Endothelial Caspase-8 prevents fatal necroptotic hemorrhage caused by commensal bacteria

Abstract

Caspase-8 transduces signals from death receptor ligands, such as tumor necrosis factor, to drive potent responses including inflammation, cell proliferation or cell death. This is a developmentally essential function because in utero deletion of endothelial Caspase-8 causes systemic circulatory collapse during embryogenesis. Whether endothelial Caspase-8 is also required for cardiovascular patency during adulthood was unknown. To address this question, we used an inducible Cre recombinase system to delete endothelial Casp8 in 6-week-old conditionally gene-targeted mice. Extensive whole body vascular gene targeting was confirmed, yet the dominant phenotype was fatal hemorrhagic lesions exclusively within the small intestine. The emergence of these intestinal lesions was not a maladaptive immune response to endothelial Caspase-8-deficiency, but instead relied upon aberrant Toll-like receptor sensing of microbial commensals and tumor necrosis factor receptor signaling. This lethal phenotype was prevented in compound mutant mice that lacked the necroptotic cell death effector, MLKL. Thus, distinct from its systemic role during embryogenesis, our data show that dysregulated microbial- and death receptor-signaling uniquely culminate in the adult mouse small intestine to unleash MLKL-dependent necroptotic hemorrhage after loss of endothelial Caspase-8. These data support a critical role for Caspase-8 in preserving gut vascular integrity in the face of microbial commensals.

Fig. 1. C8-endo mice succumb to small intestinal hemorrhage.

a The proportion of mice that became moribund and required euthanasia. Gray boxes indicate days of tamoxifen administration. Data are combined from five independent experiments (n = 23–27 mice). ****P < 0.0001 via Log-rank Mantel–cox test. b Representative images of the gastrointestinal tract from a moribund C8-endo mouse and matched C8-flox and wild-type mice. c Representative images of the luminal face of the ileum from C8-endo mice 12 or 16 days after commencing tamoxifen treatment. Open arrow indicates a nascent hemorrhagic lesion. Asterisks indicate larger more developed hemorrhagic lesions. d Relative changes to the body weight of mice. Mean ± s.d. from n = 5 mice per genotype are shown. Gray boxes indicate days of tamoxifen administration. e Representative H&E-stained sections of the small intestines of control (C8-flox) mice and C8-endo mice 10, 12 and 16 days post-tamoxifen. Yellow arrowheads indicate compromised blood vessels. Asterisk indicates hemorrhagic transformation. Arrows indicate empty crypts. Black arrowhead indicates a Goblet cell. f Immunofluorescence images of sections from C8-endo-ROSA mice five days after the first dose of tamoxifen. Right panels are enlarged micrographs of the inset white boxes in the left-most panels. Scale bars in left panels indicate 100μM. Scale bars in right panels indicate 30 μM. Arrowheads indicate GFP-negative endothelial cells. Micrographs are representative of at least six specimens per group. g Quantification of the percentage of EGFP-CD31-positive cells over the total number of CD31-positive cells across the small and large intestine of C8-endo mice. Data are the mean ± s.e.m. of >100 cells/group/mouse. Symbols represent individual C8-endo mice. ns via one-way ANOVA with Geisser-Greenhouse correction.

Fig. 2. MLKL-dependent necroptosis is driving hemorrhage in C8-endo mice.

a Micrographs (a) and quantitation (b) of cleaved Caspase-3 staining in the small bowel of a wild-type mouse 15 hours after TNF administration (WT + TNF), a control mouse (C8-flox) and a C8-endo mouse 16 days after starting tamoxifen treatment. Micrographs show the remodeled intestinal wall before (pre-bleed) and after hemorrhage (bleed) in C8-endo mice. Arrows indicate cleaved Caspase-3 in crypts. Asterisk indicates cleaved Caspase-3 within a hemorrhage. b Data are mean ± s.d. from n = 10 for WT + TNF; n = 2 for C8-flox and n = 3 for C8-endo. Over 100 villi were analyzed per mouse. Symbols represent individual mice. c The proportion of mice that became moribund and required euthanasia. Gray boxes indicate days of tamoxifen administration. Data are from one cohort (n = 4–5 mice per group) and representative of 3 independent experiments. ***p < 0.001 via Log-rank Mantel–cox test. d Lengths of the small intestine from matched control (C8-flox) mice, C8-endo-Mlkl^ko mice and moribund C8-endo mice (same cohort as in c). Data are mean ± s.e.m. *p < 0.05 via one-way ANOVA with Holm-Sidak’s multiple comparison test. Symbols represent individual mice. e H&E-stained sections of mice 16 days after starting tamoxifen treatment. f Histopathological scores of H&E-stained sections of the small intestine from moribund C8-endo and matched C8-endo-Mlkl^ko mice. Data are mean ± s.e.m. **p < 0.01 via unpaired two-sided t-test. Symbols represent individual mice.

Fig. 3. Necroptotic hemorrhage in C8-endo mice is driven by TNF and commensal bacteria.

a The proportion of mice that became moribund and required euthanasia. Gray boxes indicate days of tamoxifen administration. Data are from one cohort (n = 6 mice per group) and representative of 2 independent experiments. **p < 0.01 via Log-rank Mantel–cox test. b H&E-stained sections of mice 14 days after starting tamoxifen treatment. c Histopathological scores of H&E-stained sections of the small intestine from moribund C8-endo and matched C8-endo Tnf^−/− mice. Data are mean ± s.e.m. **p < 0.01 via unpaired two-sided t-test. Symbols represent individual mice. d The proportion of mice that became moribund and required euthanasia. Gray boxes indicate days of tamoxifen administration. C8-endo mice drank vehicle (water) or an antibiotic cocktail starting one week prior to tamoxifen treatment. Data are from one cohort (n = 7–9 per group). *p < 0.05 via Log-rank Mantel–cox test. e The relative abundance of bacterial taxonomic families (Taxa) in samples from the indicated mice treated with an antibiotic cocktail (Abx) or without antibiotics (no Abx). Data are from one experimental cohort. The local contribution to beta diversity (LCBD) is shown. f Mice were sacrificed 12 days following the first dose of tamoxifen and TNF levels quantitated from the indicated tissues by ELISA. Data are mean ± s.e.m. Symbols represent individual mice. g The proportion of mice that became moribund and required euthanasia. Gray boxes indicate days of tamoxifen administration. Arrows indicate timing of LPS-Rs or TLR7-Ag administration. Data are combined from two cohorts (n = 5–11 mice per group). *p < 0.05 and ***p < 0.001 via Log-rank Mantel–cox test.