Oral Microbially-Induced Small Extracellular Vesicles Cross the Blood-Brain Barrier.

Porphyromonas gingivalis (Pg) and its gingipain proteases contribute to Alzheimer's disease (AD) pathogenesis through yet unclear mechanisms. Cellular secretion of small extracellular vesicles or exosomes (EXO) increases with aging as part of the senescence-associated secretory phenotype (SASP). We have shown that EXO isolated from Pg-infected dendritic cells contain gingipains and other Pg antigens and transmit senescence to bystander gingival cells, inducing alveolar bone loss in mice in vivo. Here, EXO were isolated from the gingiva of mice and humans with/without periodontitis (PD) to determine their ability to penetrate the blood-brain barrier (BBB) in vitro and in vivo. PD was induced by Pg oral gavage for 6 weeks in C57B6 mice. EXO isolated from the gingiva or brain of donor Pg-infected (PD EXO) or control animals (Con EXO) were characterized by NTA, Western blot, and TEM. Gingival PD EXO or Con EXO were labeled and injected into the gingiva of uninfected WT mouse model. EXO biodistribution in brains was tracked by an in vivo imaging system (IVIS) and confocal microscopy. The effect of human PD EXO on BBB integrity and permeability was examined using TEER and FITC dextran assays in a human in vitro 3D model of the BBB. Pg antigens (RGP and Mfa-1) were detected in EXO derived from gingival and brain tissues of donor Pg-infected mice. Orally injected PD EXO from donor mice penetrated the brains of recipient uninfected mice and colocalized with hippocampal microglial cells. IL-1ß and IL-6 were expressed in human PD EXO and not in Con EXO. Human PD EXO promoted BBB permeability and penetrated the BBB in vitro. This is the first demonstration that microbial-induced EXO in the oral cavity can disseminate, cross the BBB, and may contribute to AD pathogenesis.

Engineered Human Dendritic Cell Exosomes as Effective Delivery System for Immune Modulation.

Exosomes (exos) contain molecular cargo of therapeutic and diagnostic value for cancers and other inflammatory diseases, but their therapeutic potential for periodontitis (PD) remains unclear. Dendritic cells (DCs) are the directors of immune response and have been extensively used in immune therapy. We previously reported in a mouse model of PD that custom murine DC-derived exo subtypes could reprogram the immune response toward a bone-sparing or bone-loss phenotype, depending on immune profile. Further advancement of this technology requires the testing of human DC-based exos with human target cells. Our main objective in this study is to test the hypothesis that human monocyte-derived dendritic cell (MoDC)-derived exos constitute a well-tolerated and effective immune therapeutic approach to modulate human target DC and T cell immune responses in vitro. MoDC subtypes were generated with TGFb/IL-10 (regulatory (reg) MoDCs, CD86lowHLA-DRlowPDL1high), E. coli LPS (stimulatory (stim) MoDCs, CD86highHLA-DRhighPDL1low) and buffer (immature (i) MoDCs, CD86lowHLA-DRmedPDL1low). Exosomes were isolated from different MoDC subtypes and characterized. Once released from the secreting cell into the surrounding environment, exosomes protect their prepackaged molecular cargo and deliver it to bystander cells. This modulates the functions of these cells, depending on the cargo content. RegMoDCexos were internalized by recipient MoDCs and induced upregulation of PDL1 and downregulation of costimulatory molecules CD86, HLADR, and CD80, while stimMoDCexos had the opposite influence. RegMoDCexos induced CD25+Foxp3+ Tregs, which expressed CTLA4 and PD1 but not IL-17A. In contrast, T cells treated with stimMoDCexos induced IL-17A+ Th17 T cells, which were negative for immunoregulatory CTLA4 and PD1. T cells and DCs treated with iMoDCexos were immune 'neutral', equivalent to controls. In conclusion, human DC exos present an effective delivery system to modulate human DC and T cell immune responses in vitro. Thus, MoDC exos may present a viable immunotherapeutic agent for modulating immune response in the gingival tissue to inhibit bone loss in periodontal disease.

Dendritic cells a critical link to alveolar bone loss and systemic disease risk in periodontitis: Immunotherapeutic implications.

