The alveolar bone repair process may be influenced by multiple local and systemic factors, which include immune system cells and mediators. Macrophages allegedly play important roles in the repair ...process, and the transition of an initial inflammatory M1 profile into a pro-reparative M2 profile theoretically contributes to a favorable repair outcome. In this context, considering immunoregulatory molecules as potential targets for improving bone repair, this study evaluated the role of the immunoregulatory molecule FTY720, previously described to favor the development of the M2 phenotype, in the process of alveolar bone healing in C57Bl/6 (WT) mice. Experimental groups submitted to tooth extraction and maintained under control conditions or treated with FTY720 were evaluated by microtomographic (μCT), histomorphometric, immunohistochemical and molecular analysis to characterize healing and host response features at 0, 1, 3, 7 and 14 days. Our results demonstrated that the FTY720 group presented higher bone tissue density, higher bone tissue volume, greater tissue volume fraction, greater number and thickness of trabeculae and a higher number of osteoblasts and osteoclasts than the control group. Accordingly, the bone markers BMP2, BMP7, ALPL, SOST and RANK mRNA expressions increased in the FTY720 treated group. Furthermore, the levels of FIZZ, ARG2 and IL-10 mRNA increased in the FTY720 group together with the presence of CD206+ cells, suggesting that the boost of bone formation mediated by FTY720 involves an increased polarization and activity of M2 macrophages in healing sites. Thus, our results demonstrate that FTY720 favored the process of alveolar bone repair, probably trough a strengthened M2 response, associated with an increased expression of markers osteogenic differentiation and activity markers. Immunoregulatory strategies based in the modulation of macrophage polarization profile can comprise effective tools to improve the bone repair process.
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•Bone repair is influenced by immune system cells and mediators.•Macrophages M2 pro-reparative profile theoretically contributes to a favorable repair outcome.•Immunoregulatory molecule FTY720 favors the development of the M2 phenotype.•FTY720 favored the process of alveolar bone repair along with a strengthened M2 response.
Bone tissue has a significant potential for healing, which involves a significant the interplay between bone and immune cells. While fracture healing represents a useful model to investigate ...endochondral bone healing, intramembranous bone healing models are yet to be developed and characterized. In this study, a micro-computed tomography, histomorphometric and molecular (RealTimePCRarray) characterization of post tooth-extraction alveolar bone healing was performed on C57Bl/6 WT mice. After the initial clot dominance (0 h), the development of a provisional immature granulation tissue is evident (7 d), characterized by marked cell proliferation, angiogenesis and inflammatory cells infiltration; associated with peaks of growth factors (BMP-2-4-7,TGFβ1,VEGFa), cytokines (TNFα, IL-10), chemokines & receptors (CXCL12, CCL25, CCR5, CXCR4), matrix (Col1a1-2, ITGA4, VTN, MMP1a) and MSCs (CD105, CD106, OCT4, NANOG, CD34, CD146) markers expression. Granulation tissue is sequentially replaced by more mature connective tissue (14 d), characterized by inflammatory infiltrate reduction along the increased bone formation, marked expression of matrix remodeling enzymes (MMP-2-9), bone formation/maturation (RUNX2, ALP, DMP1, PHEX, SOST) markers, and chemokines & receptors associated with healing (CCL2, CCL17, CCR2). No evidences of cartilage cells or tissue were observed, strengthening the intramembranous nature of bone healing. Bone microarchitecture analysis supports the evolving healing, with total tissue and bone volumes as trabecular number and thickness showing a progressive increase over time. The extraction socket healing process is considered complete (21 d) when the dental socket is filled by trabeculae bone with well-defined medullary canals; it being the expression of mature bone markers prevalent at this period. Our data confirms the intramembranous bone healing nature of the model used, revealing parallels between the gene expression profile and the histomorphometric events and the potential participation of MCSs and immune cells in the healing process, supporting the forthcoming application of the model for the better understanding of the bone healing process.
