Host extracellular vesicles confer cytosolic access to systemic LPS licensing non-canonical inflammasome sensing and pyroptosis.

Intracellular surveillance for systemic microbial components during homeostasis and infections governs host physiology and immunity. However, a long-standing question is how circulating microbial ligands become accessible to intracellular receptors. Here we show a role for host-derived extracellular vesicles (EVs) in this process; human and murine plasma-derived and cell culture-derived EVs have an intrinsic capacity to bind bacterial lipopolysaccharide (LPS). Remarkably, circulating host EVs capture blood-borne LPS in vivo, and the LPS-laden EVs confer cytosolic access for LPS, triggering non-canonical inflammasome activation of gasdermin D and pyroptosis. Mechanistically, the interaction between the lipid bilayer of EVs and the lipid A of LPS underlies EV capture of LPS, and the intracellular transfer of LPS by EVs is mediated by CD14. Overall, this study demonstrates that EVs capture and escort systemic LPS to the cytosol licensing inflammasome responses, uncovering EVs as a previously unrecognized link between systemic microbial ligands and intracellular surveillance.

Pyroptosis-induced inflammation and tissue damage.

Pyroptosis is a programmed necrotic cell death executed by gasdermins, a family of pore-forming proteins. The cleavage of gasdermins by specific proteases enables their pore-forming activity. The activation of the prototype member of the gasdermin family, gasdermin D (GSDMD), is linked to innate immune monitoring by inflammasomes. Additional gasdermins such as GSDMA, GSDMB, GSDMC, and GSDME are activated by inflammasome-independent mechanisms. Pyroptosis is emerging as a key host defense strategy against pathogens. However, excessive pyroptosis causes cytokine storm and detrimental inflammation leading to tissue damage and organ dysfunction. Consequently, dysregulated pyroptotic responses contribute to the pathogenesis of various diseases, including sepsis, atherosclerosis, acute respiratory distress syndrome, and neurodegenerative disorders. This review will discuss the inflammatory consequences of pyroptosis and the mechanisms of pyroptosis-induced tissue damage and disease pathogenesis.

A bacterial autotransporter impairs innate immune responses by targeting the transcription factor TFE3.

Type I interferons (IFNs) are consequential cytokines in antibacterial defense. Whether and how bacterial pathogens inhibit innate immune receptor-driven type I IFN expression remains mostly unknown. By screening a library of enterohemorrhagic Escherichia coli (EHEC) mutants, we uncovered EhaF, an uncharacterized protein, as an inhibitor of innate immune responses including IFNs. Further analyses identified EhaF as a secreted autotransporter-a type of bacterial secretion system with no known innate immune-modulatory function-that translocates into host cell cytosol and inhibit IFN response to EHEC. Mechanistically, EhaF interacts with and inhibits the MiT/TFE family transcription factor TFE3 resulting in impaired TANK phosphorylation and consequently, reduced IRF3 activation and type I IFN expression. Notably, EhaF-mediated innate immune suppression promotes EHEC colonization and pathogenesis in vivo. Overall, this study has uncovered a previously unknown autotransporter-based bacterial strategy that targets a specific transcription factor to subvert innate host defense.

A TLR4-independent critical role for CD14 in intracellular LPS sensing.

Intracellular lipopolysaccharide (LPS) sensing by the noncanonical inflammasome comprising caspase-4 or -11 governs antibacterial host defense. How LPS gains intracellular access in vivo is largely unknown. Here, we show that CD14-an LPS-binding protein with a well-documented role in TLR4 activation-plays a vital role in intracellular LPS sensing in vivo. By generating Cd14-/- and Casp11-/- mice strains on a Tlr4-/- background, we dissociate CD14's known role in TLR4 signaling from its role in caspase-11 activation and show a TLR4-independent role for CD14 in GSDMD activation, pyroptosis, alarmin release, and the lethality driven by cytosolic LPS. Mechanistically, CD14 enables caspase-11 activation by mediating cytosolic localization of LPS in a TLR4-independent manner. Overall, our findings attribute a critical role for CD14 in noncanonical inflammasome sensing of LPS in vivo and establish-together with previous literature-CD14 as an essential proximal component of both TLR4-based extracellular and caspase-11-based intracellular LPS surveillance.

Mechanisms and Consequences of Noncanonical Inflammasome-Mediated Pyroptosis.

The noncanonical inflammasome, comprising inflammatory caspases 4, 5, or 11, monitors the cytosol for bacterial lipopolysaccharide (LPS). Intracellular LPS-elicited autoproteolysis of these inflammatory caspases leads to the cleavage of the pore-forming protein gasdermin D (GSDMD). GSDMD pore formation induces a lytic form of cell death known as pyroptosis and the release of inflammatory cytokines and DAMPs, thereby promoting inflammation. The noncanonical inflammasome-dependent innate sensing of cytosolic LPS plays important roles in bacterial infections and sepsis pathogenesis. Exciting studies in the recent past have significantly furthered our understanding of the biochemical and structural basis of the caspase-4/11 activation of GSDMD, caspase-4/11's substrate specificity, and the biological consequences of noncanonical inflammasome activation of GSDMD. This review will discuss these recent advances and highlight the remaining gaps in our understanding of the noncanonical inflammasome and pyroptosis.

Intracellular immune sensing promotes inflammation via gasdermin D-driven release of a lectin alarmin.

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a ß-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

Regulation and Role of Chitotriosidase during Lung Infection with Klebsiella pneumoniae.

Chitinases and chitinase-like proteins are an evolutionary conserved group of proteins. In the absence of chitin synthesis in mammals, the conserved presence of chitinases suggests their roles in physiology and immunity, but experimental evidence to prove these roles is scarce. Chitotriosidase (chit1) is one of the two true chitinases present in mammals and the most prevalent chitinase in humans. In this study, we investigated the regulation and the role of chit1 in a mouse model of Klebsiella pneumoniae lung infection. We show that chitinase activity in bronchoalveolar lavage fluid is significantly reduced during K. pneumoniae lung infection. This reduced activity is inversely correlated with the number of neutrophils. Further, instilling neutrophil lysates in lungs decreased chitinase activity. We observed degradation of chit1 by neutrophil proteases. In a mouse model, chit1 deficiency provided a significant advantage to the host during K. pneumoniae lung infection by limiting bacterial dissemination. This phenotype was independent of inflammatory changes in chit1-/- mice as they exerted a similar inflammatory response. The decreased dissemination resulted in improved survival in chit1-/- mice infected with K. pneumoniae in the presence or absence of antibiotic therapy. The beneficial effects of chit1 deficiency were associated with altered Akt activation in the lungs. Chit1-/- mice induced a more robust Akt activation postinfection. The role of the Akt pathway in K. pneumoniae lung infection was confirmed by using an Akt inhibitor, which impaired health and survival. These data suggest a detrimental role of chit1 in K. pneumoniae lung infections.