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. 2018 Jul 17;49(1):42-55.e6.
doi: 10.1016/j.immuni.2018.06.011.

Caspase-8 Collaborates With Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock

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Free PMC article

Caspase-8 Collaborates With Caspase-11 to Drive Tissue Damage and Execution of Endotoxic Shock

Pratyusha Mandal et al. Immunity. .
Free PMC article

Abstract

The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

Keywords: Caspase; RIP kinase; TNF; endotoxic shock; extrinsic apoptosis; ileum; interferon; programmed necrosis; pyroptosis.

Conflict of interest statement

DECLARATION OF INTERESTS

Scott B. Berger, Michelle Schaeffer, Sandra Hoffman, Carol Capriotti, John Bertin and Peter J. Gough are employees of GlaxoSmithKline.

Figures

Figure 1
Figure 1. Casp8 drives endotoxic shock
(A–B) Kaplan-Meier survival plot (left) and body temperature plot (right) over time (hpc) in LPS-challenged WT (n=19), Ripk3−/− (n=15), Casp8+/−Ripk3−/− (n=8), Casp8−/−Ripk3−/− (n=26) and Casp11−/− (n=11) mice (A) or WT (n=6), Ifnar1−/− (n=8) and Irf3−/− (n=5) mice (B). (C) Kaplan-Meier survival plot for WT (n=6), littermate Casp8+/−Ripk3−/− (n=6) and Casp8−/−Ripk3−/− (n=7) as well as Casp11−/− mice (n=6) given a poly(I:C) prime (4 mg/kg) followed 6 h later with low dose LPS challenge (5 mg/kg). (D) Kaplan-Meier survival plot for WT (n=19), littermate Casp8+/−Ripk3−/− (n=20) and Casp8−/−Ripk3−/− (n=15) mice over time, h post infection (hpi) with E. coli (ATCC 25922). Groups here included balanced numbers of male and female animals. Statistical comparisons (to WT) was by Log-Rank (Mantel-Cox) test (* p<0.05; ** p<0.01). Please also see Figure S1.
Figure 2
Figure 2. Casp8 and Casp11 are dispensable for initiation of shock
(A) Crosstalk model of myeloid cell-induced inflammatory signal transduction during shock, with design of in vivo complementation assay using BM cell (BMC) transfer shown to the right. (B) Quantification of serum IFNβ in PBS- or LPS-challenged (2 hpc) WT mice (n=3), Irf3−/− mice (n=3) and Irf3−/− recipients (n=2) of WT BM transfer. (C) Percentage of WT CD45.1+ donor and Irf3−/− CD45.2+ recipient leukocytes in the lung (left) and in peripheral blood mononuclear cells (PBMC, right) from recipient mice. (D) Viability of CD45.1+ donor cells (annexin V negative or a combination of annexin V and 7AAD negative) in lungs (left) and PBMC (right) at 3.5 h post transfer. (E–J) Kaplan-Meier survival plots of LPS-challenged Irf3−/− (n=4), WT (n=3) and Casp8−/−Ripk3−/− (n=3) mice (left), and Irf3−/− recipients of BMC from WT (n=7), Irf3−/− (n=4) or Casp8−/−Ripk3−/− (n=4) mice (right) (E), Irf3−/− recipients of BMC from Myd88−/− (n=4) mice (F), Casp11−/− (n=3) mice or Irf3−/−recipients of BMC from Casp11−/− (n=4) or Ifnar1−/− (n=6) mice (G), Irf3−/− mice (n=3) or recipients (n=3) of BMC from Gsdmd−/− mice (H). Statistical comparisons as in Fig. 1 (* p<0.05; ** p<0.01, *** p<.001). Please also see Figure S2.
Figure 3
Figure 3. Casp8 drives small intestinal and splenic damage
(A–B) Representative (3 mice of each genotype in two independent experiments) images of hematoxylin and eosin (H&E) stained sections of small intestine (A) and spleen (B), from WT, Ripk3−/− and Casp8−/−Ripk3−/− mice at 9 hpc with PBS or LPS, as indicated (bar=100 μm). Arrows indicate damaged and sloughed cells with condensed nuclei. (C–D) Histological score of leukocyte infiltrate (inflammatory cells), condensed nuclei (apoptosis), atrophy (tissue damage) and edema (fluid accumulation) in small intestine (C), or extramedullary hematopoiesis (EMH) as well as congestion in spleen (D), in tissue sections from WT, Ripk3−/− and Casp8−/−Ripk3−/− mice at 9 hpc with PBS or LPS (n=3 to 5 for each group). N.D., not detected. Mean and range in scores are shown. Please also see Figure S3.
Figure 4
Figure 4. Casp8 and Casp11 collaborate for lower small intestinal injury
