ABSTRACT
Despite intensive scientific efforts, the therapy of peritonitis is presently limited to symptomatic measures, including infectious source control and broad-spectrum antibiotics. Promising therapeutic approaches to reduce morbidity and mortality are still missing. Within the early phase of abdominal sepsis, apoptosis of neutrophil granulocytes is inhibited, which is linked to tissue damage and septic shock. TNF-related apoptosis-inducing ligand (TRAIL) is a promising agent to stimulate neutrophil apoptosis. However, the underlying mechanisms have not been elucidated so far. The objective of the present study was to characterize the molecular mechanisms of TRAIL-stimulated apoptosis in early abdominal sepsis. Therefore, the murine sepsis model Colon ascendens stent peritonitis (CASP) was applied in wild type (WT) and TRAIL knock-out (TRAIL-/-) C57/BL6j mice. Neutrophil granulocytes were isolated from spleen, blood, bone marrow, and peritoneal lavage using magnetic-activated cell sorting. Neutrophil maturation was analyzed by light microscopy, and apoptotic neutrophils were quantified by fluorescence-activated cell sorting (FACS). Western blot and FACS were used to investigate expression changes in apoptotic proteins and TRAIL receptors. The impact of TRAIL-induced apoptosis was studied in vitro. In septic mice (CASP 6 h), the number of neutrophils in the BM was reduced but increased in the blood and peritoneal lavage. This was paralleled by an increased maturation of neutrophils from rod-shaped to segmented neutrophils (right shift). In vitro, extrinsic TRAIL stimulation did not alter the apoptosis level of naïve neutrophils but stimulated apoptosis in neutrophils derived from septic WT and TRAIL-/- mice. Neutrophils of the bone marrow and spleen showed enhanced protein expression of anti-apoptotic Flip, c-IAP1, and McL-1 and reduced expression levels of pro-apoptotic Bax in neutrophils, which might correlate with apoptosis inhibition in these cells. CASP increased the expression of intrinsic TRAIL in neutrophils derived from the bone marrow and spleen. This might be explained by an increased expression of the TRAIL receptors DR5, DcR1, and DcR2 on neutrophils in sepsis. No differences were observed between septic or naïve WT and TRAIL-/- mice. In conclusion, the present study shows that neutrophil granulocytes are sensitive to TRAIL-stimulated apoptosis in the early stage of abdominal sepsis, emphasizing the promising role of TRAIL as a therapeutic agent.
ABSTRACT
The TNF-superfamily member TRAIL is known to mediate selective apoptosis in tumor cells suggesting this protein as a potential antitumor drug target. However, initial successful pr-clinical results could not be translated into the clinic. Reasons for the ineffectiveness of TRAIL-targeting in tumor therapies could include acquired TRAIL resistance. A tumor cell acquires TRAIL resistance, for example, by upregulation of antiapoptotic proteins. In addition, TRAIL can also influence the immune system and thus, tumor growth. We were able to show in our previous work that TRAIL-/- mice show improved survival in a mouse model of pancreatic carcinoma. Therefore, in this study we aimed to immunologically characterize the TRAIL-/- mice. We observed no significant differences in the distribution of CD3+, CD4+, CD8+ T-cells, Tregs, and central memory CD4+ and CD8+ cells. However, we provide evidence for relevant differences in the distribution of effector memory T-cells and CD8+CD122+ cells but also in dendritic cells. Our findings suggest that T-lymphocytes of TRAIL-/- mice proliferate at a lower rate, and that the administration of recombinant TRAIL significantly increases their proliferation, while regulatory T-cells (Tregs) from TRAIL-/- mice are less suppressive. Regarding the dendritic cells, we found more type-2 conventional dendritic cells (DC2s) in the TRAIL-/- mice. For the first time (to the best of our knowledge), we provide a comprehensive characterization of the immunological landscape of TRAIL-deficient mice. This will establish an experimental basis for future investigations of TRAIL-mediated immunology.