in inflamed tissue the immunoreactivity was greater for the AT₁R compared to AT₂R in fibroblasts. When compared to healthy tissue, ACE activity was increased in human gingiva from volunteers with gingivitis. Human-gingiva homogenates generated Ang II, Ang 1-9 and Ang 1-7 when incubated with precursors. In gingiva homogenates, Ang II formation from Ang I was nearly abolished only when captopril and chymostatin were combined. Ang 1-7 formation was significantly greater when human gingiva homogenates were incubated with chymostatin alone compared to incubation without any inhibitor, only captopril, or captopril and chymostatin. In rat gingiva, RAS components were also found; their expression was not different between healthy and experimentally induced periodontitis (EP) groups. However, renin inhibition (aliskiren) and an AT₁R antagonist (losartan) significantly blocked EP-alveolar-bone loss in rats. Collectively, these data are consistent with the hypothesis that a local RAS system is not only present but is also functional in both human and rat periodontal tissue. Furthermore, blocking AT₁R and renin can significantly prevent periodontal bone loss induced by EP in rats.</p></div

Renin-Angiotensin System Components in the Gingiva Tissue of Rats with Experimentally Induced Periodontitis.

<p><b>A)</b> Results of qPCR analysis for mRNA of various RAS components extracted from the rat gingiva of either sham surgery (sham) or experimentally induced periodontitis (EP) for 14 days; tested RAS components included the following: angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE-2), angiotensin II receptor type 1A (AT_1AR), angiotensin II receptor type 1B (AT_1BR), angiotensin II receptor type 2 (AT₂R), and Mas receptor (MasR). Graph displays expression levels of the target mRNA relative to β-actin mRNA from 5 rats in duplicate (n = 5). Solid bars represent the means with SD of sham group; whereas, open bars represent animals with EP. A one-way ANOVA and Tukey’s test with statistical significance set at <i>p</i>-value < 0.05 was used. <b>B)</b> All images are at 10x magnification and scale bars indicate a distance of 100 μm. Immunoreactivity (IR) for renin (panels a to d), ACE (panels e to h), AT₁R (panels i to l) or AT₂R (panels m to p) in rat gingiva and bone tissue. Brown staining indicates positive IR. Black arrows indicate some of the positive IR in the positive controls. Negative <b>(panel a)</b> and positive <b>(panel b)</b> control for renin in rat kidney. <b>Panel c)</b> negative control for renin in mandible of rat with 14 d of EP treated with water. <b>Panel d)</b> immunoreactivity for renin in mandible of rat with 14 d of EP treated with water. Negative <b>(panel e)</b> and positive <b>(panel f)</b> control for ACE in a rat kidney. <b>Panel g)</b> negative control for ACE in mandible of rat with 14 d of EP treated with water. <b>Panel h)</b> immunoreactivity for ACE in mandible of rat with 14 d of EP treated with water. Negative <b>(panel i)</b> and positive <b>(panel j)</b> control for AT₁Rs in a rat adrenal gland. <b>Panel k)</b> negative control for AT₁Rs in mandible of rat with 14 d of EP treated with water. <b>Panel l)</b> Immunoreactivity for AT₁Rs in mandible of rat with 14 d of EP treated with water. Negative <b>(panel m)</b> and positive <b>(panel n)</b> control for AT₂Rs in a rat adrenal gland. <b>Panel o)</b> negative control for AT₂Rs in mandible of rat with 14 d of EP treated with water. <b>Panel p)</b> immunoreactivity for AT₂Rs in mandible of rat with 14 d of EP treated with water. <b>C)</b> Table indicating location of immunoreactivity observed in 2 sections of 5 rats with 14 d of EP treated with water for renin, ACE, AT₁Rs and AT₂Rs in different mandibular regions: (-) indicates negative immunoreactivity, (+) indicates positive immunoreactivity and (++) indicates abundant immunoreactivity.</p

Renin-Angiotensin System Components in Human Gingiva.

