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Due to its capacity to drive osteoclast differentiation, the receptor activator of nuclear factor kappa-β ligand (RANKL) is believed to exert a pathological influence in periodontitis. However, RANKL was initially identified as an activator of dendritic cells (DCs), expressed by T cells, and exhibits diverse effects on the immune system. Hence, it is probable that RANKL, acting as a bridge between the bone and immune systems, plays a more intricate role in periodontitis. Using ligature-induced periodontitis (LIP), rapid alveolar bone loss was detected that was later halted even though the ligature was still present. This late phase of LIP was also linked with immunosuppressive conditions in the gingiva. Further investigation revealed that the ligature prompted an immediate migration of RANK-expressing Langerhans cells (LCs) and EpCAM + DCs, the antigen-presenting cells (APCs) of the gingival epithelium, to the lymph nodes, followed by an expansion of T regulatory (Treg) cells in the gingiva. Subsequently, the ligatured gingiva was repopulated by monocyte-derived RANK-expressing EpCAM + DCs, while gingival epithelial cells upregulated RANKL expression. Blocking RANKL signaling with monoclonal antibodies significantly reduced the frequencies of Treg cells in the gingiva and prevented gingival immunosuppression. In addition, RANKL signaling facilitated the differentiation of LCs from bone marrow precursors. To further investigate the role of RANKL, we used K14-RANKL mice, in which RANKL is overexpressed by gingival epithelial cells. The elevated RANKL expression shifted the steady-state frequencies of LCs and EpCAM + DCs within the epithelium, favoring LCs over EpCAM + DCs. Following ligature placement, heightened levels of Treg cells were observed in the gingiva of K14-RANKL mice, and alveolar bone loss was significantly reduced. These findings suggest that RANKL-RANK interactions between gingival epithelial cells and APCs are crucial for suppressing gingival inflammation, highlighting a protective immunological role for RANKL in periodontitis that was overlooked due to its osteoclastogenic activity., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Aim: Polarization of macrophages (Mφ) is a well-controlled axis with considerable consequences in both the pro-inflammatory and resolution phases of inflammation. We aimed to determine if periodontal therapy may instigate M1 to M2 Mφ polarization favoring resolution of inflammation within periodontal tissues., Methods: Gingival biopsies were excised from subjects diagnosed with Stage III, Grade B periodontitis before and 4-6 weeks after nonsurgical periodontal therapy. Total RNA was isolated and pro- and anti-inflammatory markers associated with Mφ polarization assessed by RT-qPCR. Mice were subject to ligature-induced periodontitis and gingival tissues collected after 8 days in-situ or 10 days after ligature removal and M1 and M2 Mφ markers examined by RT-qPCR and flow cytometry., Results: In human samples, improvement in clinical parameters posttherapy correlates with reduced bacterial burden, downregulation in M1 (TNF-α, STAT1, CXCL10, and miR-155), and elevated levels of M2 (STAT6, TGM2, CCL22, and IL-10) Mφ markers. In a murine model of resolution of LIP, we observed reduced levels of M1 Mφ markers cox2, iNOS2, F4/80+CD80+, and F4/80+CD86+ and elevated levels of M2-like Mφ markers tgm2, arg1, F4/80+CD206+ and F4/80+CD163+ corroborated human findings., Conclusion: Resolution of periodontal inflammation is associated with M1 to M2 Mφ polarization after nonsurgical periodontal therapy. Assessment of Mφ markers can provide relevant clinical information on the successful response of periodontal therapy and may be used to target nonresponders., (© 2024 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Immune cells residing at the gingiva experience diverse and unique signals, tailoring their functions to enable them to appropriately respond to immunological challenges and maintain tissue integrity. The gingiva, defined as the mucosal barrier that surrounds and supports the teeth, is the only barrier site completely transected by a hard structure, the tooth. The tissue is damaged in early life during tooth eruption and chronically throughout life by the process of mastication. This occurs alongside challenges typical of barrier sites, including exposure to invading pathogens, the local commensal microbial community and environmental antigens. This review will focus on the immune network safeguarding gingival integrity, which is far less understood than that resident at other barrier sites. A detailed understanding of the gingiva-resident immune network is vital as it is the site of the inflammatory disease periodontitis, the most common chronic inflammatory condition in humans which has well-known detrimental systemic effects. Furthering our understanding of how the immune populations within the gingiva develop, are tailored in health, and how this is dysregulated in disease would further the development of effective therapies for periodontitis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Objectives: The gingival mucosal barrier, an important oral cavity barrier, plays a significant role in preventing pathogenic microorganism invasion and maintaining periodontal tissue health. Pathogenic microorganism invasion of the gingival mucosa produces a large number of cytokines. Among them, pyroptosis is an important player in exacerbating immune-inflammatory responses, leading to tissue destruction. However, the mechanism of pyroptosis and the immune response it triggers have not been fully elucidated. We provide an overview of recent advances in understanding gingival physical barrier pyroptosis and inflammation-induced hyperimmunity., Methods: PubMed, Web of Science databases were searched for articles, reviews, and clinical studies published until March 2024., Results: We summarised the importance of the gingival barrier in terms of the functions of different cells, described the progress in research on gingival epithelial cell and gingival fibroblast pyroptosis and the immune-inflammatory response it induces, and discussed the relationship between pyroptosis and systemic diseases, association of multiple cell death systems. Finally, we propose future directions for pyroptosis research., Conclusions: Pyroptosis often triggers a range of inflammatory immune responses that lead to associated diseases. Therefore, further study of the molecular mechanisms of pyroptosis and the immune responses is warranted., (© 2024 Wiley Periodicals LLC.)

Aims: Microbial flora of dental plaque trigger innate and adaptive immune responses. The function of antigen-presenting cells (APCs) is to bridge the innate and adaptive immune systems. The human immune system contains three main types of APCs: dendritic cells (DC) (Langerhans cells (LCs) and interstitial DCs, IDCs), macrophages and B lymphocytes. In this study, the distribution and density of all APCs in healthy and inflamed human gingival tissue were comparatively analysed., Methods: Research was conducted on gingival biopsy specimens obtained from 55 patients and classified in three groups: healthy gingiva (control group, n=10), moderate periodontal disease (PD) (n=21) and severe PD (n=24). For APCs' identification antibodies raised against CD 1a (for LCs), S 100 protein (for iDCs), CD 68 (for macrophages) and CD 20 (for B lymphocytes) were used., Results: Increased density of IDCs, macrophages and B lymphocytes in lamina propria and reduced density of LCs in the gingival epithelium were found in patients with periodontitis. Simultaneously, it was noticed an increased concentration of macrophages and B cells in the gingival epithelium in patients with PD. No statistically significant difference in the distribution and density of APC was found among patients with moderate and advanced periodontitis., Conclusions: It was hypothesised that in the periodontitis the role of antigen presentation was largely taken from LCs by the DCs, macrophages and B cells. These APCs are thought to have less protective and tolerogenic potential than LCs and this is a significant reason for alveolar bone destruction in periodontitis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3 + T cells and promoted the generation of CD4 + CD25 + FoxP3 + CD39 + CD73 + Tregs and Th17 + CD39 + lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.

