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

Abstract

Significance Cytosolic lipopolysaccharides (LPSs) induce oligomerization of caspase-4/5/11, resulting in pyroptosis, but the involvement of any other host resistance factors in this process is unknown. Galectins bind to components of pathogenic microorganisms, particularly LPSs, in a glycan-dependent manner. However, these proteins are mainly present intracellularly, and little is known regarding their functions associated with binding to components of microorganisms, including LPSs, in the cytosol. Here, we report that galectin-3 recognizes cytosolic LPSs from various bacteria and amplifies LPS-induced caspase-4/11 oligomerization and activation, causing more intense pyroptosis in a carbohydrate-dependent manner. This study defines a unique molecular mechanism based on the carbohydrate-binding and self-association properties of galectin-3, through which this glycan-binding protein enhances cytosolic LPS-induced noncanonical inflammasome activation in macrophages. 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.