Sepsis alters the megakaryocyte-platelet transcriptional axis resulting in platelet lymphotoxicity

1. 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

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

3. 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)

4. 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)

5. Contents of megakaryocytes and platelets Megakaryocyte Platelets Platelet microparticles Contain: DNA mRNA mRNA Protein Protein Protein

6. Experimental Hypothesis: • Platelet transcriptome can change in response to systemic perturbations (ie sepsis)

7. 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

8. Cecal Ligation and Puncture (CLP)

9. 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

10. cytotoxic serine protease secreted within cytotoxic granules known player in well described apoptotic pathways GzmB-Induced Cell Death

11. qT RT PCR validation Murine platelet granzyme B mRNA expression Human platelet granzyme B mRNA expression

12. Gz B upregulated in both platelets AND megakaryocytes during sepsis Megakaryocyte granzyme B mRNA expression Correlates with Platelet granzyme B mRNA expression

13. 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

14. Granzyme B upregulated in sepsis Megakaryocyte platelets Platelet microparticles GzB mRNA GzB mRNA GzB Protein GzB Protein?

15. 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

16. 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

17. 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?

18. 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

21. Classification of sepsis severity by clustering Hierarchical Clustering Explorer (http://www.cs.umd.edu/hcil/hce/)