Objective: The present study was performed to identify key biomarkers associated with immune cell infiltration in peri-implantitis through bioinformatic analyses., Methods: Six peri-implantitis soft tissue samples and six healthy gingiva samples were obtained from GSE106090, and were used to identify immune-associated differentially expressed genes (DEGs) in peri-implantitis. The candidate biomarkers associated with immune cell infiltration were examined by immunohistochemical staining., Results: We identified 2089 upregulated and 2173 downregulated genes. Upregulated DEGs were significantly associated with immune response. Ten key candidate biomarkers were identified in the PPI network, including IL1B, TLR2, TLR4, CCL4, CXCL8, IL10, IL6, CD4, CCL3, and PTPRC. The expression level of the 10 genes increased in peri-implantitis soft tissue samples compared with healthy gingiva samples. The proportion of CD4 + T cells, iTreg, and Tfh in infiltration immune cells increased in peri-implantitis soft tissue samples and were positively correlated with the expression level of candidate biomarkers TLR4, CCL3, CXCL8, and IL1B. Immunohistochemistry showed that there were more lymphocytes in peri-implantitis soft tissue samples, with an increased expression level of TLR4, CCL3, CXCL8, and IL1B., Conclusion: Identification of four novel diagnostic biomarkers was helpful for revealing the molecular mechanisms and could serve as a risk predictor for the immune microenvironment in peri-implantitis., (© 2023 The Authors. Oral Diseases published by Wiley Periodicals LLC.)

Th17 cell plasticity is associated with pathogenicity in chronic inflammation. In a model of periodontitis, McClure et al. (https://doi.org/10.1084/jem.20232015) describe location-dependent divergence in Th17 plasticity, with surprisingly limited conversion in inflamed gingiva but emergence of protective exTh17-TfH cells in draining LN that enhance protective antibody., (© 2024 McGeachy.)

Platelet-rich fibrin (PRF) is prepared by spontaneous coagulation of fractionated blood. When squeezed between two plates, PRF is separated into solid PRF membranes and a liquid exudate, the PRF serum. The question arises regarding how much the overall activity remains in the PRF membranes and what is discarded into the PRF serum. To this end, we have exposed gingival fibroblasts to lysates prepared from PRF membranes and PRF serum, followed by bulk RNA sequencing. A total of 268 up- and 136 down-regulated genes in gingival fibroblasts exposed to PRF membrane lysates were significantly regulated under the premise of a minimum log2 with 2.5-fold change and a minus log10 significance level of two, respectively. PRF serum only caused 62 up- and 32 down-regulated genes under these conditions. Among the 46 commonly up-regulated genes were CXCL1, CXCL5, CXCL6, CXCL8, IL33, IL6, and PTGS2/COX2, stanniocalcin-1-all linked to an inflammatory response. PRF membrane lysates further increased chemokines CCL2, CCL7, CXCL2, CXCL3, and IL1R1, IL1RL1, and IL1RN, as well as the paracrine factors IL11, LIF, IGF1, BMP2, BMP6, FGF2, and CCN2/CTGF, and all hyaluronan synthases. On the other hand, PRF serum increased DKK1. The genes commonly down-regulated by PRF membrane lysates and PRF serum included interferon-induced protein with tetratricopeptide repeats (IFIT1, IFIT2, IFIT3) and odd-skipped-related transcription factors (OSR1 and OSR2), as well as FGF18 and GDF15, respectively. Taken together, PRF membrane lysates, compared to PRF serum, cause a more complex response in gingival fibroblasts, but each increased chemokine expression in gingival fibroblasts.

Th17 cell plasticity is crucial for development of autoinflammatory disease pathology. Periodontitis is a prevalent inflammatory disease where Th17 cells mediate key pathological roles, yet whether they exhibit any functional plasticity remains unexplored. We found that during periodontitis, gingival IL-17 fate-mapped T cells still predominantly produce IL-17A, with little diversification of cytokine production. However, plasticity of IL-17 fate-mapped cells did occur during periodontitis, but in the gingiva draining lymph node. Here, some Th17 cells acquired features of Tfh cells, a functional plasticity that was dependent on IL-6. Notably, Th17-to-Tfh diversification was important to limit periodontitis pathology. Preventing Th17-to-Tfh plasticity resulted in elevated periodontal bone loss that was not simply due to increased proportions of conventional Th17 cells. Instead, loss of Th17-to-Tfh cells resulted in reduced IgG levels within the oral cavity and a failure to restrict the biomass of the oral commensal community. Thus, our data identify a novel protective function for a subset of otherwise pathogenic Th17 cells during periodontitis., (© 2024 McClure et al.)

Background: This study aimed to investigate the contribution of myeloid differentiation primary-response gene 88 (MyD88) on the differentiation of T helper type 17 (Th17) and regulatory T (Treg) cells and the emerging subgingival microbiota dysbiosis in Porphyromonas gingivalis-induced experimental periodontitis., Methods: Alveolar bone loss, infiltrated inflammatory cells, immunostained cells for tartrate-resistant acid phosphatase (TRAP), the receptor activator of nuclear factor-kB ligand (RANKL), and osteoprotegerin (OPG) were quantified by microcomputerized tomography and histological staining between age- and sex-matched homozygous littermates (wild-type [WT, Myd88 +/+ ] and Myd88 -/- on C57BL/6 background). The frequencies of Th17 and Treg cells in cervical lymph nodes (CLNs) and spleen were determined by flow cytometry. Cytokine expression in gingival tissues, CLNs, and spleens were studied by quantitative polymerase chain reaction (qPCR). Analysis of the composition of the subgingival microbiome and functional annotation of prokaryotic taxa (FAPROTAX) analysis were performed., Results: P. gingivalis-infected Myd88 -/- mice showed alleviated bone loss, TRAP + osteoclasts, and RANKL/OPG ratio compared to WT mice. A significantly higher percentage of Foxp3 + CD4 + T cells in infected Myd88 -/- CLNs and a higher frequency of RORγt + CD4 + T cells in infected WT mice was noted. Increased IL-10 and IL-17a expressions in gingival tissue at D14-D28 then declined in WT mice, whereas an opposite pattern was observed in Myd88 -/- mice. The Myd88 -/- mice exhibited characteristic increases in gram-positive species and species having probiotic properties, while gram-negative, anaerobic species were noted in WT mice. FAPROTAX analysis revealed increased aerobic chemoheterotrophy in Myd88 -/- mice, whereas anaerobic chemoheterotrophy was noted in WT mice after P. gingivalis infection., Conclusions: MyD88 plays an important role in inflammation-induced bone loss by modulating the dynamic equilibrium between Th17/Treg cells and dysbiosis in P. gingivalis-induced experimental periodontitis., (© 2024 American Academy of Periodontology.)

Periodontitis, delineated by the destruction of structures that support teeth, is predominantly propelled by intricate immune responses. Immunomodulatory treatments offer considerable promise for the management of this ailment; however, the modulation of the periodontal immune microenvironment to facilitate tissue regeneration presents a substantial biomedical challenge. Herein, our study investigates the role of Wilms' tumor 1-associating protein (WTAP), a critical m 6 A methyltransferase, in the immunomodulation of periodontitis and assesses its viability as a therapeutic target. We observed heightened expression of WTAP in macrophages extracted from gingival tissues impacted by periodontitis, with a strong association with M1 polarization. Via loss-of-function experiments, we demonstrated that diminishing WTAP expression precipitates a transition from M1 to M2 macrophage phenotypes amidst inflammatory conditions, thus improving the periodontal immune landscape. Further, RNA sequencing and indirect co-culture assays indicated that suppressing of WTAP expression modulates osteoimmune responses and enhances the osteogenic differentiation of bone marrow stromal cells. The local deployment of adeno-associated virus-shWTAP in murine models of periodontitis robustly validated the therapeutic promise of targeting WTAP in this disease. Collectively, our findings highlight the crucial role of WTAP in orchestrating macrophage-mediated osteoimmune responses and tissue regeneration in periodontitis, proposing novel avenues for immunotherapeutic interventions in its treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Li, Yang, Niu, Li, Hu, Sun, Chen, Hu, Huang and Deng.)

