Mice Lacking PLAP-1/Asporin Show Alteration of Periodontal Ligament Structures and Acceleration of Bone Loss in Periodontitis.

Periodontal ligament-associated protein 1 (PLAP-1), also known as Asporin, is an extracellular matrix protein expressed in the periodontal ligament and plays a crucial role in periodontal tissue homeostasis. Our previous research demonstrated that PLAP-1 may inhibit TLR2/4-mediated inflammatory responses, thereby exerting a protective function against periodontitis. However, the precise roles of PLAP-1 in the periodontal ligament (PDL) and its relationship to periodontitis have not been fully explored. In this study, we employed PLAP-1 knockout mice to investigate its roles and contributions to PDL tissue and function in a ligature-induced periodontitis model. Mandibular bone samples were collected from 10-week-old male C57BL/6 (WT) and PLAP-1 knockout (KO) mice. These samples were analyzed through micro-computed tomography (µCT) scanning, hematoxylin and eosin (HE) staining, picrosirius red staining, and fluorescence immunostaining using antibodies targeting extracellular matrix proteins. Additionally, the structure of the PDL collagen fibrils was examined using transmission electron microscopy (TEM). We also conducted tooth extraction and ligature-induced periodontitis models using both wild-type and PLAP-1 KO mice. PLAP-1 KO mice did not exhibit any changes in alveolar bone resorption up to the age of 10 weeks, but they did display an enlarged PDL space, as confirmed by µCT and histological analyses. Fluorescence immunostaining revealed increased expression of extracellular matrix proteins, including Col3, BGN, and DCN, in the PDL tissues of PLAP-1 KO mice. TEM analysis demonstrated an increase in collagen diameter within the PDL of PLAP-1 KO mice. In line with these findings, the maximum stress required for tooth extraction was significantly lower in PLAP-1 KO mice in the tooth extraction model compared to WT mice (13.89 N ± 1.34 and 16.51 N ± 1.31, respectively). In the ligature-induced periodontitis model, PLAP-1 knockout resulted in highly severe alveolar bone resorption, with a higher number of collagen fiber bundle tears and significantly more osteoclasts in the periodontium. Our results demonstrate that mice lacking PLAP-1/Asporin show alteration of periodontal ligament structures and acceleration of bone loss in periodontitis. This underscores the significant role of PLAP-1 in maintaining collagen fibrils in the PDL and suggests the potential of PLAP-1 as a therapeutic target for periodontal diseases.

Complement Is Required for Microbe-Driven Induction of Th17 and Periodontitis.

In both mice and humans, complement and Th17 cells have been implicated in periodontitis, an oral microbiota-driven inflammatory disease associated with systemic disorders. A recent clinical trial showed that a complement C3 inhibitor (AMY-101) causes sustainable resolution of periodontal inflammation, the main effector of tissue destruction in this oral disease. Although both complement and Th17 are required for periodontitis, it is uncertain how these immune components cooperate in disease development. In this study, we dissected the complement-Th17 relationship in the setting of ligature-induced periodontitis (LIP), a model that previously established that microbial dysbiosis drives Th17 cell expansion and periodontal bone loss. Complement was readily activated in the periodontal tissue of LIP-subjected mice but not when the mice were placed on broad-spectrum antibiotics. Microbiota-induced complement activation generated critical cytokines, IL-6 and IL-23, which are required for Th17 cell expansion. These cytokines as well as Th17 accumulation and IL-17 expression were significantly suppressed in LIP-subjected C3-deficient mice relative to wild-type controls. As IL-23 has been extensively studied in periodontitis, we focused on IL-6 and showed that LIP-induced IL-17 and bone loss required intact IL-6 receptor signaling in the periodontium. LIP-induced IL-6 was predominantly produced by gingival epithelial cells that upregulated C3a receptor upon LIP challenge. Experiments in human gingival epithelial cells showed that C3a upregulated IL-6 production in cooperation with microbial stimuli that upregulated C3a receptor expression in ERK1/2- and JNK-dependent manner. In conclusion, complement links the periodontal microbiota challenge to Th17 cell accumulation and thus integrates complement- and Th17-driven immunopathology in periodontitis.

