J. D. Lyons1, Z. Liang1, N. Klingensmith1, K. Fay1, C. Chen1, C. Coopersmith1 1Emory University,Surgery,Atlanta, GA, USA
Introduction:
Sepsis increases intestinal epithelial cell (IEC) death, decreases IEC proliferation, and worsens gut barrier function. The trophic hormone epidermal growth factor (EGF), given either systemically or by local tissue over-expression, has been repeatedly shown in mouse models to limit gut damage during sepsis and improve survival. Pre-existing malignancy, however, is known to worsen sepsis survival and alter patterns of gut damage in septic shock, possibly by inducing changes in circulating lymphocyte populations. We sought to determine if EGF remained beneficial in septic mice with pre-existing cancer.
Methods:
Experiments were performed on age-matched fabpi-EGF mice (animals with enterocyte-specific expression of EGF), wild-type (WT) mice, and RAG1-/- mice (animals that lack mature lymphocytes). Mice were given subcutaneous injections of a pancreatic cancer cell line (Pan02), observed for 3 weeks to allow tumor growth, and then received intra-tracheal injections of Pseudomonas aeruginosa to model sepsis from bacterial pneumonia. Mice were either sacrificed at 24 hours or followed for 7-day survival. IEC death by apoptosis was quantified histologically, counting morphologically apoptotic cells on hematoxylin-eosin staining. IEC proliferation was quantified by injecting mice with the thymidine analogue bromodeoxyuridine (BRDU) prior to sacrifice and staining for BRDU-positive cells in intestinal crypts. Gut permeability was assayed by detecting serum concentrations of fluorescent dextran (FD4), which was orally gavaged prior to sacrifice. Means from each group were compared via T-test with Welch’s correction, and survival studies were compared via Log-Rank test. P < 0.05 determined significant throughout.
Results:
Although gut EGF expression has been shown to be enterocyte-protective in healhy animals subjected to sepsis modles, Fabpi-EGF mice with cancer paradoxically had increased rates of septic IEC death compared to mice with cancer alone (29.6±15.8 apoptotic cells/100 intestinal crypts v. 14.6±6.3, n=7-8, p=0.049). Gut permeability was also surprisingly significantly increased in septic cancer mice that expressed EGF as compared to septic mice with only cancer (5990±3500 ng FD4/ml serum v. 2840±1230, n=6-10, p=0.02). Rates of IEC proliferation did not differ between groups (258.3±36.5 BRDU-positive cells/100 crypts v. 249.9±43.3, n=6-9, p=0.69). Furthermore, there was no difference in 7-day survival between fabpi-EGF mice with cancer and mice with cancer alone (35% vs 17%, n=35-36, p=0.09) though EGF therapy has previously exerted a strong survival benefit. RAG1-/- mice with cancer had significantly worsened gut cell death after sepsis than RAG1-/- mice without cancer (38.8±25.5 apoptotic cells/100 crypts v. 7.3±5.4, n=4-5, p=0.049), suggesting cancer may impact IEC death processes in sepsis independent of lymphocytes.
Conclusion:
Gut-specific expression of EGF, while protective in healthy animals, loses its ability to improve survival and appears to actually worsen septic gut damage when mice have pre-existing cancer. How this reversal comes about is unclear, but our data suggest cancer’s impacts on IEC death after sepsis may be independent of cancer’s known effects on immune cell populations.