Introduction: Macrophages (Mφs) are functionally dynamic immune cells that bridge innate and adaptive immune responses; however, the underlying epigenetic mechanisms that control Mφ plasticity and innate immune functions are not well elucidated., Objective: To identify novel functions of macrophage-enriched lncRNAs in regulating polarization and innate immune responses., Methods: Total RNA isolated from differentiating monocyte-derived M1 and M2 Mφs was profiled for lncRNAs expression using RNAseq. Impact of LRRC75A-AS1, GAPLINC and AL139099.5 knockdown was examined on macrophage differentiation, polarization markers, phagocytosis, and antigen processing by flow cytometry and florescence microscopy. Cytokine profiles were examined by multiplex bead array and cytoskeletal signaling pathway genes were quantified by PCR-based array. Gingival biopsies were collected from periodontally healthy and diseased subjects to examine lncRNAs, M1/M2 marker expression., Results: Transcriptome profiling of M1 and M2 Mφs identified thousands of differentially expressed known and novel lncRNAs. We characterized three Mφ-enriched lncRNAs LRRC75A-AS1, GAPLINC and AL139099.5 in polarization and innate immunity. Knockdown of LRRC75A-AS1 and GAPLINC downregulated the Mφ differentiation markers and skewed Mφ polarization by decreasing M1 markers without a significant impact on M2 markers. LRRC75A-AS1 and GAPLINC knockdown also attenuated bacterial phagocytosis, antigen processing and inflammatory cytokine secretion in Mφs, supporting their functional role in potentiating innate immune functions. Mechanistically, LRRC75A-AS1 and GAPLINC knockdown impaired Mφ migration by downregulating the expression of multiple cytoskeletal signaling pathways suggesting their critical role in regulating Mφ migration. Finally, we showed that LRRC75A-AS1 and GAPLINC were upregulated in periodontitis and their expression correlates with higher M1 markers suggesting their role in macrophage polarization in vivo., Conclusion: Our results show that polarized Mφs acquire a unique lncRNA repertoire and identified many previously unknown lncRNA sequences. LRRC75A-AS1 and GAPLINC, which are induced in periodontitis, regulate Mφ polarization and innate immune functions supporting their critical role in inflammation., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Mesenchymal stem cells (MSCs) have demonstrated potent immunomodulatory properties that have shown promise in the treatment of autoimmune diseases, including rheumatoid arthritis (RA). However, the inherent heterogeneity of MSCs triggered conflicting therapeutic outcomes, raising safety concerns and limiting their clinical application. This study aimed to investigate the potential of extracellular vesicles derived from human gingival mesenchymal stem cells (GMSC-EVs) as a therapeutic strategy for RA. Through in vivo experiments using an experimental RA model, our results demonstrate that GMSC-EVs selectively homed to inflamed joints and recovered Treg and Th17 cell balance, resulting in the reduction of arthritis progression. Our investigations also uncovered miR-148a-3p as a critical contributor to the Treg/Th17 balance modulation via IKKB/NF-κB signaling orchestrated by GMSC-EVs, which was subsequently validated in a model of human xenograft versus host disease (xGvHD). Furthermore, we successfully developed a humanized animal model by utilizing synovial fibroblasts obtained from patients with RA (RASFs). We found that GMSC-EVs impeded the invasiveness of RASFs and minimized cartilage destruction, indicating their potential therapeutic efficacy in the context of patients with RA. Overall, the unique characteristics - including reduced immunogenicity, simplified administration, and inherent ability to target inflamed tissues - position GMSC-EVs as a viable alternative for RA and other autoimmune diseases.

Interleukin-34 (IL-34) is a cytokine that supports the viability and differentiation of macrophages. An important cytokine for the development of epidermal immunity, IL-34, is present and plays a role in the immunity of the oral environment. IL-34 has been linked to inflammatory periodontal diseases, which involve innate phagocytes, including macrophages. Whether IL-34 can alter the ability of macrophages to effectively interact with oral microbes is currently unclear. Using macrophages derived from human blood monocytes with either the canonical cytokine colony-stimulating factor (CSF)1 or IL-34, we compared the ability of the macrophages to phagocytose, kill, and respond through the production of cytokines to the periodontal keystone pathogen Porphyromonas gingivalis. While macrophages derived from both cytokines were able to engulf the bacterium equally, IL-34-derived macrophages were much less capable of killing internalized P. gingivalis. Of the macrophage cell surface receptors known to interact with P. gingivalis, dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin was found to have the largest variation between IL-34- and CSF1-derived macrophages. We also found that upon interaction with P. gingivalis, IL-34-derived macrophages produced significantly less of the neutrophil chemotactic factor IL-8 than macrophages derived in the presence of CSF1. Mechanistically, we identified that the levels of IL-8 corresponded with P. gingivalis survival and dephosphorylation of the major transcription factor NF-κB p65. Overall, we found that macrophages differentiated in the presence of IL-34, a dominant cytokine in the oral gingiva, have a reduced ability to kill the keystone pathogen P. gingivalis and may be susceptible to specific bacteria-mediated cytokine modification., (© 2022 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we report that the clotting factor fibrin is a critical regulator of neutrophil function at the oral mucosal barrier. We demonstrate that commensal microbiota trigger extravascular fibrin deposition in the oral mucosa. Fibrin engages neutrophils through the α M β 2 integrin receptor and activates effector functions, including the production of reactive oxygen species and neutrophil extracellular trap formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis in mice and humans. Concordantly, genetic polymorphisms in PLG , encoding plasminogen, are associated with common forms of periodontal disease. Thus, fibrin is a critical regulator of neutrophil effector function, and fibrin-neutrophil engagement may be a pathogenic instigator for a prevalent mucosal disease.

Here, we show that Porphyromonas gingivalis ( Pg ), an endogenous oral pathogen, dampens all aspects of interferon (IFN) signaling in a manner that is strikingly similar to IFN suppression employed by multiple viral pathogens. Pg suppressed IFN production by down-regulating several IFN regulatory factors (IRFs 1, 3, 7, and 9), proteolytically degrading STAT1 and suppressing the nuclear translocation of the ISGF3 complex, resulting in profound and systemic repression of multiple interferon-stimulated genes. Pg -induced IFN paralysis was not limited to murine models but was also observed in the oral tissues of human periodontal disease patients, where overabundance of Pg correlated with suppressed IFN generation. Mechanistically, multiple virulence factors and secreted proteases produced by Pg transcriptionally suppressed IFN promoters and also cleaved IFN receptors, making cells refractory to exogenous IFN and inducing a state of broad IFN paralysis. Thus, our data show a bacterial pathogen with equivalence to viruses in the down-regulation of host IFN signaling., Competing Interests: The authors declare no competing interest.

