Pyroptosis is a lytic form of cell death that is induced by inflammatory caspases upon activation of the canonical or noncanonical inflammasome pathways. These caspases cleave gasdermin D (GSDMD) to generate an N-terminal GSDMD fragment, which executes pyroptosis by forming membrane pores. We found that calcium influx through GSDMD pores serves as a signal for cells to initiate membrane repair by recruiting the endosomal sorting complexes required for transport (ESCRT) machinery to damaged membrane areas, such as the plasma membrane. Inhibition of the ESCRT-III machinery strongly enhances pyroptosis and interleukin-1β release in both human and murine cells after canonical or noncanonical inflammasome activation. These results not only attribute an anti-inflammatory role to membrane repair by the ESCRT-III system but also provide insight into general cellular survival mechanisms during pyroptosis.

The human non-canonical inflammasome controls caspase-4 activation and gasdermin-Ddependent pyroptosis in response to cytosolic bacterial lipopolysaccharide (LPS). Since LPS binds and oligomerizes caspase-4, the pathway is thought to proceed without dedicated LPS sensors or an activation platform. Here we report that interferon-induced guanylate-binding proteins (GBPs) are required for non-canonical inflammasome activation by cytosolic Salmonella or upon cytosolic delivery of LPS. GBP1 associates with the surface of cytosolic Salmonella seconds after bacterial escape from their vacuole, initiating the recruitment of GBP2-4 to assemble a GBP coat. The GBP coat then promotes the recruitment of caspase-4 to the bacterial surface and caspase activation, in absence of bacteriolysis. Mechanistically, GBP1 binds LPS with high affinity through electrostatic interactions. Our findings indicate that in human epithelial cells GBP1 acts as a cytosolic LPS sensor and assembles a platform for caspase-4 recruitment and activation at LPS-containing membranes as the first step of noncanonical inflammasome signaling.

Pathogenic and commensal Gram-negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase-11 non-canonical inflammasome. Here, we show that OMV-induced inflammasome activation requires TLR4-TRIF signaling, the production of type I interferons, and the action of guanylate-binding proteins (GBPs), both in macrophages and Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram-negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell-autonomous immunity and innate immunity.

SummaryMacrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.