Tristetraprolin regulates the skeletal phenotype and osteoclastogenic potential through monocytic myeloid-derived suppressor cells.

Tristetraprolin (TTP; also known as NUP475, GOS24, or TIS11), encoded by Zfp36, is an RNA-binding protein that regulates target gene expression by promoting mRNA decay and preventing translation. Although previous studies have indicated that TTP deficiency is associated with systemic inflammation and a catabolic-like skeletal phenotype, the mechanistic underpinnings remain unclear. Here, using both TTP-deficient (TTPKO) and myeloid-specific TTPKO (cTTPKO) mice, we reveal that global absence or loss of TTP in the myeloid compartment results in a reduced bone microarchitecture, whereas gain-of-function TTP knock-in (TTPKI) mice exhibit no significant loss of bone microarchitecture. Flow cytometry analysis revealed a significant immunosuppressive immune cell phenotype with increased monocytic myeloid-derived suppressor cells (M-MDSCs) in TTPKO and cTTPKO mice, whereas no significant changes were observed in TTPKI mice. Single-cell transcriptomic analyses of bone marrow myeloid progenitor cell populations indicated a dramatic increase in early MDSC marker genes for both cTTPKO and TTPKO bone marrow populations. Consistent with these phenotypic and transcriptomic data, in vitro osteoclastogenesis analysis of bone marrow M-MDSCs from cTTPKO and TTPKO displayed enhanced osteoclast differentiation and functional capacity. Focused transcriptomic analyses of differentiated M-MDSCs showed increased osteoclast-specific transcription factors and cell fusion gene expression. Finally, functional data showed that M-MDSCs from TTP loss-of-function mice were capable of osteoclastogenesis and bone resorption in a context-dependent manner. Collectively, these findings indicate that TTP plays a central role in regulating osteoclastogenesis through multiple mechanisms, including induction of M-MDSCs that appear to regulate skeletal phenotype.

GPR40 deficiency worsens metabolic syndrome-associated periodontitis in mice.

BACKGROUND AND OBJECTIVE: G protein-coupled receptor 40 (GPR40) is a receptor for medium- and long-chain free fatty acids (FFAs). GPR40 activation improves type 2 diabetes mellitus (T2DM), metabolic syndrome (MetS), and the complications of T2DM and MetS. Periodontitis, a common oral inflammatory disease initiated by periodontal pathogens, is another complication of T2DM and MetS. Since FFAs play a key role in the pathogenesis of MetS which exacerbates periodontal inflammation and GPR40 is a FFA receptor with anti-inflammatory properties, it is important to define the role of GPR40 in MetS-associated periodontitis. MATERIALS AND METHODS: We induced MetS and periodontitis by high-fat diet and periodontal injection of lipopolysaccharide (LPS), respectively, in wild-type and GPR40-deficient mice and determined alveolar bone loss and periodontal inflammation using micro-computed tomography, histology, and osteoclast staining. We also performed in vitro study to determine the role of GPR40 in the expression of proinflammatory genes. RESULTS: The primary outcome of the study is that GPR40 deficiency increased alveolar bone loss and enhanced osteoclastogenesis in control mice and the mice with both MetS and periodontitis. GPR40 deficiency also augmented periodontal inflammation in control mice and the mice with both MetS and periodontitis. Furthermore, GPR40 deficiency led to increased plasma lipids and insulin resistance in control mice but had no effect on the metabolic parameters in mice with MetS alone. For mice with both MetS and periodontitis, GPR40 deficiency increased insulin resistance. Finally, in vitro studies with macrophages showed that deficiency or inhibition of GPR40 upregulated proinflammatory genes while activation of GPR40 downregulated proinflammatory gene expression stimulated synergistically by LPS and palmitic acid. CONCLUSION: GPR40 deficiency worsens alveolar bone loss and periodontal inflammation in mice with both periodontitis and MetS, suggesting that GPR40 plays a favorable role in MetS-associated periodontitis. Furthermore, GPR40 deficiency or inhibition in macrophages further upregulated proinflammatory and pro-osteoclastogenic genes induced by LPS and palmitic acid, suggesting that GPR40 has anti-inflammatory and anti-osteoclastogenic properties.