Extensive research in humans and animal models has begun to unravel the complex mechanisms that drive the immunopathogenesis of periodontitis. Neutrophils mount an early and rapid response to the subgingival oral microbiome, producing destructive enzymes to kill microbes. Chemokines and cytokines are released that attract macrophages, dendritic cells, and T cells to the site. Dendritic cells, the focus of this review, are professional antigen-presenting cells on the front line of immune surveillance. Dendritic cells consist of multiple subsets that reside in the epithelium, connective tissues, and major organs. Our work in humans and mice established that myeloid dendritic cells are mobilized in periodontitis. This occurs in lymphoid and nonlymphoid oral tissues, in the bloodstream, and in response to Porphyromonas gingivalis. Moreover, the dendritic cells mature in situ in gingival lamina propria, forming immune conjugates with cluster of differentiation (CD) 4+ T cells, called oral lymphoid foci. At such foci, the decisions are made as to whether to promote bone destructive T helper 17 or bone-sparing regulatory T cell responses. Interestingly, dendritic cells lack potent enzymes and reactive oxygen species needed to kill and degrade endocytosed microbes. The keystone pathogen P. gingivalis exploits this vulnerability by invading dendritic cells in the tissues and peripheral blood using its distinct fimbrial adhesins. This promotes pathogen dissemination and inflammatory disease at distant sites, such as atherosclerotic plaques. Interestingly, our recent studies indicate that such P. gingivalis-infected dendritic cells release nanosized extracellular vesicles called exosomes, in higher numbers than uninfected dendritic cells do. Secreted exosomes and inflammasome-related cytokines are a key feature of the senescence-associated secretory phenotype. Exosomes communicate in paracrine with neighboring stromal cells and immune cells to promote and amplify cellular senescence. We have shown that dendritic cell-derived exosomes can be custom tailored to target and reprogram specific immune cells responsible for inflammatory bone loss in mice. The long-term goal of these immunotherapeutic approaches, ongoing in our laboratory and others, is to promote human health and longevity.

Role of dendritic cell-mediated immune response in oral homeostasis: A new mechanism of osteonecrosis of the jaw.

Dendritic cells are an important link between innate and adaptive immune response. The role of dendritic cells in bone homeostasis, however, is not understood. Osteoporosis medications that inhibit osteoclasts have been associated with osteonecrosis, a condition limited to the jawbone, thus called medication-related osteonecrosis of the jaw. We propose that disruption of the local immune response renders the oral microenvironment conducive to osteonecrosis. We tested whether zoledronate (Zol) treatment impaired dendritic cell (DC) functions and increased bacterial load in alveolar bone in vivo and whether DC inhibition alone predisposed the animals to osteonecrosis. We also analyzed the role of Zol in impairment of differentiation and function of migratory and tissue-resident DCs, promoting disruption of T-cell activation in vitro. Results demonstrated a Zol induced impairment in DC functions and an increased bacterial load in the oral cavity. DC-deficient mice were predisposed to osteonecrosis following dental extraction. Zol treatment of DCs in vitro caused an impairment in immune functions including differentiation, maturation, migration, antigen presentation, and T-cell activation. We conclude that the mechanism of Zol-induced osteonecrosis of the jaw involves disruption of DC immune functions required to clear bacterial infection and activate T cell effector response.

Systemic Antibiotic Therapy Reduces Circulating Inflammatory Dendritic Cells and Treg-Th17 Plasticity in Periodontitis.