(A–C) Immunoblot for cleaved (Cl)-Casp (C)8; 43 and 18 kDa), Cl-C3 (22, 19 and 17 kDa) or Cl-PARP (89 kDa) as well as total C11 (43, 38.5 and 25 kDa) and uncleaved C8 (55 kDa) or C3 (31 kDa), showing mucosa from duodenum (Du) in challenged WT and Casp8−/−Ripk3−/− mice (only uncleaved 43 and 38.5 kDa forms of C11 detected) (A), PBMC and Du from WT mice (B), mucosa from Du, as well as stomach (S), proximal jejunum (Jp), distal jejunum (Jd), ileum (Ile), cecum (Ce) and colon (Co) from challenged WT mice (only 19 and 17 kDa forms of Cl-C3 detected) (C). Graphical depiction of intestinal tract segments is above C. Molecular weight markers shown to the right. (D) Immunohistochemistry (IHC), showing Cl-C3 in situ with hematoxylin counterstain, or histology following H&E stain of the indicated intestinal segments from challenged WT and Casp8−/−Ripk3−/− mice (bar=100 μm) representative of 8 mice from 4 independent experiments. Insets show magnified images of the sections (bar=40 μm). Arrows indicate Cl-C3 positive cells in IHC sections and condensed nuclei in H&E stained sections. (E) Immunoblot of mucosa from Du, Jp, Jd and Ile from WT, Casp8−/−Ripk3−/− and Casp11−/− mice at 1.5 hpc with LPS. (F) IHC of Cl-C3 in ileum from two WT and two Casp11−/− mice at 1.5 hpc with PBS or LPS representative of 5 mice from 3 independent experiments. Arrows indicate Cl-C3 positive cells. (G) Immunoblot of ileal mucosa from WT and Irf3−/− mice at 1.5 hpc with PBS or LPS, with two LPS-challenged Irf3−/− mice shown. Please also see Figure S4.
Figure 5
Figure 5. Casp8 regulates inflammatory signaling in ileum
(A) Immunoblot of ileal mucosa from unmanipulated (time=0) or from WT and Casp8−/−Ripk3−/− mice at the indicated mpc with LPS, showing expression of phosphorylated forms (p) of IκBα (40 kDa), SAPK and JNK (54 and 46 kDa, respectively), as well as Cl-C8, C8, cFLIPL (50 kDa) and cFLIPs (25 kDa), RIPK1 (75 kDa), IgH (50 kDa), C3, C11, C1 (43 kDa) and Gsdmd (55 kDa, with an * indicating the ~30 kDa cleaved form). (B) Principal component (PC) analysis (PCA) of ileal mucosa from WT, Casp8−/−Ripk3−/− and Casp11−/− mice (n=5 for each group) including 27,578 genes with detectable base mean expression where the contribution of each PC is indicated as a percentage. (C) Heat map comparing expression patterns of genes in ileal mucosa from WT, Casp8−/−Ripk3−/− and Casp11−/− mice (n=5 for each group) at 1.5 hpc, where mean log2 expression of each gene was compared to normalized expression of all genes. Blue, white and red indicate downregulated, unchanged and upregulated expression of genes, respectively. Please also see Figure S5.
Figure 6
Figure 6. TNF signaling drives small intestinal injury and endotoxemia
(A) IHC showing Cl-C3 in situ with hematoxylin counterstain in small intestinal segments of WT mice at 1.5 hpc with LPS (bar=100 μm) treated with either isotype control or anti-TNF antibody for 24 h prior to challenge. Arrows indicate Cl-C3 positive cells. (B) DEVDase (Cl-C3 and Cl-C7) activity assessed as fold increase over PBS-challenged control animals at 1.5 hpc (n=3 for each) in mucosa from intestinal segments following LPS challenge. Mean and range shown. (C–E) Kaplan-Meier survival plots for LPS-challenged WT mice treated as described in A (n=7 for each group) (C), and for Casp8DA/DARipk3+/− males (n=5, left) and females (n=6, right), littermate Casp8+/DARipk3+/− males (n=3, left) and females (n=3, right), as well as female Casp8DA/DARipk3−/− (n=4, right) mice (E). Statistical comparisons were as described in Fig. 1. Please also see Figure S6.
Figure 7
Figure 7. Casp8 and Casp11 mediate gut leakage
(A) IHC and H&E of ileum from WT, Casp8−/−Ripk3−/− and Casp11−/− mice at 6 hpc with LPS (bar=100 μm). Arrows indicate Cl-C3 positive cells. (B) IHC of ileum from Irf3−/− mice or Irf3−/− recipients of BMC from WT (n=3 each) mice at 6 to 9 hpc with LPS. (C) Detection of FD-4 in sera from WT (n=10), Ripk3−/− (n=10), Casp8−/−Ripk3−/− (n=12), Ifnar1−/− (n=6) and Casp11−/− (n=6) mice at 6 hpc. (Statistical comparisons used Wilcoxan matched pairs analysis (* p<0.05). (D) Fold increase in total eubacterial DNA, measured by quantitative PCR, in mesenteric lymph node (MLN) and blood from WT (n=3), Casp8−/−Ripk3−/− (n=3) or Casp11−/− (n=2) mice at 6 hpc with LPS compared to PBS control. (E) Model for partitioned initiation and execution steps during LPS shock. Please also see Figure S7.

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