Abstract Inflammatory cytokines contribute to periapical tissue destruction. Their activity is potentially regulated by suppressors of cytokine signaling (SOCS), which downregulate signal ...transduction as part of an inhibitory feedback loop. We investigated the expression of the cytokines tumor necrosis factor α (TNF-α); interleukin (IL)-10 and RANKL; and SOCS-1, -2, and -3 by real-time polymerase chain reaction in 57 periapical granulomas and 38 healthy periapical tissues. Periapical granulomas exhibited significantly higher SOCS-1, -2, and -3, TNF-α, IL-10, and RANKL messenger RNA levels when compared with healthy controls. Significant positive correlations were found between SOCS1 and IL-10 and between SOCS3 and IL-10. Significant inverse correlations were observed between SOCS1 and TNF-α, SOCS1 and RANKL, and SOCS3 and TNF-α. Increased SOCS-1, -2, and -3 messenger RNA levels in periapical granulomas may be related to the downregulation of inflammatory cytokines in these lesions; therefore, SOCS molecules may play a role in the dynamics of periapical granulomas development.
Angiogenesis and lymphangiogenesis are the processes of neovascularization that evolve from preexisting blood and lymphatic vessels. There are few studies on angiogenesis and none on ...lymphangiogenesis in leprosy. Thus, the role of neovascularization in the pathophysiological mechanisms of the disease was studied across the spectrum of leprosy, its reactional states and its residual lesions.
Seventy-six biopsies of leprosy skin lesions and seven healthy controls were selected. Fifty-five serum samples were used for the detection of CD105 by ELISA. Histological sections were stained with antibodies against CD31 (blood and lymphatic vessels), D2-40/podoplanin (lymphatic vessels), and CD105/endoglin (neovessels). Microvessels were counted in 100 high-power fields (400x) and the number of vessels was evaluated in relation to the extension of the inflammatory infiltrate (0-3), to the bacillary index (0-6) and to the clinical forms. Angiogenesis, as marked by CD31 and CD105, was observed across the leprosy spectrum, compared with the controls. Additionally, there was a positive correlation between these markers with extension of the infiltrate (p <0.0001). For D2/40, lymphangiogenesis was observed in the tuberculoid form (p <0.0001). There was no statistical significance for values of CD105 detected in plasma by ELISA.
Angiogenesis is present across the spectrum of leprosy and in its reactional forms. The increase in the number of vessels, as detected by CD31 and CD105 staining, is related to the extension of the inflammatory infiltrate. Samples from reactional lesions have a higher number of CD31+ and CD105+ stained vessels, which indicates their involvement in the pathophysiological mechanisms of the reactional states. The regression of lesions is accompanied by the regression of neovascularization. Drugs inhibiting angiogenesis may be relevant in the treatment of leprosy, in addition to multidrugtherapy, and in the prevention of the development of reactions.
da Fonseca DM, Trombone APF, Repeke CE, Avila‐Campos MJ, Coelho‐Castelo AAM, Silva JS, Campanelli AP, Bonato VLD, Garlet GP. Functional interferences in host inflammatory immune response by airway ...allergic inflammation restrain experimental periodontitis development in mice. J Clin Periodontol 2011; 38: 131–141. doi: 10.1111/j.1600‐051X.2010.01660.x
Aims: Periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown.
Material and Methods: Balb/C mice were submitted to the co‐induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges, and evaluated regarding PD and AL severity, immune response cytokine production at periodontal tissues, and T‐helper transcription factors in submandibular lymph nodes (LNs) and infection parameters.
Results: PD/AL co‐induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor‐α (TNF‐α), interleukin (IL)‐1β,
interferon‐γ, IL‐17A, receptor activator of nuclear factor κ‐light‐chain‐enhancer of activated B cells ligand and matrix metalloproteinase (MMP)‐13 levels in periodontal tissues, while IL‐4 and IL‐10 levels were unaltered by AL co‐induction. AL co‐induction also resulted in upregulated T‐bet and related orphan receptor γ and downregulated GATA3 levels expression in submandibular LNs when compared with PD group.
Conclusion: Our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.
The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to ...trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT bone and histology bone and subcutaneous) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome.