Periodontitis (PD) is a common dysbiotic inflammatory disease that leads to local bone deterioration and tooth loss. PD patients experience low-grade bacteremias with oral microbes implicated in the risk of heart disease, cancer, and kidney failure. Although Th17 effectors are vital to fighting infection, functional imbalance of Th17 effectors and regulatory T cells (Tregs) promote inflammatory diseases. In this study, we investigated, in a small pilot randomized clinical trial, whether expansion of inflammatory blood myeloid dendritic cells (DCs) and conversion of Tregs to Th17 cells could be modulated with antibiotics (AB) as part of initial therapy in PD patients. PD patients were randomly assigned to either 7 d of peroral metronidazole/amoxicillin AB treatment or no AB, along with standard care debridement and chlorhexidine mouthwash. 16s ribosomal RNA analysis of keystone pathogen Porphyromonas gingivalis and its consortium members Fusobacterium nucleatum and Streptococcus gordonii confirmed the presence of all three species in the reservoirs (subgingival pockets and blood DCs) of PD patients before treatment. Of the three species, P. gingivalis was reduced in both reservoirs 4-6 wk after therapy. Further, the frequency of CD1C+CCR6+ myeloid DCs and IL-1R1 expression on IL-17A+FOXP3+CD4+ T cells in PD patients were reduced to healthy control levels. The latter led to decreased IL-1ß-stimulated Treg plasticity in PD patients and improvement in clinical measures of PD. Overall, we identified an important, albeit short-term, beneficial role of AB therapy in reducing inflammatory DCs and Treg-Th17 plasticity in humans with PD.

Selective Antimicrobial Therapies for Periodontitis: Win the "Battle and the War".

Traditional antimicrobial therapies for periodontitis (PD) have long focused on non-selective and direct approaches. Professional cleaning of the subgingival biofilm by instrumentation of dental root surfaces, known as scaling and root planning (SRP), is the mainstay of periodontal therapy and is indisputably effective. Non-physical approaches used as adjuncts to SRP, such as chemical and biological agents, will be the focus of this review. In this regard, traditional agents such as oral antiseptics and antibiotics, delivered either locally or systemically, were briefly reviewed as a backdrop. While generally effective in winning the "battle" against PD in the short term, by reducing its signs and symptoms, patients receiving such therapies are more susceptible to recurrence of PD. Moreover, the long-term consequences of such therapies are still in question. In particular, concern about chronic use of systemic antibiotics and their influence on the oral and gut microbiota is warranted, considering antibiotic resistance plasmids, and potential transfer between oral and non-oral microbes. In the interest of winning the "battle and the war", new more selective and targeted antimicrobials and biologics for PD are being studied. These are principally indirect, blocking pathways involved in bacterial colonization, nutrient acquisition, inflammation or cellular invasion without directly killing the pathogens. This review will focus on current and prospective antimicrobial therapies for PD, emphasizing therapies that act indirectly on the microbiota, with clearly defined cellular and molecular targets.

Enterococcus faecalis Induces Differentiation of Immune-Aberrant Dendritic Cells from Murine Bone Marrow-Derived Stem Cells.

Enterococcus faecalis, long implicated in serious systemic infections and failure of root canal treatment, is a persistent inhabitant of oral periapical lesions. Dendritic cells (DCs) and other innate immune cells patrol the oral mucosa for infecting microbes. Dendritic cells are efficient at capturing microbes when immature, whereupon they can transform into potent antigen-presenting cells upon full maturation. Autophagy, a sophisticated intracellular process first described for elimination of damaged organelles, regulates DC maturation and other important immune functions of DCs. The present study examined how E. faecalis influences the differentiation of murine bone marrow-derived stem cells (BMSCs) into functional DCs in the presence of the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Although the viability and differentiation of DCs were not affected by E. faecalis, expression of the autophagy-related proteins ATG7, Beclin1, and LC3bI/II were significantly suppressed in an mTOR-dependent manner. Ultrastructurally, E. faecalis was identified in single-membrane vacuoles, some of which were in the process of binary fission. Bacterium-containing autophagosomes were absent within the cytoplasm. Accessory molecules (major histocompatibility complex class II [MHC-II], CD80, and CD86) and anti-inflammatory cytokine (transforming growth factor ß1 [TGF-ß1]) were suppressed in E. faecalis-induced DCs, while IL-1ß, tumor necrosis factor alpha (TNF-α), and IL-12 levels were upregulated. When pulsed with ovalbumin (OVA), the E. faecalis-induced DCs showed reduction in CD4+ OVA-specific OT-II T cell proliferation. It is concluded that E. faecalis promotes the differentiation of bone marrow stem cells into CD11c-positive DCs with aberrant immune functions while retaining the capability of proinflammatory cytokine induction.

The influence of vitamin D supplementation on local and systemic inflammatory markers in periodontitis patients: A pilot study.

