Activation of NLRC4 downregulates TLR5-mediated antibody immune responses against flagellin

Abstract

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.

Flagellin-rPAc vaccine inhibits biofilm formation but not proliferation ofS. mutans

“…Antibody titers in serum and saliva were determined by ELISA as previously described. 13,[31][32][33] Briefly, polystyrene 96-well microplates (Greiner, Germany) were coated with 100 ml rPAc or KF (5 mg/ml) at 37 C for 3 h, and then blocked with PBS containing 1% bovine serum albumin (BSA) overnight at 4 C. Serial dilutions of serum or saliva samples were added in each well and incubated at 37 C for 2 h. After 6washes with PBS containing 0.5% Tween-20 (PBST), the plates were incubated with 100 ml of alkaline phosphatase-conjugated goat antimouse/rat heavy chain IgG or IgA at 37 C for 1 h. After 6 washes of PBST, 100 ml phosphate substrate (p-nitrophenylphosphate) was added into each well for color development. Endpoint titers were expressed as the reciprocal of the last dilution giving an optical density at 405 nm of 0.1 greater than that of control after 30 min of incubation.…”

Amino acids 89–96 of Salmonella flagellin: a key site for its adjuvant effect independent of the TLR5 signaling pathway

“…F lagellin, the main component of bacterial flagella, is a well-known pathogen-associated molecular pattern (PAMP) that can be recognized by the cell surface-localized Toll-like receptor 5 (TLR5) and the cytosolic NOD-like receptor protein 4 (NLRC4) inflammasome receptor NAIP5/6. 1 Hosts can detect flagellated bacteria using the dual sensors, TLR5 and NAIP5/6, to recognize flagellin, and these sensors then initiate innate immune responses to protect the host from bacterial infection. Recognition by extracellular TLR5 can trigger the MyD88-dependent signaling pathway and activate nuclear factor κB, leading to the upregulation of several proinflammatory cytokines.…”

Autoinflammatory mutation in NLRC4 reveals a leucine-rich repeat (LRR)–LRR oligomerization interface

“… 10 There has also been a report of activation of NLRC4 resulting in dampening of Toll-like receptor 5–induced antibody response to flagella, raising the possibility of an associated immunodeficiency. 38 However, patient P2 cleared the organism with appropriate antimicrobial therapy as repeat CSF cultures were negative. Therefore it is difficult to conclude that the response to this flagellated organism was impaired.…”