Inhibition of acid sphingomyelinase by imipramine abolishes the synergy between metabolic syndrome and periodontitis on alveolar bone loss.

BACKGROUND AND OBJECTIVE: Clinical studies have shown that metabolic syndrome (MetS) exacerbates periodontitis. However, the underlying mechanisms remain largely unknown. Since our animal study has shown that high-fat diet-induced MetS exacerbates lipopolysaccharide (LPS)-stimulated periodontitis in mouse model and our in vitro study showed that acid sphingomyelinase (aSMase) plays a key role in the amplification of LPS-triggered pro-inflammatory response by palmitic acid (PA) in macrophages, we tested our hypothesis that inhibitor of aSMase attenuates MetS-exacerbated periodontitis in animal model. Furthermore, to explore the potential underlying mechanisms, we tested our hypothesis that aSMase inhibitor downregulates pro-inflammatory and pro-osteoclastogenic gene expression in macrophages in vitro. MATERIAL AND METHODS: We induced MetS and periodontitis in C57BL/6 mice by feeding high-fat diet (HFD) and periodontal injection of A. actinomycetemcomitans LPS, respectively, and treated mice with imipramine, a well-established inhibitor of aSMase. Micro-computed tomography (micro-CT), tartrate-resistant acid phosphatase staining, histological and pathological evaluations as well as cell cultures were performed to evaluate alveolar bone loss, osteoclast formation, periodontal inflammation and pro-inflammatory gene expression. RESULTS: Analysis of metabolic parameter showed that while HFD induced MetS by increasing bodyweight, insulin resistance, cholesterol and free fatty acids, imipramine reduced free fatty acids but had no significant effects on other metabolic parameters. MicroCT showed that either MetS or periodontitis significantly reduced bone volume fraction (BVF) of maxilla and the combination of MetS and periodontitis further reduced BVF. However, imipramine increased BVF in mice with both MetS and periodontitis to a level similar to that in mice with periodontitis alone, suggesting that imipramine abolished the synergy between MetS and periodontitis on alveolar bone loss. Consistently, results showed that imipramine inhibited osteoclast formation and periodontal inflammation in mice with both MetS and periodontitis. To elucidate the mechanisms by which imipramine attenuates MetS-exacerbated periodontitis, we showed that imipramine inhibited the upregulation of pro-inflammatory cytokines and transcription factor c-FOS as well as ceramide production by LPS plus PA in macrophages. CONCLUSION: This study has shown that imipramine as an inhibitor of aSMase abolishes the synergy between MetS and periodontitis on alveolar bone loss in animal model and inhibits pro-inflammatory and pro-osteoclastogenic gene expression in macrophages in vitro. This study provides the first evidence that aSMase is a potential therapeutic target for MetS-exacerbated periodontitis.

Myeloid-derived suppressor cells in obesity-associated periodontal disease: A conceptual model.

Periodontitis is a common chronic inflammatory disease characterized by destruction of the supporting structures of the teeth. Severe periodontitis is highly prevalent-affecting 10%-15% of adults-and carries several negative comorbidities, thus reducing quality of life. Although a clear relationship exists between severity of obesity and incidence of periodontal disease, the biologic mechanisms that support this link are incompletely understood. In this conceptual appraisal, a new "two-hit" model is presented to explain obesity-exacerbated periodontal bone loss. This proposed model recognizes a previously unappreciated aspect of myeloid-derived suppressor cell population expansion, differentiation, and activity that can participate directly in periodontal bone loss, providing new mechanistic and translational perspectives.

Acid sphingomyelinase deficiency exacerbates LPS-induced experimental periodontitis.