OBJECTIVES: Vitamin D deficiency/insufficiency is a worldwide public health issue that has been linked to numerous inflammatory disorders, including periodontitis. There is increasing support for a role for adequate vitamin D levels in overall health. Populations with darker skin color have a higher prevalence of vitamin D deficiency/insufficiency and periodontitis. The purpose of this small pilot study was to investigate the influence of 12 weeks of 25(OH)D vitamin D supplementation (VDS) on mediators of systemic inflammation in dark-skinned, periodontitis patients. MATERIALS AND METHODS: A total of 23 patients with moderate to severe periodontitis were randomly assigned to the vitamin D group or placebo group and received intensive single visit scaling and root planning to elicit a systemic inflammatory response. RESULTS: Vitamin D supplementation increased serum 25(OH)D levels approximately 2-fold over baseline levels; moreover, VDS group had reduced peripheral blood CD3 and CD3+CD8+ cytotoxic T lymphocyte (CTLs) counts and reduced pro-inflammatory salivary cytokines. In contrast, VDS group had higher levels of the autophagy-related proteins and other proteins crucial for anti-microbial autophagy in whole blood PBMCs. CONCLUSION: In conclusion, VDS has multiple benefits for reducing systemic inflammation and promoting induction of autophagy-related proteins related to anti-microbial functions.

Microbially-Induced Exosomes from Dendritic Cells Promote Paracrine Immune Senescence: Novel Mechanism of Bone Degenerative Disease in Mice.

As the aging population grows, chronic age-related bone degenerative diseases become more prevalent and severe. One such disease, periodontitis (PD), rises to 70.1% prevalence in Americans 65 years and older. PD has been linked to increased risk of other age-related diseases with more serious mortality and morbidity profiles such as Alzheimer's disease and cardiovascular disease, but the cellular and biological mechanisms remain unclear. Recent in vitro studies from our group indicate that murine dendritic cells (DCs) and T cells are vulnerable to immune senescence. This occurs through a distinct process involving invasion of DCs by dysbiotic pathogen Porphyromonas gingivalis (Pg) activating the senescence associated secretory phenotype (SASP). Exosomes of the Pg-induced SASP transmit senescence to normal bystander DC and T cells, ablating antigen presentation. The biological significance of these findings in vivo and the mechanisms involved were examined in the present study using young (4-5mo) or old (22-24mo) mice subjected to ligature-induced PD, with or without dysbiotic oral pathogen and injection of Pg-induced DC exosomes. Senescence profiling of gingiva and draining lymph nodes (LN) corroborates role of advanced age and PD in elevation of senescence biomarkers beta galactosidase (SA-ß-Gal), p16 INK4A p21Waf1/Clip1, IL6, TNFα, and IL1ß, with attendant increase in alveolar bone loss, reversed by senolytic agent rapamycin. Immunophenotyping of gingiva and LN revealed that myeloid CD11c+ DCs and T cells are particularly vulnerable to senescence in vivo under these conditions. Moreover, Pg-induced DC exosomes were the most potent inducers of alveolar bone loss and immune senescence, and capable of overcoming senescence resistance of LN T cells in young mice. We conclude that immune senescence, compounded by advanced age, and accelerated by oral dysbiosis and its induced SASP exosomes, plays a pivotal role in the pathophysiology of experimental periodontitis.

Exogenous and Endogenous Dendritic Cell-Derived Exosomes: Lessons Learned for Immunotherapy and Disease Pathogenesis.

Immune therapeutic exosomes, derived exogenously from dendritic cells (DCs), the 'directors' of the immune response, are receiving favorable safety and tolerance profiles in phase I and II clinical trials for a growing number of inflammatory and neoplastic diseases. DC-derived exosomes (EXO), the focus of this review, can be custom tailored with immunoregulatory or immunostimulatory molecules for specific immune cell targeting. Moreover, the relative stability, small size and rapid uptake of EXO by recipient immune cells offer intriguing options for therapeutic purposes. This necessitates an in-depth understanding of mechanisms of EXO biogenesis, uptake and routing by recipient immune cells, as well as their in vivo biodistribution. Against this backdrop is recognition of endogenous exosomes, secreted by all cells, the molecular content of which is reflective of the metabolic state of these cells. In this regard, exosome biogenesis and secretion is regulated by cell stressors of chronic inflammation and tumorigenesis, including dysbiotic microbes, reactive oxygen species and DNA damage. Such cell stressors can promote premature senescence in young cells through the senescence associated secretory phenotype (SASP). Pathological exosomes of the SASP amplify inflammatory signaling in stressed cells in an autocrine fashion or promote inflammatory signaling to normal neighboring cells in paracrine, without the requirement of cell-to-cell contact. In summary, we review relevant lessons learned from the use of exogenous DC exosomes for immune therapy, as well as the pathogenic potential of endogenous DC exosomes.

