Lenabasum Reduces Porphyromonas gingivalis–Driven Inflammation

Inflammation - The aim of this study was to evaluate the potential anti-inflammatory and anti-resorptive effects of lenabasum in the context of Porphyromonas gingivalis (Pg)–induced...     

Simple Monolayer Differentiation of Murine Cardiomyocytes via Nutrient Deprivation-Mediated Activation of β-Catenin

Methods to generate murine cardiomyocytes from pluripotent stem cells (PSCs) in vitro are resource and time intensive. All current protocols require exogenously provided soluble factors and almost all utilize embryoid body formation to modulate pathways associated with mesoderm specification and cardiomyocyte differentiation. Here, we developed a simple protocol without EBs and without exogenous soluble factors that enabled cardiomyocyte differentiation of a murine induced PSC line based on controlled nutrient deprivation in 2D monolayer cultures. We showed that this protocol reproducibly imposed metabolic stress and consequently modulated active β-catenin levels to yield functional cardiomyocytes. The yield of cardiomyocytes and calcium handling kinetics were comparable to existing approaches. However, this approach did not produce consistent results between murine PSC lines suggesting signaling pathways linking nutrient deprivation to β-catenin activation are not universally conserved and may be a remnant of the parent population from which the induced PSCs were derived.     

Identification of a Gingipain-Sensitive Surface Ligand of Porphyromonas gingivalis That Induces Toll-Like Receptor 2- and 4-Independent NF-κB Activation in CHO Cells

Porphyromonas gingivalis is a major periodontal pathogen that has the pathogenic proteinases Arg-specific gingipain and Lys-specific gingipain. We previously found that a cell surface component on P. gingivalis is able to induce Toll-like receptor 2 (TLR2)- and TLR4-independent signaling in 7.19 cells and that this component can be degraded by gingipains. In this study, we purified this component from the P. gingivalis gingipain-null mutant KDP136 and obtained two candidate proteins. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis showed that the proteins, with molecular masses of 123 and 43 kDa, were encoded by PGN_0748 and PGN_0728 (pgm6), respectively, in the P. gingivalis ATCC 33277 genome sequence. The PGN_0748-encoded protein, which we refer to as gingipain-sensitive ligand A (GslA), reacted with antiserum that could effectively inhibit the activity of KDP136 to induce NF-κB activation in 7.19 cells, but Pgm6 did not. To further determine what protein is responsible for the NF-κB activation, we constructed gslA, pgm6, and pgm6 pgm7 deletion mutants from KDP136. When 7.19 cells were exposed to those mutants, the gslA deletion mutant did not induce NF-κB activation, whereas the pgm6 and pgm6 pgm7 deletion mutants did. Furthermore, NF-κB activation in 7.19 cells induced by KDP136 was partially inhibited by antiserum against a recombinant protein expressed from the 5′-terminal third of gslA. These results indicate that GslA is one of the factors that induce NF-κB activation in 7.19 cells. Interestingly, the gslA gene was present in four of seven P. gingivalis strains tested. This restricted distribution might be associated with the virulence potential of each strain.Porphyromonas gingivalis is an anaerobic gram-negative bacterium that is frequently isolated from advanced periodontal lesions (25). The number of P. gingivalis cells is closely associated with the depth of periodontal pockets and is significantly reduced after treatment (7). Thus, this organism is thought to play an important role in the development and progression of periodontitis.P. gingivalis has two major cysteine proteinases, Arg-specific gingipain and Lys-specific gingipain. These proteinases have been reported to cleave various host immune effector molecules, such as immunoglobulin G (IgG) and IgM (22); several cytokines and cytokine receptors (1, 2, 10, 11, 17); and a pattern recognition receptor, CD14 (21, 23). These modifications of host immune regulatory molecules enable P. gingivalis to escape from the host immune system. This activity of gingipain seems to play an important role in the colonization of P. gingivalis in the oral cavity.Besides degradation of the host molecules, we previously found that gingipains could degrade a ligand expressed on the P. gingivalis cell surface (8). A CHO cell-derived nuclear factor (NF)-κB reporter cell line, 7.19, was stimulated with wild-type (ATCC 33277) and gingipain-deficient P. gingivalis (KDP136) bacterial cells. Since bacterial cells possess a number of ligands for Toll-like receptor 2 (TLR2) and TLR4, 7.19 cells, which lack both TLR2- and TLR4-signaling pathways, enable analysis of TLR2- and TLR4-independent signaling (18). Interestingly, 7.19 cells were activated by gingipain-null mutant KDP136 but not by its parental strain ATCC 33277, suggesting that the ligand of P. gingivalis was degraded by gingipains in the wild-type bacterial cells. In fact, the ability of KDP136 to induce activation of NF-κB in 7.19 cells was diminished after treatment of the bacterial cells with gingipains. In a previous study (8), we partially purified components with the ability to activate NF-κB in 7.19 cells from KDP136. The activities of the components were also diminished by treatment with gingipains.The aim of the present study was to purify and identify the gingipain-sensitive ligand from gingipain-deficient P. gingivalis cells. We tried further purification and obtained two proteins encoded by protein-coding sequence (CDS) PGN_0748 and CDS PGN_0728 (pgm6) in the P. gingivalis ATCC 33277 genome sequence (14). We then constructed CDS mutants from KDP136 and determined which protein is responsible for the activity that induces NF-κB activation in 7.19 cells.     