Activated-mast cells (MCs) within gingival-tissue of chronic-periodontitis (CP) patients, release various inflammatory-factors. Bradykinin is a nine-amino-acid peptide and pro-inflammatory mediator, produced through factor-XII-cascade or tryptase-cascade. The ability of MC-chymase in bradykinin generation has not been discussed yet. This study investigated the salivary levels of MC-chymase, high molecular weight kininogen (HMWK) and bradykinin of CP patients; examined the potential of MC-proteases in bradykinin production using biochemistry-models; and explored the effects of bradykinin on gingival fibroblasts (GFs). Saliva-samples were collected; MC-protease activities were detected; HMWK cleavage was assessed by western-blot and SDS-PAGE; bradykinin levels were measured using immunoassay. Primary GFs were extracted and cultured with or without bradykinin; cell-viability, gelatine-zymography and flow-cytometry were applied. Immunocytochemistry and western-blot were used to detect intracellular protein expressions of bradykinin-stimulated GFs. The data showed that the salivary-levels of MC-proteases, bradykinin, HMWK, and lactoferrin of CP-patients were increased. HMWK was cleaved by MC-chymase in-vitro, resulting in bradykinin generation. Bradykinin promoted cell proliferation, cell cycle and matrix-metalloproteinase-2(MMP-2) activity, and increased intracellular expressions of nuclear-factor-kappa-B(NF-κB), focal-adhesion-kinase(FAK), transforming-growth-factor-β(TGF-β), P38, P53 of GFs. MC-chymase promotes bradykinin production to stimulate GFs and to continue inflammation during CP development. A new BK-generation cascade found in this study provides a new basis for the pathogenesis of CP and the mechanism of continuous inflammation. The activation of MC-chymase/bradykinin-generation cascade depends on HMWK level and MC-chymase activity under inflammatory condition. MC-chymase contributes to bradykinin production, mediating the cross-talks between MCs and GFs. MC-chymase can be used as a therapeutic target and a salivary biomarker in this case., (Copyright © 2021. Published by Elsevier B.V.)

The subgingival biofilm attached to tooth surfaces triggers and maintains periodontitis. Previously, late-onset periodontitis has been considered a consequence of dysbiosis and a resultant polymicrobial disruption of host homeostasis. However, a multitude of studies did not show "healthy" oral microbiota pattern, but a high diversity depending on culture, diets, regional differences, age, social state etc. These findings relativise the aetiological role of the dysbiosis in periodontitis. Furthermore, many late-onset periodontitis traits cannot be explained by dysbiosis; e.g. age-relatedness, attenuation by anti-ageing therapy, neutrophil hyper-responsiveness, and microbiota shifting by dysregulated immunity, yet point to the crucial role of dysregulated immunity and neutrophils in particular. Furthermore, patients with neutropenia and neutrophil defects inevitably develop early-onset periodontitis. Intra-gingivally injecting lipopolysaccharide (LPS) alone causes an exaggerated neutrophil response sufficient to precipitate experimental periodontitis. Vice versa to the surplus of LPS, the increased neutrophil responsiveness characteristic for late-onset periodontitis can effectuate gingiva damage likewise. The exaggerated neutrophil extracellular trap (NET) response in late-onset periodontitis is blameable for damage of gingival barrier, its penetration by bacteria and pathogen-associated molecular patterns (PAMPs) as well as stimulation of Th17 cells, resulting in further neutrophil activation. This identifies the dysregulated immunity as the main contributor to periodontal disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vitkov, Muñoz, Schoen, Knopf, Schauer, Minnich, Herrmann and Hannig.)

Peri-implantitis could lead to progressive bone loss and implant failure; however, the mechanism of peri-implantitis remains unclear. Based on emerging evidence, pyroptosis, a novel proinflammatory programmed death, contributes to different oral infectious diseases. In the present study, we investigated the involvement of cleaved caspase-3 and gasdermin E (GSDME) in peri-implantitis and established a pyroptosis model in vitro. By collecting and examining the inflamed biopsies around peri-implantitis, we found that the pyroptosis-related markers (caspase-3, GSDME, and IL-1β) were enhanced relative to levels in control individuals. Furthermore, human gingival epithelium cells (HGECs) induced by tumor necrosis factor-α (TNF-α) exhibited pyroptosis morphological changes (cell swelling and balloon-shaped bubbles) and upregulated expression of pyroptosis-related markers. Pretreated with Ac-DEVD-CHO (a caspase-3 inhibitor) or GSDME small interference RNA (siRNA) were found to attenuate pyroptosis in HGECs. In conclusion, our findings revealed a high expression of caspase-3 and GSDME in the inflamed biopsies of peri-implantitis and confirmed that the caspase-3/GSDME pathway mediates TNF-α-triggered pyroptosis in human gingival epithelium cells, which provides a new target for peri-implantitis treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Periodontitis is a highly prevalent chronic inflammatory disease leading to periodontal tissue breakdown and subsequent tooth loss, in which excessive host immune response accounts for most of the tissue damage and disease progression. Despite of the imperative need to develop host modulation therapy, the inflammatory responses and cell population dynamics which are finely tuned by the pathological microenvironment in periodontitis remained unclear. To investigate the local microenvironment of the inflammatory response in periodontitis, 10 periodontitis patients and 10 healthy volunteers were involved in this study. Single-cell transcriptomic profilings of gingival tissues from two patients and two healthy donors were performed. Histology, immunohistochemistry, and flow cytometry analysis were performed to further validate the identified cell subtypes and their involvement in periodontitis. Based on our single-cell resolution analysis, we identified HLA-DR-expressing endothelial cells and CXCL13 + fibroblasts which are highly associated with immune regulation. We also revealed the involvement of the proinflammatory NLRP3 + macrophages in periodontitis. We further showed the increased cell-cell communication between macrophage and T/B cells in the inflammatory periodontal tissues. Our data generated an intriguing catalog of cell types and interaction networks in the human gingiva and identified new inflammation-promoting cell subtypes involved in chronic periodontitis, which will be helpful in advancing host modulation therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Qian, Huang, Chen, Mo, Zhou, Zhao, Li and Lai.)

Objective: The purpose of this study was to observe the effect of hyperocclusion on the remodeling of gingival tissues and detect the related signaling pathways., Design: Hyperocclusion models were established by tooth extraction in mice. The mice were sacrificed at 3, 7, 14, 28, or 56 days after the surgery, and the left mandibular first molars with gingival tissues were isolated and examinations were focused on the gingival tissues. Apoptotic cells were examined using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technology. Proliferating cells, p65, inflammatory cytokines, and β-catenin were detected using immunohistochemical methods., Results: A series of apoptosis and proliferation responses were triggered in stressed gingival tissues. It was observed that the levels of p65, proinflammatory factors including interleukin-1β and tumor necrosis factor-α in extraction group were higher compared with those from mice with intact dentition, and peaked on days 14, 14 and 7 respectively. The expression of β-catenin was increased under hyperocclusion situations, peaked on day 14, and declined to the initial levels over time., Conclusions: The results of this study suggest that hyperocclusion causes remodeling of the gingival tissues by activating a series of adaptive responses. Both nuclear factor kappa B and Wnt/β-catenin signaling pathways may be responsible for those adaptive responses though the exact mechanism is not clear., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Titanium is often used in the medical field and in dental implants due to its biocompatibility, but it has a high rate of leading to peri-implantitis, which progresses faster than periodontitis. Therefore, in the present study, the expression of cytokines from gingival epithelial cells by nanotitania was investigated, which is derived from titanium in the oral cavity, and the additional effect of Porphyromonas gingivalis (periodontopathic bacteria) lipopolysaccharide (PgLPS) was investigated. Ca9-22 cells were used as a gingival epithelial cell model and were cultured with nanotitania alone or with PgLPS. Cytokine expression was examined by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. In addition, cellular uptake of nanotitania was observed in scanning electron microscopy images. The expression of interleukin (IL)-6 and IL-8 significantly increased in Ca9-22 cells by nanotitania treatment alone, and the expression was further increased by the presence of PgLPS. Nanotitania was observed to phagocytose Ca9-22 cells in a dose- and time-dependent manner. Furthermore, when the expression of IL-11, related to bone resorption, was investigated, a significant increase was confirmed by stimulation with nanotitania alone. Therefore, nanotitania could be associated with the onset and exacerbation of peri-implantitis, and the presence of periodontal pathogens may worsen the condition. Further clinical reports are needed to confirm these preliminary results.