C3-targeted host-modulation approaches to oral inflammatory conditions.

Periodontitis is an inflammatory disease caused by biofilm accumulation and dysbiosis in subgingival areas surrounding the teeth. If not properly treated, this oral disease may result in tooth loss and consequently poor esthetics, deteriorated masticatory function and compromised quality of life. Epidemiological and clinical intervention studies indicate that periodontitis can potentially aggravate systemic diseases, such as, cardiovascular disease, type 2 diabetes mellitus, rheumatoid arthritis, and Alzheimer disease. Therefore, improvements in the treatment of periodontal disease may benefit not only oral health but also systemic health. The complement system is an ancient host defense system that plays pivotal roles in immunosurveillance and tissue homeostasis. However, complement has unwanted consequences if not controlled appropriately or excessively activated. Complement overactivation has been observed in patients with periodontitis and in animal models of periodontitis and drives periodontal inflammation and tissue destruction. This review places emphasis on a promising periodontal host-modulation therapy targeting the complement system, namely the complement C3-targeting drug, AMY-101. AMY-101 has shown safety and efficacy in reducing gingival inflammation in a recent Phase 2a clinical study. We also discuss the potential of AMY-101 to treat peri-implant inflammatory conditions, where complement also seems to be involved and there is an urgent unmet need for effective treatment.

Developmental endothelial locus-1 protects from hypertension-induced cardiovascular remodeling via immunomodulation.

The causative role of inflammation in hypertension-related cardiovascular diseases is evident and calls for development of specific immunomodulatory therapies. We tested the therapeutic efficacy and mechanisms of action of developmental endothelial locus-1 (DEL-1), an endogenous antiinflammatory factor, in angiotensin II- (ANGII-) and deoxycorticosterone acetate-salt-induced (DOCA-salt-induced) cardiovascular organ damage and hypertension. By using mice with endothelial overexpression of DEL-1 (EC-Del1 mice) and performing preventive and interventional studies by injecting recombinant DEL-1 in mice, we showed that DEL-1 improved endothelial function and abrogated aortic adventitial fibrosis, medial thickening, and loss of elastin. DEL-1 also protected the mice from cardiac concentric hypertrophy and interstitial and perivascular coronary fibrosis and improved left ventricular function and myocardial coronary perfusion. DEL-1 prevented aortic stiffness and abolished the progression of hypertension. Mechanistically, DEL-1 acted by inhibiting αvß3 integrin-dependent activation of pro-MMP2 in mice and in human isolated aorta. Moreover, DEL-1 stabilized αvß3 integrin-dependent CD25+FoxP3+ Treg numbers and IL-10 levels, which were associated with decreased recruitment of inflammatory cells and reduced production of proinflammatory cytokines in cardiovascular organs. The demonstrated effects and immune-modulating mechanisms of DEL-1 in abrogation of cardiovascular remodeling and progression of hypertension identify DEL-1 as a potential therapeutic factor.

Prolyl-hydroxylase inhibitor-induced regeneration of alveolar bone and soft tissue in a mouse model of periodontitis through metabolic reprogramming.

