Fungal Zymosan and Mannan Activate the Cryopyrin Inflammasome

Some fungal species are opportunistic pathogens that can cause infection in people with compromised immune systems. Activation of caspase-1 and the subsequent secretion of mature interleukin (IL)-1β is a major signaling pathway of the innate immune system, but how yeasts induce caspase-1 activation is unknown. We show here that stimulation of macrophages and dendritic cells with heat-killed Saccharomyces cerevisiae or the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1β secretion when combined with ATP. Macrophages deficient for the inflammasome adaptor ASC were defective in caspase-1 activation and IL-1β secretion, suggesting involvement of an ASC-dependent inflammasome. Indeed, caspase-1 activation was abrogated in macrophages lacking the NOD-like (NLR) protein Cryopyrin/Nalp3 and in wild type macrophages pretreated with the pannexin-1 inhibitor probenecid. IL-1β secretion further required the Toll-like receptor (TLR) adaptors MyD88 and TRIF, and partially relied on TLR2. We previously showed that bacterial molecules such as lipopolysaccharide (LPS) and peptidoglycan induce activation of caspase-7 through the Cryopyrin inflammasome. Similarly, Cryopyrin and ASC were required for activation of caspase-7 in macrophages stimulated with zymosan or mannan and ATP. These results demonstrate that the conserved fungal components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1β secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.Pathogen recognition by the innate immune system relies on a limited number of fixed germline-encoded receptors, which have evolved to identify so-called pathogen-associated molecular patterns (PAMPs),² conserved microbial structures not shared by the host and essential for their survival (1). Examples of PAMPs are LPS from Gram-negative bacteria, peptidoglycan (PGN) from Gram-positive bacteria, and zymosan and mannan from fungi. Several structurally and functionally diverse classes of pattern-recognition receptors (PRRs) exist that induce various host defense pathways, including the Toll-like receptors (TLRs) located in the plasma membrane and intracellular organelles and the more recently identified intracellular family of NOD-like receptors (NLRs) (2).Previous studies have shown that gain-of-function mutations within the NLR protein Cryopyrin/NALP3 are associated with three autoinflammatory disorders characterized by skin rashes and prolonged episodes of fever in the absence of any apparent infection (3, 4). These hereditary periodic fever syndromes are Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FACS), and neonatal-onset multisystem inflammatory disease (NOMID), and they are collectively referred to as the Cryopyrin/NALP3-associated periodic syndromes (CAPS). Subsequent studies revealed that the Cryopyrin/Nalp3 plays a crucial role in the assembly of a large (700 kDa) cytosolic protein complex termed the “inflammasome” (5–7). The bipartite adaptor protein ASC bridges the interaction between Cryopyrin/Nalp3 and caspase-1 in the inflammasome; thus allowing the recruitment and autoproteolytic activation of the cysteine protease (2). Activated caspase-1 subsequently mediates the maturation and secretion of the pro-inflammatory cytokines interleukin (IL)-1β and IL-18 (8–10). Interestingly, the Cryopyrin/Nalp3 inflammasome mediates caspase-1 activation in response to a variety of bacterial PAMPs such as LPS and PGN when combined with a second stimulus such as the P2X₇ receptor ligand ATP (11–14). Cryopyrin/Nalp3 also mediates caspase-1 activation and IL-1β secretion in macrophages stimulated with viral RNA and ATP (15) or exposed to crystalline substances including uric acid, silica and asbestos (16–18). In contrast, the related NLR protein Ipaf is required for caspase-1 activation in macrophages infected with the intracellular pathogens Salmonella, Legionella, and Shigella (19–21).Although the roles of specific inflammasomes in response to bacterial and viral PAMPs have been described, the inflammasome complexes that recognize fungal PAMPs to induce caspase-1 activation and IL-1β secretion are unknown. Here we show that heat-killed Saccharomyces cerevisiae and the purified cell wall components zymosan and mannan induced caspase-1 activation and IL-1β secretion from macrophages and dendritic cells upon co-stimulation with ATP. Macrophages deficient for the inflammasome adaptor ASC or the NLR protein Cryopyrin/Nalp3 were defective in zymosan- and mannan-induced caspase-1 activation and IL-1β secretion, whereas TNF-α secretion remained unaffected. Although macrophages lacking the TLR adaptors MyD88 or TRIF still activated caspase-1, zymosan- and mannan-induced secretion of IL-1β was significantly hampered. These results demonstrate that the conserved fungal cell wall components zymosan and mannan require ASC and Cryopyrin for caspase-1 activation and IL-1β secretion and suggest an important role for the Cryopyrin inflammasome during fungal infections.