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

Disclosure

• The authors have documented that they have nothing to disclose.

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

Cecal Ligation and Puncture (CLP)

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

Classification of sepsis severity by clustering

Hierarchical Clustering Explorer (http://www.cs.umd.edu/hcil/hce/)