sepsis alters the megakaryocyte- platelet transcriptional axis resulting in platelet lymphotoxicity...
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Sepsis alters the megakaryocyte-platelet transcriptional axis resulting in platelet lymphotoxicity
M. Sharron , 1 A.S Benton, 1 A. A. Wiles, 1 N. Sabzevheri, 2 E. P. Hoffman, 1,2 R. J. Freishtat 1,2
1Children's National Medical Center, Washington, DC; 2George
Washington University, Washington, DC
Sepsis
• Whole-body inflammatory state and the presence of a known or suspected infection.
• 215,000 deaths annually in US (Angus et al 2001)
• Little change in morbidity and mortality over last 20 yrs
• ~30% to 40% mortality (Granja et al, 2004; Angus et al, 2004)
Sepsis, Lymphopenia and Platelets
• Lymphopenia during sepsis associated with poor outcomes (Hotchkiss, et al 1999,2003)
• lymphopenia in sepsis due to apoptosis (Hotchkiss, et al 2006)
• Platelets accumulate in spleen, lung, liver, other end organs in sepsis (Drake et al, 1993; Shibazaki et al 1996)
• Platelet microparticles are cytotoxic in a variety of cell types. (Cognasse, F, et al 2007, Von Hundelshausen P, et al, 2007, Danese S, et al 2004)
Contents of megakaryocytes and platelets
MegakaryocytePlatelets
Contain: DNA
mRNA mRNA
Protein Protein Protein
Platelet microparticles
Experimental Hypothesis:
• Platelet transcriptome can change in response to systemic perturbations
(ie sepsis)
Parallel studies in humans and mice
• Broad effort to define the progression of sepsis at the molecular level
• Mouse model of sepsis (CLP- Cecal Ligation and Puncture)
• Isolation of peripheral blood cell fractions throughout the progression of sepsis in human pediatric populations
Platelet mRNA expression profiling in murine sepsis
• Differential regulation of 59 probe sets (56 genes)
• 6 cell death (GO:0008219)
Septic mice via CLP
Septic humans in PICU
• Granzyme B alone upregulated
GzmB-Induced Cell Death
• cytotoxic serine protease secreted within cytotoxic granules
• known player in well described apoptotic pathways
qT RT PCR validation
Murine platelet granzyme B mRNA expression
Human platelet granzyme B mRNA expression
Gz B upregulated in both platelets AND megakaryocytes during sepsis
• Megakaryocyte granzyme B mRNA expression
Correlates with
• Platelet granzyme B mRNA expression
Intracellular granzyme B protein expression in platelets
• granzyme B protein in platelets from healthy and septic children.
• Day1 (49.7%) and Day 3 (44.3%) in a severely septic patient
Granzyme B upregulated in sepsis
Megakaryocyte plateletsPlatelet microparticles
GzB mRNA GzB mRNA
GzB Protein GzB Protein?
Granzyme B essential for platelet induced apoptosis
Platelet/lymphocyte co-incubation
• Granzyme B necessary for platelets to induce lymphocyte apoptosis
• Presence of platelet activator (TNF α) not necessary for apoptosis
Discussion
Sepsis induces change in megakaryocyte-platelet transcriptional axis
Platelets up-regulate granzyme B in murine and human sepsis
Granzyme B from platelets induces lymphocyte apoptosis
Novel paradigm in sepsis: platelets are active lymphotoxic agents
Future Directions
• Apoptosis in additional cell types?- including lung, kidney, liver, spleen, heart
• Mechanism by which Granzyme B causes apoptosis – contact vs non contact dependent? Micro-particle mediated?
Acknowledgements
• Funding support provided to RJF by grants:– 1K23RR020069 and 1M01RR020359-
010049 from the National Institutes of Health, Bethesda, Maryland
– Board of Visitors and Research Advisory Council of Children’s National Medical Center, Washington, DC