Bone injuries and fractures reliably heal through a process of regeneration with restoration to original structure and function when the gap between adjacent sides of a fracture site is small. However, when there is significant volumetric loss of bone, bone regeneration usually does not occur. In the present studies, we explore a particular case of volumetric bone loss in a mouse model of human periodontal disease (PD) in which alveolar bone surrounding teeth is permanently lost and not replaced. This model employs the placement a ligature around the upper second molar for 10 days leading to inflammation and bone breakdown and faithfully replicates the bacterially-induced inflammatory etiology of human PD to induce bone degeneration. After ligature removal, mice are treated with a timed-release formulation of a small molecule inhibitor of prolylhydroxylases (PHDi; 1,4-DPCA) previously shown to induce epimorphic regeneration of soft tissue in non-regenerating mice. This PHDi induces high expression of HIF-1α and is able to shift the metabolic state from OXPHOS to aerobic glycolysis, an energetic state used by stem cells and embryonic tissue. This regenerative response was completely blocked by siHIF1a. In these studies, we show that timed-release 1,4-DPCA rapidly and completely restores PD-affected bone and soft tissue with normal anatomic fidelity and with increased stem cell markers due to site-specific stem cell migration and/or de-differentiation of local tissue, periodontal ligament (PDL) cell proliferation, and increased vascularization. In-vitro studies using gingival tissue show that 1,4-DPCA indeed induces de-differentiation and the expression of stem cell markers but does not exclude the role of migrating stem cells. Evidence of metabolic reprogramming is seen by the expression of not only HIF-1a, its gene targets, and resultant de-differentiation markers, but also the metabolic genes Glut-1, Gapdh, Pdk1, Pgk1 and Ldh-a in jaw periodontal tissue.

Mouse Model of Loeys-Dietz Syndrome Shows Elevated Susceptibility to Periodontitis via Alterations in Transforming Growth Factor-Beta Signaling.

Loeys-Dietz syndrome (LDS) is a syndromic connective tissue disorder caused by a heterozygous missense mutation in genes that encode transforming growth factor (TGF)-ß receptor (TGFBR) 1 and 2. We encountered a patient with LDS, who had severe periodontal tissue destruction indicative of aggressive periodontitis. The patient had a missense mutation in the glycine and serine-rich domain of TGFBR1 exon 3. This G-to-T mutation at base 563 converted glycine to valine. We established an LDS model knock-in mouse that recapitulated the LDS phenotype. Homozygosity of the mutation caused embryonic lethality and heterozygous knock-in mice showed distorted and ruptured elastic fibers in the aorta at 24 weeks of age and died earlier than wildtype (WT) mice. We stimulated mouse embryonic fibroblasts (MEFs) from the knock-in mouse with TGF-ß and examined their responses. The knock-in MEFs showed downregulated Serpine 1 mRNA expression and phosphorylation of Smad2 to TGF-ß compared with WT MEFs. To clarify the influence of TGF-ß signaling abnormalities on the pathogenesis or progression of periodontitis, we performed pathomolecular analysis of the knock-in mouse. There were no structural differences in periodontal tissues between WT and LDS model mice at 6 or 24 weeks of age. Micro-computed tomography revealed no significant difference in alveolar bone resorption between WT and knock-in mice at 6 or 24 weeks of age. However, TGF-ß-related gene expression was increased significantly in periodontal tissues of the knock-in mouse compared with WT mice. Next, we assessed a mouse periodontitis model in which periodontal bone loss was induced by oral inoculation with the bacterial strain Porphyromonas gingivalis W83. After inoculation, we collected alveolar bone and carried out morphometric analysis. P. gingivalis-induced alveolar bone loss was significantly greater in LDS model mice than in WT mice. Peritoneal macrophages isolated from Tgfbr1 G188V/+ mice showed upregulation of inflammatory cytokine mRNA expression induced by P. gingivalis lipopolysaccharide compared with WT macrophages. In this study, we established an LDS mouse model and demonstrated that LDS model mice had elevated susceptibility to P. gingivalis-induced periodontitis, probably through TGF-ß signal dysfunction. This suggests that TGF-ß signaling abnormalities accelerate the pathogenesis or progression of periodontitis.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Mice lacking PLAP-1/asporin counteracts high fat diet-induced metabolic disorder and alveolar bone loss by controlling adipose tissue expansion.