Porphyromonas gingivalis Lipopolysaccharide Activates Canonical Wnt/β-Catenin and p38 MAPK Signalling in Stem Cells from the Apical Papilla

As dental precursor cells, stem cells from the apical papilla (SCAP) are capable of forming roots and undergoing apexogenesis, which are impaired upon exposure to bacterial infection. Porphyromonas gingivalis is a common Gram-negative bacterium that is involved in pulpal and periapical infection. The purpose of this study was to investigate the effects of P. gingivalis lipopolysaccharide (LPS) on the Wnt/β-catenin and p38 mitogen-activated protein kinase (MAPK) signalling pathways in SCAP. As indicated by the IL-1β and TNF-α mRNA levels, P. gingivalis LPS induced the expression of pro-inflammatory cytokines in a dose-dependent manner. In addition, activation of the p38 MAPK and Wnt/β-catenin pathways was confirmed by the augmentation of phospho-p38 and β-catenin protein expression and increased expression of c-myc and cyclin D1 mRNA. Despite no significant increase in β-catenin mRNA expression, increased phosphorylation of glycogen synthase kinase (GSK)-3β suggested that GSK-3β was responsible for the accumulation of β-catenin in the cytoplasm and translocation to the nucleus. Previous studies have shown that GSK-3β plays a critical role in crosstalk between the Wnt/β-catenin and p38 MAPK pathways. In the present study, we showed that the level of p38 phosphorylation decreased upon pretreatment with a p38 MAPK inhibitor for 1 h before stimulating SCAP with 10 μg/ml P. gingivalis LPS. However, the levels of GSK-3β and β-catenin phosphorylation in the cytoplasm and nucleus were not significantly altered. Our results suggest that the p38 MAPK and canonical Wnt/β-catenin signalling pathways are activated by P. gingivalis LPS in SCAP, but we have no evidence that p38 MAPK is upstream of GSK-3β in the Wnt/β-catenin signalling pathway.     

Induction of β-Defensin Expression by Porphyromonas gingivalis-Infected Human Gingival Graft Transplanted in nu/nu Mouse Subdermis

It is important to understand the onset of periodontal disease in terms of bacterial infection and host factors. Host-bacteria interactions can be elicited in human cultured cells and animal models, but these models provide only limited biological information about human host reactions against bacterial attacks. Development of an in vivo model using human gingival tissue is needed. We established an in vivo model using nu/nu mice and evaluated host defense following bacterial infection in human gingiva. Human gingival samples were collected from periodontitis patients and transplanted in nu/nu mouse subdermis. After 2 weeks, human characteristics were confirmed by positive immunohistochemical reactions for human-specific markers. We used this model to investigate human β-defensin-2 (hBD-2), an antimicrobial peptide that contributes to initial defense against bacterial invasion. Using real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry, we investigated whether hBD-2 expression was induced in human gingiva as a response to Porphyromonas gingivalis as a periodontal pathogen. Two hours after infection with bacteria, we detected increased expression of hBD-2 mRNA, which was localized in the epithelium of human gingiva. Using our in vivo model, we concluded that increased hBD-2 may play an important role in early defense from bacterial infection in human gingival epithelium.     

Inhibition of IL-8–Mediated MAPK Activation in Human Neutrophils by β1 Integrin Ligands

Chemokine and integrin receptors must work in concert when circulating leukocytes mobilize toward a site of tissue inflammation or infection. In a previous study, we reported that ligation of the ₅₁ integrin with a 120-kDa cell-binding fibronectin fragment (120-kDa FN) in suspensions of human polymorphonuclear leukocytes (PMNLs) inhibited chemotaxis toward the chemokine called interleukin-8 (IL-8). Binding of chemokines to their receptors on leukocytes leads to the activation of heterotrimeric G proteins that initiate multiple signaling cascades, including p38 and p42/p44 mitogen-activated protein kinase (MAPK) pathways. In the present study, we examine the potential interaction of ₁ integrin ligation on chemokine-mediated MAPK signaling in human PMNLs. We demonstrate that blockade of the p42/p44 MAPK signaling pathway by the inhibitor PD98059 suppresses IL-8–mediated PMNL chemotaxis. Furthermore, when PMNLs are pretreated with 120-kDa FN or an activating antibody to ₁ integrins (TS2/16), IL-8–mediated phosphorylation of p42/p44 MAPK is also inhibited. In contrast, pretreating PMNL with a specific ligand (laminin-1) for the ₆₁ integrin does not suppress IL-8–mediated phosphorylation of p42/p44 MAPK. These observations demonstrate a desensitization of IL-8–mediated p42/p44 MAPK signaling in response to ligation of the ₅₁ integrin in PMNL. Also, they suggest an interplay between integrin and chemokine signaling during PMNL migration through the extracellular matrix.     

