Advances of exosomes in periodontitis treatment

Exos are secreted by almost all types of cells, including mesenchymal stem cells (MSCs), dendritic cells (DCs), B cells, T cells, and mast cells, and widely exist in many body fluids such as plasma, urine, breast milk, semen, amniotic fluid, and saliva [11]. Exos are among the three major types of extracellular vesicles (EVs). The size of the Exos is between 50 and 150 nm and the density is between 1.15 and 1.19 g / ml [12]. The biogenesis and release of Exos are different from other of other microbubbles. While other microbubbles are released directly through the plasma membrane, Exos originating from endocytosis [13]. First, the membrane of secretory cells is sunken inward to produce endocytic vesicles. Multiple endocytic vesicles combine to form early nucleosomes, and then miRNA, mRNA and DNA are packaged in the cytoplasm to form late endocytosis vesicles. The late endocytic vesicles germinate inward to form intracavitary vesicles (ILVs). The aggregation of ILVs in late endosomes create multivesicular bodies (MVBs), which are formed by the inward invagination of the endosomal limiting membrane. Finally, some MVBs with low cholesterol were degraded by lysosomes, and some MVBs, rich in cholesterol, release extracellular bodies through the fusion of the cell membrane with itself [13‐16]. The active formation of Exos is dependent on the endosomal sorting complex required for transport (ESCRT; ESCRT-0, I, II, III and Vps4) and its accessory proteins (Alix, TSG101, HSC70, and HSP90β). They recognize ubiquitinated transmembrane proteins and incorporate endosomal proteins into MVBs. [17, 18]. Passive formation of Exos is independent of ESCRT and involves lipids (ceramide), tetrapeptides (CD63) and heat shock proteins which induce cell membrane budding and promote MVB formation [19, 20]. In addition, certain components, such as four-transmembrane domain proteins and lipid rafts, have been reported to participate in the formation of some exosomes [21, 22]. In addition to the classic pathway, there is a much more immediate route of exosome biogenesis. T cells and erythroleukemia cell lines can release exosomes from the plasma membrane directly, and the Exos produced by these two pathways cannot be distinguished [23, 24]. Furthermore, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins and their effectors such as Rab GTPases (Rab27a, Rab27b and Rab35) play a significant role in exosome secretion [14].

The biogenesis of Exos is affected by many external factors, including cell type, serum conditions, cytokines, and growth factors. The heterogeneity of Exos is based on their specific morphology, content, and function [25, 26]. Encapsulated by lipid membranes derived from parental cells, Exos show inherent histocompatibility and tissue orientation mediated by surface molecules such as integrin and glycans [27]. In addition, Exos contain four transmembrane proteins (CD9, CD63, CD81 and CD82), heat shock proteins, lipoproteins, and some transport-related proteins. These proteins can not only provide markers for Exos identification but also locate Exos in specific target cells [28, 29]. Exos contain substances to mother cells, such as RNA, free fatty acids, proteins, surface receptors, and cytokines, which act as local and systemic intercellular communication media [30, 31]. Owing to these characteristics, Exos play a significant role in various biological functions and regulate many physiological and pathological processes in numerous diseases [32] (Fig. 2).

The selection and improvement of isolation strategies should be determined according to the kind of biological fluids Exos are isolated from. Effective isolation strategies should be able to concentrate the signals of the Exos to be analyzed and avoid contamination with other molecules, such as lipoproteins, non-vesicular protein aggregates, and other EVs, which are similar to Exos in terms of size and density. At present, there are many techniques for separating and purifying Exos from biological fluids and in vitro cell cultures, including ultracentrifugation, size-based separation, Exos precipitation, immunoaffinity capture-based techniques, and microfluidic isolation [33‐36]. Among these, differential centrifugation is the most widely used and basic method for Exos separation, and it is a feasible strategy to combine two or more methods to improve the yield, purity, and efficiency of Exos extracrion. Various techniques based on biophysical or biological characteristics have been used to verify upstream Exos separation methods and classify Exos subgroups for downstream analysis, such as nanoparticle tracking analysis (NTA), western blotting (WB), flow cytometry (FC), and atomic force microscopy (AFM) [35‐37] (Fig. 3).

Because the components of Exos can be reprogrammed according to disease status, Exos are increasingly being evaluated as potential diagnostic biomarkers for the diagnosis and prognosis of diseases. These characteristics have made Exos a focus of oral disease research in recent years, including periodontitis [39].