Adipose tissue fibrosis with chronic inflammation is a hallmark of obesity-related metabolic disorders, and the role of proteoglycans in developing adipose tissue fibrosis is of interest. Periodontal disease is associated with obesity; however, the underlying molecular mechanisms remain unclear. Here we investigated the roles of periodontal ligament associated protein-1 (PLAP-1)/asporin, a proteoglycan preferentially and highly expressed in the periodontal ligament, in obesity-related adipose tissue dysfunction and adipocyte differentiation. It was found that PLAP-1 is also highly expressed in white adipose tissues. Plap-1 knock-out mice counteracted obesity and alveolar bone resorption induced by a high-fat diet. Plap-1 knock-down in 3T3-L1 cells resulted in less lipid accumulation, and recombinant PLAP-1 enhanced lipid accumulation in 3T3-L1 cells. In addition, it was found that primary preadipocytes isolated from Plap-1 knock-out mice showed lesser lipid accumulation than the wild-type (WT) mice. Furthermore, the stromal vascular fraction of Plap-1 knock-out mice showed different extracellular matrix gene expression patterns compared to WT. These findings demonstrate that PLAP-1 enhances adipogenesis and could be a key molecule in understanding the association between periodontal disease and obesity-related metabolic disorders.

An injectable hydrogel-formulated inhibitor of prolyl-4-hydroxylase promotes T regulatory cell recruitment and enhances alveolar bone regeneration during resolution of experimental periodontitis.

The hypoxia-inducible factor 1α (HIF-1α) is critically involved in tissue regeneration. Hence, the pharmacological prevention of HIF-1α degradation by prolyl hydroxylase (PHD) under normoxic conditions is emerging as a promising option in regenerative medicine. Using a mouse model of ligature-induced periodontitis and resolution, we tested the ability of an injectable hydrogel-formulated PHD inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA/hydrogel), to promote regeneration of alveolar bone lost owing to experimental periodontitis. Mice injected subcutaneously with 1,4-DPCA/hydrogel at the onset of periodontitis resolution displayed significantly increased gingival HIF-1α protein levels and bone regeneration, as compared to mice treated with vehicle control. The 1,4-DPCA/hydrogel-induced increase in bone regeneration was associated with elevated expression of osteogenic genes, decreased expression of pro-inflammatory cytokine genes, and increased abundance of FOXP3+ T regulatory (Treg) cells in the periodontal tissue. The enhancing effect of 1,4-DPCA/hydrogel on Treg cell accumulation and bone regeneration was reversed by AMD3100, an antagonist of the chemokine receptor CXCR4 that mediates Treg cell recruitment. In conclusion, the administration of 1,4-DPCA/hydrogel at the onset of periodontitis resolution promotes CXCR4-dependent accumulation of Treg cells and alveolar bone regeneration, suggesting a novel approach for regaining bone lost due to periodontitis.

The DEL-1/ß3 integrin axis promotes regulatory T cell responses during inflammation resolution.

FOXP3+CD4+ regulatory T cells (Tregs) are critical for immune homeostasis and respond to local tissue cues, which control their stability and function. We explored here whether developmental endothelial locus-1 (DEL-1), which, like Tregs, increases during resolution of inflammation, promotes Treg responses. DEL-1 enhanced Treg numbers and function at barrier sites (oral and lung mucosa). The underlying mechanism was dissected using mice lacking DEL-1 or expressing a point mutant thereof, or mice with T cell-specific deletion of the transcription factor RUNX1, identified by RNA sequencing analysis of the DEL-1-induced Treg transcriptome. Specifically, through interaction with αvß3 integrin, DEL-1 promoted induction of RUNX1-dependent FOXP3 expression and conferred stability of FOXP3 expression upon Treg restimulation in the absence of exogenous TGF-ß1. Consistently, DEL-1 enhanced the demethylation of the Treg-specific demethylated region (TSDR) in the mouse Foxp3 gene and the suppressive function of sorted induced Tregs. Similarly, DEL-1 increased RUNX1 and FOXP3 expression in human conventional T cells, promoting their conversion into induced Tregs with increased TSDR demethylation, enhanced stability, and suppressive activity. We thus uncovered a DEL-1/αvß3/RUNX1 axis that promotes Treg responses at barrier sites and offers therapeutic options for modulating inflammatory/autoimmune disorders.