The Effect of l‐Arginine on Porphyromonas gingivalis‐Induced Phagocytosis of a Murine Macrophage‐like Raw 264.7 Cell Line

The aim of this study was to determine the effect of l‐arginine on Porphyromonas gingivalis‐induced phagocytosis by RAW 264.7 cells. The cells were pretreated with l‐arginine or d‐arginine prior to incubation with either unopsonized or opsonized P. gingivalis. In other experiments, the cells were pretreated with l‐arginine and various concentrations of NMLA (N^G‐monomethyl‐l‐arginine) prior to incubation with the bacteria. The phagocytosis was microscopically assessed and determined by the phagocytic index. The results showed that l‐arginine, but not d‐arginine enhances the ability of RAW264.7 cells to engulf the bacteria. The upregulatory effect of l‐arginine on P. gingivalis‐induced phagocytosis was abolished by NMLA. The results of the present study suggest that l‐arginine may upregulate the P. gingivalis‐induced phagocytic activity of RAW264.7 cells, perhaps, via modulation of nitric oxide synthase.     

Loss of β-Catenin Induces Multifocal Periosteal Chondroma-Like Masses in Mice

Osteochondromas and enchondromas are the most common tumors affecting the skeleton. Osteochondromas can occur as multiple lesions, such as those in patients with hereditary multiple exostoses. Unexpectedly, while studying the role of β-catenin in cartilage development, we found that its conditional deletion induces ectopic chondroma-like cartilage formation in mice. Postnatal ablation of β-catenin in cartilage induced lateral outgrowth of the growth plate within 2 weeks after ablation. The chondroma-like masses were present in the flanking periosteum by 5 weeks and persisted for more than 6 months after β-catenin ablation. These long-lasting ectopic masses rarely contained apoptotic cells. In good correlation, transplants of β-catenin-deficient chondrocytes into athymic mice persisted for a longer period of time and resisted replacement by bone compared to control wild-type chondrocytes. In contrast, a β-catenin signaling stimulator increased cell death in control chondrocytes. Immunohistochemical analysis revealed that the amount of detectable β-catenin in cartilage cells of osteochondromas obtained from hereditary multiple exostoses patients was much lower than that in hypertrophic chondrocytes in normal human growth plates. The findings in our study indicate that loss of β-catenin expression in chondrocytes induces periosteal chondroma-like masses and may be linked to, and cause, the persistence of cartilage caps in osteochondromas.Osteochondromas and enchondromas are the most common tumors affecting the skeleton.^1,2 Osteochondromas are cartilage-covered masses that form near the growth plate and bone surface, whereas enchondromas form within the growth plate and bone marrow. Both types of benign tumors can cause mechanical impairment of movement and also pain due to impingement or compression of nerves and blood vessels, particularly when they are present at multiple sites.^3,4 These benign tumors may become malignant.^5–7 The potential for malignant progression is greater in patients with syndromes, such as Ollier disease, Maffucci syndrome, or hereditary multiple exostoses (HME), the latter also known as multiple osteochondroma.^5–7 Current treatments largely rely on surgical excision.^3,8 Both benign and malignant cartilage tumors are generally resistant to chemotherapy and radiotherapy.^5,9 Thus, a better understanding of the cellular and molecular mechanisms underlying cartilage tumor formation and growth is critical for the development of new therapeutic strategies and treatments.Recent studies have indicated that several genes play important roles in cartilage tumor formation.^4,5 Hopyan et al¹⁰ found mutations in parathyroid hormone receptor 1 (PTHR1) in patients with multiple enchondromas. These authors generated mice harboring the same PTHR1 mutations that displayed a similar enchondroma formation. In addition, they found that the PTHR1 mutations caused constitutive activation of hedgehog signaling in cultured chondrocytes and that overexpression of Gli2, a downstream molecule of hedgehog signaling, induced enchondromas in mice.¹⁰ In the follow-up studies, however, it was found that certain cohorts of enchondromatosis patients do not have PTHR1 mutations¹¹ and that enchondroma formation may actually be independent of hedgehog signaling.¹² Thus, the pathogenesis of enchondroma formation remains to be clarified.Mutations in EXT1 and EXT2 genes have been associated with hereditary multiple exostoses (HME) (multiple osteochondroma).^5–7 Mutations in these genes are often missense or frame shift and cause synthesis of lower levels of (and shorter) heparan sulfate chains.^4,5 This is because EXT1 and EXT2 encode Golgi-associated enzymes are responsible for the polymerization of the chains.¹³ Insufficiency of heparan sulfate-rich proteoglycans is thought to be a cause of osteochondroma formation.^4,5,13 Heparan sulfate proteoglycans are important for the regulation of many signaling pathways that include hedgehog, bone morphogenetic protein, fibroblast growth factor, and Wnt pathways.^13,14 All of these pathways are critical regulators for chondrogenic differentiation and chondrocyte differentiation.^15,16 It is likely that dysregulation of these signaling pathways resulting from heparan sulfate deficiency may trigger abnormal behavior of growth plate chondrocytes or induce ectopic chondrogenic differentiation, leading to ectopic cartilage formation. Recently, several Ext mutant mouse lines have been established.^17–19 All of these transgenic mouse lines show multifocal ectopic cartilaginous masses with microscopical and structural similarities to osteochondromas found in HME patients.^17–19 The cellular and molecular mechanisms underlying EXT mutation-associated chondroma formation, however, remains largely unclear.The Wnt/β-catenin signaling pathway is essential for regulation of normal cartilage development, maintenance of permanent cartilage, and growth plate function.^20–24 Previous reports have shown that inactivation of this signaling pathway impairs cartilage and skeletal development. Conditional ablation of the β-catenin gene in limb skeletogenic cells induces a delay in endochondral bone formation and the formation of abnormal cartilaginous masses during embryonic development.^25–27 In addition, overexpression of a Wnt antagonist strongly inhibits both hypertrophy of chondrocytes and progression of endochondral ossification.²⁸ Recently, we generated compound transgenic mice in which we induced postnatal conditional ablation of β-catenin in cartilage.²⁴ We found that the resulting β-catenin deficiency impaired growth plate function and skeletal growth. In addition, the mice developed ectopic cartilaginous masses located near the bone surface but not within the bone marrow. In the present study, we characterized the pathohistology of these ectopic cartilaginous masses and investigated their possible cell origin and fate, and also related the findings to human osteochondromas.     

