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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.

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-β-Gal, p16 INK4A , p53, and p21 Waf1/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-1β, 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.

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 TGFβ1 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, TGFβ1 in regDCs EXO sustained downstream signaling in acceptor DCs. Blocking experiments suggested that sustaining TGFβ1 signaling require initial interaction of regDCs EXO with TGFβ1R followed by internalization of regDCs EXO with TGFβ1-TGFβ1R 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 TGFβ1 in-vitro. In conclusion, these results in mice may have important immunotherapeutic implications for lung inflammatory disorders.

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 ( ) 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 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 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 infected DC cells. In summary, we have identified the underlying mechanism by which promotes its own survival and that of its host DCs.

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-1β and IL-12 and increase in regulatory cytokine IL-10. No changes in TNF-α or TGF-β1 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.

The occurrence and severity of periodontitis (PD) tend to increase with age, and yet the underlying mechanisms remain unclear. Immune senescence is known to be triggered in mice and humans as they age. Experimental PD in mice has been shown to induce senescence biomarkers p16 INK4a and p21, dysfunction of antigen-presenting cells (APCs), and activation of the senescence-associated secretory phenotype (SASP). However, the causal links of senescence to experimental PD are not yet established. This study aims to elucidate the role of senescence in experimental PD at a causal level. The P16-3MR mouse model harbors the p16INK4a (Cdkn2a) promoter, driving in vivo expression of synthetic Renilla luciferase, monomeric red fluorescent protein (mRFP), and herpes simplex virus-1 thymidine kinase (HSV-TK). This facilitates in vivo identification of p16 INK4a activation at the cellular level and the consequences of selective elimination of p16INK4a-positive cells by ganciclovir (GCV) treatment. Mice were treated with/without GCV for two weeks during ligature-induced PD. In vivo bioluminescence imaging quantified p16INK4a activation, while Western blot and immunofluorescence analyses assessed key senescence and inflammatory markers (p16, p21, p53, Cyclin D1, p-H2A.X, IL17, and IL1β). Alveolar bone volume was analyzed by micro-CT and histomorphometry. Our findings demonstrate that clearance of senescent cells in mice subjected to experimental PD alleviates inflammation and mitigates bone loss. These results suggest a causal role for senescence in PD pathology, raising the future prospect of senolytic agents for therapeutic intervention in PD.

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.

(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.

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.

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-1β 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.