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Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis

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Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis Akiko Iwata1, R. Angelo de Claro2, Vicki L. Morgan-Stevenson1, Joan C. Tupper2, Barbara R. Schwartz2, Li Liu2, Xiaodong Zhu2, Katherine C. Jordan2, Robert K. Winn1, John M. Harlan2* 1 Department of Surgery, University of Washington, Seattle, Washington, United States of America, 2 Department of Medicine, University of Washington, Seattle, Washington, United States of America Abstract Background: Severe sepsis and septic shock are major causes of morbidity and mortality worldwide. In experimental sepsis there is prominent apoptosis of various cell types, and genetic manipulation of death and survival pathways has been shown to modulate organ injury and survival. Methodology/Principal Findings: We investigated the effect of extracellular administration of two anti-apoptotic members of the BCL2 (B-cell lymphoma 2) family of intracellular regulators of cell death in a murine model of sepsis induced by cecal ligation and puncture (CLP). We show that intraperitoneal injection of picomole range doses of recombinant human (rh) BCL2 or rhBCL2A1 protein markedly improved survival as assessed by surrogate markers of death. Treatment with rhBCL2 or rhBCL2A1 protein significantly reduced the number of apoptotic cells in the intestine and heart following CLP, and this was accompanied by increased expression of endogenous mouse BCL2 protein. Further, mice treated with rhBCL2A1 protein showed an increase in the total number of neutrophils in the peritoneum following CLP with reduced neutrophil apoptosis. Finally, although neither BCL2 nor BCL2A1 are a direct TLR2 ligand, TLR2-null mice were not protected by rhBCL2A1 protein, indicating that TLR2 signaling was required for the protective activity of extracellularly adminsitered BCL2A1 protein in vivo. Conclusions/Significance: Treatment with rhBCL2A1 or rhBCL2 protein protects mice from sepsis by reducing apoptosis in multiple target tissues, demonstrating an unexpected, potent activity of extracellularly administered BCL2 BH4-domain proteins. Citation: Iwata A, de Claro RA, Morgan-Stevenson VL, Tupper JC, Schwartz BR, et al. (2011) Extracellular Administration of BCL2 Protein Reduces Apoptosis and Improves Survival in a Murine Model of Sepsis. PLoS ONE 6(2): e14729. doi:10.1371/journal.pone.0014729 Editor: Lisa F. P. Ng, Singapore Immunology Network, Singapore Received May 25, 2010; Accepted January 31, 2011; Published February 24, 2011 Copyright: ß 2011 Iwata et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: National Institutes of Health grants GM042686 (RKW, JMH) and GM071398 (JMH) supported this work. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The University of Washington holds intellectual property related to the use of BCL2 proteins as therapeutic agents in diseases associated with cell death and inflammation. AI, VM-S, JCT, JMH, and RKW are employees of the University of Washington and inventors on patent applications submitted by the University of Washington related to this intellectual property. The authors confirm their adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: [email protected] Introduction survival pathways has marked effects in murine models of sepsis (e.g., [12]). These observations suggest that pharmacological Severe sepsis and its major complications of multiple organ inhibition of apoptosis by blocking cell death pathways or dysfunction and shock are major causes of morbidity and mortality promoting cell survival pathways could provide a new therapeutic worldwide. Although there have been substantial improvements in approach to sepsis in man [5]. antimicrobial therapy and supportive critical care, severe sepsis BCL2 and BCL2A1 (A1, Bfl-1) are both anti-apoptotic remains the leading cause of death among hospitalized patients in members of the larger BCL2 family of intracellular proteins. non-coronary intensive care units. Unfortunately, decades of Transgenic over-expression of BCL2 in T-cells and B-cells [13], clinical trials with a variety of drug candidates have largely failed intestinal epithelium [14] and myeloid cells [15] all resulted in a to reduce mortality [1,2]. Consequently, there is an urgent need to decrease in apoptosis in those cell types and improved survival in understand better the pathophysiology of severe sepsis and identify septic mice. Thus, increased intracellular BCL2 in multiple new therapeutic targets. lineages is able to confer protection in experimental