Alcaligenes are opportunistic commensal bacteria that reside in gut-associated lymphoid tissues such as Peyer's patches (PPs); however, how they create and maintain their homeostatic environment, without inducing an excessive inflammatory response remained unclear. We show here that Alcaligenes-derived lipopolysaccharide (Alcaligenes LPS) acts as a weak agonist of toll-like receptor 4 and promotes IL-6 production from dendritic cells, which consequently enhances IgA production. The inflammatory activity of Alcaligenes LPS was weaker than that of Escherichia coli-derived LPS and therefore no excessive inflammation was induced by Alcaligenes LPS in vitro or in vivo. Alcaligenes LPS also showed adjuvanticity, inducing antigen-specific immune responses without excessive inflammation. These findings reveal the presence of commensal bacteria-mediated homeostatic inflammatory conditions within PPs that produce optimal IgA induction without causing pathogenic inflammation and suggest that Alcaligenes LPS could be a safe and potent adjuvant.

Alcaligenes faecalis is the predominant Gramnegative bacterium inhabiting gut-associated lymphoid tissues, Peyersp atches.W ep reviously reported that an A. faecalis lipopolysaccharide (LPS) acted as aweak agonist for Toll-like receptor 4( TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as ap otent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as as afe adjuvant. In this study,w ec haracterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis.W esynthesized three lipid Amolecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1-and 4'-phosphates.H exaacylated A. faecalis lipid As howed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit adiscrete interleukin-6 release in human cell lines and mice.It was thus found to be the active principle of the LOS/LPS and apromising vaccine adjuvant candidate.

The Toll-like receptor 4 (TLR4)/myeloid differentiation factor-2 (MD-2) complex is essential for LPS recognition and induces innate immune responses against Gram-negative bacteria. As activation of TLR4/MD-2 is also critical for the induction of adaptive immune responses, TLR4/MD-2 agonists have been developed as vaccine adjuvants, but their efficacy has not yet been ascertained. Here, we demonstrate that a funiculosin (FNC) variant, FNC-RED, and FNC-RED and FNC derivatives are agonists for both murine and human TLR4/MD-2. FNC-RED induced nuclear factor-κB (NF-κB) activation via murine TLR4/MD-2, whereas FNC had no TLR4/MD-2 stimulatory activity. Biacore analysis revealed that FNC-RED binds to murine TLR4/MD-2 but not murine radioprotective 105 (RP105)/myeloid differentiation factor-1 (MD-1), another LPS sensor. FNC-RED induced CD14-independent expressions of pro-inflammatory cytokines and co-stimulatory molecules in murine macrophages and dendritic cells. In contrast, FNC-RED stimulation was reduced in CD14-dependent LPS responses, including dimerization and internalization of TLR4/MD-2 and IFN-β expression. FNC-RED-induced IL-12p40 production from murine dendritic cells was dependent on NF-κB but not MAPK pathway. In addition, fetal bovine serum augmented lipid A-induced NF-κB activation but blocked FNC-RED-mediated responses. Two synthetic phosphate group-containing FNC-RED and FNC derivatives, FNC-RED-P01 and FNC-P01, respectively, activated human TLR4/MD-2, unlike FNC-RED. Finally, computational analysis revealed that this species-specific activation by FNC-RED and FNC-RED-P01 resulted from differences in electrostatic surface potentials between murine and human TLR4/MD-2. We conclude that FNC-RED and its synthetic derivative represent a novel category of murine and human TLR4/MD-2 agonist.

Alcaligenes faecalis is the predominant Gramnegative bacterium inhabiting gut-associated lymphoid tissues, Peyersp atches.W ep reviously reported that an A. faecalis lipopolysaccharide (LPS) acted as aweak agonist for Toll-like receptor 4( TLR4)/myeloid differentiation factor-2 (MD-2) receptor as well as ap otent inducer of IgA without excessive inflammation, thus suggesting that A. faecalis LPS might be used as as afe adjuvant. In this study,w ec haracterized the structure of both the lipooligosaccharide (LOS) and LPS from A. faecalis.W esynthesized three lipid Amolecules with different degrees of acylation by an efficient route involving the simultaneous introduction of 1-and 4'-phosphates.H exaacylated A. faecalis lipid As howed moderate agonistic activity towards TLR4-mediated signaling and the ability to elicit adiscrete interleukin-6 release in human cell lines and mice.It was thus found to be the active principle of the LOS/LPS and apromising vaccine adjuvant candidate.