Bacterial flagellin is a unique pathogen-associated molecular pattern (PAMP), which can be recognized by surface localized Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor (NLR) protein 4 (NLRC4) receptors. Activation of the TLR5 and/or NLRC4 signaling pathways by flagellin and the resulting immune responses play important roles in anti-bacterial immunity. However, it remains unclear how the dual activities of flagellin that activate the TLR5 and/or NLRC4 signaling pathways orchestrate the immune responses. In this study, we assessed the effects of flagellin and its mutants lacking the ability to activate TLR5 and NLRC4 alone or in combination on the adaptive immune responses against flagellin. Flagellin that was unable to activate NLRC4 induced a significantly higher antibody response than did wild-type flagellin. The increased antibody response could be eliminated when macrophages were depleted in vivo. The activation of NLRC4 by flagellin downregulated the flagellin-induced and TLR5-mediated immune responses against flagellin.
Cellular & Molecular ImmunologyKeywords: flagellin; NLRC4; TLR5; macrophage; adaptive immunity INTRODUCTION Bacterial flagellin is one of a small number of protein pathogenassociated molecular patterns (PAMPs), which can be recognized by cell surface Toll-like receptor 5 (TLR5) 1 and the cytosolic NOD-like receptor protein 4 (NLRC4) inflammasome receptor NAIP5. 2,3 Flagellin consists of two highly conserved domains (D0 and D1) and one central hypervariable domain (D2/D3). [4][5][6] The conserved D0 and D1 domains are required for the immune activity of flagellin as a PAMP. The Nterminal amino acids 90-97 (QRVRELAV) of D1 form a highly conserved motif that is essential for both high-affinity binding and signaling to TLR5. 7,8 The C-terminal 35 amino acids of flagellin in the D0 domain are responsible for the interaction between flagellin and NLRC4. The substitution of three conserved leucine residues L502, L504, and L505 for alanine in the 35 C-terminal amino acids could abolish the ability of flagellin to activate NLRC4. 9 Flagellin-mediated activation of TLR5 activates inflammatory genes via the MyD88 pathway, whereas flagellin-activated NLRC4 triggers the assembly of the inflammasome, which culminates in caspase-1 activation, IL-1b/IL-18 secretion, and cellular pyroptosis. 10,11