Porphyromonas gingivalis Provokes Exosome Secretion and Paracrine Immune Senescence in Bystander Dendritic Cells.

Periodontitis is a disease of ageing or inflammaging, and is comorbid with other more severe age-related chronic diseases. With advanced age comes an increase in accumulation of senescent cells that release soluble and insoluble pro-inflammatory factors collectively termed the senescence associated secretory phenotype (SASP). In the present report, we examined whether immune cells typical of those at the oral mucosa-microbe interface, are vulnerable to cellular senescence (CS) and the role of dysbiotic oral pathogen Porphyromonas gingivalis. Bone marrow-derived dendritic cells (DCs) from young (yDCs) and old (oDCs) mice were co-cultured in vitro with CS inducer doxorubicin or P.gingivalis (Pg), plus or minus senolytic agent rapamycin. CS profiling revealed elevated CS mediators SA-ß-Gal, p16 INK4A, p53, and p21Waf1/Clip1 in oDCs, or yDCs in response to doxorubicin or P. gingivalis, reversible with rapamycin. Functional studies indicate impaired maturation function of oDCs, and yDC exposed to P. gingivalis; moreover, OVA-driven proliferation of CD4+ T cells from young OTII transgenic mice was impaired by oDCs or yDCs+Pg. The SASP of DCs, consisting of secreted exosomes and inflammasome-related cytokines was further analyzed. Exosomes of DCs cocultured with P. gingivalis (PgDCexo) were purified, quantitated and characterized. Though typical in terms of size, shape and phenotype, PgDCexo were 2-fold greater in number than control DCs, with several important distinctions. Namely, PgDCexo were enriched in age-related miRNAs, and miRNAs reported to disrupt immune homeostasis through negative regulation of apoptosis and autophagy functions. We further show that PgDCexo were enriched in P. gingivalis fimbrial adhesin protein mfa1 and in inflammasome related cytokines IL-1ß, TNFα and IL-6. Functionally PgDCexo were readily endocytosed by recipient yDCs, amplifying functional impairment in maturation and ability to promote Ova-driven proliferation of OTII CD4+ T cells from young mice. In conclusion P. gingivalis induces premature (autocrine) senescence in DCs by direct cellular invasion and greatly amplifies senescence, in paracrine, of bystander DCs by secretion of inflammatory exosomes. The implications of this pathological pathway for periodontal disease in vivo is under investigation in mouse models.

Matrix-Bound Zolzoledronate Enhances the Biofilm Colonization of Hydroxyapatite: Effects on Osteonecrosis.

(1) Background: The aim of this study was to test whether matrix-bound zoledronate (zol) molecules enhanced the oral biofilm colonization of a mineralized matrix, rendering the alveolar bone more susceptible to medication-related osteonecrosis of the jaw (MRONJ) following invasive dental procedures. (2) Methods: We tested the effect of matrix-bound zol on the growth and attachment of Porphyromonas gingivalis (Pg), Fusobacterium nucleatum (Fn) and Actinomyces israelii (Ai), and whether the nitrogen-containing component of zol contributed to such effect. The role of oral bacteria in the induction of osteonecrosis was then tested using an extra-oral bone defect model. (3) Results: The attachment of biofilm to hydroxyapatite discs increased when the discs were pre-treated with zol. Bacterial proliferation was not affected. Matrix-bound zol was more potent than non-nitrogen-containing etidronate in enhancing the colonization. Stimulation was dampened by pre-treating the bacteria with histidine. The delivery of oral biofilm to a tibial defect caused osteonecrosis in zol-treated rats. (4) Conclusions: We conclude that matrix-bound zol enhances the oral biofilm colonization of hydroxyapatite. This enhancement depended on the presence of the nitrogen-containing group. The oral biofilm rendered the extra-oral bone susceptible to medication-related osteonecrosis, suggesting that it has an important role in the induction of MRONJ.

