Abundant Secretion of Bioactive Interleukin-1β by Human Macrophages Induced byActinobacillus actinomycetemcomitansLeukotoxin

Abstract

Actinobacillus actinomycetemcomitans produces a leukotoxin that selectively kills human leukocytes. Recently, we reported that macrophages are highly sensitive to leukotoxin and that their lysis involves activation of caspase 1. In this study, we show that leukotoxin also induces the production and release of proinflammatory cytokines from human macrophages. The macrophages were challenged with leukotoxin or lipopolysaccharide (LPS) from A. actinomycetemcomitans or LPS from Escherichia coli, and the production and secretion of interleukin-1␤ (IL-1␤), IL-6, and tumor necrosis factor alpha (TNF-␣) were determined at the mRNA and protein levels by reverse transcription-PCR and enzyme-linked immunosorbent assay, respectively. Leukotoxin (1 to 30 ng/ml) induced abundant production and secretion of IL-1␤, while the effects on IL-6 and TNF-␣ production were limited. Leukotoxin (1 ng/ml) caused a 10-times-higher release of IL-1␤ than did LPS (100 ng/ml). The secreted IL-1␤ was mainly the bioactive 17-kDa protein. At higher concentrations (>30 ng/ml), leukotoxin caused secretion of mainly inactive cytokine, the 31-kDa pro-IL-1␤. The presence of specific antibodies to IL-1␤ or of a caspase 1 inhibitor blocked the secretion and production of the cytokine. Supernatants of leukotoxin-challenged macrophages stimulated bone resorption when tested in a mouse calvarial model. The activity could be blocked by an IL-1 receptor antagonist or specific antibodies to IL-1␤. We concluded that A. actinomycetemcomitans leukotoxin can trigger abundant production and secretion of bioactive IL-1␤ by human macrophages, which is mediated by activation of caspase 1.

Oral biofilm challenge regulates the RANKL-OPG system in periodontal ligament and dental pulp cells

“…Putative periodontal pathogens have individually the capacity to induce the production of this cytokine [32][33][34][35]. In the present study, the biofilm challenge caused a significant up-regulation of IL-1ß expression in DP cells, but not in PDL cells.…”

Aggregatibacter actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes

“…actinomycetemcomitans does not affect the intracellular levels of pro-Caspase-1 [20], although its leukotoxin can promote Caspase-1 activation [17] and enhanced IL-1β production. In the absence of leukotoxin, transcribed pro-IL-1β is withheld intracellularly [20,21].…”

“…Hence, it appears that A. actinomycetemcomitans is sensed by inflammasomes irrespective of these two major virulence factors, albeit for the activation of the effector molecule Caspase-1 and subsequent IL-1β secretion, leukotoxin is required [17,20]. The relative effect of A. actinomycetemcomitans leukotoxin and Cdt on IL-1β and IL-18 expressions by the cells was also considered in this study.…”

“…To this extent, supragingival and subgingival biofilms were shown to differentially regulate the expression of the NLRP3 and AIM2 inflammasomes in gingival fibroblasts, denoting the different pathogenic potential of these two biofilm variants for periodontal diseases [58]. actinomycetemcomitans does not affect the intracellular levels of pro-Caspase-1 [20], although its leukotoxin can promote Caspase-1 activation [17] and enhanced IL-1β production. In the absence of leukotoxin, transcribed pro-IL-1β is withheld intracellularly [20,21].…”

“…Leukotoxin, predominantly associated with the bacterial surface [14,15], can selectively kill human leukocytes by triggering apoptosis or lysis [16][17][18][19]. The lysis of human monocytes / macrophages by leukotoxin is mediated by the activation of the cysteine proteinase Caspase-1, which is also associated with the secretion of bio-active IL-1β [17,20,21]. In addition, the strong association between presence of the highly leuokotoxic clone (JP2) of A. actinomycetemcomitans and disease progression indicates an important pathogenic role of the leukotoxin [22].…”