Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns that can elicit a host defense response through binding to cell-surface Toll-like receptor 4 (TLR4). Systemic inflammatory response syndrome is induced by overstimulation of the innate immune response via LPSs, resulting in severe multiple organ failure, which is a major cause of death worldwide in intensive care units (1). LPS-induced dimerization of TLR4 initiates signal transduction involving the NF-κB– and MyD88-dependent and -independent pathways, thereby contributing to various inflammatory responses (2). Another set of the immune repertoire, which resides in the cytosol and comprises NLRP1, NLRP3, NAIP/NLRC4, and AIM2, is known as the inflammasome. Inflammasomes can be activated in response to a number of well-defined pathogen-derived ligands and physiological aberrations, which in turn trigger caspase-1–mediated pyroptotic death (3, 4). This process has been associated with strengthening the host defense program to eliminate intracellular bacteria.Recently, a cytosolic LPS-sensing pathway involving caspase-4/5 in humans and caspase-11 in mice was termed the noncanonical inflammasome pathway, and this pathway is independent of TLR4 (5–8). LPSs from extracellular bacteria can enter the cytoplasm and trigger caspase-4/5/11–dependent responses. LPSs can be delivered into the cytosol when LPS-containing outer membrane vesicles (OMVs) from gram-negative bacteria are taken up by the cells or when intracellular bacteria escape from the phagosomes that are damaged by host resistant factors such as guanylate-binding protein and HMGB1 or microbe-derived hemolysins (9–12). LPSs comprise three regions: lipid A, core oligosaccharide, and O-polysaccharide (also termed O-antigen). The lipid A moiety binds directly to the caspase-4/5/11 caspase activation and recruitment domain (CARD, also known as prodomain), leading to caspase oligomerization and activation (7). This event likely mimics the proximity-induced dimerization model of initiator caspase activation (13). Furthermore, caspase-4/5/11 executes downstream signaling events via gasdermin D. Activated inflammatory caspase proteolytically cleaves gasdermin D to create an N-terminal fragment that self-oligomerizes and then inserts into the cell membrane to form pores, causing lytic cell death (14–17). Various stimuli have been identified in the caspase-1–mediated canonical-inflammasome signaling pathway (3, 4), but the detailed mechanism underlying noncanonical inflammasome activation mediated by caspase-4/5/11 remains unclear.Galectins, a family of β-galactoside–binding proteins, can decode host-derived complex glycans and are involved in various biological responses (18–23). Galectins are nucleocytoplasmic proteins synthesized without a classical signal sequence, although they can be secreted through unconventional pathways (19, 21, 23, 24). Recent studies have revealed prominent roles of cytosolic galectins in host defense programs (12, 25, 26). The proposed molecular mechanisms involve the binding of galectins to host glycans exposed to the cytosolic milieu upon endosomal or phagosomal membrane damage. In addition to binding host glycans, galectins also recognize microbial glycans, particularly LPSs (27–30). However, the contribution of galectins to the host response through binding to cytosolic LPSs is unknown.Galectin-3 is an ∼30-kDa protein that contains a carbohydrate-recognition domain (CRD) connected to N-terminal proline, glycine, and tyrosine-rich tandem repeats. Upon binding to multivalent glycoconjugates through its CRD, the protein forms oligomers, which is attributable to the self-association property of its N-terminal region (31, 32). Galectin-3 binds to LPSs of various gram-negative bacteria by recognizing their carbohydrate residues (33–36).Although structural information is scarce (37), existing information suggests that ligand-induced oligomerization of caspase CARD is necessary for the activation of inflammatory caspases (7, 38). Therefore, we hypothesized that galectin-3 may be an intracellular LPS sensor that participates in LPS-induced CARD-mediated inflammatory caspase activation. Specifically, highly ordered arrays of LPS–galectin-3 complexes may amplify caspase-4/5/11 oligomerization and activation. Here, we investigated the formation of galectin-3–LPS–caspase-4/11 complexes in cell-based and cell-free systems. Our findings provide evidence regarding a role of galectin-3 as an intracellular mediator in noncanonical inflammasome activation through LPS glycan recognition.