BACKGROUND: Mutation of the gene for acid sphingomyelinase (ASMase) causes Niemann-Pick disease. However, the effect of ASMase deficiency on periodontal health is unknown. Periodontal disease is a disease resulting from infection and inflammation of periodontal tissue and alveolar bone that support the teeth. The goal of this study was to determine the role of ASMase deficiency in periodontal inflammation and alveolar bone loss. METHODS: We induced periodontitis in wild-type and ASMase-deficient (ASMase-/- ) mice with periodontal lipopolysaccharide (LPS) injection and compared the alveolar bone loss and periodontal inflammation between these mice. RESULTS: Results showed that ASMase deficiency did not significantly change metabolic parameters, but exacerbated LPS-induced alveolar bone loss, osteoclastogenesis, and periodontal tissue inflammation. To understand the mechanisms by which ASMase deficiency aggravates LPS-induced periodontitis, we analyzed sphingolipids in periodontal tissues. Results showed that ASMase deficiency led to increases in not only sphingomyelin, but also ceramide (CER), a bioactive sphingolipid known to promote inflammation. Results further showed that ASMase deficiency increased CER de novo synthesis. CONCLUSION: ASMase deficiency exacerbated LPS-induced alveolar bone loss and periodontal inflammation. ASMase deficiency leads to an unexpected CER increase by stimulating de novo synthesis CER, which is likely to be involved in the ASMase deficiency-exacerbated periodontitis.

Activation of vitamin D in the gingival epithelium and its role in gingival inflammation and alveolar bone loss.

BACKGROUND AND OBJECTIVE: Both chronic and aggressive periodontal disease are associated with vitamin D deficiency. The active form of vitamin D, 1,25(OH)2 D3 , induces the expression of the antimicrobial peptide LL-37 and innate immune mediators in cultured human gingival epithelial cells (GECs). The aim of this study was to further delineate the mechanism by which vitamin D enhances the innate defense against the development of periodontal disease (PD). MATERIALS AND METHODS: Wild-type C57Bl/6 mice were made deficient in vitamin D by dietary restriction. Cultured primary and immortalized GEC were stimulated with 1,25(OH)2 D3 , followed by infection with Porphyromonas gingivalis, and viable intracellular bacteria were quantified. Conversion of vitamin D3 to 25(OH)D3 and 1,25(OH)2 D3 was quantified by ELISA. Effect of vitamin D on basal IL-1α expression in mice was determined by topical administration to the gingiva of wild-type mice, followed by qRT-PCR. RESULTS: Dietary restriction of vitamin D led to alveolar bone loss and increased inflammation in the gingiva in the mouse model. In primary human GEC and established human cell lines, treatment of GEC with 1,25(OH)2 D3 inhibited the intracellular growth of P. gingivalis. Cultured GEC expressed two 25-hydroxylases (CYP27A1 and CYP2R1), as well as 1-α hydroxylase, enabling conversion of vitamin D to both 25(OH)D3 and 1,25(OH)2 D3 . Topical application of both vitamin D3 and 1,25(OH)2 D3 to the gingiva of mice led to rapid inhibition of IL-1α expression, a prominent pro-inflammatory cytokine associated with inflammation, which also exhibited more than a 2-fold decrease from basal levels in OKF6/TERT1 cells upon 1,25(OH)2 D3 treatment, as determined by RNA-seq. CONCLUSION: Vitamin D deficiency in mice contributes to PD, recapitulating the association seen in humans, and provides a unique model to study the development of PD. Vitamin D increases the activity of GEC against the invasion of periodontal pathogens and inhibits the inflammatory response, both in vitro and in vivo. GEC can convert inactive vitamin D to the active form in situ, supporting the hypothesis that vitamin D can be applied directly to the gingiva to prevent or treat periodontal disease.

Myeloid-Derived Suppressor Cells at the Intersection of Inflammaging and Bone Fragility.