At the gene level, Exos are enriched in specific microRNAs (miRNAs), that can provide disease-specific diagnostic signatures [40]. When compared to healthy controls, plasma-derived exosomal miRs (miR-1304-3p and miR-200c-3p) and snoRs (SNORD57 and SNODB1771) from periodontitis patients are differentially expressed and could be the valuable biomarkers for periodontitis diagnosis [41]. In addition, the level of programmed death-ligand 1 (PD-L1) mRNA in salivary Exos may have the potential to diagnose periodontitis and is relative to the severity of periodontitis [42].

At the protein level, detection and analysis of salivary exosomal proteins in young adults with severe periodontitis (SP) suggested that C6 proteins, which participate in the immune response during the development of periodontitis, were expressed only in the SP group [43].

In addition, levels of CD9 and CD81 Exos in periodontitis patients were significantly lower than those in the healthy controls. Because the concentration of CD9/CD81 Exos in saliva is significantly and negatively correlated with clinical measurements, it may be of great significance in the pathogenesis of periodontal disease [44]. Other advances in salivary Exos in the diagnosis of periodontitis are well-reviewed in the literature [45].

Exos have been reported to provide a novel perspective and potential therapeutic approach for treating periodontitis and improving alveolar resorption. Exos derived from 3D-cultured MSCs restored not only the Th17 cell/Treg balance through the miR-1246/Nfat5 axis, but also the immune responses in the inflamed periodontium [46]. Dental pulp stem cell-Exo (DPSC-Exos) can facilitate the conversion of macrophages from a pro-inflammatory phenotype (M1)to an anti-inflammatory phenotype (M2) and promote the healing of alveolar bone in mice with periodontitis, the mechanism of which could be associated with miR-1246 in DPSC-Exos [47]. Exos purified from human leukocyte antigen haplotype homo dental pulp cell lines (HHH-DPCs) stimulated the migration of human DPCs and mouse osteoblastic and significantly suppressed osteoclast formation in vitro [48]. Exos secreted from healthy periodontal ligament stem cells (PDLSCs) promote osteogenic differentiation of PDLSCs derived from periodontitis tissue. Healthy PDLSC-Exos (h-PDLSC-Exos) treatment resulted in accelerated bone formation in alveolar bone defects in rat models of periodontitis. Mechanistically, h-PDLSC-Exos suppressed the overactivation of canonical Wnt signaling to recover the osteogenic differentiation capacity of inflammatory PDLSCs [49]. Exos derived from TNF-α-preconditioned gingival mesenchymal stem cells (GMSCs) could significantly regulate inflammation and osteoclastogenesis, which could provide a therapeutic approach for periodontitis [50]. Human exfoliated deciduous teeth (SHED)-derived Exos (SHED-Exos) restored bone loss in mouse periodontitis model and promoted bone marrow stromal cells (BMSCs) osteogenesis, differentiation, and bone formation [51]. SHED-Exos contribute to periodontal bone regeneration by promoting neovascularization and new bone formation, possibly through the AMPK signaling pathway [52]. Exos from reparative M2 macrophages reduced alveolar bone resorption in mice with periodontitis via the IL-10/IL-10R pathway [53]. Exos derived from adipose-derived stem cells (ADSC-Exos) represent a promising adjunctive treatment to SRP in rats [54]. Exosomal miR-25-3p in saliva contributes to the development and progression of diabetes-associated periodontitis. The discovery of other miR-25-3p targets may provide critical insights into the development of drugs to treat periodontitis by regulating γδ T cell-mediated local inflammation [55]. The in vivo effects of Exos on periodontitis are summarized in Table 1.

Neutrophils are short-lived cells in the innate immune system. They play an important role in pathogen resistance by producing reactive oxygen species (ROS). Therefore, effective strategies to improve the viability and function of neutrophils may be beneficial for treating infections and immune deficiency diseases. MSC-Exos have a protective effect on neutrophil function and lifespan [56], and could significantly reduce the terminal complement activation complex C5b-9 to inhibit neutrophils accumulation [57]. Exos isolated from ADSCs (ADSC-Exos) can decrease neutrophil apoptosis and increase phagocytosis [58]. Exos isolated from LPS-treated macrophages can induce cytokine production and neutrophils migration [59].

