The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

The B Cell-Stimulatory Cytokines BLyS and APRIL Are Elevated in Human Periodontitis and Are Required for B Cell-Dependent Bone Loss in Experimental Murine Periodontitis.

B-lineage cells (B lymphocytes and plasma cells) predominate in the inflammatory infiltrate of human chronic periodontitis. However, their role in disease pathogenesis and the factors responsible for their persistence in chronic lesions are poorly understood. In this regard, two cytokines of the TNF ligand superfamily, a proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), are important for the survival, proliferation, and maturation of B cells. Thus, we hypothesized that APRIL and/or BLyS are upregulated in periodontitis and contribute to induction of periodontal bone loss. This hypothesis was addressed in both human and mouse experimental systems. We show that, relative to healthy controls, the expression of APRIL and BLyS mRNA and protein was upregulated in natural and experimental periodontitis in humans and mice, respectively. The elevated expression of these cytokines correlated with increased numbers of B cells/plasma cells in both species. Moreover, APRIL and BLyS partially colocalized with κ L chain-expressing B-lineage cells at the epithelial-connective tissue interface. Ligature-induced periodontitis resulted in significantly less bone loss in B cell-deficient mice compared with wild-type controls. Ab-mediated neutralization of APRIL or BLyS diminished the number of B cells in the gingival tissue and inhibited bone loss in wild-type, but not in B cell-deficient, mice. In conclusion, B cells and specific cytokines involved in their growth and differentiation contribute to periodontal bone loss. Moreover, APRIL and BLyS have been identified as potential therapeutic targets in periodontitis.

Milk fat globule epidermal growth factor 8 inhibits periodontitis in non-human primates and its gingival crevicular fluid levels can differentiate periodontal health from disease in humans.

AIM: We have previously shown that the secreted glycoprotein milk fat globule epidermal growth factor 8 (MFG-E8) has anti-inflammatory and anti-osteoclastogenic properties. Our objective was to investigate the potential of MFG-E8 as a diagnostic or therapeutic agent in periodontitis. MATERIALS AND METHODS: Periodontitis was induced in non-human primates (NHPs) by placing ligatures around posterior teeth on both halves of the mandible for a split-mouth design: one side was treated with MFG-E8-Fc and the other with Fc control. Disease was assessed by clinical periodontal examinations, radiographic analysis of bone loss, and analysis of cytokine mRNA expression in gingival biopsy samples. Gingival crevicular fluid (GCF) was collected from human healthy volunteers or subjects with gingivitis, chronic moderate periodontitis, or chronic severe periodontitis. Additionally, GCF was collected from a subset of severe periodontitis patients following scaling and root planing (SRP) and after pocket reduction surgery. GCF was analysed to quantify MFG-E8 and periodontitis-relevant cytokines using multiplex assays. RESULTS: In NHPs, sites treated with MFG-E8-Fc exhibited significantly less ligature-induced periodontal inflammation and bone loss than Fc control-treated sites. In humans, the GCF levels of MFG-E8 were significantly higher in health than in periodontitis, whereas the reverse was true for the proinflammatory cytokines tested. Consistently, MFG-E8 was elevated in GCF after both non-surgical (SRP) and surgical periodontal treatment of periodontitis patients. CONCLUSION: MFG-E8 is, in principle, a novel therapeutic agent and biomarker of periodontitis.

Role of complement in host-microbe homeostasis of the periodontium.

Complement plays a key role in immunity and inflammation through direct effects on immune cells or via crosstalk and regulation of other host signaling pathways. Deregulation of these finely balanced complement activities can link infection to inflammatory tissue damage. Periodontitis is a polymicrobial community-induced chronic inflammatory disease that can destroy the tooth-supporting tissues. In this review, we summarize and discuss evidence that complement is involved in the dysbiotic transformation of the periodontal microbiota and in the inflammatory process that leads to the destruction of periodontal bone. Recent insights into the mechanisms of complement involvement in periodontitis have additionally provided likely targets for therapeutic intervention against this oral disease.

Regulation of osteoclast homeostasis and inflammatory bone loss by MFG-E8.

