Extracellular Histones Enhance LPS-induced Cytokine Production

Over the last twenty years, the debilitating clinical condition known as sepsis has tripled in frequency to become the tenth leading cause of death in the United States. Extracellular histones were previously reported to mediate cell damage and organ dysfunction during hyperinflammatory response, a characteristic feature of sepsis. Lipopolysaccharide (LPS) is an endotoxin found in Gramnegative bacteria, which account for most sepsis cases. In this study we evaluate the histone-to-LPS relationship and investigate the molecular mechanisms responsible for histone-mediated hyper-inflammation. Cytokine levels as a result of histone plus LPS stimulation were compared via ELISA assays. The impact of histone classes (H1, H2A, H2B, H3, H4) and kinase inhibitors (SB202190, U0126, LY294002) on cytokine levels were also tested. Western blotting and Coomassie blue staining were used to detect possible protein receptors. Histone plus LPS stimulation synergistically yielded cytokine levels over 1000-fold greater than separate histone or LPS stimulation, respectively. Cells given H2A and H2B on average contained cytokine (TNF-α, IL-8) levels over 1000-fold higher when compared to LPS treatment. Treatments with kinase inhibitors indicate that ERK1/2 and p38 are associated with histone signaling in cytokine production. Two target proteins were indicated with MW 37 kD and ~90 kD, which will require further investigation. The roles of ERK1/2 and p38 in inflammatory signaling also need further study to determine the molecular mechanisms responsible for histone-mediated hyper-inflammatory responses in septic patients and ultimately to develop therapeutic interventions for sepsis. Introduction Sepsis, a typical example of systemic inflammatory response syndrome (SIRS) caused by infection, is a major challenge in the intensive care unit (ICU), where it is one of the leading causes of death1. Sepsis is increasingly prevalent with an overall 2.3fold increase in cases from 2000 to 20072. Early warning signs are often nonspecific and inconspicuous, exacerbating late diagnosed treatment3. As the number of sepsis hospitalizations increases, mortality rates remain high, far exceeding those of common medical conditions such as myocardial infarction and cardiovascular accident2. Despite advanced medical care, severe sepsis leads to roughly 225,000 annual deaths among 750,000 patients in the US alone1. While sepsis results from various infections, such as bacterial infection and fungal infection, Gram-negative bacterial infection is the most common cause of sepsis, and outbreaks of Escherichia Extracellular Histones Enhance LPS-induced Cytokine Production Peter Zhou1,3*, Serena McCalla2, and Jun Xu4 Student1, Teacher2: Jericho High School, Jericho, New York Intern3, Mentor/Research Assistant Member4: Oklahoma Medical Research Foundation, Oklahoma 73104 *Correspondence: [email protected] INTERNSHIP ARTICLE coli (E. coli), the most common Gram-negative bacterium, are currently a major issue among global health concerns, causing roughly 73,480 illnesses, 2,168 hospitalizations, and 61 deaths annually in the United States alone4,5,6. Lipopolysaccharide (LPS), an endotoxin found on the surface of Gram-negative bacteria such as E. coli, is known to activate the innate immune system and elicits strong immune responses in humans via Toll-like receptor (TLR) signaling pathways6,7. Consequently, LPS challenge is currently widely implemented in research investigating the hyperinflammatory response exhibited in sepsis8. In addition, previous studies have reported marked increase of histone levels during LPS-induced septic shock3,9. During an innate immune response, neutrophils can release histones, which serve to kill microbes caught in neutrophil extracellular traps (NETs)10. However histones found in NETs, especially H3 and H4, are also cytotoxic to tissue11. Xu et al. (2011) revealed that antibodies used to block extracellular histones could rescue animals from LPS-mediated death. Thus histones released in response to bacterial challenge mediate endothelial dysfunction, organ failure, and death during sepsis12. Therefore, during massive cellular activation triggered by either infectious pathogens or non-infectious stimuli, extracellular histones are released, inducing a cytokine storm and amplifying inflammation through inflammatory receptors such as TLR4 in SIRS13. Binding of LPS to TLR4 on the surface of monocytes triggers the recruitment of adaptor molecules, such as MyD88 and activates various kinases, including IRAK-4 and TRAF614. Mitogen-activated protein kinase (MAPK) pathways are then activated, leading to the activation of transcription factors, including NF-κB15. MAPKs have been implicated as key regulators of the production of pro-inflammatory cytokines16. Extracellular signal-regulated kinases (ERKs) and p38K are two major classes of MAPKs that play important roles in transducing inflammation17. Schabbauer et al. (2004)15 reported that the phosphatidyl-inositol-3 kinase (PI3K) pathway suppresses LPS signaling, effectively regulating LPS-induced inflammatory response. Both MAPK and PI3K signaling pathways may be blocked via available specific inhibitors, making them key molecular targets for therapeutic intervention. With the recent finding of histones mediating cell death in sepsis, a unique connection between histones and LPS seems likely9. We, therefore, hypothesized that histones actively interact with LPS to facilitate cytokine production. More importantly, despite numerous investigations performed studying the roles MAPK and PI3K pathways play in sepsis, the molecular mechanisms responsible for histone-mediated hyper-inflammation in septic patients remain unclear11. Herein, through ELISA and Western blot analysis, we examine the specific effects of histones on LPS-