sepsis. One of the hallmarks of experimental sepsis is increased apop- There is growing evidence that some proteins with well-defined tosis of a number of cell types. Although apoptosis of immune cells intracellular functions can exert new activities when released has been investigated most extensively [3,4], there is also apoptosis extracellularly, engaging the innate immune system to act as of other cell types [5], including endothelial cells [6,7], neutrophils danger signals to promote host defense or repair, i.e., alarmins [8], hepatocytes [9], cardiomyocytes [10], and intestinal epithelial [16] or danger-associated molecular patterns (DAMPs) [17], many cells [11]. Moreover, genetic manipulation of cell death and of which are hydrophobic molecules [18]. For example, high PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e14729 BCL2 Protein in Sepsis mobility group box 1 (HMGB1) is a nucleoprotein that is released rhBCL2A1 protein also provides striking protection in experi- extracellularly when cells undergo necrosis or, in some cell types, mental sepsis induced by CLP, and that this protective effect is by a non-canonical secretory pathway. High levels of HMGB1 associated with marked reduction of apoptosis in heart, intestine, accumulate in animals [19] and humans with severe sepsis [20,21]. and peritoneal neutrophils. These studies extend the range of Administration of antibodies against HMGB1 results in increased experimental models of tissue and organ injury in which BCL2 survival and protection against organ damage in septic animals BH4-domain proteins exert a protective effect, further highlighting [22]. However, even for this prototypic DAMP the mechanism(s) their novel and potent extracellular activity. by which it acts in vivo remains uncertain as highly purified HMGB1 lacks significant activity in vitro [23-25]. Recently, we Results reported that the intracellular BCL2 and BCL2A1 proteins and peptides derived from them that contain the BH4-domain function Treatment with extracellular rhBCL2A1 or rhBCL2 protein as cytoprotective DAMPs, conferring tissue protection when improves survival after CLP administered extracellularly [26]. At very low, cytokine-like doses Mice were subjected to CLP and followed for 7 days as extracellular administration of rhBCL2 or rhBCL2A1 protein or described in Methods. An assessment table was used as a surrogate peptides containing the BH4 domain of BCL2 or BCL2A1 marker for death and the animals euthanized according to reduced apoptosis and tissue damage in stringent murine models approved criteria. In the initial experiments, mice were treated of hind limb and myocardial ischemia-reperfusion injury. Further, with 1 mg(,40 picomoles) of rhBCL2A1 or saline vehicle at 18 in preliminary studies rhBCL2A1 protein also reduced hypertro- hours prior to CLP and then every 12 hours for 3 consecutive days phy and fibrosis in pressure overload-induced heart failure [26]. for a total of eight doses. Treatment with the BH4-domain protein, Here, we show that extracellular administration of rhBCL2 or rhBCL2A1, was protective (Fig. 1A), whereas treatment with the Figure 1. Treatment with rhBCL2A1 or rhBCL2 protein improves survival following CLP. Mice were subjected to CLP and followed for 7 days. An assessment table was used as a surrogate marker for death and the animals euthanized according to approved criteria. (A) Mice were treated by i.p. injection of 1 mg rhBCL2A1 or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to saline. *p = 0.012. (B) Mice were treated by i.p. injection of 1 mg rhBim or saline 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBim did not improve survival compared to saline. (C) Mice were treated by i.p. injection of 1 mg rhBCL2 or 0.5 mg of rhUbiquitin at 18 hours prior to CLP, at time of CLP, and then every 12 hours for 3 consecutive days. Treatment with rhBCL2 conferred significant protection compared to rhUbiquitin *p = 0.003. (D) Mice were treated by i.p. injection of 1 mg rhBCL2A1 or rhBim at 18 hours prior to CLP and then every 12 hours for 3 consecutive days. Treatment with rhBCL2A1 conferred significant protection compared to rhBim. *p = 0.005. doi:10.1371/journal.pone.0014729.g001 PLoS ONE | www.plosone.org 2 February 2011 | Volume 6 | Issue 2 | e14729 BCL2 Protein in Sepsis BH3-domain protein, rhBim, was not (Fig. 1B). In separate neutrophils in the peritoneal lavage fluid (Fig. 5B). We did not experiments, rhBCL2A1 and rhBCL2 were compared directly observe any changes in circulating neutrophil counts, and G-CSF with other recombinant
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