Airway structural cells regulate TLR5-mediated mucosal adjuvant activity

Abstract

Antigen-presenting cell (APC) activation is enhanced by vaccine adjuvants. Most vaccines are based on the assumption that adjuvant activity of Toll-like receptor (TLR) agonists depends on direct, functional activation of APCs. Here, we sought to establish whether TLR stimulation in non-hematopoietic cells contributes to flagellin's mucosal adjuvant activity. Nasal administration of flagellin enhanced T-cell-mediated immunity, and systemic and secretory antibody responses to coadministered antigens in a TLR5-dependent manner. Mucosal adjuvant activity was not affected by either abrogation of TLR5 signaling in hematopoietic cells or the presence of flagellin-specific, circulating neutralizing antibodies. We found that flagellin is rapidly degraded in conducting airways, does not translocate into lung parenchyma and stimulates an early immune response, suggesting that TLR5 signaling is regionalized. The flagellin-specific early response of lung was regulated by radioresistant cells expressing TLR5 (particularly the airway epithelial cells). Flagellin stimulated the epithelial production of a small set of mediators that included the chemokine CCL20, which is known to promote APC recruitment in mucosal tissues. Our data suggest that (i) the adjuvant activity of TLR agonists in mucosal vaccination may require TLR stimulation of structural cells and (ii) harnessing the effect of adjuvants on epithelial cells can improve mucosal vaccines.

Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5

“…6E). As reported (39,40), an S. Typhimurium flagellin mutant lacking only the two carboxy-terminal amino acids (fliC ) also failed to activate Naip5-and Nlrc4-dependent inflammasome responses (Fig. 6 C-E).…”

“…Gentamycin (50 μg/mL, 10131-027, Life Technologies) was added, and cell lysates were prepared 30 min later. In other experiments, recombinant H. pylori flagellin (flaA, HPA-5040, Austral Biologicals), or purified S. Typhimurium flagellin (fliC, 5 μg/mL) produced in E. coli (40) or 293T cells (fliC 293T ) was delivered in the cytosol using streptolysin O (SLO, 25 μg/mL, S5265-25KU, Sigma), as described (17). In some experiments, cells were pretreated with Ac-YVAD-cmk (50 μM, ALX-260-028-M001, Enzo Life Sciences) for 30 min before S. Typhimurium infection or fliC transfection.…”

A Toll-Like Receptor 5 Agonist Improves the Efficacy of Antibiotics in Treatment of Primary and Influenza Virus-Associated Pneumococcal Mouse Infections

“…The role of the neutrophils in the therapeutic effect of the combination treatments and the influence of antimicrobial molecules remain to be elucidated. Furthermore, there is strong evidence to suggest that the primary targets of intranasally administered flagellin are the airway epithelial cells (20,21,38). The epithelial cells express TLR5 strongly and respond both in vitro and in vivo to flagellin by upregulating the transcription of genes encoding host antibacterials and phagocyte-specific chemokines (21,(38)(39)(40).…”

“…S1C to E in the supplemental material). Indeed, flagellin enhanced the transcription of Ccl20 and Cxcl1 (both surrogate markers of TLR5-mediated lung stimulation [21]) in animals from 0 to 24 h postinfection. These results indicated that flagellin is able to boost innate signaling even in the context of ongoing pneumococcal infection.…”

“…We and others have shown that nasal administration of flagellin also confers prophylactic neutrophil-dependent protection against lung infection with Streptococcus pneumoniae (18). Notably, our recent studies highlighted the fact that the flagellin-induced innate immune response in the respiratory tract specifically requires TLR5 signaling in the airway epithelium and is independent of the cytosolic Nodlike receptor family caspase activation and recruitment domaincontaining protein 4 (NLRC4) (20,21).…”