Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants

Abstract

Aluminium adjuvants, typically referred to as 'alum', are the most commonly used adjuvants in human and animal vaccines worldwide, yet the mechanism underlying the stimulation of the immune system by alum remains unknown. Toll-like receptors are critical in sensing infections and are therefore common targets of various adjuvants used in immunological studies. Although alum is known to induce the production of proinflammatory cytokines in vitro, it has been repeatedly demonstrated that alum does not require intact Toll-like receptor signalling to activate the immune system. Here we show that aluminium adjuvants activate an intracellular innate immune response system called the Nalp3 (also known as cryopyrin, CIAS1 or NLRP3) inflammasome. Production of the pro-inflammatory cytokines interleukin-1beta and interleukin-18 by macrophages in response to alum in vitro required intact inflammasome signalling. Furthermore, in vivo, mice deficient in Nalp3, ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain) or caspase-1 failed to mount a significant antibody response to an antigen administered with aluminium adjuvants, whereas the response to complete Freund's adjuvant remained intact. We identify the Nalp3 inflammasome as a crucial element in the adjuvant effect of aluminium adjuvants; in addition, we show that the innate inflammasome pathway can direct a humoral adaptive immune response. This is likely to affect how we design effective, but safe, adjuvants in the future.

Figure 1. Aluminium-containing adjuvants stimulate macrophages to produce the pro-inflammatory cytokines IL-1β and IL-18 in a Nalp3 inflammasome-dependent manner

a, Macrophages were stimulated with 50 ng ml⁻¹ LPS for 18 h and then 500 µg ml⁻¹ Imject alum (‘Alum1’) or aluminium hydroxide gel (‘Alum2’) for 8 h. IL-1β released into culture supernatants was measured by ELISA with a minimum detection level of 200 pg ml⁻¹. b, LPS-primed macrophages were stimulated with the indicated amount of Imject alum for 8 h and analysed as in a. c, Unprimed or LPS-primed WT, ASC-deficient, Nalp3-deficient, caspase-1-deficient (Casp1) and Ipaf-deficient macrophages were stimulated with Imject alum (500 µg ml⁻¹) for 8 h, and the IL-1β released into the culture supernatants was measured by ELISA. d, WT or Nalp3-deficient macrophages were stimulated as in c, and the IL-18 released was measured by ELISA. e, WT or Nalp3-deficient LPS-primed macrophages were stimulated with either Imject alum, CFA (120 µg ml⁻¹) or IFA (30 µl ml⁻¹) for 8 h and analysed as in a. Determinations were performed in triplicate and are expressed as means and s.d.; data are from one of at least three independent experiments.

Figure 2. Caspase-1 activation by aluminium adjuvants requires Nalp3 and ASC

a, b, LPS-primed or unprimed macrophages from WT mice (a, b), Nalp3-deficient mice and caspase-1-deficient (Casp1) mice (a) and from ASC-deficient mice (b) were stimulated with Imject alum (500 µg ml⁻¹) for the indicated durations, and cell lysates were immunoblotted with antibodies against the p10 subunit of caspase-1. Macrophages from caspase-1 knockout mice were stimulated as indicated to provide a reference for some of the non-specific bands seen with this antibody. Indeed, the band at 15 kDa in the Nalp3 knockout samples in a and in all samples in b is distinct from the p10 band representing active caspase-1. c, LPS-primed macrophages from WT or Nalp3-deficient mice were stimulated with Imject alum (500 µg ml⁻¹) for the indicated durations, and the IL-1β released into culture supernatants was measured by ELISA. Results are shown as means ± s.d. from one of three independent experiments.

Figure 3. Alum requires intact endocytic macrophage machinery and causes potassium-gradient-dependent IL-1β secretion without causing significant cell death

a, LPS-primed peritoneal macrophages were treated with either colchicine (28 µg ml⁻¹) or cytochalasin B (10 µM) for 1 h before the addition of Imject alum (500 µg ml⁻¹), ATP (5 mM) or MSU (200 µg ml⁻¹). b, Lactate dehydrogenase (LDH) release was measured from LPS-primed WT, Nalp3-deficient and caspase-1-deficient (Casp1) macrophage culture supernatants stimulated with the indicated amounts of Imject alum. c, Unprimed or LPS-primed WT macrophages were stimulated for 8 h with either Imject alum (500 µg ml⁻¹) or MSU (200 µg ml⁻¹) in the presence or absence of 2 U ml⁻¹ uricase. d, LPS-primed macrophages from WT or P2X7R-deficient (P2X7) mice were stimulated with Imject alum (500 µg ml⁻¹) or ATP (5 mM) and samples were analysed as in a. e, Unprimed or LPS-primed WT or Nalp3-deficient macrophages were stimulated with Imject alum in serum-free buffer with either 150 mM NaCl or 150 mM KCl and analysed as in a. Determinations were performed in triplicate and are expressed as means and s.d.; data are from one of at least three independent experiments.

Figure 4. Antibody production and T_H2-dependent inflammation induced by aluminium adjuvants are decreased in the absence of Nalp3, ASC and caspase-1

a, WT, Nalp3-deficient, ASC-deficient or caspase-1-deficient (Casp1) mice (three to five mice per group) 6–8 weeks old were injected intraperitoneally with ovalbumin adsorbed on Imject alum on day 0 and again on day 10. Mice were challenged intranasally with ovalbumin on days 21, 22 and 23. Sera were collected from mice on day 25 and analysed for ovalbumin-specific IgG1 by ELISA as described previously. Asterisk, P < 0.03; nonparametric Mann–Whitney U-test. b, WT, Nalp3-deficient, ASC-deficient or MyD88-deficient mice (three to five mice per group) were primed subcutaneously with ovalbumin in CFA on day 0 and on day 10 in IFA. Sera were collected on day 21 and analysed for ovalbumin-specific IgG2c by ELISA. c, Three to five mice per group were primed and challenged as in a; bronchoalveolar lavage was collected on day 25 and analysed as described previously (see Methods) (total cell number; means and s.d. are shown). Asterisk, P < 0.03; nonparametric Mann–Whitney U-test. d, Lung draining (hilar) lymph nodes were collected from WT and Nalp3-deficient mice primed and challenged as in a and pooled within each group for restimulation; cells were restimulated in vitro with (+) or without (−) 200 µg ml⁻¹ ovalbumin and mitomycin-C-treated splenocytes for 48 h. Supernatants were analysed for IL-5 (filled bars) or IFN-γ (open bars).