The glycoprotein milk fat globule-epidermal growth factor factor 8 (MFG-E8) is expressed in several tissues and mediates diverse homeostatic functions. However, whether it plays a role in bone homeostasis has not been established. In this study, we show for the first time, to our knowledge, that osteoclasts express and are regulated by MFG-E8. Bone marrow-derived osteoclast precursors from MFG-E8-deficient (Mfge8(-/-)) mice underwent increased receptor activator of NF-κB ligand-induced osteoclastogenesis, leading to enhanced resorption pit formation compared with wild-type controls. Consistently, exogenously added MFG-E8 inhibited receptor activator of NF-κB ligand-induced osteoclastogenesis from mouse or human osteoclast precursors. Upon induction of experimental periodontitis, an oral inflammatory disease characterized by loss of bone support of the dentition, Mfge8(-/-) mice exhibited higher numbers of osteoclasts and more bone loss than did wild-type controls. Accordingly, local microinjection of anti-MFG-E8 mAb exacerbated periodontal bone loss in wild-type mice. Conversely, microinjection of MFG-E8 inhibited bone loss in experimental mouse periodontitis. In comparison with wild-type controls, Mfge8(-/-) mice also experienced >60% more naturally occurring chronic periodontal bone loss. In conclusion, MFG-E8 is a novel homeostatic regulator of osteoclasts that could be exploited therapeutically to treat periodontitis and perhaps other immunological disorders associated with inflammatory bone loss.

Genetic and intervention studies implicating complement C3 as a major target for the treatment of periodontitis.

Chronic periodontitis is induced by a dysbiotic microbiota and leads to inflammatory destruction of tooth-supporting connective tissue and bone. The third component of complement, C3, is a point of convergence of distinct complement activation mechanisms, but its involvement in periodontitis was not previously addressed. We investigated this question using two animal species models, namely, C3-deficient or wild-type mice and nonhuman primates (NHPs) locally treated with a potent C3 inhibitor (the compstatin analog Cp40) or an inactive peptide control. In mice, C3 was required for maximal periodontal inflammation and bone loss, and for the sustenance of the dysbiotic microbiota. The effect of C3 on the microbiota was therefore different from that reported for the C5a receptor, which is required for the initial induction of dysbiosis. C3-dependent bone loss was demonstrated in distinct models, including Porphyromonas gingivalis-induced periodontitis, ligature-induced periodontitis, and aging-associated periodontitis. Importantly, local treatment of NHPs with Cp40 inhibited ligature-induced periodontal inflammation and bone loss, which correlated with lower gingival crevicular fluid levels of proinflammatory mediators (e.g., IL-17 and RANKL) and decreased osteoclastogenesis in bone biopsy specimens, as compared with control treatment. To our knowledge, this is the first time, for any disease, that complement inhibition in NHPs was shown to inhibit inflammatory processes that lead to osteoclastogenesis and bone loss. These data strongly support the feasibility of C3-targeted intervention for the treatment of human periodontitis.

Toll-Like Receptor 9-Mediated Inflammation Triggers Alveolar Bone Loss in Experimental Murine Periodontitis.

Chronic periodontitis is a local inflammatory disease induced by a dysbiotic microbiota and leading to destruction of the tooth-supporting structures. Microbial nucleic acids are abundantly present in the periodontium, derived through release after phagocytic uptake of microbes and/or from biofilm-associated extracellular DNA. Binding of microbial DNA to its cognate receptors, such as Toll-like receptor 9 (TLR9), can trigger inflammation. In this study, we utilized TLR9 knockout (TLR9(-/-)) mice and wild-type (WT) controls in a murine model of Porphyromonas gingivalis-induced periodontitis and report the first in vivo evidence that TLR9 signaling mediates the induction of periodontal bone loss. P. gingivalis-infected WT mice exhibited significantly increased bone loss compared to that in sham-infected WT mice or P. gingivalis-infected TLR9(-/-) mice, which were resistant to bone loss. Consistent with this, the expression levels of interleukin 6 (IL-6), tumor necrosis factor (TNF), and receptor-activator of nuclear factor kappa B ligand (RANKL) were significantly elevated in the gingival tissues of the infected WT mice but not in infected TLR9(-/-) mice compared to their levels in controls. Ex vivo studies using splenocytes and bone marrow-derived macrophages revealed significantly diminished cytokine production in TLR9(-/-) cells relative to the cytokine production in WT cells in response to P. gingivalis, thereby implicating TLR9 in inflammatory responses to this organism. Intriguingly, compared to the cytokine production in WT cells, TLR9(-/-) cells exhibited significantly decreased proinflammatory cytokine production upon challenge with lipopolysaccharide (LPS) (TLR4 agonist) or Pam3Cys (TLR2 agonist), suggesting possible cross talk between TLR9, TLR4, and TLR2. Collectively, our results provide the first proof-of-concept evidence implicating TLR9-triggered inflammation in periodontal disease pathogenesis, thereby identifying a new potential therapeutic target to control periodontal inflammation.