Age-related alteration of the immune system with aging, or immunosenescence, plays a major role in several age-associated conditions, including loss of bone integrity. Studies over the past several years have clearly established the immune system is chronically activated with advanced aging, termed inflammaging, and is characterized by elevated levels of proinflammatory cytokines in response to physiological or environmental cues that essentially result in an arrested immune system that maintains a low-level state of activation. This age-associated inflammation impacts several biological systems including the innate immune system, where aging results in a skewing of the hematopoiesis toward the myeloid lineage, including the expansion of myeloid-derived suppressor cells (MDSCs). This heterogeneous population of myeloid cells classically displays immunosuppressive capacity but they also have the ability to directly differentiate into osteoclasts. This review explores the possibility of inflammaging to be involved in reduction of bone microarchitecture and loss of bone mass/strength through the expansion of MDSCs and the osteoclastogenic capacity and activity.

Mean annual attachment, bone level, and tooth loss: A systematic review.

BACKGROUND: Rate of progression of periodontitis has been used to inform the design of classifications of periodontal diseases. However, the evidence underpinning this topic is unclear and no systematic review has yet been conducted. OBJECTIVES: The focused question for this systematic review was: in adults, what is the progression of periodontitis in terms of clinical attachment loss, radiographic bone loss, and tooth loss? DATA SOURCES: Highly sensitive electronic search was conducted for published data in MEDLINE, EMBASE, LILACS, and unpublished grey literature in OpenGrey up to February 2016. Reference lists of retrieved studies for full-text screening and reviews were hand-searched for potentially eligible studies. STUDY ELIGIBILITY CRITERIA AND PARTICIPANTS: Prospective, longitudinal observational studies with follow-up of at least 12 months and presenting data on the primary outcome, change in clinical attachment level, in adults (age ≥18 years). Secondary outcomes, tooth loss and bone level change, were only assessed in studies reporting the primary outcome. Studies investigating specific disease populations or only on treated periodontitis patients were excluded. STUDY APPRAISAL AND SYNTHESIS METHODS: Risk of bias and methodology were assessed using the Newcastle-Ottawa Scale with two additional questions on security of outcome assessment. Studies were pooled by abstracting or estimating mean annual attachment or bone level change and annual tooth loss. Random effects meta-analysis was conducted with investigation of effect of potential modifiers where possible. RESULTS: A total 11,482 records were screened for eligibility; 33 publications of 16 original studies reporting on more than 8,600 participants were finally included as eligible for the review. The studies represented populations from both developing and developed economies. Mean annual attachment loss was 0.1 mm per year (95% CI 0.068, 0.132; I2  = 99%) and mean annual tooth loss was 0.2 teeth per year (95% CI 0.10, 0.33; I2  = 94%). Observational analysis of highest and lowest mean attachment change quintiles suggested substantial differences between groups with minimal annual change in the lowest quintile and an average deterioration of 0.45 mm mean attachment loss per year in the highest group. This value increased to 0.6 mm per year with periodontitis alone. There was surprisingly little effect of age or gender on attachment level change. Geographic location, however, was associated with more than three times higher mean annual attachment loss in Sri Lanka and China (0.20 mm, 95% CI 0.15, 0.27; I2  = 83%) vs North America and Europe (0.056 mm, 95% CI 0.025, 0.087; I2  = 99%) P < 0.001. LIMITATIONS: There were a limited number of studies (N = 16), high variability of design in key study components (sampling frames, included ages, data analyses), and high statistical heterogeneity that could not be explained. CONCLUSIONS: Within the limitations of the research, the data show that mean annual attachment level change varies considerably both within and between populations. Overall, the evidence does not support or refute the differentiation between forms of periodontal diseases based upon progression of attachment level change.

Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri-Implant Diseases and Conditions.