Frontline Science: Activation of metabolic nuclear receptors restores periodontal tissue homeostasis in mice with leukocyte adhesion deficiency-1.

ß2 Integrins mediate neutrophil-endothelial adhesion and recruitment of neutrophils to sites of inflammation. The diminished expression of ß2 integrins in patients with mutations in the ITGB2 (CD18) gene (leukocyte adhesion deficiency-Type 1; LAD1) results in few or no neutrophils in peripheral tissues. In the periodontium, neutrophil paucity is associated with up-regulation of IL-23 and IL-17, which drive inflammatory bone loss. Using a relevant mouse model, we investigated whether diminished efferocytosis (owing to neutrophil scarcity) is associated with LAD1 periodontitis pathogenesis and aimed to develop approaches to restore the missing efferocytosis signals. We first showed that CD18-/- mice phenocopied human LAD1 in terms of IL-23/IL-17-driven inflammatory bone loss. Ab-mediated blockade of c-Mer tyrosine kinase (Mer), a major efferocytic receptor, mimicked LAD1-associated up-regulation of gingival IL-23 and IL-17 mRNA expression in wild-type (WT) mice. Consistently, soluble Mer-Fc reversed the inhibitory effect of efferocytosis on IL-23 expression in LPS-activated Mϕs. Adoptive transfer of WT neutrophils to CD18-/- mice down-regulated IL-23 and IL-17 expression to normal levels, but not when CD18-/- mice were treated with blocking anti-Mer Ab. Synthetic agonist-induced activation of liver X receptors (LXR) and peroxisome proliferator-activated receptors (PPAR), which link efferocytosis to generation of homeostatic signals, inhibited the expression of IL-23 and IL-17 and favorably affected the bone levels of CD18-/- mice. Therefore, our data link diminished efferocytosis-associated signaling due to impaired neutrophil recruitment to dysregulation of the IL-23-IL-17 axis and, moreover, suggest LXR and PPAR as potential therapeutic targets for treating LAD1 periodontitis.

The secreted protein DEL-1 activates a ß3 integrin-FAK-ERK1/2-RUNX2 pathway and promotes osteogenic differentiation and bone regeneration.

The integrin-binding secreted protein developmental endothelial locus-1 (DEL-1) is involved in the regulation of both the initiation and resolution of inflammation in different diseases, including periodontitis, an oral disorder characterized by inflammatory bone loss. Here, using a mouse model of bone regeneration and in vitro cell-based mechanistic studies, we investigated whether and how DEL-1 can promote alveolar bone regeneration during resolution of experimental periodontitis. Compared with WT mice, mice lacking DEL-1 or expressing a DEL-1 variant with an Asp-to-Glu substitution in the RGD motif ("RGE point mutant"), which does not interact with RGD-dependent integrins, exhibited defective bone regeneration. Local administration of DEL-1 or of its N-terminal segment containing the integrin-binding RGD motif, but not of the RGE point mutant, reversed the defective bone regeneration in the DEL-1-deficient mice. Moreover, DEL-1 (but not the RGE point mutant) promoted osteogenic differentiation of MC3T3-E1 osteoprogenitor cells or of primary calvarial osteoblastic cells in a ß3 integrin-dependent manner. The ability of DEL-1 to promote in vitro osteogenesis, indicated by induction of osteogenic genes such as the master transcription factor Runt-related transcription factor-2 (Runx2) and by mineralized nodule formation, depended on its capacity to induce the phosphorylation of focal adhesion kinase (FAK) and of extracellular signal-regulated kinase 1/2 (ERK1/2). We conclude that DEL-1 can activate a ß3 integrin-FAK-ERK1/2-RUNX2 pathway in osteoprogenitors and promote new bone formation in mice. These findings suggest that DEL-1 may be therapeutically exploited to restore bone lost due to periodontitis and perhaps other osteolytic conditions.