Enterococcus faecalis shifts macrophage polarization toward M1-like phenotype with an altered cytokine profile.

Background: The macrophage is an innate immune defense cell involved in pathogen recognition and clearance. Aim: In view of the diversity of the macrophage phenotype and function, the present study investigated how Enterococcus faecalis infection affects the differentiation, phenotype and cytokine profile of macrophages. Methods: Murine bone marrow-derived stem cells were co-cultured with E. faecalis before and after differentiation. Macrophage M0 polarization towards M1 or M2 was initiated at day 6 by addition of LPS and INF-γ, or IL-4 and IL-13, respectively. Results: E. faecalis did not inhibit macrophage differentiation and were identified within macrophages. Viability of the macrophages infected with E. faecalis prior to differentiation was enhanced, evidenced by apoptosis inhibition, as was expression of CD38 and IRF5 proteins, indicators of M1-like polarization. These M1-like macrophages expressed an aberrant cytokine mRNA profile, with reduction in inflammatory cytokines IL-1ß and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-ß1 were detected, compared with the control groups. This atypical M1-like phenotype was retained even upon stimulation with growth factors that normally trigger their development into M2 macrophages. Conclusions: These findings suggested that E. faecalis infection of bone marrow-derived stem cells during differentiation into macrophages induces an atypical M1-like phenotype associated with intracellular bacterial survival.

Proteomic Characterization, Biodistribution, and Functional Studies of Immune-Therapeutic Exosomes: Implications for Inflammatory Lung Diseases.

Dendritic cell (DC)-derived exosomes (DC EXO), natural nanoparticles of endosomal origin, are under intense scrutiny in clinical trials for various inflammatory diseases. DC EXO are eobiotic, meaning they are well-tolerated by the host; moreover, they can be custom-tailored for immune-regulatory or -stimulatory functions, thus presenting attractive opportunities for immune therapy. Previously we documented the efficacy of immunoregulatory DCs EXO (regDCs EXO) as immunotherapy for inflammatory bone disease, in an in-vivo model. We showed a key role for encapsulated TGFß1 in promoting a bone sparing immune response. However, the on- and off-target effects of these therapeutic regDC EXO and how target signaling in acceptor cells is activated is unclear. In the present report, therapeutic regDC EXO were analyzed by high throughput proteomics, with non-therapeutic EXO from immature DCs and mature DCs as controls, to identify shared and distinct proteins and potential off-target proteins, as corroborated by immunoblot. The predominant expression in regDC EXO of immunoregulatory proteins as well as proteins involved in trafficking from the circulation to peripheral tissues, cell surface binding, and transmigration, prompted us to investigate how these DC EXO are biodistributed to major organs after intravenous injection. Live animal imaging showed preferential accumulation of regDCs EXO in the lungs, followed by spleen and liver tissue. In addition, TGFß1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFß1 signaling require initial interaction of regDCs EXO with TGFß1R followed by internalization of regDCs EXO with TGFß1-TGFß1R complex. Finally, these regDCs EXO that contain immunoregulatory cargo and showed biodistribution to lungs could downregulate the main severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target receptor, ACE2 on recipient lung parenchymal cells via TGFß1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

Dendritic cell derived exosomes loaded with immunoregulatory cargo reprogram local immune responses and inhibit degenerative bone disease in vivo.