Pyogranulomatous Pneumonia in Goats Caused by an Undescribed Porphyromonas Species, “Porphyromonas katsikii”

A yet-undescribed bacterial species, tentatively named “Porphyromonaskatsikii,” was isolated from individuals of a small goat herd with pyogranulomatous pneumonia during an outbreak of acute respiratory disease. The isolated bacteria grew in the form of black-pigmented colonies after 14 days of incubation under anaerobic conditions at 37°C on a tryptic soy blood agar medium. The bacteria were identified as a yet-undescribed Porphyromonas species by determination of the nucleotide sequence of the rrs 16S rRNA gene, and this species was tentatively named Porphyromonaskatsikii. PCR amplification with specific primers for this yet-undescribed species revealed the presence of P. katsikii in the lung tissue of all affected animals, while no PCR signals were evidenced from the lungs of healthy goats or from goats with pasteurellosis caused by Mannheimia haemolytica. These data indicate P. katsikii as the causative agent of acute respiratory distress. P. katsikii is phylogenetically related to Porphyromonassomerae and Porphyromonas levii, which cause pathologies in humans and animals, respectively. P. katsikii was not detected by PCR from samples of the gingival pockets or of the faces of healthy goats.     

Intercellular Contact Protein β-Catenin in Neurons of the Habenula of the Rat Brain

Neuroscience and Behavioral Physiology - Studies were performed on frontal brain sections (n = 5) from Wistar rats using immunocytochemical methods. This is the first report on the detection of the...     

MicroRNA-214 induces dendritic cell switching from tolerance to immunity by targeting β-Catenin signaling

MicroRNAs (miRNAs) are known to function as negative gene regulators. Recently, miRNAs have been shown to regulate immunity processes; however, the mechanism is unclear. The role of microRNA-214 (miR-214) in dendritic cell (DC) maturation has not been investigated. We found that the miR-214 level was correlated with the maturation of DCs and inflammatory cytokine secretion, as depressed miR-214 levels induced DC tolerance. We also identified β-catenin as a target gene of miR-214 and demonstrated its association with Treg cell differentiation. MiR-214 regulates gene expression by binding to the 3’UTR of β-catenin. The results suggest that β-catenin is a critical regulator of tolerance in DCs via miR-214. The expression of miR-214 could be a potential therapeutic strategy in organ transplantation or autoimmunity patients.