This study reports the influence of peste des petits ruminants (PPR) vaccination on the clinico-pathological outcomes of PPR in the face of an outbreak. Twenty-two West African dwarf goats procured for a different study started showing early signs of PPR during acclimatization. In response, PPR vaccine was administered either intranasally with phytogenic mucoadhesive gum (Group A; n = 6) or without gum (Group B; n = 6); subcutaneously (Group C; n = 6) or not vaccinated (Group D; n = 4) and studied for 21 days. The clinical scores, hematology, serology and pathology scores were evaluated. Clinical signs of PPR were present in all groups, presenting a percentage mortality of 33%; 33%; 64% and 100% for Groups A, B, C, and D, respectively. Polycythemia and mild leukopenia were observed in all groups, and all animals were seropositive by day 7 post-vaccination. The lung consolidation scores were low in Groups A and B, compared to Group C. Histopathological lesions consistent with PPR was observed in the lymphoid organs, gastrointestinal tract, and lungs with the presence of PPR antigen as detected by immunohistochemistry. The findings suggest that intranasal vaccination with or without mucoadhesive gum may influence the outcome of PPR infection more than the subcutaneous route in the face of an outbreak.

Follicular helper T cells (Tfh) cells have been identified in the circulation and in tertiary lymphoid structures in chronic inflammation. Gingival tissues with periodontitis reflect chronic inflammation, so genomic footprints of Tfh cells should occur in these tissues and may differ related to aging effects. Macaca mulatta were used in a ligature-induced periodontitis model [adult group (aged 12-23 years); young group (aged 3-7 years)]. Gingival tissue and subgingival microbiome samples were obtained at matched healthy ligature-induced disease and clinical resolution sites. Microarray analysis examined Tfh genes (n = 54) related to microbiome characteristics documented using 16S MiSeq. An increase in the major transcription factor of Tfh cells, BCL6, was found with disease in both adult and young animals, while master transcription markers of other T cell subsets were either decreased or showed minimal change. Multiple Tfh-related genes, including surface receptors and transcription factors, were also significantly increased during disease. Specific microbiome patterns were significantly associated with profiles indicative of an increased presence/function of Tfh cells. Importantly, unique microbial complexes showed distinctive patterns of interaction with Tfh genes differing in health and disease and with the age of the animals. An increase in Tfh cell responsiveness occurred in the progression of periodontitis, affected by age and related to specific microbial complexes in the oral microbiome. The capacity of gingival Tfh cells to contribute to localized B cell activation and active antibody responses, including affinity maturation, may be critical for controlling periodontal lesions and contributing to limiting and/or resolving the lesions., (© 2021 British Society for Immunology.)

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host-pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1β-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.

Bacterial biofilm (dental plaque) plays a key role in caries etiopathogenesis and chronic periodontitis in humans. Dental plaque formation is determined by exopolysaccharides (EPSs) produced by cariogenic and periopathogenic bacteria. The most frequent cariogenic bacteria include oral streptococci (in particular S. mutans ) and lactobacilli (most frequently L. acidophilus ). In turn, the dominant periopathogen in periodontitis is Porphyromonas gingivalis. Development of dental caries is often accompanied with gingivitis constituting the mildest form of periodontal disease. Basic cellular components of the gingiva tissue are fibroblasts the damage of which determines the progression of chronic periodontitis. Due to insufficient knowledge of the direct effect of dental plaque on metabolic activity of the fibroblasts, this work analyses the effect of EPSs produced by S. mutans and L. acidophilus strains (H 2 O 2 -producing and H 2 O 2 -not producing) on ATP levels in human gingival fibroblasts (HGF-1) and their viability. EPSs produced in 48-hours bacterial cultures were isolated by precipitation method and quantitatively determined by phenol - sulphuric acid assay. ATP levels in HGF-1 were evaluated using a luminescence test, and cell viability was estimated using fluorescence test. The tests have proven that EPS from S. mutans did not affect the levels of ATP in HGF-1. Whereas EPS derived from L. acidophilus strains, irrespective of the tested strain, significantly increased ATP levels in HGF-1. The analysed EPSs did not affect the viability of cells. The tests presented in this work show that EPSs from cariogenic bacteria have no cytotoxic effect on HGF-1. At the same time, the results provide new data indicating that EPSs from selected oral lactobacilli may have stimulating effect on the synthesis of ATP in gingival fibroblasts which increases their energetic potential and takes a protective effect., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Hematopoietic stem cells reside in the bone marrow, where they generate the effector cells that drive immune responses. However, in response to inflammation, some hematopoietic stem and progenitor cells (HSPCs) are recruited to tissue sites and undergo extramedullary hematopoiesis. Contrasting with this paradigm, here we show residence and differentiation of HSPCs in healthy gingiva, a key oral barrier in the absence of overt inflammation. We initially defined a population of gingiva monocytes that could be locally maintained; we subsequently identified not only monocyte progenitors but also diverse HSPCs within the gingiva that could give rise to multiple myeloid lineages. Gingiva HSPCs possessed similar differentiation potentials, reconstitution capabilities, and heterogeneity to bone marrow HSPCs. However, gingival HSPCs responded differently to inflammatory insults, responding to oral but not systemic inflammation. Combined, we highlight a novel pathway of myeloid cell development at a healthy barrier, defining a gingiva-specific HSPC network that supports generation of a proportion of the innate immune cells that police this barrier., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2021 Krishnan et al.)

Systemic lupus erythematosus (SLE) is a complex chronic autoimmune disease characterized by tissue damage and widespread inflammation in response to environmental challenges. Deposition of immune complexes in kidneys glomeruli are associated with lupus nephritis, determining SLE diagnosis. Periodontitis is a chronic inflammatory disease characterized by clinical attachment and bone loss, caused by a microbial challenge - host response interaction. Deposition of immune complex at gingival tissues is a common finding in the course of the disease. Considering that, the primary aim of this study is to investigate the deposition of immune complexes at gingival tissues of SLE patients compared to systemically healthy ones, correlating it to periodontal and systemic parameters. Twenty-five women diagnosed with SLE (SLE+) and 25 age-matched systemically healthy (SLE-) women were included in the study. Detailed information on overall patient's health were obtained from file records. Participants were screened for probing depth (PD), clinical attachment loss (CAL), gingival recession (REC), full-mouth bleeding score (FMBS) and plaque scores (FMPS). Bone loss was determined at panoramic X-ray images as the distance from cementenamel junction to alveolar crest (CEJ-AC). Gingival biopsies were obtained from the first 15 patients submitted to surgical periodontal therapy of each group, and were analyzed by optical microscopy and direct immunofluorescence to investigate the deposition of antigen-antibody complexes. Eleven (44%) patients were diagnosed with active SLE (SLE-A) and 14 (56%) with inactive SLE (LES-I). Mean PD, CAL and FMBS were significantly lower in SLE+ than SLE-( p < 0.05; Mann Whitney). The chronic use of low doses of immunosuppressants was associated with lower prevalence of CAL >3 mm. Immunofluorescence staining of markers of lupus nephritis and/or proteinuria was significantly increased in SLE+ compared to SLE-, even in the presence of periodontitis. These findings suggest that immunomodulatory drugs in SLE improves periodontal parameters. The greater deposition of antigen-antibody complexes in the gingival tissues of patients diagnosed with SLE may be a marker of disease activity, possibly complementing their diagnosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pires, Nogueira, Nunes, Degand, Pessoa, Damante, Zangrando, Greghi, de Rezende and Sant'Ana.)

