Cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by NLRP3

Abstract

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-1beta and IL-18. In this study we show that, in human and mouse macrophages, alum-induced secretion of IL-1beta, 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.

Figure 1. Alum activates the NLRP3-Inflammasome

Wild type, ASC^−/−, NLRP3^−/−, or NLRC4^−/− BMM were stimulated for 9 hours with LPS (5 ng/ml) in presence or absence of aluminum hydroxide (AlHy, 130 µg/ml), or for 4 hours with L. monocytogenes or S. typhymurium (L.m., S.t.,, MOI=10). (A) IL-1β or IL-6 were measured in conditioned supernatants.(B) The presence of the immature and mature forms of IL-1β and caspase-1 in cell culture supernatants (Sup) or cell lysates (Lys) were analyzed by immunoblot. One experiment representative of five (WT and NLRP3^−/−) or two (ASC^−/−, NLRC4^−/−) is shown. Values are mean ± SEM. Asterisks denote significant difference versus WT. p<0.001

Figure 2. ASC- and NLRP3-knockdown shRNA prevents inflammasome activation by alum in THP-1 cell lines

(A) Expression of ASC or NLRP3 was measured in different THP-1 cell lines by RNase protection assay. NLRP3-a and NLRP3-b are two probes of different sizes both detecting NLRP3. (B) Control-, ASC-, and NLRP3-knockdown THP-1 cell lines were stimulated with LPS (10 ng/ml) in presence of 5 mM ATP, aluminum hydroxide (AlHy, 130 µg/ml), L. monocytogenes, or S typhymurium for 10 hours. IL-1β was measured in conditioned supernatants. One experiment representative of five is shown. Values are mean ± SEM. Asterisks denotes significant difference versus Control-shRNA cell line. p<0.001 (C) THP-1 knockdown cell lines were stimulated as indicated and IL-33 expression was analyzed by immunoblot in cell supernatants.

Figure 3. Inflammasome activation by particulate adjuvants is mediated by NLRP3

Human PBMC (A) or wild type and NLRP3^−/− BMM (B) were stimulated for 9 hours with LPS (5 ng/ml) in presence or absence of aluminum hydroxide (AlHy, 130 µg/ml), QuilA (5 µg/ml), or chitosan (200 µg/ml). IL-1β or IL-18 were measured in conditioned supernatants. Z,Z-YVAD caspase-1 inhibitor. One experiment representative of two is shown. Values are mean ± SEM. Asterisks denote significant difference versus treatment with LPS alone or treatment in presence of Z-YVAD, as indicated *, p<0.001. **,p<0.005

Figure 4. Alum adjuvant effect is mediated by NLRP3

Wild type (n=5) and NLRP3-deficient mice (n=5) were vaccinated (A) with alum-adsorbed diphtheria toxoid (DT), or (B) with ovalbumin adsorbed to alum or Complete Freund adjuvant (CFA). Boost injections were administered at day 14 Total IgE or DT- and ovalbumin-specific IgG₁ were measured in sera obtained at the indicated time points (A) or at 14 days (B). Values are mean ± SEM *, p<0.005. **, p<0.01. ***, p<0.05. N.D., not detectable.