Project Abstract Despite recent advances in antibiotic therapy and intensive care, sepsis remains the most common cause of death in the intensive care unit, annually claiming >225,000 victims in the U.S. alone. The pathogenesis of sepsis remains obscure, but is partly attributable to dys-regulated inflammation propagated by “early” cytokines (e.g., TNF and IL-1β), but sustained by “intermediate” (e.g., SAA) and “late” (e.g., HMGB1) mediators. We recently discovered that LPS and SAA upregulated the expression of connexin 43 (Cx43) and/or pannexin 1 (Panx1) hemichannels to facilitate ATP-dependent PKR activation and HMGB1 release, but did not know whether LPS and SAA also induced procathepsin L (pCTS-L) secretion to mediate lethal bacterial infections (LBI). Our preliminary data indicated that LPS and SAA induced a marked expression and secretion of pCTS- L in both murine macrophage and human monocyte cultures. Consequently, pCTS-L was not detectable in the circulation of healthy animals or human subjects, but significantly elevated in the blood of septic animals and patients. Highly purified recombinant pCTS-L stimulated primary human monocytes to release various chemokines, as well as pro- (e.g., TNF and IL-1β) and anti-inflammatory cytokines (e.g., IL-10) in vitro, and exacerbated endotoxemic lethality in vivo. In contrast, pCTS-L-neutralizing antibodies significantly rescued mice from lethal sepsis, suggesting pCTS-L as another late mediator of LBI. Meanwhile, a semi-high throughput screening of a NatProduct Collection of 800 natural products and a US Drug Collection of 1360 bioactive compounds led to the finding of a few lead compounds [including lanosterol (LAN) and progesterone (PRO)] with striking structural resemblance and similar pCTS-L-inhibiting activities. Although LAN is an abundant secondary metabolite in some medicinal plants including wolfberry and aveloz, it also serves as a substrate for the synthesis of PRO in animals and humans. These exciting findings prompted the current proposal to investigate a novel role of pCTS-L in LBI, as well as intricate mechanisms by which a natural product, LAN, and its derivative, PRO, inhibit pCTS-L-induced inflammation. The experiments outlined in Aim 1 will test the hypothesis that pCTS-L systemically accumulates in the circulation of septic patients and correlates with other surrogate markers of sepsis. In Aim 2, we will test the hypothesis that alterations of pCTS-L levels (by supplementation of pCTS-L or genetic knockout) or activities (by use of neutralizing IgGs or natural inhibitor LAN or PRO) divergently influence the outcomes of LBI. The experiments outlined in Aim 3 will test the hypothesis that LAN and/or PRO inhibit the pCTS-L-induced inflammation through impairing the TLR4/ RAGE-dependent hemichannel and PKR activation in vitro, and confer protection against LBI partly by attenuating systemic inflammation and associated dysregulated coagulation in vivo. Collectively, this project wil...