Chronic bone degenerative diseases represent a major threat to the health and well-being of the population, particularly those with advanced age. This study isolated exosomes (EXO), natural nano-particles, from dendritic cells, the "directors" of the immune response, to examine the immunobiology of DC EXO in mice, and their ability to reprogram immune cells responsible for experimental alveolar bone loss in vivo. Distinct DC EXO subtypes including immune-regulatory (regDC EXO), loaded with TGFB1 and IL10 after purification, along with immune stimulatory (stimDC EXO) and immune "null" immature (iDCs EXO) unmodified after purification, were delivered via I.V. route or locally into the soft tissues overlying the alveolar bone. Locally administrated regDC EXO showed high affinity for inflamed sites, and were taken up by both DCs and T cells in situ. RegDC EXO-encapsulated immunoregulatory cargo (TGFB1 and IL10) was protected from proteolytic degradation. Moreover, maturation of recipient DCs and induction of Th17 effectors was suppressed by regDC EXO, while T-regulatory cell recruitment was promoted, resulting in inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss. This work is the first demonstration of DC exosome-based therapy for a degenerative alveolar bone disease and provides the basis for a novel treatment strategy.

Disruption of Immune Homeostasis in Human Dendritic Cells via Regulation of Autophagy and Apoptosis by Porphyromonas gingivalis.

As fundamental processes of immune homeostasis, autophagy, and apoptosis must be maintained to mitigate risk of chronic inflammation and autoimmune diseases. Periodontitis is a chronic inflammatory disease characterized by oral microbial dysbiosis, and dysregulation of dendritic cell (DC) and T cell responses. The aim of this study was to elucidate the underlying mechanisms by which the oral microbe Porphyromonas gingivalis (P. gingivalis) manipulates dendritic cell signaling to perturb both autophagy and apoptosis. Using a combination of Western blotting, flow cytometry, qRT-PCR and immunofluorescence analysis, we show a pivotal role for the minor (Mfa1) fimbriae of P. gingivalis in nuclear/cytoplasmic shuttling of Akt and FOXO1 in human monocyte-derived DCs. Mfa1-induced Akt nuclear localization and activation ultimately induced mTOR. Activation of the Akt/mTOR axis downregulated intracellular LC3II, also known as Atg8, required for autophagosome formation and maturation. Use of allosteric panAkt inhibitor MK2206 and mTOR inhibitor rapamycin confirmed the role of Akt/mTOR signaling in autophagy inhibition by P. gingivalis in DCs. Interestingly, this pathway was also linked to induction of the anti-apoptotic protein Bcl2, decreased caspase-3 cleavage and decreased expression of pro-apoptotic proteins Bax and Bim, thus promoting longevity of host DCs. Addition of ABT-199 peptide to disrupt the interaction of antiapoptotic Bcl2 and its proapoptotic partners BAK/BAX restored apoptotic death to P. gingivalis-infected DC cells. In summary, we have identified the underlying mechanism by which P. gingivalis promotes its own survival and that of its host DCs.

Polymicrobial synergy within oral biofilm promotes invasion of dendritic cells and survival of consortia members.

Years of human microbiome research have confirmed that microbes rarely live or function alone, favoring diverse communities. Yet most experimental host-pathogen studies employ single species models of infection. Here, the influence of three-species oral microbial consortium on growth, virulence, invasion and persistence in dendritic cells (DCs) was examined experimentally in human monocyte-derived dendritic cells (DCs) and in patients with periodontitis (PD). Cooperative biofilm formation by Streptococcus gordonii, Fusobacterium nucleatum and Porphyromonas gingivalis was documented in vitro using growth models and scanning electron microscopy. Analysis of growth rates by species-specific 16s rRNA probes revealed distinct, early advantages to consortium growth for S. gordonii and F. nucleatum with P. gingivalis, while P. gingivalis upregulated its short mfa1 fimbriae, leading to increased invasion of DCs. F. nucleatum was only taken up by DCs when in consortium with P. gingivalis. Mature consortium regressed DC maturation upon uptake, as determined by flow cytometry. Analysis of dental plaques of PD and healthy subjects by 16s rRNA confirmed oral colonization with consortium members, but DC hematogenous spread was limited to P. gingivalis and F. nucleatum. Expression of P. gingivalis mfa1 fimbriae was increased in dental plaques and hematogenous DCs of PD patients. P. gingivalis in the consortium correlated with an adverse clinical response in the gingiva of PD subjects. In conclusion, we have identified polymicrobial synergy in a three-species oral consortium that may have negative consequences for the host, including microbial dissemination and adverse peripheral inflammatory responses.