Abstract: Orthodontic treatment can lead to microbial-induced gingival inflammation and aseptic periodontal inflammations. The aim of this study was to investigate the relationship between salivary pro-inflammatory cytokines levels with gingival health status and oral microbe loads among patients undergoing orthodontic treatment.The present investigation was a cross-sectional study among a sample of 111 consecutive orthodontic patients (mean age 18.4 ± 4.4 years). Clinical examinations were conducted to assess the gingival health status employing the Modified Gingival Index, Gingival Bleeding Index, and Plaque Index. Salivary microbiological assessments of total aerobic and anaerobic bacteria count, streptococci count, and lactobacilli count were undertaken. Saliva immunological assessments included Interleukin-1Beta (IL-1β) and macrophage migration inhibitory factor (MIF) ELISA assays.The mean ± standard deviation of salivary IL-1β was 83.52 ± 85.62 pg/ml and MIF was 4.12 ± 0.96 ng/ml. Moderate positive correlations were found between salivary IL-1β levels and total aerobic and anaerobic bacteria count, streptococci count, and lactobacilli count (r = 0.380-0.446, P < .001), and weak positive correlations between salivary MIF levels and total salivary aerobic and anaerobic bacteria counts (r = 0.249-0.306, P < .01) were observed. A positive correlation was found between salivary IL-1β levels and Bleeding Index (r = 0.216, P < .05).The level of salivary IL-1β positively correlates with oral bacterial load among orthodontic patients; the relationship between inflammatory cytokines and oral microflora deserved further study., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data show that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α, and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1-primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections., (© 2021 Federation of American Societies for Experimental Biology.)

Objectives: An increased prevalence of periodontitis and perturbation of the oral microbiome has been identified in patients with rheumatoid arthritis (RA). The periodontal pathogen Porphyromonas gingivalis may cause local citrullination of proteins, potentially triggering anti-citrullinated protein antibody production. However, it is not known if oral dysbiosis precedes the onset of clinical arthritis. This study comprehensively characterised the oral microbiome in anti-cyclic citrullinated peptide (anti-CCP) positive at-risk individuals without clinical synovitis (CCP+at risk)., Methods: Subgingival plaque was collected from periodontally healthy and diseased sites in 48 CCP+at risk, 26 early RA and 32 asymptomatic healthy control (HC) individuals. DNA libraries were sequenced on the Illumina HiSeq 3000 platform. Taxonomic profile and functional capability of the subgingival microbiome were compared between groups., Results: At periodontally healthy sites, CCP+at risk individuals had significantly lower microbial richness compared with HC and early RA groups (p=0.004 and 0.021). Microbial community alterations were found at phylum, genus and species levels. A large proportion of the community differed significantly in membership (523 species; 35.6%) and structure (575 species; 39.1%) comparing CCP+at risk and HC groups. Certain core species, including P. gingivalis , had higher relative abundance in the CCP+at risk group. Seventeen clusters of orthologous gene functional units were significantly over-represented in the CCP+at risk group compared with HC (adjusted p value <0.05)., Conclusion: Anti-CCP positive at-risk individuals have dysbiotic subgingival microbiomes and increased abundance of P. gingivalis compared with controls. This supports the hypothesis that the oral microbiome and specifically P. gingivalis are important in RA initiation., Competing Interests: Competing interests: ZC reports scholarship from the China Scholarship Council during the conduct of the study. Dr TD and JM reports grants from Colgate Palmolive, outside the submitted work. KM reports grants from the National Institute for Health Research (NIHR) Leeds Biomedical Research Unit and grants from Leeds Biomedical Research Centre during the conduct of the study. DD reports grants from NIHR, grants from Wellcome Trust, during the conduct of the study; grants from Colgate Palmolive, outside the submitted work. PE reports grants and personal fees from Pfizer, Merck Sharp & Dohme, AbbVie, Bristol-Myers Squibb, Roche, Samsung, Sandoz, and Eli Lilly and Company; and personal fees from Novartis and UCB outside the submitted work., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Our previous study demonstrated that IL-10 secreting B (B10) cells alleviate inflammation and bone loss in experimental periodontitis. The purpose of this study is to determine whether antigen-specificity is required for the local infiltration of B10 cells. Experimental periodontitis was induced in the recipient mice by placement of silk ligature with or without the presence of live Porphyromonas gingivalis (P. gingivalis). Donor mice were pre-immunized by intraperitoneal (IP) injection of formalin-fixed P. gingivalis, or PBS as non-immunized control. Spleen B cells were purified and treated with LPS and CpG for 48 h to expand the B10 population in vitro. Fluorescence-labelled B10 cells were transferred into the recipient mice by tail vein injection and were tracked on day 0, 3, 5 and 10 using IVIS Spectrum in vivo imaging system. The number of B10 cells and P. gingivalis-binding B cells were significantly increased after in vitro treatment of LPS and CpG. On day 5, the fluorescence intensity in gingival tissues was the highest in mice transferred with B10 cells from pre-immunized donor mice. Gingival expression of IL-6, TNF-α, RANKL/OPG ratio and periodontal bone loss in recipient mice were significantly reduced, and the expression of IL-10 and the number of CD19+ B cells were significantly increased after pre-immunized B10 cell transfer in the presence of antigen, compared to those with non-immunized B10 cell transfer or no antigen presence. This study suggests that antigen specificity dictate the local infiltration of B10 cells into periodontal tissue and these antigen-specific B10 cells promote anti-inflammatory responses., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, has been associated with periodontal disease in companion animals and its virulence has been attributed to various factors, including lipopolysaccharide (LPS), protease and fimbriae. Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns, such as peptidoglycan, lipids, lipoproteins, nucleic acid and LPS. Following P. gulae infection, some inflammatory responses are dependent on both TLR2 and TLR4. In addition, a recent clinical study revealed that acute and persistent inflammatory responses enhance the expressions of TLR2 and TLR4 in the oral cavity. In this study, we investigated the interaction between P. gulae LPS and human gingivalis epithelial cells (Ca9-22 cells). P. gulae LPS was found to increase TLR2 and TLR4 mRNA expressions and protein productions, and enhanced inflammatory responses, such as COX 2 , TNF-ɑ, IL-6 and IL-8. Stimulated Ca9-22 cells exhibited phosphorylation of ERK1/2 and p38, and their inhibitors diminished inflammatory responses, while knockdown of the TLR2 and/or TLR4 genes with small interfering RNA (siRNA) prevented inflammatory responses. Moreover, p38 and ERK1/2 phosphorylation was decreased in TLR2 and TLR4 gene knockdown cells. These findings suggest that P. gulae LPS activates p38 and ERK1/2 via TLR2 and TLR4, leading to inflammatory responses in human gingival epithelial cells., (© 2020 John Wiley & Sons Ltd.)

Competing Interests: Competing interests: None declared.

Unlike epidermal Langerhans cells (LCs) that originate from embryonic precursors and are self-renewed locally, mucosal LCs arise and are replaced by circulating bone marrow (BM) precursors throughout life. While the unique lifecycle of epidermal LCs is associated with an age-dependent decrease in their numbers, whether and how aging has an impact on mucosal LCs remains unclear. Focusing on gingival LCs we found that mucosal LCs are reduced with age but exhibit altered morphology with that observed in aged epidermal LCs. The reduction of gingival but not epidermal LCs in aged mice was microbiota-dependent; nevertheless, the impact of the microbiota on gingival LCs was indirect. We next compared the ability of young and aged BM precursors to differentiate to mucosal LCs. Mixed BM chimeras, as well as differentiation cultures, demonstrated that aged BM has intact if not superior capacity to differentiate into LCs than young BM. This was in line with the higher percentages of mucosal LC precursors, pre-DCs, and monocytes, detected in aged BM. These findings suggest that while aging is associated with reduced LC numbers, the niche rather than the origin controls this process in mucosal barriers.