<p><b>A)</b> qPCR analysis of the following RAS components extracted from human gingiva from healthy (open bars), gingivitis (gray bars) and periodontitis (black bars) groups: angiotensinogen (AGT), angiotensin converting enzyme (ACE), angiotensin converting enzyme 2 (ACE-2), renin, angiotensin II receptor type 1 (AT₁R), and Mas receptor (MasR), (n = 7). Graphs displays relative expression levels of the target mRNA relative to RPL-13 mRNA. The means were compared using a 1-way ANOVA and Tukey’s test. <b>B)</b> Immunoreactivity of AT₁R and AT₂R in the gingiva from healthy volunteers (open bars) and volunteers with gingivitis and periodontitis combined (black bars). The means were compared using a 1-way ANOVA and Ang with gingivitis and periodontitis is indicated by *. <b>C)</b> Representative photographs of immunoreactivity for either AT₁R (Ca and Ce) or AT₂R (Ce and Cf) in the gingiva from either healthy volunteers (Ca and Cd) or volunteers with gingivitis and periodontitis (Ce and Cf). Photographs Cb and Cd are from tissue incubated with nonimmune serum in Healthy Tissue. Scale bars indicate a distance of 20μm.</p

Bone Loss in Rats with Experimentally Induced Periodontitis.

<p><b>A)</b> Graph with representative images indicating the amount of area measured between the cementoenamel junction (CEJ) to the alveolar bone crest (ABC) of the first molar after 14 d of experimentally induced periodontal disease (EP) or fictitious induction (sham). Rats were treated with vehicle (water), losartan (50 mg/kg), aliskiren (30 mg/kg) or enalapril (10 mg/kg). All groups n = 5. Statistical significance was determined by ANOVA with interaction analysis and Tukey’s test. Statistical significance (<i>p</i>-value < 0.05) is indicated by * <i>vs</i>. periodontal disease rats treated with water. <b>B to F)</b> Representative images of each of the following: sham group treated with water <b>(B)</b>, EP group treated with water <b>(C)</b>, aliskiren <b>(D)</b>, losartan <b>(E</b>) or enalapril <b>(F</b>). The samples pictured have the following respective CEJ to ABC area: (B) 2.1 mm², (C) 3.3 mm², (D) 2.1 mm², (E) 2.1 mm², (F) 3.1 mm².</p

Catalog numbers for primers used for human qPCR—RAS genes assays (Applied Biosystems).

<p>Catalog numbers for primers used for human qPCR—RAS genes assays (Applied Biosystems).</p

Schematic Representation of the Renin-Angiotensin System.

<p>Angiotensin-I converting enzyme, ACE; angiotensin I converting enzyme 2, ACE-2; angiotensin II 1 receptor, AT₁R; angiotensin II 2 receptor, AT₂R; aminopeptidase, AP; aminopeptidase A, APA; aminopeptidase N, APN; carboxypeptidase, CPP; endopeptidase, EP; Mas receptor, MasR; Mas-related gene type D receptor, MrgDR; neprilysin, NEP; (pre)prorenin receptor, PRR.</p

ACE Activity and Generation of Renin-Angiotensin System Components from Human Gingiva.

<p><b>A)</b> Fluorimetric assay of ACE activity in gingiva homogenates from donors with healthy gingiva (open bar), gingivitis (gray bar) or periodontitis (black bar). The means were compared using a 1-way ANOVA and Tukey’s test. * indicates significant difference from healthy group. <b>B to E)</b> HPLC assay of homogenates from all three groups after incubation with either captopril (10μM), chymostatin (100μM), both captopril (10μM) and chymostatin (100μM), or nothing. * indicates significant differences from all other healthy groups (open bars); † indicates significant differences from all other gingivitis groups (gray bars); ‡ indicates significant differences from all other periodontitis groups (black bars); unless otherwise noted with brackets (<i>e</i>.<i>g</i>. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134601#pone.0134601.g003" target="_blank">Fig 3C</a>). <b>B)</b> Indicates the amount of Ang II formed when incubated with Ang I. <b>C)</b> Indicates the amount of Ang 1–9 formed when incubated with Ang I. <b>D)</b> Indicates the amount of Ang 1–7 formed when incubated with Ang I. <b>E)</b> Indicates the amount of Ang 1–7 formed when incubated with Ang II.</p