No 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 1,2 . 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-1β 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.Reprints and permissions information is available at www.nature.com/reprints.Correspondence and requests for materials should be addressed to R.A.F. (richard.flavell@yale.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature. [12][13][14][15] . We formed the hypothesis that the particulate nature of alum might be recognized by NLRs, much like crystalline MSU. To test whether alum activates the Nalp3 inflammasome, we used primary peritoneal macrophages from mice deficient in critical signalling components of the Nalp3 inflammasome. Because inflammasome activation requires two signals for the production of mature IL-1β, we first primed macrophages with lipopolysaccharide (LPS) and then exposed them to aluminium adjuvants. Consistent with previous reports [13][14][15] , aluminium adjuvants induced the production of IL-1β and IL-18 from wild-type (C57BL/6; WT) primary murine macrophages ( Fig. 1a, d), bone-marrow-derived macrophages ( Supplementary Fig. 1a) and bone-marrowderived dendritic cells ( Supplementary Fig. 1b) in vitro. IL-1β secretion was dependent on the dose of alum (Fig. 1b) and peaked between 8 and 10 h of stimulation with alum in WT macrophages, but continued out to 48 h ( Fig. 2c and data not shown). HHS Public AccessIn contrast, macrophages from animals deficient in Nalp3, ASC or caspase-1 failed to produce IL-1β or IL-18 on stimulation with multiple types of aluminium adjuvant (Fig. 1c, d, Supplementary Fig. 1b and data not shown). Another member ...

Inhalation of crystalline silica and asbestos is known to cause the progressive pulmonary fibrotic disorders silicosis and asbestosis, respectively. Although alveolar macrophages are believed to initiate these inflammatory responses, the mechanism by which this occurs has been unclear. Here we show that the inflammatory response and subsequent development of pulmonary fibrosis after inhalation of silica is dependent on the Nalp3 inflammasome. Stimulation of macrophages with silica results in the activation of caspase-1 in a Nalp3-dependent manner. Macrophages deficient in components of the Nalp3 inflammasome were incapable of secreting the proinflammatory cytokines interleukin (IL)-1␤ and IL-18 in response to silica. Similarly, asbestos was capable of activating caspase-1 in a Nalp3-dependent manner. Activation of the Nalp3 inflammasome by silica required both an efflux of intracellular potassium and the generation of reactive oxygen species. This study demonstrates a key role for the Nalp3 inflammasome in the pathogenesis of pneumoconiosis.asbestosis ͉ NLRP3 ͉ caspase-1 ͉ interleukin-1␤

Epithelial tight junctions contain size- and charge-selective pores that control the paracellular movement of charged and noncharged solutes. Claudins influence the charge selectivity and electrical resistance of junctions, but there is no direct evidence describing pore composition or whether pore size or density differs among cell types. To characterize paracellular pores independent of influences from charge selectivity, we profiled the `apparent permeabilities' (Papp) of a continuous series of noncharged polyethylene glycols (PEGs) across monolayers of five different epithelial cell lines and porcine ileum. We also characterized Papp of high and low electrical resistance MDCK cell monolayers expressing heterologous claudins. Papp profiling confirms that the paracellular barrier to noncharged solutes can be modeled as two distinct pathways: high-capacity small pores and a size-independent pathway allowing flux of larger solutes. All cell lines and ileum share a pore aperture of radius 4 Å. Using Papp of a PEG of radius 3.5 Å to report the relative pore number provides the novel insight that pore density along the junction varies among cell types and is not necessarily related to electrical resistance. Expression of claudin-2 results in a selective increase in pore number but not size and has no effect on the permeability of PEGs that are larger than the pores; however, neither knockdown of claudin-2 nor overexpression of several other claudins altered either the number of small pores or their size. We speculate that permeability of all small solutes is proportional to pore number but that small electrolytes are subject to further selectivity by the profile of claudins expressed, explaining the dissociation between the Papp for noncharged solutes and electrical resistance. Although claudins are likely to be components of the small pores, other factors might regulate pore number.