Polyunsaturated fatty acid-derived specialized pro-resolving lipid mediators (SPMs) play an important role in modulating inflammation. The aim of the study was to compare profiles of SPMs, SPM related lipid mediators and SPM receptor gene expression in gingiva of subjects with periodontitis to healthy controls. A total of 28 subjects were included; 13 periodontally healthy and 15 periodontitis before or after non-surgical periodontal therapy. Gingival tissues were collected from two representative posterior teeth prior to and 8 weeks after scaling and root planning; only once in the healthy group. Lipid mediator-SPM metabololipidomics was performed to identify metabolites in gingiva. qRT-PCR was performed to assess relative gene expression (2 -ΔΔCT ) of known SPM receptors. Intergroup comparisons were made using Wilcoxon tests. Thirty-six omega-6 or omega-3 fatty acid-derived lipid mediators and seven receptor genes were identified in gingiva. Profiles of lipid mediators and receptor gene expression were significantly different between the three groups. Levels of six lipid mediators, 5-HETE, 15-HETE, 15(S)-HEPE, 4-HDHA, 7-HDHA, and 17-HDHA in periodontitis before treatment were significantly higher than in periodontitis after treatment. The expression of BLT1 in the healthy group was significantly higher than periodontitis subjects before and after treatment. The expression of GPR18 in periodontitis before treatment was significantly higher than in periodontitis after treatment while the expression of GPR32 in periodontitis before treatment was significantly lower than in periodontitis after treatment. Elevated levels of SPM biosynthetic pathway markers in periodontitis subjects before treatment indicated inflammation induced pro-resolution activity in gingiva, but receptors for these molecules were deficient in periodontitis pre-treatment suggesting that failure of resolution of inflammation contributes to excess, chronic inflammation in periodontitis., (Copyright © 2020 Ferguson, Bokka, Maddipati, Ayilavarapu, Weltman, Zhu, Chen, Zheng, Angelov, Van Dyke and Lee.)

Periodontitis is known to be initiated by periodontal microbiota derived from biofilm formation. The microbial dysbiotic changes in the biofilm trigger the host immune and inflammatory responses that can be both beneficial for the protection of the host from infection, and detrimental to the host, causing tissue destruction. During this process, recognition of Pathogen-Associated Molecular Patterns (PAMPs) by the host Pattern Recognition Receptors (PRRs) such as Toll-like receptors (TLRs) play an essential role in the host-microbe interaction and the subsequent innate as well as adaptive responses. If persistent, the adverse interaction triggered by the host immune response to the microorganisms associated with periodontal biofilms is a direct cause of periodontal inflammation and bone loss. A large number of T and B lymphocytes are infiltrated in the diseased gingival tissues, which can secrete inflammatory mediators and activate the osteolytic pathways, promoting periodontal inflammation and bone resorption. On the other hand, there is evidence showing that immune regulatory T and B cells are present in the diseased tissue and can be induced for the enhancement of their anti-inflammatory effects. Changes and distribution of the T/B lymphocytes phenotype seem to be a key determinant of the periodontal disease outcome, as the functional activities of these cells not only shape up the overall immune response pattern, but may directly regulate the osteoimmunological balance. Therefore, interventional strategies targeting TLR signaling and immune regulatory T/B cells may be a promising approach to rebalance the immune response and alleviate bone loss in periodontal disease. In this review, we will examine the etiological role of TLR signaling and immune cell osteoclastogenic activity in the pathogenesis of periodontitis. More importantly, the protective effects of immune regulatory lymphocytes, particularly the activation and functional role of IL-10 expressing regulatory B cells, will be discussed.

The possible role of B-cell growth and differentiation-related cytokines on the pathogenesis of diabetes-related periodontitis has not been addressed so far. The aim of this study was to evaluate the effects of diabetes mellitus (DM) on the gene expression of proliferation-inducing ligand (APRIL) and B-lymphocyte stimulator (BLyS), two major cytokines associated to survival, differentiation and maturation of B cells in biopsies from gingival tissue with periodontitis. Gingival biopsies were obtained from subjects with periodontitis (n = 17), with periodontitis and DM (n = 19) as well as from periodontally and systemically healthy controls (n = 10). Gene expressions for APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were evaluated using qPCR. The expressions APRIL, BLyS, RANKL, OPG, TRAP and DC-STAMP were all higher in both periodontitis groups when compared to the control group (p < 0.05). Furthermore, the expressions of BLyS, TRAP and RANKL were significantly higher in the subjects with periodontitis and DM when compared to those with periodontitis alone (p < 0.05). The mRNA levels of BLyS correlated positively with RANKL in the subjects with periodontitis and DM (p < 0.05). BLyS is overexpressed in periodontitis tissues of subjects with type 2 DM, suggesting a possible role of this cytokine on the pathogenesis DM-related periodontitis.

Objective: Exosomes have been shown to play a strong role in intercellular communication. While GMSCs have been extensively studied, less research exists on exosomes derived from GMSCs, especially on how exosomes affect macrophages. This study aimed to investigate the impact of GMSC-derived exosomes on macrophage polarization and phenotype under inflammatory conditions., Methods: Exosomes were isolated from GMSCs-conditioned media by ultracentrifugation (UC) and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot (WB). In vitro, GMSC-derived exosomes were co-incubated with macrophages for 24 h in the absence or presence of M1 polarizing conditions in the six-well plate. The protein and mRNA expression levels of M1 and M2 macrophage markers were detected and the supernatants were collected for an enzyme-linked immunosorbent assay (ELISA)., Results: Exosomes were successfully isolated from GMSCs. Macrophages co-cultured with exosomes showed significantly decreased levels of the M1 markers Tumor Necrosis Factor-α (TNF-α), Interleukin-12 (IL-12), CD86 and Interleukin-1β (IL-1β). By contrast, M2 marker Interleukin-10 (IL-10) levels moderately increased. Meanwhile, similar results were acquired in the cell culture supernatants., Conclusion: GMSC-derived exosomes may promote M1 macrophage transformation into M2 macrophages, reducing the pro-inflammatory factors produced by M1 macrophages., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Candida albicans is a pathogenic fungus capable of switching its morphology between yeast-like cells and filamentous hyphae and can associate with bacteria to form mixed biofilms resistant to antibiotics. In these structures, the fungal milieu can play a protective function for bacteria as has recently been reported for C. albicans and a periodontal pathogen- Porphyromonas gingivalis . Our current study aimed to determine how this type of mutual microbe protection within the mixed biofilm affects the contacting host cells. To analyze C. albicans and P. gingivalis persistence and host infection, several models for host-biofilm interactions were developed, including microbial exposure to a representative monocyte cell line (THP1) and gingival fibroblasts isolated from periodontitis patients. For in vivo experiments, a mouse subcutaneous chamber model was utilized. The persistence of P. gingivalis cells was observed within mixed biofilm with C. albicans . This microbial co-existence influenced host immunity by attenuating macrophage and fibroblast responses. Cytokine and chemokine production decreased compared to pure bacterial infection. The fibroblasts isolated from patients with severe periodontitis were less susceptible to fungal colonization, indicating a modulation of the host environment by the dominating bacterial infection. The results obtained for the mouse model in which a sequential infection was initiated by the fungus showed that this host colonization induced a milder inflammation, leading to a significant reduction in mouse mortality. Moreover, high bacterial counts in animal organisms were noted on a longer time scale in the presence of C. albicans , suggesting the chronic nature of the dual-species infection.

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14 + CD16 + ) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis toward a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated., (Copyright © 2020 Almubarak, Tanagala, Papapanou, Lalla and Momen-Heravi.)

