Thursday, April 25, 2024
4/25/2024
Sepsis is a life-threatening illness due to the human body’s extreme inflammatory response to infection. There are >1.5 million cases of sepsis and >250,000 sepsis-related deaths each year in the United States. However, there are no effective therapies. Hyperactive inflammasomes are involved in the pathogenesis of sepsis and could be therapeutically targeted to save lives. Inflammasomes are large cytosolic multiprotein complexes formed in response to infections and cellular stresses that drive auto-activation of inflammatory caspases (Caspase-1, -4, -5), production of inflammatory mediators [interleukin (IL) -1 and -18)], and pyroptosis, a form of cell death. Canonical inflammasomes are assembled by a pattern recognition receptor (PRR), an adaptor ASC (optional) and Caspase-1. Non-canonical inflammasomes are activated by direct binding of a bacterial endotoxin lipopolysaccharide (LPS) to Caspase-4/5 (mouse Caspase-11), which oligomerizes and cleaves Gasdermin D (GSDMD). Active GSDMD induces pore formation in the plasma membrane and cell death. Although much has been learned about the regulatory mechanisms for canonical inflammasomes, little is known for non-canonical inflammasomes. To address this gap, we recently identified an ubiquitin regulatory X (UBX) domain-containing protein 1 (UBXN1) involved in non-canonical inflammasome-mediated sepsis, and believe that validating its role in this process will reveal new treatment strategies. To begin uncovering its function, we generated the first inducible global Ubxn1 knockout mouse model—a critical tool, as early global knockout is embryonically lethal. Using this model, we found that Ubxn1-/- mice were highly resistant to lethal LPS endotoxemia and cecal- ligation-and-puncture (CLP)-elicited polymicrobial sepsis, compared to sex- and age-matched Ubxn1+/+ littermates. Accordingly, the levels of IL-1, IL-18, and GSDMD/Caspase-11 activation were reduced in Ubxn1-/- mice. Of note, GSDMD and Caspase-11 activation in response to LPS was reduced in primary Ubxn1-/- macrophages as well. This UBXN1 function is conserved in human cells. Both Caspase-4/GSDMD activation and pyroptosis were impaired in IFN-¿-primed, LPS-transfected UBXN1-/- HeLa cells. Moreover, UBXN1 interacted with LPS and Caspase-4. These results support our hypothesis that UBXN1 positively regulates non- canonical inflammasome signaling and thus contributes to sepsis pathogenesis. We will prove this by pinpointing the molecular mechanism of UBXN1 action on the Caspase-4/11-LPS, and by characterizing the physiological role of UBXN1 in CLP sepsis. UBXN1 protein is highly conserved between rodents and humans (93% identical), so is its function in inflammasome signaling. By using the CLP method, our studies should provide pre-clinical evidence relevant to human sepsis. Successful completion of this R21 will lay a solid foundation for more in-depth mechanistic and functional studies in a future R01 application.
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