Pressure Cycling Technology Assisted Mass Spectrometric Quantification of Gingival Tissue Reveals Proteome Dynamics during the Initiation and Progression of Inflammatory Periodontal Disease.

Understanding the progression of periodontal tissue destruction is at the forefront of periodontal research. The authors aimed to capture the dynamics of gingival tissue proteome during the initiation and progression of experimental (ligature-induced) periodontitis in mice. Pressure cycling technology (PCT), a recently developed platform that uses ultra-high pressure to disrupt tissues, is utilized to achieve efficient and reproducible protein extraction from ultra-small amounts of gingival tissues in combination with liquid chromatography-tandem mass spectrometry (MS). The MS data are processed using Progenesis QI and the regulated proteins are subjected to METACORE, STRING, and WebGestalt for functional enrichment analysis. A total of 1614 proteins with ≥2 peptides are quantified with an estimated protein false discovery rate of 0.06%. Unsupervised clustering analysis shows that the gingival tissue protein abundance is mainly dependent on the periodontitis progression stage. Gene ontology enrichment analysis reveals an overrepresentation in innate immune regulation (e.g., neutrophil-mediated immunity and antimicrobial peptides), signal transduction (e.g., integrin signaling), and homeostasis processes (e.g., platelet activation and aggregation). In conclusion, a PCT-assisted label-free quantitative proteomics workflow that allowed cataloging the deepest gingival tissue proteome on a rapid timescale and provided novel mechanistic insights into host perturbation during periodontitis progression is applied.

Complement-Dependent Mechanisms and Interventions in Periodontal Disease.

Periodontitis is a prevalent inflammatory disease that leads to the destruction of the tooth-supporting tissues. Current therapies are not effective for all patients and this oral disease continues to be a significant public health and economic burden. Central to periodontal disease pathogenesis is a reciprocally reinforced interplay between microbial dysbiosis and destructive inflammation, suggesting the potential relevance of host-modulation therapies. This review summarizes and discusses clinical observations and pre-clinical intervention studies that collectively suggest that complement is hyperactivated in periodontitis and that its inhibition provides a therapeutic benefit. Specifically, interception of the complement cascade at its central component, C3, using a locally administered small peptidic compound (Cp40/AMY-101) protected non-human primates from induced or naturally occurring periodontitis. These studies indicate that C3-targeted intervention merits investigation as an adjunctive treatment of periodontal disease in humans.

Macrophage ß2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis.

ß2-integrins promote neutrophil recruitment to infected tissues and are crucial for host defense. Neutrophil recruitment is defective in leukocyte adhesion deficiency type-1 (LAD1), a condition caused by mutations in the CD18 (ß2-integrin) gene. Using a model of Citrobacter rodentium (CR)-induced colitis, we show that CD18-/- mice display increased intestinal damage and systemic bacterial burden, compared to littermate controls, ultimately succumbing to infection. This phenotype is not attributed to defective neutrophil recruitment, as it is shared by CXCR2-/- mice that survive CR infection. CR-infected CD18-/- mice feature prominent upregulation of IL-17 and downregulation of IL-22. Exogenous IL-22 administration, but not endogenous IL-17 neutralization, protects CD18-/- mice from lethal colitis. ß2-integrin expression on macrophages is mechanistically linked to Rac1/ROS-mediated induction of noncanonical-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome-dependent IL-1ß production, which promotes ILC3-derived IL-22. Therefore, ß2-integrins are required for protective IL-1ß-dependent IL-22 responses in colitis, and the identified mechanism may underlie the association of human LAD1 with colitis.

DEL-1 promotes macrophage efferocytosis and clearance of inflammation.

