Young- Jung

Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia induce cell death and release of endogenous danger signals

OBJECTIVE The aim of this study was to analyze whether periodontopathogens induced inflammatory cell death and the release of diverse endogenous danger molecules in THP-1-derived macrophages. METHODS The macrophages were treated with Treponema denticola, Porphyromonas gingivalis, and Tannerella forsythia. Activation of caspase-1 and caspase-4 was detected by Western blotting. Cell death of bacteria-stimulated macrophages was examined using a lactate dehydrogenase (LDH) assay and propidium iodide (PI)/annexin V (AV) staining. Levels of endogenous danger signals, including adenosine triphosphate (ATP), uric acid, heat shock protein 60 (HSP60), high-mobility group box protein 1 (HMGB1), and fibronectin in the culture supernatants were determined using an ATP bioluminescence assay kit, a uric acid assay kit, and Western blotting, respectively. RESULTS T. denticola, P. gingivalis, and T. forsythia induced activation of caspase-1 and caspase-4. The LDH assay and PI/AV staining showed that all three pathogens induced pyroptotic cell death. All three bacteria induced release of ATP, which is an important ligand for inflammasome activation; the increase in ATP ultimately leads to caspase-1 activation. T. denticola induced release of HSP60 and fibronectin, while T. forsythia induced release of HMGB1 in addition to HSP60 and fibronectin. None of the endogenous molecules except for fibronectin were detected in P. gingivalis-infected cells, possibly due to degradation of these factors by the proteolytic activity of the bacteria. Interestingly, P. gingivalis induced uric acid release. CONCLUSION Inflammatory cell death and endogenous danger molecules released from cells infected with periodontopathogens may play critical roles in the pathogenesis and progression of periodontitis by augmenting immune and inflammatory responses.

Enterococcus faecalis activates caspase-1 leading to increased interleukin-1 beta secretion in macrophages.

INTRODUCTION Recent studies of inflammasome activation have focused on the pathogenesis of diverse inflammatory and autoimmune diseases. Inflammasome activation results in caspase-1 activation, which is required for processing of prointerleukin (IL)-1 beta to its secreted form as well as a proinflammatory cell death (ie, pyroptosis). The purpose of this study was to analyze whether Enterococcus faecalis associated with endodontic infection induces inflammasome activation. METHODS THP-1 macrophages were treated with E. faecalis in the presence or absence of caspase-1 inhibitors. Caspase-1 activation, pro-IL-1 beta expression, and IL-1 beta secretion were detected by immunoblotting, real-time reverse-transcription polymerase chain reaction, and enzyme-linked immunosorbent assay, respectively. Cell death was measured by lactate dehydrogenase release and propidium iodide staining. Adenosine triphosphate (ATP) release was measured by an ATP bioluminescence assay kit. RESULTS E. faecalis induced caspase-1 activation and pro-IL-1 beta expression, which resulted in IL-1 beta secretion in macrophages. E. faecalis significantly induced ATP release, which is a mechanism of Nod-like receptor family protein 3 (NLRP3) inflammasome activation, whereas oxATP treatment inhibited E. faecalis-induced caspase-1 activation. E. faecalis significantly increased lactate dehydrogenase release and propidium iodide uptake, which are characteristics of pyroptosis. CONCLUSIONS Our results show that E. faecalis may contribute to the progression of pulpal inflammation by stimulating excessive secretion of IL-1 beta and cell death.

Contradictory roles of Porphyromonas gingivalis gingipains in caspase-1 activation

Porphyromonas gingivalis utilizes its major proteases, Arg gingipains (RgpA and RgpB) and Lys gingipain (Kgp), for dysregulation of host immune systems. The aim of this study was to investigate the roles of gingipains in caspase‐1 activation and its sequelae in P. gingivalis‐infected macrophages. Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of interleukin‐1β and lactate dehydrogenase without detectable active caspase‐1 in the culture supernatants. The proteins released from caspase‐1‐activated cells were rapidly degraded by gingipains. However, P. gingivalis with gingipains induced higher intracellular caspase‐1 activity in the infected cells than the gingipain‐null mutant, which was associated with ATP release from the infected cells. In addition, growing the gingipain‐null mutant with gingipains enhanced caspase‐1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgps increased the caspase‐1‐activating potential of wild‐type P. gingivalis, indicating an inhibitory effect of the collaborative action of Kgp and Rgps. These results illuminate the contradictory roles of gingipains in the manipulation of host defence systems by P. gingivalis, as they act by both stimulating and inhibiting innate immune responses.

Caspase-4 activation by a bacterial surface protein is mediated by cathepsin G in human gingival fibroblasts

Caspase-4 is an inflammatory caspase; however, its mechanism of activation is poorly understood. In this study, we demonstrate that Td92, a surface protein of the periodontal pathogen Treponema denticola and a homolog of the Treponema pallidum surface protein Tp92, activates caspase-4 and induces pyroptosis in primary cultured human gingival fibroblasts (HGFs) via cathepsin G activation. Cathepsin G inhibition or siRNA knockdown of cathepsin G inhibited Td92-induced caspase-4 activation and cell death. Td92-induced cell death was significantly inhibited by siRNA knockdown of gasdermin D. Td92 treatment resulted in the binding of cathepsin G to caspase-4 and the coaggregation of these two molecules. In addition, Td92 induced IL-1α expression and secretion, and this was inhibited by caspase-4 knockdown. Cytochalasin D did not block Td92-induced caspase-4 activation, suggesting that Td92 internalization is not required for caspase-4 activation. Our results demonstrate that cathepsin G is directly engaged in caspase-4 activation by a bacterial ligand, which is responsible for cell death and IL-1α secretion in HGFs.

Inflammasome activators induce fibronectin expression and release in macrophages

Extracellular fibronectin (Fn) can activate pro‐inflammatory pathways and serves as an endogenous danger signalling molecule; thus, it has been suggested as a biomarker for several diseases. In the present study, we found that pathogen‐derived activators of the inflammasomes induce the expression and secretion of Fn in macrophages through a mechanism involving adenosine triphosphate and caspase‐1 activation. We also found that plasma Fn induces caspase‐1 activation and cell death in macrophages, epithelial cells, and fibroblasts. Together, these results indicate that Fn plays a critical role in inflammasome‐activated cells by amplifying caspase‐1 activation and inducing inflammatory cell death.