Riyoko Tamai

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

Alendronate augments interleukin-1β release from macrophages infected with periodontal pathogenic bacteria through activation of caspase-1

Nitrogen-containing bisphosphonates (NBPs) are anti-bone-resorptive drugs with inflammatory side effects that include osteomyelitis and osteonecrosis of the jaw. Oral bacteria have been considered to be a trigger for these NBP-associated jaw bone diseases. The present study examined the effects of alendronate (a typical NBP) and clodronate (a non-NBP) on the production of proinflammatory cytokines by macrophages infected with Porphyromonas gingivalis and Tannerella forsythia, which are important pathogens of periodontal diseases. Pretreatment with alendronate augmented IL-1beta, but not TNFalpha, production by macrophages infected with P. gingivalis or T. forsythia. This augmentation of IL-1beta production was inhibited by clodronate. Furthermore, caspase-1, a promoter of IL-1beta production, was activated by treatment with alendronate, and caspase-1 inhibitor reduced the production of IL-1beta induced by alendronate and P. gingivalis. These results suggest that NBPs augment periodontal pathogenic bacteria-induced IL-1beta release via caspase-1 activation, and this phenomenon may contribute to the development of NBP-associated inflammatory side effects including jaw osteomyelitis. Co-treatment with clodronate may prevent and/or reduce these inflammatory effects induced by NBPs.

Porphyromonas gingivalis with either Tannerella forsythia or Treponema denticola induces synergistic IL-6 production by murine macrophage-like J774.1 cells.

BACKGROUND Chronic periodontitis is caused by mixed bacterial infection. Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are frequently detected in deep periodontal pockets. We demonstrate that these bacteria induce proinflammatory cytokine production by the mouse macrophage-like cell line J774.1. MATERIALS AND METHODS J774.1 cells were incubated with and without bacteria for 24h in 96-well flat-bottomed plates. The culture supernatants were analyzed by enzyme-linked immunosorbent assay for secreted mouse interleukin (IL)-6, monocyte chemoattractant protein-1, IL-23, IL-1 beta and tumor necrosis factor-alpha. The cytokine concentrations were determined using a standard curve prepared for each assay. RESULTS Mixed infection with P. gingivalis and either T. forsythia or T. denticola at 10(5)CFU/ml acted synergistically to increase IL-6 production, but not monocyte chemoattractant protein-1, IL-23, IL-1 beta or tumor necrosis factor-alpha production. Gingipain inhibitors KYT-1 and KYT-36 inhibited IL-6 production by J774.1 cells incubated with 10(5)CFU/ml of mixed bacteria. CONCLUSION These results suggest that P. gingivalis with either T. forsythia or T. denticola directly induces synergistic IL-6 protein production and that gingipains play a role in this synergistic effect.

Candida albicans and Candida parapsilosis Rapidly Up-Regulate Galectin-3 Secretion by Human Gingival Epithelial Cells

Galectin-3 is a β-galactoside-binding C-type lectin that plays an important role in innate immunity. The purpose of this study was to determine whether Candida albicans and Candida parapsilosis up-regulate galectin-3 secretion by human gingival epithelial cells and gingival fibroblasts. Ca9-22, a human gingival epithelial cell line, and human gingival fibroblasts were incubated in the presence or absence of C. albicans or C. parapsilosis without serum. Levels of secreted human galectin-3 in culture supernatants were measured by enzyme-linked immunosorbent assay. We also pretreated Ca9-22 cells with cytochalasin D (an actin polymerization inhibitor), ALLN (a calpain inhibitor) and LY294002 [a phosphatidylinositol-3 kinase (PI3K) inhibitor] to determine whether the up-regulation of galectin-3 secretion was mediated by cytoskeletal changes, protease activity, or PI3K signaling. Galectin-3 secretion was significantly and rapidly up-regulated by live C. albicans and C. parapsilosis, as well as heat-killed C. albicans. In addition, cytochalasin D, LY294002 and ALLN did not inhibit the up-regulation in galectin-3 secretion. These results suggest that both live and heat-killed C. albicans and C. parapsilosis may increase the activity of the innate immune system and invasion by other microorganisms via up-regulation of galectin-3 secretion.

