Claudia Cristina Biguetti

IL‐4/CCL22/CCR4 Axis Controls Regulatory T‐Cell Migration That Suppresses Inflammatory Bone Loss in Murine Experimental Periodontitis

Inflammatory bone resorption is a hallmark of periodontitis, and Tregs and Th2 cells are independently associated with disease progression attenuation. In this study, we employed an infection‐triggered inflammatory osteolysis model to investigate the mechanisms underlying Treg and Th2 cell migration and the impact on disease outcome. Aggregatibacter actinomycetemcomitans–infected C57Bl/6 (wild‐type [WT]) mice develop an intense inflammatory reaction and alveolar bone resorption, and Treg and Th2 cell migration is temporally associated with disease progression attenuation. Tregs extracted from the lesions preferentially express CCR4 and CCR8, whereas Th2 cells express CCR3, CCR4, and CCR8. The absence of CCR5 and CCR8 did not significantly impact the migration of Tregs and Th2 cells or affect the disease outcome. CCR4KO mice presented a minor reduction in Th2 cells in parallel with major impairment of Treg migration, which was associated with increased inflammatory bone loss and higher proinflammatory and osteoclastogenic cytokine levels. The blockade of the CCR4 ligand CCL22 in WT mice resulted in an increased inflammatory bone loss phenotype similar to that in the CCR4KO strain. Adoptive transfer of CCR4+ Tregs to the CCR4KO strain revert the increased disease phenotype to WT mice–like levels; also, the in situ production of CCL22 in the lesions is mandatory for Tregs migration and the consequent bone loss arrest. The local release of exogenous CCL22 provided by poly(lactic‐co‐glycolic acid) (PLGA) microparticles promotes migration of Tregs and disease arrest in the absence of endogenous CCL22 in the IL‐4KO strain, characterized by the lack of endogenous CCL22 production, defective migration of Tregs, and exacerbated bone loss. In summary, our results show that the IL‐4/CCL22/CCR4 axis is involved in the migration of Tregs to osteolytic lesion sites, and attenuates development of lesions by inhibiting inflammatory migration and the production of proinflammatory and osteoclastogenic mediators.

Cytokine Networks Regulating Inflammation and Immune Defense in the Oral Cavity

The host/pathogen interaction in infectious oral diseases is characterized by complex and precisely orchestrated host response mechanisms aimed to protect the host against the microbial challenge with minimal collateral damage to host cells and tissues. Central to the host response in this battlefront is the expression of cytokines. The resulting cytokine networks, which ultimately regulate the host response at multiple levels, thereby determine the clinical outcome of the disease and explain most of its defining features from a mechanistic viewpoint. This review intends to present a structured view of the intricate cytokine networks that regulate inflammation and immune defense in the oral cavity, guiding the reader throughout the evolving paradigms that describe how the simultaneous action of multiple cytokines shapes the nature of the immune response to oral infection.

Mesenchymal Stem Cells as Active Prohealing and Immunosuppressive Agents in Periapical Environment: Evidence from Human and Experimental Periapical Lesions

INTRODUCTION Previous studies describe contrasting molecular profiles of active and inactive periapical granulomas characterized by distinct expression of cytokines, osteoclastogenic factors, and wound healing markers. Although the molecular mechanisms underlying such a dichotomy remain unknown, in this study we investigated the potential involvement of mesenchymal stem cells (MSCs) in determining human and murine periapical lesion activity and outcomes. METHODS Periapical granulomas (n = 83) and control samples (n = 24) were comparatively assessed for the expression levels of 11 mesenchymal stem cell (MSC) markers using real-time polymerase chain reaction. Experimental periapical lesions induced in mice were evaluated for MSC marker expression and the effects of AMD3100 treatment on lesion outcomes. RESULTS MCS marker expression was prevalent in periapical granulomas compared with that in controls, whereas CD29, CD73, CD90, CD146, CD166, NANOG, Stro-1, and CXCR4 expressions were higher in inactive than in active lesions. Experimental periapical lesion inactivity was also associated with an increased expression of MSC markers. The inhibition of MSC mobilization to the periapex by AMD3100 resulted in increased lesion sizes; decreased expression of MSCs and wound healing markers; and increased expression of interleukin 1 beta (IL-17β), interleukin 17 (IL-17), tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), and nuclear factor kappa-B ligand (RANKL). CONCLUSIONS Our results show that MSC markers are overexpressed in inactive human and experimental periapical lesions and that MSC mobilization results in the attenuation of experimental lesion progression associated with immunosuppressive and prohealing mechanisms.