Complement Involvement in Periodontitis: Molecular Mechanisms and Rational Therapeutic Approaches.

The complement system is a network of interacting fluid-phase and cell surface-associated molecules that trigger, amplify, and regulate immune and inflammatory signaling pathways. Dysregulation of this finely balanced network can destabilize host-microbe homeostasis and cause inflammatory tissue damage. Evidence from clinical and animal model-based studies suggests that complement is implicated in the pathogenesis of periodontitis, a polymicrobial community-induced chronic inflammatory disease that destroys the tooth-supporting tissues. This review discusses molecular mechanisms of complement involvement in the dysbiotic transformation of the periodontal microbiome and the resulting destructive inflammation, culminating in loss of periodontal bone support. These mechanistic studies have additionally identified potential therapeutic targets. In this regard, interventional studies in preclinical models have provided proof-of-concept for using complement inhibitors for the treatment of human periodontitis.

Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis.

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Local complement-targeted intervention in periodontitis: proof-of-concept using a C5a receptor (CD88) antagonist.

When excessively activated or deregulated, complement becomes a major link between infection and inflammatory pathology including periodontitis. This oral inflammatory disease is associated with a dysbiotic microbiota, leads to the destruction of bone and other tooth-supporting structures, and exerts an adverse impact on systemic health. We have previously shown that mice deficient either in complement C5a receptor (C5aR; CD88) or TLR2 are highly and similarly resistant to periodontitis, suggesting that a cross-talk between the two receptors may be involved in the disease process. In this paper, we show that C5aR and TLR2 indeed synergize for maximal inflammatory responses in the periodontal tissue and uncover a novel pharmacological target to abrogate periodontitis. Using two different mouse models of periodontitis, we show that local treatments with a C5aR antagonist inhibited periodontal inflammation through downregulation of TNF, IL-1ß, IL-6, and IL-17 and further protected against bone loss, regardless of the presence of TLR2. These findings not only reveal a crucial cooperation between C5aR and TLR2 in periodontal inflammation but also provide proof-of-concept for local targeting of C5aR as a powerful candidate for the treatment of human periodontitis.

TREM-1 Is Upregulated in Experimental Periodontitis, and Its Blockade Inhibits IL-17A and RANKL Expression and Suppresses Bone loss.

AIM: Triggering receptor expressed on myeloid cells-1 (TREM-1) is a modifier of local and systemic inflammation. There is clinical evidence implicating TREM-1 in the pathogenesis of periodontitis. However, a cause-and-effect relationship has yet to be demonstrated, as is the underlying mechanism. The aim of this study was to elucidate the role of TREM-1 using the murine ligature-induced periodontitis model. METHODS: A synthetic antagonistic LP17 peptide or sham control was microinjected locally into the palatal gingiva of the ligated molar teeth. RESULTS: Mice treated with the LP17 inhibitor developed significantly less bone loss as compared to sham-treated mice, although there were no differences in total bacterial load on the ligatures. To elucidate the impact of LP17 on the host response, we analyzed the expression of a number of immune-modulating genes. The LP17 peptide altered the expression of 27/92 genes ≥ two-fold, but only interleukin (IL)-17A was significantly downregulated (4.9-fold). Importantly, LP17 also significantly downregulated the receptor activator of nuclear factor kappa-B-ligand (RANKL) to osteoprotegerin (OPG) ratio that drives osteoclastic bone resorption in periodontitis. CONCLUSION: Our findings show for the first time that TREM-1 regulates the IL-17A-RANKL/OPG axis and bone loss in experimental periodontitis, and its therapeutic blockade may pave the way to a novel treatment for human periodontitis.

A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans.

Periodontitis is one of the most common human inflammatory diseases, yet the mechanisms that drive immunopathology and could be therapeutically targeted are not well defined. Here, we demonstrate an expansion of resident memory T helper 17 (TH17) cells in human periodontitis. Phenocopying humans, TH17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral TH17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)-dependent manner, periodontitis-associated expansion of TH17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. TH17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of TH17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in TH17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human TH17 cell defects were associated with diminished periodontal inflammation and bone loss, despite increased prevalence of recurrent oral fungal infections. Our study highlights distinct functions of TH17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis.

DEL-1 restrains osteoclastogenesis and inhibits inflammatory bone loss in nonhuman primates.

