Alum is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. We have recently shown that alum activates caspase-1 and induces secretion of mature IL-1β and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1β, IL-18, and IL-33 is mediated by the NLR (nucleotide-binding domain leucine-rich repeat-containing) protein NLRP3 and its adaptor ASC, but not by NLRC4. Other particulate adjuvants, such as QuilA and chitosan, induce inflammasome activation in a NLRP3-dependent fashion, suggesting that activation of the NLRP3-inflammasome may be a common mechanism of action of particulate adjuvants. Importantly, we demonstrate that Ag-specific Ab production elicited by vaccines that contain alum is significantly impaired in NLRP3-deficient mice. Our results demonstrate for the first time a role for the NLRP3-inflammasome during development of the immune response elicited by alum-enhanced vaccination and suggest that therapeutic intervention aimed at NLRP3 may improve adjuvant efficacy.

Aluminum hydroxide (Alum) is the only adjuvant approved for routine use in humans, although the basis for its adjuvanticity remains poorly understood. In this study, we show that Alum activates caspase-1 and induce secretion of mature IL-1β and IL-18. Human PBMC or dendritic cells stimulated with pure TLR4 and TLR2 agonists released only traces of IL-1β or IL-18, despite the fact that the IL-1β mRNA was readily induced by both TLR agonists. In contrast, cells costimulated with TLR agonists plus Alum released large amount of IL-1β and IL-18. Alum-induced IL-1β and IL-18 production was not due to enhancement of TLR signaling but rather reflected caspase-1 activation and in mouse dendritic cells occurred in a MyD88-independent fashion. Secretion of other proinflammatory cytokines such as IL-8 was not affected by Alum treatments. However, TLR-induced production of IL-10 was increased and that of IFN-γ-inducible protein decreased by Alum cotreatment. Considering the immunostimulatory activities of these cytokines and the ability of IL-1β to act as adjuvant, our results suggest a mechanism for the adjuvanticity of Alum.

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Cells undergoing necrosis release endogenous danger signals that possess proinflammatory potential. In this study we show that mature IL-1β and IL-18 are released by necrotic cells but not by apoptotic cells. We identify 7-bromoindirubin-3′-oxime, an indirubin oxime derivative that induces necrosis, as a potent inducer of caspase-1 activation and release of mature IL-1β and IL-18. Inflammasome activation was triggered by other necrosis-inducing treatments but was not observed in response to apoptosis-inducing stimuli. Necrosis-induced inflammasome activation was mediated by the NLRP3 and ASC molecules. Release of IL-18 and IL-1β in response to necrosis-inducing stimuli was observed in THP-1 macrophages and the MSTO-211H human mesothelioma cell line independently of LPS priming. Using the in vivo model of naphthalene-induced airway epithelial cell injury, we showed that necrosis activates the ASC inflammasome in vivo. Our study identifies a new mechanism through which necrosis generates proinflammatory molecules that contributes to the sterile inflammatory response.

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipidated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9 -17 in the extracellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Francisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.