1. * * * * * * * Results Abstract Description of intervention/study
P2Y2 Purinergic Receptor Gene Deletion Protects Mice from Polymicrobial Sepsis
Athis Arunachalam1, Bryan Tackett, Janielle Maynard and Sundararajah Thevananther
Fellow, Section of Neonatology, Department of Pediatrics, Baylor College of Medicine/Texas Children’s Hospital, Houston, TX
Results
Introduction
(Background and Purpose/objectives)
Sepsis continues to be a major cause of morbidity and mortality across all age groups.
Molecular mechanisms and key humoral mediators of uncontrolled inflammatory response in sepsis remains unknown.
ATP is released into the extracellular milieu from injured cells, infiltrating neutrophils and macrophages.
Extracellular ATP via activation of P2Y2 purinergic receptors plays a pivotal role in inflammation and immunomodulation.
Despite its ubiquitous expression, role of P2Y2 purinergic receptor function in the induction of systemic inflammation and multi-organ injury associated with sepsis has not been well characterized.
Hypothesis
Extracellular ATP-mediated activation of P2Y2 purinergic receptor is essential for the induction of inflammatory cascades and multi-organ injury in polymicrobial sepsis.
Fig 3: Liver Injury is Attenuated in P2Y2-/- mice (21hrs) WT
P2Y2-/-
SHAM
CLP
Liver tissue sections (21 hr post-CLP or Sham, H& E staining), 60X field of view. Hemorrhagic necrosis (white arrows), leukocyte infiltration (black arrows).
Fig 4: Liver, Lung and Kidney Pro-inflammatory Cytokine/Chemokine mRNA Expression is Attenuated in P2Y2-/- mice (21hrs)
Abstract
Liver
Lung
Kidney
Background: Extracellular ATP, via activation of P2Y2 purinergic receptor, plays a pivotal role in inflammation. However, the functional significance of P2Y2 receptor activation is not well understood in sepsis. The overall goal is to test the hypothesis that P2Y2 receptor activation is essential for the induction of inflammatory cascades and multi-organ injury secondary to sepsis. Methods: Sepsis was induced by cecal ligation and puncture (CLP) in adult (10-18 weeks; C57BL6/J) wild type (WT) and P2Y2 knock out (P2Y2-/-) mice. Serum was analyzed for pro-inflammatory cytokine and chemokine levels (MultiplexTM Ma Cytokine Assay ). Tissues (liver, lungs,kidney), blood and peritoneal fluid were collected at 6, 12, and 21 hrs. after CLP. Tissues were analyzed for leukocyte infiltration and pro-inflammatory cytokine/chemokine mRNA expression. WBC counts and bacterial cultures from blood and peritoneal fluid were determined. Results: WT mice subjected to CLP were moribund (100%) between hr. However, P2Y2-/- mice survived longer (70% survival at 7 days). In response to CLP, induction of serum cytokine and chemokine was significantly attenuated (IL-6 and MIP-2) in P2Y2-/- as compared to WT mice at 21 hr. Additionally, attenuation of markers of liver, lung and kidney injury were noted in the P2Y2-/- mice as compared to WT. Attenuated bacterial growth was also noted in P2Y2-/- mice in blood and peritoneum. Conclusion: P2Y2 receptor function is critical for sepsis-induced inflammation and mortality in mice. * *
TNFα
MCP-1
Description of intervention/study *
Age matched adult (10-18 weeks) wild type (WT) male and P2Y2-/-(KO) mice were subjected to cecal ligation and puncture (CLP), a well-established experimental model for the study of polymicrobial sepsis.
Blood and Tissues (liver, lungs and kidneys) were collected at 6, 12 and 21h post-CLP. In survival experiment mice were monitored up to 7days post CLP.
Liver tissue sections were analyzed for injury and leukocyte infiltration (H&E), pro-inflammatory cytokine and chemokine mRNA expression (quantitative RT-PCR) of liver, lung and kidney were determined.
Serum was analyzed for pro-inflammatory cytokine and chemokine levels (MultiplexTM Map Cytokine Assay), liver (ALT, total and direct bilirubin) and renal (blood urea nitrogen, creatinine) function.
Blood and Peritoneal fluid were analyzed for bacterial growth and leukocyte levels.
Fig 5: Attenuated Bacterial Growth in P2Y2-/- mice
Blood
Peritoneal Fluid * * *
Results
Fig 6A: Altered Peritoneal Leukocyte Infiltration in P2Y2-/- mice
Fig 1: P2Y2-/- mice are Resistant to Sepsis-induced Mortality
Fig 6B: Sepsis induced Neutropenia is NOT sustained in P2Y2-/- mice * WT
P2Y2-/-
p<0.05, WT-CLP vs P2Y2-CLP, (all slides)
Kaplan-Meier survival curve. WT-CLP vs P2Y2-/- CLP.
(p < 0.05; n=10).
Conclusions
Fig 2: Serum Pro-inflammatory Cytokine/Chemokine Induction is Attenuated in P2Y2-/- mice
P2Y2 purinergic receptor activation is critical for sepsis induced mortality in mice.
P2Y2 receptor function plays a major role in the pathogenesis of sepsis via its role in the induction of early hyper-inflammatory phase, which precedes multi-organ injury.
These findings will help elucidate the molecular mechanisms of purinergic signaling in sepsis.
Our data highlight a novel role for P2 purinergic receptors as potential therapeutic targets in the management of sepsis WT
P2Y2-/-
* p<0.05, WT-CLP vs P2Y2-CLP, n=3 (SHAM)-10 (CLP)
References
Angus, D.C., et al., Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med, (7): p
Chen, Y., et al., ATP release guides neutrophil chemotaxis via P2Y2 and A3 receptors. Science, (5806): p
Ayata, C.K., et al., Purinergic P2Y(2) receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury. Gastroenterology, (6): p e4.
Boucher, I., et al., The P2Y2 receptor mediates the epithelial injury response and cell migration. Am J Physiol Cell Physiol, (2): p. C
Texas Pediatric Society Electronic Poster Contest