Stimulation of human Toll-like receptor (TLR) 2 and TLR6 with membrane lipoproteins ofMycoplasma fermentansinduces apoptotic cell death after NF-κB activation

Abstract

SummaryMycoplasmal membrane diacylated lipoproteins not only initiate proinflammatory responses through Tolllike receptor (TLR) 2 and TLR6 via the activation of the transcriptional factor NF-k k k k B, but also initiate apoptotic responses. The aim of this study was to clarify the apoptotic machineries. Mycoplasma fermentans lipoproteins and a synthetic lipopeptide, MALP-2, showed cytocidal activity towards HEK293 cells transfected with a TLR2-encoding plasmid. The activity was synergically augmented by co-expression of TLR6, but not by co-expression of other TLRs. Under the condition of co-expression of TLR2 and TLR6, the lipoproteins could induce maximum NF-k k k k B activation and apoptotic cell death in the cells 6 h and 24 h after stimulation respectively. Dominant-negative forms of MyD88 and FADD, but not IRAK-4, reduced the cytocidal activity of the lipoproteins. In addition, both dominant-negative forms also downregulated the activation of both NF-k k k k B and caspase-8 in the cells. Additionally, the cytocidal activity was sufficiently attenuated by a selective inhibitor of p38 MAPK. These findings suggest that mycoplasmal lipoproteins can trigger TLR2-and TLR6-mediated sequential bifurcate responses: NF-k k k k B activation as an early event, which is partially mediated by MyD88 and FADD; and apoptosis as a later event, which is regulated by p38 MAPK as well as by MyD88 and FADD.

Heterodimer‐specific TLR2 stimulation results in divergent functional outcomes in B‐cell precursor acute lymphoblastic leukemia

“…Our results are consistent with three previous reports of TLR2 stimulation directly initiating proapoptotic signaling; in THP-1 myeloid leukemia cells with Pam 3 CSK 4 [39] and in TLR2-overexpressed HEK293 cells with MALP-2 or Pam 3 CSK 4 [40,41]. While induction of cell death often depends on the balance between proapoptotic and prosurvival signals [68], the exact signaling networks that induce cell survival or cell death after engagement of TLRs are not yet completely understood [69].…”

“…Since TLR2 ligands have also been reported to induce tumor cell death in disparate models [39,40], we next compared the effects of TLR2/1 versus TLR2/6 ligation on ALL cell viability. Stimulation of TLR2/1 with Pam 3 CSK 4 induced significant direct cell death in ALL cell lines (p < 0.001) and primary ALL cells (p < 0.001) ( Fig TLR2 ligand-induced cell death has been reported to be mediated either through caspase activation or reactive oxygen species (ROS) production, depending on what cell type was studied [39,40].…”

“…Stimulation of TLR2/1 with Pam 3 CSK 4 induced significant direct cell death in ALL cell lines (p < 0.001) and primary ALL cells (p < 0.001) ( Fig TLR2 ligand-induced cell death has been reported to be mediated either through caspase activation or reactive oxygen species (ROS) production, depending on what cell type was studied [39,40]. For cell line 380, which yielded the most significant response of the cell lines, Pam 3 CSK 4 -induced apoptosis detected by Annexin-V binding was detectable after 2 h (data not shown)…”

“…Since TLR2 ligands have also been reported to induce tumor cell death in disparate models [39,40], we next compared the effects of TLR2/1 versus TLR2/6 ligation on ALL cell viability. Stimulation of TLR2/1 with Pam 3 CSK 4 induced significant direct cell death in ALL cell lines (p < 0.001) and primary ALL cells (p < 0.001) ( Fig.…”

“…While the pattern of CD40 upregulation is consistent with our previous report, the magnitude of the increase observed is greater in this study [35]. We attribute this inconsistency to the use of only short-term cultured cells obtained directly from the commercial repository, an approach that limits variation resulting from genetic drift in the cell lines.Since TLR2 ligands have also been reported to induce tumor cell death in disparate models [39,40], we next compared the effects of TLR2/1 versus TLR2/6 ligation on ALL cell viability. Stimulation of TLR2/1 with Pam 3 CSK 4 induced significant direct cell death in ALL cell lines (p < 0.001) and primary ALL cells (p < 0.001) ( Fig TLR2 ligand-induced cell death has been reported to be mediated either through caspase activation or reactive oxygen species (ROS) production, depending on what cell type was studied [39,40].…”