Flagellin, as a TLR5 agonist, is an established mucosal adjuvant for enhancing mucosal IgA responses by i.n. immunization. Nasal epithelial cells (NECs) are the first sentinel cells to be exposed to antigen and adjuvant in i.n. immunization, and it is suggested that they play an important role in the mucosal adjuvant activity of flagellin. However, the molecular mechanism leading to modulation and the response by flagellin-activated NECs remain unknown. We aimed to identify the soluble mediator(s) from flagellin-activated NECs that modulate the functions of airway dendritic cells (DCs) and enhance subsequent IgA response. In vitro studies showed that compared with the TLR4 agonist LPS, flagellin directly triggered slight up-regulation of CD80 on airway DCs but was insufficient to affect CD86 expression and DC-mediated IgA response. With the use of an in vitro system for culturing mouse primary NECs (mNECs), we demonstrated that flagellin-activated mNECs could functionally modulate airway DCs, which manifested as significant up-regulation of CD80/CD86 and enhancement of IgA production. The functional modulation of airway DCs was dependent on TLR5 activation of mNECs rather than direct TLR5 activation of airway DCs. With the use of cytokine array and antibody-blocking assays, we further identified that GM-CSF, a cytokine secreted from TLR5-activated mNECs, contributes to the activation of mNECs to airway DCs and subsequent IgA enhancement. In vivo blocking experiments confirmed that GM-CSF is an important factor in recombinant flagellin derived from Salmonella typhi (FliC)-induced airway DC activation and antigen-specific IgA enhancement. Our data directly demonstrate that nasal epithelial GM-CSF contributes to TLR5-mediated modulation of airway DCs and a subsequent IgA response.

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.

Flagellin is a potent activator of a broad range of cell types that are involved in innate and adaptive immunity. Therefore, it is a good adjuvant candidate for vaccines, and it might function as a biological protectant against both major acute radiation syndrome during cancer radiotherapy and a mitigator of radiation emergencies. However, accumulating evidence has implicated flagellin in the occurrence of some inflammatory diseases, such as acute lung inflammation, cardiovascular collapse and inflammatory bowel disease. The aim of this study was to elucidate whether only flagellin-TLR5 signaling activation plays a role in the pathophysiology of liver or whether some other flagellin activity also contributes to liver injury either via bacterial infections or during clinical applications. Recombinant flagellin proteins with or without TLR5-stimulating activity were used to evaluate the role of flagellin-TLR5 signaling in liver injury in wild-type and TLR5 KO mice. Gross lesions and large areas of hepatocellular necrosis were observed in liver tissue 12 h after the intraperitoneal administration of 100 or 200 µg flagellin (FliC) in a dose- and time-dependent manner in wild-type mice, but not in TLR5 KO mice. Deletion of the N-terminal or TLR5 binding domain of flagellin inhibited flagellin-induced inflammatory responses and the subsequent acute liver function abnormality and damage. These data confirmed that flagellin is an essential determinant of liver injury and demonstrated that the over-activation of TLR5 signaling by high-dose flagellin caused acute inflammatory responses, neutrophil accumulation and oxidative stress in the liver, which contributes to the progression and severity of flagellin-induced liver injury.

For subunit vaccines, adjuvants play a key role in shaping the magnitude, persistence and form of targeted antigen-specific immune response. Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application. Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA, RNA, peptides and proteins. Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system, i.e. the cytokine production, was studied. They induced the production of the proinflammatory cytokines IL-8 (Caco-2 cells) and IL-1β (bone marrow-derived macrophages; BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice. The immunostimulation was more pronounced than with free flagellin.