These results suggested that depleting macrophages in mice abolished the differences in the immune responses induced by the flagellin variants FliC-L3A and FliC, which further confirmed that macrophages were the main effector cell that suppressed the TLR5-mediated adaptive immune response against flagellin by activating NLRC4. Open Acipimox in a separate window Figure 5 The different flagellin-specific antibody responses induced by FliC-L3A and FliC were abolished after macrophage depletion. might elicit different innate and adaptive immune responses qualitatively and quantitatively.15 Finally, flagellin is a potent immunogen that can induce strong adaptive immune responses against itself.16,17,18 Innate immunity is responsible for detecting bacterial invasion and infection by using the dual sensors TLR5 and NLRC4 to recognize flagellin, and then initiating immune responses to protect the host from infection. The activation of these sensors might have significantly different outputs depending on the structural properties, amount released, and distribution of flagellin. However, it is unclear whether the activation of TLR5 and/or NLRC4 is beneficial to the host in its defense against bacterial infection. For example, TLR5-deficient mice are less prone to infection with than are wild-type (WT) mice,19 and NLRC4 activation might protect mice from the mucosal and systemic dissemination of salmonella.20 But, Letran observed no significant difference in the susceptibility of mice to bacterial infection in WT and TLR5-deficient mice.21 On the other hand, could downregulate NLRC4 expression to prevent the inflammasome response and thereby promote bacterial persistence and dissemination.22 NLRC4-dependent IL-1 production by intestinal phagocytes could discriminate pathogenic from commensal bacteria, thereby contributing to the immune defense against enteric bacterial infection.10 Therefore, the activation of both of TLR5 and NLRC4 by flagellin should be taken into consideration regarding the interaction between pathogenic bacteria and immune responses. Further studies on the interaction and cross-talk between TLR5 and NLRC4 will be valuable to increase understanding of the complexities of the innate immune recognition of flagellated pathogens and also critical for the reasonable design of flagellin-based vaccines. In the present study, we assessed the ability of flagellin and its mutants lacking ability to activate either TLR5 or NLRC4, or both TLR5 and NLRC4 to stimulate the immune responses against flagellin. Abolishing NLRC4 activation by flagellin increased the antibody responses against flagellin significantly compared with WT flagellin. This increased antibody response could Acipimox be eliminated when macrophages were depleted in mice. These data revealed an interaction between NLRC4 and TLR5-mediated activity that affects the immune responses against flagellin. It is possible that NLRC4-mediated activation negatively regulates the TLR5-mediated immune response against flagellin due to the decreased amount of macrophages and corresponding cytokines secretion. MATERIALS AND Acipimox METHODS Animals Female C57BL/6 mice aged 6C8 weeks were purchased from Beijing Laboratory Animal Research Center and housed under specific pathogen-free (SPF) conditions Acipimox in the Animal Center of Wuhan Institute of Virology (WIV), Chinese Academy of Sciences (CAS). All animals were divided randomly into different groups before the immunization experiments. Animal studies were performed according to Regulations for the Administration of Affairs Concerning Experimental Animals in China (1988), and the Guidelines for Animal Care and Use, WIV, CAS (permit number WIVA09201203). All animal studies and methods conformed to ARRIVE guidelines. Construction, expression, and purification of recombinant FliC, FliC90-97, FliC-L3A, and FliC90-97:L3A WT flagellin protein and its three modified proteins FliC Acipimox (WT), FliC90-97 (unable to activate TLR5), FliC-L3A (unable to activate NLRC4 due HEY1 to mutation of C-terminal L502, L504, and L505 to A), and FliC90-97:L3A (unable to activate both TLR5 and NLRC4) were constructed as described previously.23 Appropriate oligonucleotide primers containing restriction enzyme sites were designed based upon the full-length fiagellin gene from subsp. (GenBank Accession No. 1070204) to construct truncated, deleted, and/or chimeric BL21 (DE3), selected, and their sequences were confirmed using DNA sequencing (Invitrogen/Life Technologies). Transformed BL21 (DE3) containing recombinant flagellin expression constructs were grown and induced as described previously.24 The resulting recombinant proteins were prepared and purified using affinity chromatography on a Ni-NTA column (Qiagen, Venlo, Limburg, the Netherlands). The concentrations of the purified proteins were determined using a Bradford protein assay. The purified proteins were verified by western blotting using an anti-His-tag monoclonal antibody (Qiagen) followed by a secondary alkaline phosphatase (AP)-conjugated goat anti-mouse IgG antibody (Southern Biotech). The antibodyCantigen complexes were visualized using SuperSignal West Pico Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific, Waltham, MA, USA) and imaged on a Versadoc 3000 imager (Bio-Rad, Hercules, CA, USA). Lipopolysaccharide (LPS) was removed from the purified recombinant proteins using the Affinity Pak Detoxi Gel Endotoxin Removing gel (Pierce), and the residual LPS content of the protein was determined using a Limulus assay (Associates of Cape Cod, East Falmouth, MA, USA). The endotoxin value of the purified recombinant flagellin preparation was 0.03 EU/mg. Any contamination with other PAMPs was assessed using the purified proteins to stimulate the flagellin-nonresponsive RAW264.7 cells25,26.