The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity

Abstract

Aluminum hydroxide (alum) is the most widely used adjuvant in human vaccines, but the immune mechanisms that are activated by alum remain poorly understood. Alum has recently been shown to promote caspase-1 activation and IL-1beta secretion, but the cellular pathways involved remain elusive. Here we report that the release of IL-1beta triggered by alum is abrogated in macrophages deficient in the NLR family, pyrin domain containing 3 (Nlrp3) protein and the apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc) but not the NLR family, CARD domain containing 4 (Nlrc4) protein. The requirement of the Nlrp3 inflammasome was specific for IL-1beta in that secretion of TNF-alpha was independent of Nlrp3 or Asc. Consistently, processing of pro-caspase-1 induced by alum was abolished in macrophages lacking Nlrp3 or Asc. Unlike caspase-1 processing and IL-1beta secretion triggered by LPS, alum-mediated activation of the inflammasome did not require exogenous ATP. Importantly, induction of IgG production against human serum albumin by alum was unimpaired in mice deficient in Nlrp3. These results indicate that alum induces IL-1beta via the Nlrp3 inflammasome but this activity is dispensable for alum-mediated adjuvant activity.

Figure 1

Alum-induced IL-1β production is Nlpr3-dependent. Bone-marrow derived macrophages form WT, Nlrp3-KO, Nlrc4-KO, and Asc-KO mice were stimulated with LPS (1 μg/ml), alum (250 μg/ml) or LPS + alum for 6 hrs (A, B). Cell-free supernatants were analyzed for production of IL-1β by ELISA A and TNF-α (B). Results are representative of three separate experiments.

Figure 2

Alum-induced caspase-1 activation is Nlpr3- and Asc-dependent. Bone-marrow derived macrophages from WT, Nlrp3-KO, Nlrc4-KO, Asc-KO were stimulated for 6 hrs with alum (250 μg/ml), LPS (1 μg/ml), or LPS + alum (A); or stimulated for 6 hrs with LPS (1 μg/ml) ± ATP (5 mM) for the last 30 minutes (B). Extracts were prepared from cell and culture supernatants and immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit. Results are representative of three separate experiments.

Figure 3

Alum-induced Ig antibody production is Nlpr3-independent. 12-week old mice were immunized with a mixture of human serum albumin (HSA) (100 μg in PBS) and alum by intraperitoneal injection on day 0 and boosted with antigen on day 21. Blood samples were obtained on day 35 and antibody titers measured in serum. Each point represents the serum antibody titer to HSA for an individual mouse. Results are representative of three independent experiments. NS, not significant