A new periodontitis classification scheme has been adopted, in which forms of the disease previously recognized as "chronic" or "aggressive" are now grouped under a single category ("periodontitis") and are further characterized based on a multi-dimensional staging and grading system. Staging is largely dependent upon the severity of disease at presentation as well as on the complexity of disease management, while grading provides supplemental information about biological features of the disease including a history-based analysis of the rate of periodontitis progression; assessment of the risk for further progression; analysis of possible poor outcomes of treatment; and assessment of the risk that the disease or its treatment may negatively affect the general health of the patient. Necrotizing periodontal diseases, whose characteristic clinical phenotype includes typical features (papilla necrosis, bleeding, and pain) and are associated with host immune response impairments, remain a distinct periodontitis category. Endodontic-periodontal lesions, defined by a pathological communication between the pulpal and periodontal tissues at a given tooth, occur in either an acute or a chronic form, and are classified according to signs and symptoms that have direct impact on their prognosis and treatment. Periodontal abscesses are defined as acute lesions characterized by localized accumulation of pus within the gingival wall of the periodontal pocket/sulcus, rapid tissue destruction and are associated with risk for systemic dissemination.

Mitogen-Activated Protein Kinase 2 Signaling Shapes Macrophage Plasticity in Aggregatibacter actinomycetemcomitans-Induced Bone Loss.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontal disease, which is characterized by inflammation-driven alveolar bone loss. A. actinomycetemcomitans activates the p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase 2 (MK2) stress pathways in macrophages that are involved in host responses. During the inflammatory process in periodontal disease, chemokines are upregulated to promote recruitment of inflammatory cells. The objective of this study was to determine the role of MK2 signaling in chemokine regulation during A. actinomycetemcomitans pathogenesis. Utilizing a murine calvarial model, Mk2+/+ and Mk2-/- mice were treated with live A. actinomycetemcomitans bacteria at the midsagittal suture. MK2 positively regulated the following macrophage RNA: Emr1 (F4/80), Itgam (CD11b), Csf1r (M-CSF Receptor), Itgal (CD11a), Tnf, and Nos2 Additionally, RNA analysis revealed that MK2 signaling regulated chemokines CCL3 and CCL4 in murine calvarial tissue. Utilizing the chimeric murine air pouch model, MK2 signaling differentially regulated CCL3 and CCL4 in the hematopoietic and nonhematopoietic compartments. Bone resorption pits in calvaria, observed by micro-computed tomography, and osteoclast formation were decreased in Mk2-/- mice compared to Mk2+/+ mice after A. actinomycetemcomitans treatment. In conclusion, these data suggest that MK2 in macrophages contributes to regulation of chemokine signaling during A. actinomycetemcomitans-induced inflammation and bone loss.

Critical role of MKP-1 in lipopolysaccharide-induced osteoclast formation through CXCL1 and CXCL2.

UNLABELLED: Osteoclast (OC) progenitors (OCP) have been defined in the bone marrow (BM) as CD3(-)CD45R(B220)(-)GR1(-)CD11b(lo/)(-)CD115(+) (dOCP) and more recently in the peripheral blood (PB) as Lym(-)Ly6G(-)CD11b(+)Ly6C(+). These progenitors respond to stimuli, including LPS from periopathogenic Aggregatibacter actinomycetemcomitans, activating MAPK signaling, resulting in cytokine/chemokine-mediated osteoclastogenesis. Intracellular negative signaling pathways, including MAPK phosphatase-1 (MKP-1, gene Dusp1) deactivate MAPK pathways (p-p38 and p-JNK) and reduce inflammatory cytokines/chemokines. OBJECTIVE: To delineate the role of MKP-1 in chemokine-mediated OC formation using defined OC progenitor populations. Given its role in innate immune inflammatory signaling, we hypothesize that MKP-1 regulates LPS-induced OC formation from BM OCP through deregulated chemokines. METHODS: BM and PB from WT and Dusp1(-/-) female mice (8-12weeks) was obtained and sorted into defined progenitor populations. BM sorted dOCP were primed with MCSF and RANKL (48h), blocked with vehicle or chemokine blocking antibodies and stimulated with LPS (48-96h). TRAP assay and OC activity were measured for OC formation and activity following treatments. NanoString Array and qPCR were utilized for gene expression analysis. RESULTS: Dusp1(-/-) dOCPs formed more and larger osteoclasts from CD11b(hi) and dOCP compared to matched WT (P<0.05 each). PB-derived dOCP produced larger and more functional osteoclasts from Dusp1(-/-) mice compared to WT controls. NanoString array data revealed significant deregulation in chemokine expression from Dusp1(-/-) versus WT cells. qPCR validation of target genes revealed that Dusp1 deficient CD11b(+) populations display 1.5-3.5-fold greater expression of CXCL1 and 2-3-fold greater expression of CXCL2 compared to WT in CD11b(hi) and dOCP (P<0.05 each). Antibody blocking studies using anti-CXCL1 and CXCL2 antibodies blunted osteoclastogenesis in Dusp1(-/-) cells. CONCLUSION: MKP-1 negatively regulates chemokine-driven OC formation and subsequent bone resorption in response to LPS stimulation. Collectively, these data provide useful insight into mechanisms potentially leading to the development of therapeutic treatment of periodontal disease.

