Abstract
Sepsis is a life-threatening condition that requires urgent care. Thus, the identification of specific and sensitive biomarkers for its early diagnosis and management are of clinical importance. The alarmin prothymosin alpha (proTα) and its decapeptide proTα(100-109) are immunostimulatory peptides related to cell death. In this study, we generated bacterial models of sepsis in mice using two Klebsiella pneumoniae strains (L-78 and ATCC 43816) and monitored sepsis progression using proTα(100-109) as a biomarker. Serum concentration of proTα(100-109) gradually increased as sepsis progressed in mice infected with L-78, a strain which, unlike ATCC 43816, was phagocytosed by monocytes/macrophages. Analysis of splenocytes from L-78-infected animals revealed that post-infection spleen monocytes/macrophages were gradually driven to caspase-3-mediated apoptosis. These results were verified in vitro in L-78-infected human monocytes/macrophages. Efficient phagocytosis of L-78 by monocytes stimulated their apoptosis and the concentration of proTα(100-109) in culture supernatants increased. Human macrophages strongly phagocytosed L-78, but resisted cell death. This is the first report suggesting that high levels of proTα(100-109) correlate, both in vitro and in vivo, with increased percentages of cell apoptosis. Moreover, we showed that low levels of proTα(100-109) early post-infection likely correlate with sepsis resolution and thus, the decapeptide could eventually serve as an early surrogate biomarker for predicting bacteria-induced sepsis outcome.
Highlights
Sepsis is a lifethreatening response to infection, leading to excessive tissue damage, organ failure, and death
Animals were infected with the clinically isolated strain L-78, which is of low virulence (50% lethal dose [LD50] > 108 colony-forming units [CFU]), highly resistant to antibiotics and endocytosed by monocytes/ macrophages, and with the ATCC 43816 strain, which is highly infective (LD50 = 5 x 103 CFU), sensitive to antibiotics and not endocytosed
To rule out obvious non-specific interactions between prothymosin alpha (proTα)(100109)-specific Abs and any cross-reacting substance present in murine serum, we assessed if the primary structure of the decapeptide can be depicted within the amino acid sequence of currently known mouse proteins or proteins known to be encoded in K. pneumoniae (Supplementary Table S1)
Summary
Sepsis (from Greek σῆψις, decay) is a lifethreatening response to infection, leading to excessive tissue damage, organ failure, and death. Excessive immune cell activation leads to release of inflammatory mediators, determined usually in blood samples and used as biomarkers of infection. ProΤα(100-109) is generated upon truncation of proTα by activated caspase-3, in cells undergoing apoptosis [6]. Both molecules extracellularly act as alarmins, ligate TLR-4, induce the maturation of dendritic cells, and mount TH1-biased immune responses in vitro [7,8,9]. We analyzed K. pneumoniae infection in mice and in human cells in vitro, focusing on the mechanisms via which these bacteria kill innate immune cells and lead to the generation of proTα(100-109). We further validated our results in a “moderate” model of sepsis in mice, and showed that early post-infection (pi) proTα(100-109) serum levels can predict mortality of individual mice infected with K. pneumoniae
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