Resolution of inflammation is essential for tissue homeostasis and represents a promising approach to inflammatory disorders. Here we found that developmental endothelial locus-1 (DEL-1), a secreted protein that inhibits leukocyte-endothelial adhesion and inflammation initiation, also functions as a non-redundant downstream effector in inflammation clearance. In human and mouse periodontitis, waning of inflammation was correlated with DEL-1 upregulation, whereas resolution of experimental periodontitis failed in DEL-1 deficiency. This concept was mechanistically substantiated in acute monosodium-urate-crystal-induced inflammation, where the pro-resolution function of DEL-1 was attributed to effective apoptotic neutrophil clearance (efferocytosis). DEL-1-mediated efferocytosis induced liver X receptor-dependent macrophage reprogramming to a pro-resolving phenotype and was required for optimal production of at least certain specific pro-resolving mediators. Experiments in transgenic mice with cell-specific overexpression of DEL-1 linked its anti-leukocyte-recruitment action to endothelial cell-derived DEL-1 and its efferocytic/pro-resolving action to macrophage-derived DEL-1. Thus, the compartmentalized expression of DEL-1 facilitates distinct homeostatic functions in an appropriate context that can be harnessed therapeutically.

Fibroblast growth factor-2 inhibits CD40-mediated periodontal inflammation.

Fibroblast growth factor-2 (FGF-2) stimulates periodontal regeneration by a broad spectrum of effects on periodontal ligament (PDL) cells, such as proliferation, migration, and production of extracellular matrix. A critical factor in the success of periodontal regeneration is the rapid resolution of inflammatory responses in the tissue. We explored an anti-inflammatory effect of FGF-2 during periodontal regeneration and healing. We found that FGF-2 on mouse periodontal ligament cells (MPDL22) markedly downregulated CD40 expression, a key player of inflammation. In addition, FGF-2 inhibited CD40 signaling by the non-canonical nuclear factor-kappa B2 (NFκB2) pathway, resulting in decreased production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which have the potential to recruit immune cells to inflamed sites. Furthermore, in vivo treatment of FGF-2 enhanced healing of skin wounds by counteracting the CD40-mediated inflammation. These results reveal that FGF-2 has an important function as a negative regulator of inflammation during periodontal regeneration and healing.

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.

Safety profile after prolonged C3 inhibition.

The central component of the complement cascade, C3, is involved in various biological functions, including opsonization of foreign bodies, clearance of waste material, activation of immune cells, and triggering of pathways controlling development. Given its broad role in immune responses, particularly in phagocytosis and the clearance of microbes, a deficiency in complement C3 in humans is often associated with multiple bacterial infections. Interestingly, an increased susceptibility to infections appears to occur mainly in the first two years of life and then wanes throughout adulthood. In view of the well-established connection between C3 deficiency and infections, therapeutic inhibition of complement at the level of C3 is often considered with caution or disregarded. We therefore set out to investigate the immune and biochemical profile of non-human primates under prolonged treatment with the C3 inhibitor compstatin (Cp40 analog). Cynomolgus monkeys were dosed subcutaneously with Cp40, resulting in systemic inhibition of C3, for 1 week, 2 weeks, or 3 months. Plasma concentrations of both C3 and Cp40 were measured periodically and complete saturation of plasma C3 was confirmed. No differences in hematological, biochemical, or immunological parameters were identified in the blood or tissues of animals treated with Cp40 when compared to those injected with vehicle alone. Further, skin wounds showed no signs of infection in those treated with Cp40. In fact, Cp40 treatment was associated with a trend toward accelerated wound healing when compared with the control group. In addition, a biodistribution study in a rhesus monkey indicated that the distribution of Cp40 in the body is associated with the presence of C3, concentrating in organs that accumulate blood and produce C3. Overall, our data suggest that systemic C3 inhibition in healthy adult non-human primates is not associated with a weakened immune system or susceptibility to infections.