In vitro osteoclastogenesis assessment using murine myeloid-derived suppressor cells.

The study of myeloid-derived suppressor cells (MDSCs) has been commonly reported in the context of cancer immunology. MDSCs play a key role in cancer growth and progression by inhibiting both innate and adaptive immunity. In addition to the immunosuppressive function of MDSCs in cancer, a novel function of MDSCs as osteoclast precursors has recently been attracting attention. Because monocytic-MDSCs (M-MDSCs) are derived from the same myeloid lineage as macrophages, which are osteoclast progenitors, M-MDSCs can undergo differentiation into osteoclasts, contributing to bone destruction not only in the cancer microenvironment but also in inflammatory conditions including obesity and osteoarthritis. Herein, we present details of the technique to evaluate osteoclasts in vitro, as well as specific techniques to isolate M-MDSCs and identify them. This protocol can be easily adapted to isolate M-MDSCs from most pathologic conditions for easy evaluation.

Analysis of the effect of CCR7 on the microenvironment of mouse oral squamous cell carcinoma by single-cell RNA sequencing technology.

BACKGROUND: Studies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC. METHODS: In this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization. RESULTS: In the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells. CONCLUSIONS: CCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.

CCR7 affects the tumor microenvironment by regulating the activation of naïve CD8+ T cells to promote the proliferation of oral squamous cell carcinoma.

BACKGROUND: Head and neck cancer is the sixth most common malignancy worldwide, and oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, being one of the leading causes of cancer morbidity and mortality worldwide. CC Chemokine receptor 7(CCR7) is a multifunctional G protein-coupled trans-membrane chemokine that affects immune cell chemotaxis, migration, and cancer progression through its interaction with its ligands C-C motif chemokine ligand 19(CCL19) and C-C motif chemokine ligand 21(CCL21). Numerous studies have demonstrated the involvement of CCR7 in the malignant progression of a variety of cancers, reflecting the pro-cancer properties of CCR7. The Cancer Genome Atlas data suggests CCR7 has elevated expression in oral cancer. Specifically, CCR7 expression in tumor microenvironment (TME) may regulate the ability of some immune cells to engage in anti-tumor immune responses. Since CD8+ T cells have become a key immunotherapeutic target, the role of CCR7 in antitumor immune response of naïve CD8+ T cells in TME has not been thoroughly investigated. METHODS: A CCR7 knockout mouse model was constructed, and the mechanism of ccr7 on the regulation of tumor microenvironment by naïve CD8+ T cells was verified under the guidance of single-cell RNA sequencing combined with in vivo animal experiments and in vitro cell experiments. RESULTS: CCR7 is knocked out with impaired tumor growth and altered CD8+ T cell profiles, revealing the importance of this protein in OSCC. CONCLUSIONS: Inhibition of CCR7 enhances CD8+ T cell activation, proliferation, and anti-tumor function, suggesting its potential as a therapeutic target.

