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-1?), 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-1?) 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 will improve our understanding of the role of pCTS-L in LBI, and shed light on the intricate mechanism underlying natural product LAN and PRO-mediated protection against lethal bacterial infections.
The pathogenesis of sepsis remains obscure, but is partly attributable to dys-regulated systemic inflammation propagated by ?early? cytokines (e.g., TNF and IL-1?), but sustained by ?late? mediators such as HMGB1. The elucidation of procathepsin L (pCTS-L) as another possible ?late? mediator of lethal bacterial infections, and the intricate mechanism underlying a medicinal herbal product (lanosterol) and its animal derivative (progesterone)-mediated inhibition of pCTS-L-induced inflammation will significantly improve our understanding of the mechanisms underlying the pathogenesis of sepsis, as well as the protective effects of alternative natural therapies.
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