MSC-Exos can modify the polarization of the pro-inflammatory phenotype (M1 macrophages) to the anti-inflammatory phenotype (M2 macrophages) via shuttling miR-182 [60]. Exos derived from BMSCs (BMSC-Exos) can increase M2 macrophages [61], and BMSC-Exos have been reported to inhibit M1 macrophages and promote M2 macrophages in a murine alveolar macrophage cell line by inhibiting cellular glycolysis [62]. FNDC5 pre-conditioned BMSC-Exos have also been confirmed to play an anti-inflammatory role and promote M2 macrophages via NF-κB signaling pathway and the Nrf2/HO-1 axis [63]. Exos from human umbilical cord mesenchymal stem cells (hUCMSC-Exos) facilitated CD163 + M2 macrophages [64] and promoted M2 macrophages in LPS-stimulated RAW 264.7 via tumor necrosis factor receptor-associated factor 1 (TRAF1) [65]. ADSC-Exos can significantly upregulate the mRNA expression of M2 macrophages [66], induce M2 macrophages through the transactivation of Arg-1 by Exos-carried active STAT3 [67], and alleviate LPS induced inflammation by regulating Nrf2/HO-1 expression (68). Exos from GMSCs (GMSC-Exos) and dental pulp stem cells (DPSC-Exos) can promote the transformation of macrophages from M1 to M2 [47, 69]. TNF-α stimulated GMSC-Exos have also been reported to induce anti-inflammatory M2 macrophage polarization [47, 50].

MSC-Exos decreased DC surface marker expression in cells treated with LPS and decreased lymphocyte proliferation in the presence of MSC-Exos treated DCs, suggesting that MSC-Exos may play a key role in DC-induced immune responses [70]. hUCMSC-Exos suppressed the maturation and activation of DC and decreased the expression of IL-23, which is particularly important for promoting the pathogenicity of Th17 cells [71]. Exos from RegDC (RegDC-Exos) suppress the maturation of DCs and promote the recruitment of Treg cells, resulting in the inhibition of bone resorptive cytokines and reduction in osteoclastic bone loss [72]. Exos from lymphatic endothelial cells (LEC-Exos) promote the directional migration of human DCs in complex tissue environments in a CX3CL1/fractalkine-dependent fashion [73].

MSC-Exos decreased T lymphocyte proliferation and the percentage of CD4 + and CD8 + T cell subsets in a dose-dependent manner while increasing Treg cell populations [74]. MSC-Exos promote the proliferation and immune-suppression capacity of Tregs by upregulating IL-10 and TGF-β1[75], and inhibit the differentiation of Th2 cells by regulating the miR-146a-5p/SERPINB2 pathway [76]. PDLSC-Exos alleviated inflammatory microenvironment and maintained Th17/Treg balance via the Th17/Treg/miR‐155‐5p/SIRT1 regulatory network [77]. CD137-modified endothelial cell-Exos (EC-Exos) promote Th17 cell differentiation via the NF-КB pathway by regulating IL-6 expression [78].

MSC-Exos had the beneficial effect of reducing plasmablasts and incresing Breg-like cells in lymph nodes [74].

Exosomal miR-1260b of TNF-α-preconditioned GMSC-Exos was found to inhibit osteoclastogenic activity by targeting the Wnt5a-mediated RANKL pathway [50]. RegDC-Exos inhibit the production of bone resorptive cytokines and bone loss in osteoclasts [72]. Cyclic mechanical stretch (CMS)-treated BMSC-Exos can impair osteoclast differentiation by inhibiting the RANKL-NF-κB signaling pathway [79]. ADSC-Exos can reduce bone resorption and recover bone loss by suppressing NLRP3 inflammasome activation in osteoclasts [80] and by antagonizing osteocyte-mediated osteoclastogenesis [81]. ADSC-Exos combined with microRNA-146a (miR-146a-Exo) were reported to restrain bone resorption by inhibiting pro-inflammatory cytokine production in high glucose-treated osteoclasts [82]. Exos derived from osteoblasts can inhibit osteoclast differentiation via the miR-503-3p/Hpse axis [83]. Exos from endothelial progenitor cells (EPC-Exos) can promote bone repair by enhancing the recruitment and differentiation of osteoclast precursors via LncRNA-MALAT1 [84].

The effects of Exos on the host cells involved in periodontitis are summarized in Table 2.