DEL-1 (developmental endothelial locus-1) is an endothelial cell-secreted protein that regulates LFA-1 (lymphocyte function-associated antigen-1) integrin-dependent leukocyte recruitment and inflammation in various tissues. We identified a novel regulatory mechanism of DEL-1 in osteoclast biology. Specifically, we showed that DEL-1 is expressed by human and mouse osteoclasts and regulates their differentiation and resorptive function. Mechanistically, DEL-1 inhibited the expression of NFATc1, a master regulator of osteoclastogenesis, in a Mac-1 integrin-dependent manner. In vivo mechanistic analysis has dissociated the anti-inflammatory from the anti-bone-resorptive action of DEL-1 and identified structural components thereof mediating these distinct functions. Locally administered human DEL-1 blocked inflammatory periodontal bone loss in nonhuman primates-a relevant model of human periodontitis. The ability of DEL-1 to regulate both upstream (inflammatory cell recruitment) and downstream (osteoclastogenesis) events that lead to inflammatory bone loss paves the way to a new class of endogenous therapeutics for treating periodontitis and perhaps other inflammatory disorders.

Antagonistic effects of IL-17 and D-resolvins on endothelial Del-1 expression through a GSK-3ß-C/EBPß pathway.

Del-1 is an endothelial cell-secreted anti-inflammatory protein. In humans and mice, Del-1 expression is inversely related to that of IL-17, which inhibits Del-1 through hitherto unidentified mechanism(s). Here we show that IL-17 downregulates human endothelial cell expression of Del-1 by targeting a critical transcription factor, C/EBPß. Specifically, IL-17 causes GSK-3ß-dependent phosphorylation of C/EBPß, which is associated with diminished C/EBPß binding to the Del-1 promoter and suppressed Del-1 expression. This inhibitory action of IL-17 can be reversed at the GSK-3ß level by PI3K/Akt signalling induced by D-resolvins. The biological relevance of this regulatory network is confirmed in a mouse model of inflammatory periodontitis. Intriguingly, resolvin-D1 (RvD1) confers protection against IL-17-driven periodontal bone loss in a Del-1-dependent manner, indicating an RvD1-Del-1 axis against IL-17-induced pathological inflammation. The dissection of signalling pathways regulating Del-1 expression provides potential targets to treat inflammatory diseases associated with diminished Del-1 expression, such as periodontitis and multiple sclerosis.

Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases.

Defective neutrophil recruitment in leukocyte adhesion deficiency type I disease causes local IL-17-driven inflammatory bone loss.

Leukocyte adhesion deficiency type I (LAD-I), a disease syndrome associated with frequent microbial infections, is caused by mutations on the CD18 subunit of ß2 integrins. LAD-I is invariably associated with severe periodontal bone loss, which historically has been attributed to the lack of neutrophil surveillance of the periodontal infection. We provide an alternative mechanism by showing that the cytokine interleukin-17 (IL-17) plays a major role in the oral pathology of LAD-I. Defective neutrophil recruitment in LAD-I patients or in LFA-1 (CD11a/CD18)-deficient mice--which exhibit the LAD-I periodontal phenotype--was associated with excessive production of predominantly T cell-derived IL-17 in the periodontal tissue, although innate lymphoid cells also contributed to pathological IL-17 elevation in the LFA-1-deficient mice. Local treatment with antibodies to IL-17 or IL-23 in LFA-1-deficient mice not only blocked inflammatory periodontal bone loss but also caused a reduction in the total bacterial burden, suggesting that the IL-17-driven pathogenesis of LAD-I periodontitis leads to dysbiosis. Therefore, our findings support an IL-17-targeted therapy for periodontitis in LAD-I patients.

Optimization of the ligature-induced periodontitis model in mice.

Periodontitis is a prevalent oral inflammatory disease that leads to alveolar bone loss and may exert an adverse impact on systemic health. Experimental animal models are critical tools to investigate mechanisms of periodontal pathogenesis and test new therapeutic approaches. The ligature-induced periodontitis model has been used frequently in relatively large animals, including non-human primates, to assess the host response and its effects on the tooth-supporting tissues (gingiva and bone) under well-controlled conditions. Although mice constitute the most convenient and versatile model for mechanistic immunological research (plethora of genetically engineered strains and immunological reagents), the tiny size of the murine oral cavity has presented technical challenges for ligature placement. In this report, we present a straightforward method for ligating the second maxillary molar tooth, and, moreover, identified the most appropriate sites for evaluating inflammatory bone loss in a valid and reproducible manner. These optimizations are expected to facilitate the use of the mouse ligature-induced periodontitis model and consequently contribute to better understanding of the immunopathological mechanisms of periodontitis.