D. S. Heffernan1, T. T. Chun1, C. Chung1, A. Ayala1, J. Lomas-Neira1 1Brown University School Of Medicine,Surgical Research,Providence, RI, USA
Introduction:
We have previously demonstrated a central role for invariant Natural Killer T-cells (iNKT-cells) in modulating the immune response to, and mortality from, intra-abdominal sepsis. Across a spectrum of diseases iNKT-cells can affect the neutrophil response. End organ pulmonary dysfunction, is well recognized as contributing to sepsis related morbidity and mortality. The Angiopoietin (Ang)-1 and Ang-2 pathways are central to the pulmonary vascular endothelial cell response to injury, mediated via neutrophil response and gap junction integrity. Ang-1 promotes anti-inflammatory, pro-survival and anti-permeability reponses, whereas Ang-2 signaling promotes vessel destabilization, vessel leakage and inflammation. Given this, we hypothesize that iNKT-cells mediate their sepsis survival effect via direct/indirect actions on the Ang-1/Ang-2 pathway.
Methods:
Sepsis, induced by Cecal Ligation and Puncture(CLP), versus sham laparotomy, in 8-12 week old C57BL/6 wild type(WT) and iNKT-knockout (iNKT-/-) mice. 24 hours later, blood was collected to measure oxygenation (PaO2/FiO2) and lungs were harvested. Histology, cytokine and Western blot analysis was undertaken for Ang-1/Ang-2, their ligands (Tie-1/Tie-2) as well as Vinculin (marker of endothelial integrity).
Results:
Sepsis did not induce gross histological damage in either WT or iNKT-/- mouse lungs. Further, there was no difference in oxygenation levels (PaO2/FiO2) following sepsis between WT and iNKT-/- mice. However, although sepsis induced a minimal pulmonary neutrophil influx in WT mice, within the iNKT-/- septic mice, a marked influx of neutrophils was noted. Whereas sepsis in WT was associated with increases in KC and MIP-2 levels, no changes were noted in iNKT-/- mice. Within WT, sepsis induced a decline in the pro-survival, anti-inflammatory Ang-1 levels, whereas in iNKT-/- mice, no change in Ang-1 expression was noted. Sepsis was not associated with any change in Ang-2 expression within either WT or iNKT-/- mice. With respect to Angiopoietin receptors, within WT mice sepsis induced a marked increase in Tie-1 and a marked decrease in Tie-2 expression. However, within iNKT-/- mice there was no change in either Tie-1 or Tie-2 expression. Regarding vinculin, as a marker of gap junction integrity, within WT, sepsis induced a decrease in Vinculin levels, whereas no change was noted following sepsis within iNKT-/- mice.
Conclusion:
iNTK-cells affect the Angiopoietin pathway within the lungs, a pathway critical to maintaining the pulmonary vascular endothelial response to critical illness. Given currently available agents to both up- and down-regulate iNKT-cells, these findings offer a potentially novel therapeutic target for the amelioration of this morbid state.