Diverse Toll-Like Receptors Mediate Cytokine Production by Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans in Macrophages

Abstract

ABSTRACTToll-like receptors (TLRs) orchestrate a repertoire of immune responses in macrophages against various pathogens.Fusobacterium nucleatumandAggregatibacter actinomycetemcomitansare two important periodontal pathogens. In the present study, we investigated TLR signaling regulating cytokine production of macrophages in response toF. nucleatumandA. actinomycetemcomitans. TLR2 and TLR4 are redundant in the production of cytokines (interleukin-6 [IL-6] and tumor necrosis factor alpha [TNF-α]) inF. nucleatum- andA. actinomycetemcomitans-infected macrophages. The production of cytokines by macrophages in response toF. nucleatumandA. actinomycetemcomitansinfection was impaired in MyD88-deficient macrophages. Moreover, cytokine concentrations were lower in MyD88-deficient macrophages than in TLR2/TLR4 (TLR2/4) double-deficient cells. An endosomal TLR inhibitor, chloroquine, reduced cytokine production in TLR2/4-deficient macrophages in response toF. nucleatumandA. actinomycetemcomitans, and DNA fromF. nucleatumorA. actinomycetemcomitansinduced IL-6 production in bone marrow-derived macrophages (BMDMs), which was abolished by chloroquine. Western blot analysis revealed that TLR2/4 and MyD88 were required for optimal activation of NF-κB and mitogen-activated protein kinases (MAPKs) in macrophages in response toF. nucleatumandA. actinomycetemcomitans, with different kinetics. An inhibitor assay showed that NF-κB and all MAPKs (p38, extracellular signal-regulated kinase [ERK], and Jun N-terminal protein kinase [JNK]) mediateF. nucleatum-induced production of cytokines in macrophages, whereas NF-κB and p38, but not ERK and JNK, are involved inA. actinomycetemcomitans-mediated cytokine production. These findings suggest that multiple TLRs may participate in the cytokine production of macrophages against periodontal bacteria.