Alendronate regulates cytokine production induced by lipid A through nuclear factor-κB and Smad3 activation in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE Nitrogen-containing bisphosphonates (NBPs) are widely used as anti-bone-resorptive drugs. However, use of NBPs results in inflammatory side-effects, including jaw osteomyelitis. In the present study, we examined the effects of alendronate, a typical NBP, on cytokine production by human peripheral blood mononuclear cells (PBMCs) and gingival fibroblasts incubated with lipid A. METHODS The PBMCs and gingival fibroblasts were pretreated with or without alendronate for 24 h. Cells were then incubated in the presence or absence of lipid A for a further 24 h. Levels of secreted human interleukin (IL)-1β, IL-6, IL-8 and monocyte chemoattractant protein-1 (MCP-1) in culture supernatants were measured by ELISA. We also examined nuclear factor-κB (NF-κB) activation in both types of cells by ELISA. Activation of Smad3 in the cells was assessed by flow cytometry. In addition, we performed an inhibition assay using SIS3, a specific inhibitor for Smad3. RESULTS Pretreatment of PBMCs with alendronate promoted lipid A-induced production of IL-1β and IL-6, but decreased lipid A-induced IL-8 and MCP-1 production. In human gingival fibroblasts, alendronate pretreatment increased lipid A-induced production of IL-6 and IL-8, and increased NF-κB activation in gingival fibroblasts but not PBMCs stimulated with lipid A. In contrast, alendronate activated Smad3 in both types of cells. Finally, SIS3 inhibited alendronate-augmented IL-6 and IL-8 production by human gingival fibroblasts but up-regulated alendronate-decreased IL-8 production by PBMCs. CONCLUSION These results suggest that alendronate-mediated changes in cytokine production by gingival fibroblasts occur via regulation of NF-κB and Smad3 activity.

Etidronate down-regulates Toll-like receptor (TLR) 2 ligand-induced proinflammatory cytokine production by inhibiting NF-κB activation

BACKGROUND Etidronate is a non-nitrogen-containing bisphosphonate (non-NBP) used for anti-bone resorptive therapy as well as having inhibitory effects on atherosclerotic plaques. The present study examined the effects of etidronate on the production of proinflammatory cytokines and chemokines by the macrophage-like cell line, J774.1, incubated with Pam3Cys-Ser-(Lys)4 (Pam3CSK4, a Toll-like receptor (TLR) 2 agonist) and lipid A (a TLR4 agonist). METHODS J774.1 cells and human monocytic THP-1 cells were pretreated with or without etidronate for 5min, and then incubated with or without Pam3CSK4 or lipid A for 24h. Levels of secreted interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1α (MIP-1α) in culture supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Cytotoxicity was determined by LDH activity in the supernatants. We also examined the effects of etidronate on the activation of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) in J774.1 cells by ELISA and Western blotting. RESULTS Treatment of J774.1 cells with etidronate down-regulated TLR2 ligand-induced production of IL-6, TNF-α, MCP-1, and MIP-1α. Etidronate also inhibited Pam3CSK4-induced MCP-1 and TNF-α production by THP-1 cells. However, etidronate did not induce cytotoxicity and reduced lipid A-induced cytotoxicity in J774.1 cells. In addition, this agent did not down-regulate TLR4 ligand-induced proinflammatory cytokine production. Furthermore, etidronate inhibited the translocation of NF-κB but not p38 MAPK in J774.1 cells stimulated with Pam3CSK4 or lipid A. CONCLUSION Etidronate likely inhibits proinflammatory cytokine production in J774.1 cells by suppressing NF-κB activation in the TLR2 and not the TLR4 pathway.

Gastrointestinal colonisation and systemic spread of Candida albicans in mice treated with antibiotics and prednisolone

Normally, Candida albicans is a commensal microbe that resides in the human oral cavity, gut and vagina. However, the fungus can cause mucosal and systemic infections in immunocompromised individuals. The mechanism by which local mucosal infections progress to systemic candidiasis is poorly understood. Here, a murine model of gastrointestinal (GI) candidiasis was developed by inoculation of the oral cavity, followed by treatment with tetracycline (TC) and prednisolone (PSL). Temporal progression from a local infection of the oral cavity to a systemic infection was then monitored. Histological analysis of tissues from mice treated with both TC and PSL revealed massive infiltration of the tongue and stomach by hyphae. PSL increased the fungal burden in the tongue, stomach and small intestine, and facilitated dissemination to the spleen, kidney and liver within 3 days post-infection. Treatment with both TC and PSL supressed interferon (IFN)-γ and interleukin (IL)-17 (cytokines that play key roles in host defence against fungal infection) levels in the tongue, which were induced by C. albicans infection. In addition, the mucosal layer of the small intestine of mice treated with both TC and PSL was almost destroyed by the fungal infection; this may be a critical event that allows passage of the fungus across the mucosa and into the systemic circulation. Thus, this mouse model is useful for studying mechanisms underlying progression of C. albicans from a local infection of the oral cavity to a systemic infection in immunocompromised individuals.

Alendronate augments lipid A-induced IL-1β release and Smad3/NLRP3/ASC-dependent cell death

