Antimicrobial Peptides for Gram-Negative Sepsis: A Case for the Polymyxins

nology, is a common and serious clinical problem. While fewer than 100 cases were reported prior to 1920 (Felty and Keefer, 1924), it is now the 13th leading cause of overall mortality (Gelfand and Shapiro, 1993) and the number one cause of deaths in the intensive care unit accounting for some 200,000 fatalities in the US annually. The incidence continues to rise in the US (Martin et al., 2003; Figure 1) and worldwide (Moss and Martin, 2004), perhaps due to increased invasive procedures, immunosuppression, and cytotoxic chemotherapy. Mortality associated with sepsis, unfortunately, has essentially remained unchanged at about 45% (Cross and Opal, 1994), despite tremendous strides in antimicrobial chemotherapy, pointing to the absence of therapeutic strategies aimed specifically at the pathophysiology of sepsis. The pathophysiology of the disease is characterized by a systemic inflammatory response syndrome (SIRS), culminating in its frequently fatal sequel, multiple organ dysfunction syndrome (MODS). The systemic inflammatory response is a consequence of dysregulated activation of innate immune effector mechanisms (Castellheim et al., 2009). Counterregulatory mechanisms that are subsequently deployed to dampen the initial overexuberant systemic inflammatory responses are also thought to contribute to the pathophysiology due to late-stage immunosuppressive (hypoinflammatory) phenomena, which render the host unable to eradicate the offending pathogen (Hotchkiss and Karl, 2003; Hotchkiss et al., 2009). The primary trigger of SIRS in the Gramnegative septic shock syndrome is thought to be endotoxin, a constituent of the outer membrane of all Gram-negative bacteria. Endotoxins consist of a polysaccharide portion and a lipid called lipid A, and are therefore also called lipopolysaccharides (LPS). The polysaccharide portion consists of an O-antigen-specific polymer of repeating oligosaccharide units, the composition of which is highly varied among Gram-negative bacteria. A relatively wellconserved core hetero-oligosaccharide covalently bridges the O-antigen-specific chain with lipid A (Rietschel et al., 1994). Total synthesis of the structurally highly conserved lipid A has been shown to be the active moiety of LPS (Rietschel et al., 1987). Whereas LPS itself is chemically inert, the presence of LPS in blood (endotoxemia), often a consequence of antibiotic therapy of preexisting bacterial infections (Holzheimer, 2001), is recognized by Tolllike receptor 4 (TLR4; Beutler and Poltorak, 2001; Palsson-McDermott and O’Neill, 2004; Hennessy et al., 2010), a member of a large super-family of pattern recognition receptors (Jounai et al., 2012; Newton and Dixit, 2012; Olive, 2012). Endotoxemia and its sequelae may arise even in the absence of Gram-negative bacterial infections, conditions such as trauma (Saadia et al., 1990), burns (Jones II et al., 1991), and splanchnic ischemia during cardiac surgery (Rocke et al., 1987) increase intestinal permeability, resulting in the spill-over into the portal circulation of LPS from the colon which is abundantly colonized by Gram-negative bacteria. The sensing of LPS, “read by our tissues as the very worst of bad news” (Thomas, 1975), results in a cascade of exaggerated host responses, manifesting in the clinical syndrome characterized by endothelial damage, coagulopathy, loss of vascular tone, myocardial dysfunction, tissue hypoperfusion, and multiple-system organ failure (Balk and Bone, 1989; Bone et al., 1992; Bone, 1993). LPS activates almost every component of the cellular and humoral (plasma protein) limbs of the immune system, resulting in the production of a plethora of proinflammatory mediators, important among which are not only early-phase cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 (Dinarello, 1991, 1996) but also late-phase endogenous mediators such as high mobility group box 1 protein (HMGB1; (Wang et al., 1999; Andersson and Tracey, 2011). These cytokines and other mediators act in concert, amplifying the resultant generalized inflammatory processes. Our understanding of basic mechanisms underlying the cellular response to LPS has increased vastly in recent years. These advances will likely offer novel therapeutic possibilities in the future. However, after more than two decades of intensive effort at evaluating more than 30 investigational compounds, specific therapeutic options for sepsis have remained elusive. Drotrecogin alfa (XigrisTM, recombinant human activated protein C), an anticoagulant that ameliorates disseminated intravascular coagulation was approved in November 2001 by the FDA, but recently withdrawn due to lack of efficacy (Ranieri et al., 2012; Wenzel and Edmond, 2012). Clinical trials aimed at blocking various proinflammatory mediators including TNF-α, IL-1β, platelet-activating factor, and prostaglandins produced by the activated cellular components have all been disappointing (Zeni et al., 1997), suggesting that targeting downstream cellular inflammatory processes once immune activation has already progressed is unlikely to be of benefit. It follows, therefore, that the paradigm of proximal, upstream intervention using molecules that specifically block the recognition of LPS by TLR4 would offer attractive therapeutic targets. As mentioned Antimicrobial peptides for Gram-negative sepsis: a case for the polymyxins