Differential expression of mitogen activating protein kinases in periodontitis.

AIM: Following toll-like receptor (TLR) engagement, lipopolysaccharide (LPS) can stimulate the expression of pro-inflammatory cytokines thus activating the innate immune response. The production of inflammatory cytokines results, in part, from the activation of kinase-induced signalling cascades and transcriptional factors. Of the four distinct classes of mitogen-activated protein kinases (MAPK) described in mammals, p38, c-Jun N-terminal activated kinases (JNK1-3) and extracellular activated kinases (ERK1,2) are the best studied. Previous data have established that p38 MAPK signalling is required for inflammation and bone loss in periodontal disease pre-clinical animal models. MATERIALS & METHODS: In this study, we obtained healthy and diseased periodontal tissues along with clinical parameters and microbiological parameters. Excised fixed tissues were immunostained with total and phospho-specific antibodies against p38, JNK and ERK kinases. RESULTS: Intensity scoring from immunostained tissues was correlated with clinical periodontal parameters. Rank correlations with clinical indices were statistically significantly positive (p-value < 0.05) for total p38 (correlations ranging 0.49-0.68), phospho-p38 (range 0.44-0.56), and total ERK (range 0.52-0.59) levels, and correlations with JNK levels also supported association (range 0.42-0.59). Phospho-JNK and phospho-ERK showed no significant positive correlation with clinical parameters of disease. CONCLUSION: These data strongly implicate p38 MAPK as a major MAPK involved in human periodontal inflammation and severity.

Periodontal disease is associated with increased gut colonization of pathogenic Haemophilus parainfluenzae in patients with Crohn's disease.

Intestinal colonization of the oral bacterium Haemophilus parainfluenzae has been associated with Crohn's disease (CD) severity and progression. This study examines the role of periodontal disease (PD) as a modifier for colonization of H. parainfluenzae in patients with CD and explores the mechanisms behind H. parainfluenzae-mediated intestinal inflammation. Fifty subjects with and without CD were evaluated for the presence of PD, and their oral and fecal microbiomes were characterized. PD is associated with increased levels of H. parainfluenzae strains in subjects with CD. Oral inoculation of H. parainfluenzae elicits strain-dependent intestinal inflammation in murine models of inflammatory bowel disease, which is associated with increased intestinal interferon-γ (IFN-γ)+ CD4+ T cells and disruption of the host hypusination pathway. In summary, this study establishes a strain-specific pathogenic role of H. parainfluenzae in intestinal inflammation and highlights the potential effect of PD on intestinal colonization by pathogenic H. parainfluenzae strains in patients with CD.

Is monocyte chemotactic protein 1 elevated in aseptic loosening of TKA? A pilot study.

BACKGROUND: Failure of TKA from aseptic loosening is a growing concern, as TKA is performed with increasing frequency. Loosening is multifactorial and may be associated with elevated inflammatory cytokines in addition to biomechanical failure. QUESTIONS/PURPOSES: We asked whether proinflammatory cytokines and chemokines are elevated in synovial fluid from patients undergoing revision surgery as compared to those with osteoarthritis (OA) or rheumatoid arthritis (RA). METHODS: We obtained synovial fluid samples from 20 patients: six with aseptic loosening of TKA (all with bone loss), 10 with primary OA, and four with RA. A panel of cytokines/chemokines was screened using a SearchLight(®) Array (Pierce Biotechnology, Rockford, IL, USA) in one revision sample. Using these data, we assayed the synovial fluids for monocyte chemotactic protein 1 (MCP-1) by ELISA. RESULTS: We observed an increase in synovial MCP-1 levels in samples from patients planned for TKA revision compared to those with OA or RA. In patients undergoing revision arthroplasty, the mean (± SD) MCP-1 concentration was 21,233 ± 18,966 pg/mL (range, 1550-50,657 pg/mL; n = 6). In patients with OA, the mean MCP-1 level was 3012 ± 3321 pg/mL. In patients with RA, the mean MCP-1 concentration was 690 ± 561 pg/mL. CONCLUSIONS: All patients undergoing revision TKA showed elevated concentrations of MCP-1 compared to patients with OA and RA, suggesting MCP-1 may serve as a potential marker or predictor of bone loss in patients undergoing revision surgery. CLINICAL RELEVANCE: MCP-1 may be a novel biomarker in patients showing early symptoms of aseptic loosening of TKA.