Abstract Alendronate (ALN) is a nitrogen‐containing bisphosphonate (NBP) that inhibits bone resorption. NBPs have inflammatory side effects, and ALN augments bacteria‐induced interleukin (IL)‐1&bgr; production. The present study aimed to examine whether ALN induces pyroptosis, a form of cell death associated with IL‐1&bgr; release, in macrophage‐like J774.1 cells incubated with lipid A, a component of gram‐negative bacteria. Pretreatment of J774.1 cells with ALN increased lipid A‐induced IL‐1&bgr; production and cell death, but not IL‐6 and TNF‐&agr; production. Ac‐YVAD‐CHO, a caspase‐1 inhibitor, inhibited ALN‐augmented IL‐1&bgr; production induced by lipid A, although it did not affect ALN‐induced cell death. Moreover, Ac‐IETD‐CHO, a caspase‐8 inhibitor, and Z‐VAD‐FMK, a pan‐caspase inhibitor, did not inhibit ALN‐induced cell death, suggesting that the effects of ALN are exerted independently of caspase activation. We also demonstrate that a Smad3 inhibitor (SIS3) suppressed ALN‐augmented IL‐1&bgr; production. Moreover, SIS3 attenuated ALN‐augmented release of LDH and caspase‐1. These results suggest that ALN augments IL‐1&bgr; production, cell death, and caspase‐1 release in a manner dependent on Smad3. We then investigated whether ALN‐augmented IL‐1&bgr; production and cell death are dependent on apoptosis‐associated speck‐like protein containing a CARD (ASC) and NOD‐like receptor pyrin domain containing‐3 (NLRP3), which are associated with Smad3 activation. Both anti‐ASC and anti‐NLRP3 antibodies suppressed ALN‐induced cell death and caspase‐1 release, but only anti‐ASC antibody inhibited ALN‐augmented IL‐1&bgr; production. Our findings suggest that ALN‐augmented IL‐1&bgr; production and cell death require Smad3 and ASC activation, and that SIS3 and anti‐ASC antibodies may serve as palliative agents for necrotizing inflammatory diseases caused by ALN. Graphical abstract Figure. Unlabelled Image HighlightsAlendronate (ALN) augmented lipid A‐induced IL‐1&bgr; release by J774.1 cells.ALN did not up‐regulate IL‐6 and TNF‐&agr; production induced by lipid A.Caspase‐1 was related to ALN‐augmented IL‐1&bgr; release and ALN‐induced cell death.ALN induced pyroptosis and caspase‐independent cell death by J774.1 cells via Smad3/NLRP3/ASC.ALN increased the release of caspase‐1 by J774.1 cells via Smad3/NLRP3/ASC.

Extracellular galectin-1 enhances adhesion to and invasion of oral epithelial cells by Porphyromonas gingivalis

Galectin-1 and galectin-3 are C-type lectin receptors that bind to lipopolysaccharide in the cell wall of gram-negative bacteria. In this study, we investigated the effects of galectin-1 and galectin-3 on adhesion to and invasion of the human gingival epithelial cell line Ca9-22 by Porphyromonas gingivalis, a periodontal pathogenic gram-negative bacterium. Recombinant galectin-1, but not galectin-3, enhanced P. gingivalis adhesion and invasion, although both galectins bound similarly to P. gingivalis. Flow cytometry also revealed that Ca9-22 cells express low levels of galectin-1 and moderate levels of galectin-3. Ca9-22 cells in which galectin-3 was knocked-down did not exhibit enhanced P. gingivalis adhesion and invasion. Similarly, specific antibodies to galectin-1 and galectin-3 did not inhibit P. gingivalis adhesion and invasion. These results suggest that soluble galectin-1, but not galectin-3, may exacerbate periodontal disease by enhancing the adhesion to and invasion of host cells by periodontal pathogenic bacteria.

Effects of Nitrogen-containing Bisphosphonates on the Response of Human Peripheral Blood Mononuclear Cells and Gingival Fibroblasts to Bacterial Components

Abstract Nitrogen-containing bisphosphonates (NBPs) are widely used as anti-bone resorptive drugs; however, NBPs have inflammatory side effects, including osteomyelitis and osteonecrosis of the jaw. In this paper, we review the effects of alendronate, a typical NBP, on cytokine production by mouse and human cells incubated with Porphyromonas gingivalis or lipid A. Pretreatment of J774.1 cells, a mouse macrophage-like cell line, with alendronate augments P. gingivalis -induced interleukin (IL)-1β production, but decreases the production of Toll-like receptor (TLR) ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α). Furthermore, caspase-1, a promoter of IL-1β production, is activated by treatment with alendronate; however, alendronate directly activates Smad3. Pretreatment of human peripheral blood mononuclear cells (PBMCs) with alendronate promotes P. gingivalis -induced production of IL-1β and IL-6, but decreases P. gingivalis -induced IL-8 and MCP-1 production. We observed similar results in PBMCs treated with alendronate followed by lipid A. Pretreatment with alendronate did not increase NF-κB activation in PBMCs stimulated with lipid A. In human gingival fibroblasts, alendronate pretreatment increased lipid A-induced production of IL-6 and IL-8. In addition, pretreatment with alendronate increased NF-κB and Smad3 activation in gingival fibroblasts stimulated with lipid A, and SIS3, a specific inhibitor of Smad3, significantly inhibited alendronate-increased IL-6 and IL-8 production. These results suggest that alendronate-mediated changes in cytokine production by cells occur via regulation of transcriptional activity, including NF-κB and Smad3, and that this agent may exacerbate periodontitis and jaw osteomyelitis, which are chronic inflammatory diseases caused by high levels of oral bacterial components.