Analysis of Multiple Cytokine Polymorphisms in Individuals with Untreated Deep Carious Lesions Reveals IL1B (rs1143643) as a Susceptibility Factor for Periapical Lesion Development

INTRODUCTION It has been proposed that individual genetic predisposition may contribute to persistent apical periodontitis. Cytokines are associated with levels of inflammation and are involved in caries, pulpal, and periapical tissue destruction. We hypothesized that polymorphisms in cytokine genes may contribute to an individuals increased susceptibility to apical tissue destruction in response to deep carious lesions. METHODS Subjects with deep carious lesions with or without periapical lesions (≥3 mm) were recruited at the University of Pittsburgh, Pittsburgh, PA, and the University of Texas at Houston, Houston, TX. Genomic DNA samples of 316 patients were sorted into 2 groups: 136 cases with deep carious lesions and periapical lesions (cases) and 180 cases with deep carious lesions but no periapical lesions (controls). Nine single-nucleotide polymorphisms in IL1B, IL6, TNF, RANK, RANKL, and OPG genes were selected for genotyping. Genotypes were generated by end point analysis using TaqMan chemistry (Invitrogen, Carlsbad, CA) in a real-time polymerase chain reaction instrument. Allele and genotype frequencies were compared among cases and controls using the PLINK program (http://pngu.mgh.harvard.edu/purcell/plink/). Ninety-three human periapical granulomas and 24 healthy periodontal ligament tissues collected postoperatively were used for messenger RNA expression analyses of IL1B. RESULTS A single-nucleotide polymorphism in IL1B (rs1143643) showed allelic (P = .02) and genotypic (P = .004) association with cases of deep caries and periapical lesions. We also observed altered transmission of IL1B marker haplotypes (P = .02) in these individuals. IL1B was highly expressed in granulomas (P < .001). CONCLUSIONS Variations in IL1B may be associated with periapical lesion formation in individuals with untreated deep carious lesions. Future studies could help predict host susceptibility to developing periapical lesions.

TBX21-1993T/C (rs4794067) polymorphism is associated with increased risk of chronic periodontitis and increased T-bet expression in periodontal lesions, but does not significantly impact the IFN-g transcriptional level or the pattern of periodontophatic bacterial infection

Th1-polarized host response, mediated by IFN-γ, has been associated with increased severity of periodontal disease as well as control of periodontal infection. The functional polymorphism TBX21-1993T/C (rs4794067) increases the transcriptional activity of the TBX21 gene (essential for Th1 polarization) resulting in a predisposition to a Th-1 biased immune response. Thus, we conducted a case-control study, including a population of healthy controls (H, n = 218), chronic periodontitis (CP, n = 197), and chronic gingivitis patients (CG, n = 193), to investigate if genetic variations in TBX21 could impact the development of Th1 responses, and consequently influence the pattern of bacterial infection and periodontitis outcome. We observed that the polymorphic allele T was significantly enriched in the CP patients compared to CG subjects, while the H controls demonstrated and intermediate genotype. Also, investigating the putative functionality TBX21-1993T/C in the modulation of local response, we observed that the transcripts levels of T-bet, but not of IFN-γ, were upregulated in homozygote and heterozygote polymorphic subjects. In addition, TBX21-1993T/C did not influence the pattern of bacterial infection or the clinical parameters of disease severity, being the presence/absence of red complex bacteria the main factor associated with the disease status and the subrogate variable probing depth (PD) in the logistic regression analysis.

Characterization of a Vascular Endothelial Growth Factor–loaded Bioresorbable Delivery System for Pulp Regeneration

Introduction: Vascular endothelial growth factor (VEGF) is a signal protein that stimulates angiogenesis and vasculogenesis and has been used in tissue regeneration and pulp regeneration experimental models. The purpose of this study was to develop a delivery system composed of a biodegradable fiber and controlled release of VEGF to promote cell viability and secure an adequate blood supply for the survival of human stem cells of the apical papilla (SCAP) favoring endodontic regenerative procedures. Methods: We developed a polydioxanone fiber, 50 &mgr;m in diameter, loaded with VEGF at a linear concentration of 12.2 ng/cm. Cytotoxic effects of the VEGF‐loaded fiber (VF) on SCAP and mouse fibroblasts were assessed by using a multiparametric assay kit (XTT‐NR‐CVDE [Xenometrix, Allschwil, Switzerland]). We evaluated VF‐induced mRNA expression of downstream growth factors by using a human growth factor Taqman array in real‐time polymerase chain reaction. We also assessed the in vivo subcutaneous reaction of C57BL/6 mice to implants of VF alone and human root fragments (10 mm in length) filled with VF after 10, 20, and 45 days. Statistical analyses were performed by using analysis of variance and Student t tests or non‐parametric alternatives. Results: Enzyme‐linked immunosorbent assay verified detectable concentrations of released VEGF in solution for 25 days. No cytotoxicity was observed on SCAP and mouse fibroblasts treated with VEGF. In addition, VEGF treatment also induced the expression of additional growth factors with roles in tissue and blood vessel formation and neuroprotective function. Implantation of VF and root fragments filled with VF showed biocompatibility in vivo, promoting new blood vessels and connective tissue formation into the root canal space with negligible inflammation. Conclusions: Our results show that the VF used in this study is biocompatible and may be a promising scaffold for additional optimization and use in endodontic regenerative procedures. Highlights:A vascular endothelial growth factor fiber was developed to secure blood supply to stem cells of the apical papilla.Fiber showed biocompatibility in vivo and in vitro.Fiber induced expression of additional growth factors.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool

INTRODUCTION The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. METHODS Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti-glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). RESULTS Treg inhibition by anti-glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. CONCLUSIONS Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

Effects of angiotensin II type I receptor blocker losartan on orthodontic tooth movement

INTRODUCTION Drugs that block the renin-angiotensin system (RAS) are widely used for treating hypertension, heart and kidney failure, and the harmful effects of diabetes. Components of the RAS have been identified in various organs, but little is known of their effects on bone remodeling. The aim of this study was to evaluate whether the blockage of the RAS influences strain-induced bone remodeling in a model of orthodontic tooth movement. METHODS An orthodontic appliance was placed in C57BL6/J mice that were randomly divided into 2 groups: vehicle-treated mice (VH) and mice treated with losartan (an angiotensin II receptor blocker). Orthodontic tooth movement and the number of tartrate-resistant acid phosphatase-positive cells were determined by histopathologic analysis. The expression of mediators involved in bone remodeling was evaluated by quantitative real-time polymerase chain reaction. Blood pressure was measured before and during the experimental period. RESULTS Orthodontic tooth movement and tartrate-resistant acid phosphatase-positive cells were significantly reduced in the losartan group compared with the VH group. mRNA levels of osteoclast markers (RANK, RANKL, cathepsin K, and metalloproteinase 13) were lower in the losartan mice than in the VH group, whereas the expressions of osteoblast markers and negative regulators of bone resorption (periostin, dentin matrix protein, alkaline phosphatase, collagen 1A1, semaphorin 3A3, metalloproteinase 2, and osteoprotegerin) were higher in the VH group. CONCLUSIONS Blockage of the RAS system decreases osteoclast differentiation and activity and, consequently, results in decreased strain-induced bone remodeling in orthodontic tooth movement.

Effects of green tea and bisphosphonate association on dental socket repair of rats

OBJECTIVES To evaluate the effects of green tea intake and zoledronic acid intravenous therapy on teeth socket repair. DESIGN Sixty male albinus Wistar rats were divided into 4 groups: C-Control, intravenous (IV) 0.9% saline solution (SS), GT-1% green tea in drinking water and IV SS, BP-IV zoledronic acid (BP), and BP+GT-IV BP and 1% green tea. 0.035mg/kg of BP was administered every two weeks. After ten weeks, right upper molars were extracted and the green tea started to be offered for GT and BP+GT. After 7, 14, and 28days the animals were euthanized. RESULTS Histopathology analysis revealed lack of socket repair in BP and BP+GT groups, which presented significant increased number of polimorphonuclear leukocytes at day 28, in comparison with C (p<0.05). No significant differences were detected between C and the experimental groups at the same period (p<0.05) when considering mononuclear leukocytes. Immunolabeling revealed that the association of BP and GT caused a slight disturbance in OPG/RANKL system and retarded Runx-2 labeling. Although strong TRAP labeling was observed, most of the positive cells in BP and BP+GT groups were not located on bone surface. CONCLUSIONS Socket healing of rats treated with BP and regular drinking green tea presented no relevant differences in comparison to those treated with BP alone.

CCR5δ32 (rs333) polymorphism is associated with decreased risk of chronic and aggressive periodontitis: A case-control analysis based in disease resistance and susceptibility phenotypes

Abstract 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&Dgr;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&Dgr;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&Dgr;32 in the expression of the biomarkers TNF&agr;, IL‐1&bgr;, IL‐10, IL‐6, IFN‐&ggr; and T‐bet, and key periodontal pathogens P. gingivalis, T. forsythia, and T. denticola. In the association analysis of resistant versus susceptible subjects, CCR5&Dgr;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 TNF&agr; 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.