Porphyromonas gingivalis indirectly elicits intestinal inflammation by altering the gut microbiota and disrupting epithelial barrier function through IL9-producing CD4+ T cells.

Recent epidemiological studies have shown that inflammatory bowel disease is associated with periodontal disease. The oral-gut microbiota axis is a potential mechanism intersecting the two diseases. Porphyromonas gingivalis is currently considered a keystone oral pathogen involved in periodontal disease pathogenesis and disease progression. Recent studies have shown that oral ingestion of P. gingivalis leads to intestinal inflammation. However, the molecular underpinnings of P. gingivalis-mediated gut inflammation have remained elusive. In this study, we show that the oral administration of P. gingivalis indeed leads to ileal inflammation and alteration in gut microbiota with significant reduction in bacterial alpha diversity despite the absence of P. gingivalis in the lower gastrointestinal tract. Utilizing an antibiotic-conditioned mouse model, cecal microbiota transfer experiments were performed to demonstrate that P. gingivalis-induced dysbiotic gut microbiota is sufficient to reproduce gut pathology. Furthermore, we observed a significant expansion in small intestinal lamina propria IL9+ CD4+ T cells, which was negatively correlated with both bacterial and fungal alpha diversity, signifying that P. gingivalis-mediated intestinal inflammation may be due to the subsequent loss of gut microbial diversity. Finally, we detected changes in gene expression related to gut epithelial barrier function, showing the potential downstream effect of intestinal IL9+ CD4+ T-cell induction. This study for the first time showed the mechanism behind P. gingivalis-mediated intestinal inflammation where P. gingivalis indirectly induces intestinal IL9+ CD4+ T cells and inflammation by altering the gut microbiota. Understanding the mechanism of P. gingivalis-mediated intestinal inflammation may lead to the development of novel therapeutic approaches to alleviate the morbidity from inflammatory bowel disease patients with periodontal disease.

T-Cell Infiltration and Immune Checkpoint Expression Increase in Oral Cavity Premalignant and Malignant Disorders.

The immune cell niche associated with oral dysplastic lesion progression to carcinoma is poorly understood. We identified T regulatory cells (Treg), CD8+ effector T cells (Teff) and immune checkpoint molecules across oral dysplastic stages of oral potentially malignant disorders (OPMD). OPMD and oral squamous cell carcinoma (OSCC) tissue sections (N = 270) were analyzed by immunohistochemistry for Treg (CD4, CD25 and FoxP3), Teff (CD8) and immune checkpoint molecules (PD-1 and PD-L1). The Treg marker staining intensity correlated significantly (p < 0.01) with presence of higher dysplasia grade and invasive cancer. These data suggest that Treg infiltration is relatively early in dysplasia and may be associated with disease progression. The presence of CD8+ effector T cells and the immune checkpoint markers PD-1 and PD-L1 were also associated with oral cancer progression (p < 0.01). These observations indicate the induction of an adaptive immune response with similar Treg and Teff recruitment timing and, potentially, the early induction of exhaustion. FoxP3 and PD-L1 levels were closely correlated with CD8 levels (p < 0.01). These data indicate the presence of reinforcing mechanisms contributing to the immune suppressive niche in high-risk OPMD and in OSCC. The presence of an adaptive immune response and T-cell exhaustion suggest that an effective immune response may be reactivated with targeted interventions coupled with immune checkpoint inhibition.