Periodontitis is an inflammatory disease caused by host immune response, resulting in a loss of periodontium and alveolar bone. Immune cells, such as T cells and macrophages, play a critical role in the periodontitis onset. Halofuginone, a natural quinazolinone alkaloid, has been shown to possess anti-fibrosis, anti-cancer, and immunomodulatory properties. However, the effect of halofuginone on periodontitis has never been reported. In this study, a ligature-induced mice model of periodontitis was applied to investigate the potential beneficial effect of halofuginone on periodontitis. We demonstrated that the administration of halofuginone significantly reduced the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in vivo, and markedly suppressed immune cell infiltration into the infected sites. Furthermore, we also observed that halofuginone treatment blocked the T-helper 17 (Th17) cell differentiation in vivo and in vitro. We demonstrated for the first time that halofuginone alleviated the onset of periodontitis through reducing immune responses.

Objective: Preterm Low-Birth-Weight (PLBW) is frequently associated with periodontal disease. However, the mechanism is still unknown. The present study was performed to examine the possible link between periodontal infections and PLBW in post-partum women utilizing clinical parameters and CD4+ and CD8 + T lymphocytes ratio in gingival granulation tissue., Materials: The tissues used in this study consisted of 35 gingival granulation tissue biopsies from 35 mothers of healthy infants (HTBW), 35 biopsies of gingival granulation tissue from 35 mothers of PLBW within one month postpartum and gingival tissue biopsies from 7 control individual with no periodontal disease (HC). CD4+ and CD8 + T lymphocyte ratios in a unit area of the gingival granulation tissue were determined by hystometrically. Statistical analysis was performed by using Kruskal-Wallis and Mann-Whitney U tests., Results: CD8 + T lymphocytes were more prevalent in the PLBW group than in the HTBW and HC group (P < 0.05). The CD4+/CD8+ ratio in the PLBW group was lower than those of the other groups (p < 0.05). There were no statistically significant differences in CD4 + T lymphocytes counts between all groups (P > 0.05)., Conclusion: Within the limits of this study it can be concluded that CD8 + T lymphocytes in gingival tissue may play important roles in the pathogenesis of periodontitis and PLBW., (Copyright © 2019. Published by Elsevier Ltd.)

During inflammation of the gums, resident cells of the periodontium, gingival fibroblasts (GFs), interact with heterogeneous cell populations of the innate and adaptive immune system that play a crucial role in protecting the host from pathogenic infectious agents. We investigated the effects of chronic inflammation, by exposing peripheral blood mononuclear cells (PBMCs), peripheral blood lymphocyte (PBL) cultures, and GF-PBMC cocultures to Toll-like receptor 2 (TLR2) and TLR4 activators for 21 days and assessed whether this influenced leukocyte retention, survival, and proliferation. Chronic stimulation of PBMC-GF cocultures with TLR2 and TLR4 agonists induced a reduction of NK (CD56+CD3-), T (CD3+), and B (CD19+) cells, whereas the number of TLR-expressing monocytes were unaffected. TLR2 agonists doubled the T cell proliferation, likely of a selective population, given the net decrease of T cells. Subsequent chronic exposure experiments without GF, using PBMC and PBL cultures, showed a significantly ( p < 0.0001) increased proinflammatory cytokine production of TNF-α and IL-1β up to 21 days only in TLR2-activated PBMC with concomitant T cell proliferation, suggesting a role for monocytes. In conclusion, chronic TLR activation mediates the shift in cell populations during infection. Particularly, TLR2 activators play an important role in T cell proliferation and proinflammatory cytokine production by monocytes, suggesting that TLR2 activation represents a bridge between innate and adaptive immunity.

Inflammation is a common feature of many neurodegenerative diseases. The treatment of stem cells as a therapeutic approach to repair damage in the central nervous system represents a valid alternative. In this study, using Next-Generation Sequencing (NGS) technology, we analyzed the transcriptomic profile of human Gingival Mesenchymal Stem Cells (hGMSCs) treated with Moringin [4-(α-l-ramanosyloxy)-benzyl isothiocyanate] (hGMSCs-MOR) or with Cannabidiol (hGMSCs-CBD) at dose of 0.5 or 5 µM, respectively. Moreover, we compared their transcriptomic profiles in order to evaluate analogies and differences in pro- and anti-inflammatory pathways. The hGMSCs-MOR selectively downregulate TNF-α signaling from the beginning, reducing the expression of TNF-α receptor while hGMSCs-CBD limit its activity after the process started. The treatment with CBD downregulates the pro-inflammatory pathway mediated by the IL-1 family, including its receptor while MOR is less efficient. Furthermore, both the treatments are efficient in the IL-6 signaling. In particular, CBD reduces the effect of the pro-inflammatory JAK/STAT pathway while MOR enhances the pro-survival PI3K/AKT/mTOR. In addition, both hGMSCs-MOR and hGMSCs-CBD improve the anti-inflammatory activity enhancing the TGF-β pathway.

Bone marrow mesenchymal stem/stromal cells (BM-MSCs) have immunoregulatory properties and have been used as immune regulators for the treatment of graft-versus-host disease (GVHD). Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to BM-MSCs for potential clinical applications because of their accessibility and easy preparation. The aim of this in vitro study was to compare MSCs from dental pulp (DP-MSCs), gingival tissue (G-MSCs), and periodontal ligament (PDL-MSCs) in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3 + T cells to determine which MSCs would be the most appropriate for in vivo immunoregulatory applications. BM-MSCs were included as the gold standard. Our results demonstrated, in vitro, that MSCs from DP, G, and PDL showed immunoregulatory properties similar to those from BM, in terms of the cellular proliferation inhibition of both CD4 + - and CD8 + -activated T-cells. This reduced proliferation in cell co-cultures correlated with the production of interferon-γ and tumor necrosis factor alpha (TNF-α) and the upregulation of programmed death ligand 1 (PD-L1) in MSCs and cytotoxic T-cell-associated Ag-4 (CTLA-4) in T-cells and increased interleukin-10 and prostaglandin E 2 production. Interestingly, we observed differences in the production of cytokines and surface and secreted molecules that may participate in T-cell immunosuppression in co-cultures in the presence of DT-MSCs compared with BM-MSCs. Importantly, MSCs from four sources favored the generation of T-cell subsets displaying the regulatory phenotypes CD4 + CD25 + Foxp3 + and CD4 + CD25 + CTLA-4 + . Our results in vitro indicate that, in addition to BM-MSCs, MSCs from all of the dental sources analyzed in this study might be candidates for future therapeutic applications.

The gingival epithelium is a physical and immunological barrier to the microbiota of the oral cavity, which interact through soluble mediators with the immune cells that patrol the tissue at the gingival epithelium. We sought to develop a three-dimensional gingivae-biofilm interface model using a commercially available gingival epithelium to study the tissue inflammatory response to oral biofilms associated with "health", "gingivitis" and "periodontitis". These biofilms were developed by sequential addition of microorganisms to mimic the formation of supra- and sub-gingival plaque in vivo. Secondly, to mimic the interactions between gingival epithelium and immune cells in vivo, we integrated peripheral blood mononuclear cells and CD14 + monocytes into our three-dimensional model and were able to assess the inflammatory response in the immune cells cultured with and without gingival epithelium. We describe a differential inflammatory response in immune cells cultured with epithelial tissue, and more so following incubation with epithelium stimulated by "gingivitis-associated" biofilm. These results suggest that gingival epithelium-derived soluble mediators may control the inflammatory status of immune cells in vitro, and therefore targeting of the epithelial response may offer novel therapies. This multi-cellular interface model, both of microbial and host origin, offers a robust in vitro platform to investigate host-pathogens at the epithelial surface.