Previous studies demonstrate that the balance between pro- and anti-inflammatory mediators determines the stable or progressive nature of periapical granulomas by modulating the balance of the ...osteoclastogenic factor RANKL and its antagonist OPG. However, the cytokine networks operating in the development of periapical lesions are quite more complex than what the simple pro- versus anti-inflammatory mediators' paradigm suggests. Here we simultaneously investigated the patterns of Th1, Th2, Th9, Th17, Th22, Thf, Tr1 and Tregs cytokines/markers expression in human periapical granulomas.
The expression of TNF-α, IFN-γ, IL-17A, IL23, IL21, IL-33, IL-10, IL-4, IL-9, IL-22, FOXp3 markers (via RealTimePCR array) was accessed in active/progressive (N=40) versus inactive/stable (N=70) periapical granulomas (as determined by RANKL/OPG expression ratio), and also to compare these samples with a panel of control specimens (N=26). A cluster analysis of 13 cytokine levels was performed to examine possible clustering between the cytokines in a total of 110 granulomas.
The expression of all target cytokines was higher in the granulomas than in control samples. TNF-α, IFN-γ, IL-17A and IL-21 mRNA levels were significantly higher in active granulomas, while in inactive lesions the expression levels of IL-4, IL-9, IL-10, IL-22 and FOXp3 were higher than in active granulomas. Five clusters were identified in inactive lesion groups, being the variance in the expression levels of IL-17, IL-10, FOXp3, IFN-γ, IL-9, IL-33 and IL-4 statistically significant (KW p<0.05). Three clusters were identified in active lesions, being the variance in the expression levels of IL-22, IL-10, IFN-γ, IL-17, IL-33, FOXp3, IL-21 and RANKL statistically significant (KW p<0.05).
There is a clear dichotomy in the profile of cytokine expression in inactive and active periapical lesions. While the widespread cytokine expression seems to be a feature of chronic lesions, hierarchical cluster analysis demonstrates the association of TNF-α, IL-21, IL-17 and IFN-γ with lesions activity, and the association of FOXP3, IL-10, IL-9, IL-4 and IL-22 with lesions inactivity.
Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for ...growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1(nu)). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.
Despite the successful clinical application of titanium (Ti) as a biomaterial, the exact cellular and molecular mechanisms responsible for Ti osseointegration remains unclear, especially because of ...the limited methodological tools available in this field.
In this study, we present a microscopic and molecular characterization of an oral implant osseointegration model using C57Bl/6 mice.
Forty-eight male wild-type mice received a Ti implant on the edentulous alveolar crest and the peri-implant sites were evaluated through microscopic (μCT, histological and birefringence) and molecular (RealTimePCRarray) analysis in different points in time after surgery (3, 7, 14 and 21 days).
The early stages of osseointegration were marked by an increased expression of growth factors and MSC markers. Subsequently, a provisional granulation tissue was formed, with high expression of VEGFb and earlier osteogenic markers (BMPs, ALP and Runx2). The immune/inflammatory phase was evidenced by an increased density of inflammatory cells, and high expression of cytokines (TNF, IL6, IL1) chemokines (CXCL3, CCL2, CCL5 and CXC3CL1) and chemokine receptors (CCR2 and CCR5). Also, iNOS expression remained low, while ARG1 was upregulated, indicating predominance of a M2-type response. At later points in time, the bone matrix density and volume were increased, in agreement with a high expression of Col1a1 and Col21a2. The remodelling process was marked by peaks of MMPs, RANKL and OPG expression at 14 days, and an increased density of osteoclasts. At 21 days, intimate Ti/bone contact was observed, with expression of final osteoblast differentiation markers (PHEX, SOST), as well as red spectrum collagen fibers.
This study demonstrated a unique molecular view of oral osseointegration kinetics in C57Bl/6 mice, evidencing potential elements responsible for orchestrating cell migration, proliferation, ECM deposition and maturation, angiogenesis, bone formation and remodeling at the bone-implant interface in parallel with a novel microscopic analysis.
Abstract 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.
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