Intestinal genetic inactivation of caspase-8 diminishes migration of enterocytes

Abstract

Aim: To verify the hypothesis that caspase-8 (Casp8), which regulates cellular apoptosis and necroptosis, is critically involved in enterocyte migration.

Methods: Casp8-silenced Caco2 cells were used in migration assays. In addition, enterocyte-specific Casp8 heterozygous (Casp8(+/∆int)) or homozygous knockout mice (Casp8(∆int)) were generated by crossing genetically modified mice carrying loxP recombination sites in intron 2 and 4 of the murine Casp8 gene with transgenic animals expressing a cre-transgene under control of the villin promoter in a pure C57/BL6 genetic background. The nucleoside analog BrdU was injected i.p. in male Casp8(+/∆int) and Casp8(∆int) animals 4 h, 20 h, or 40 h before performing morphometric studies. Locations of anti-BrdU-immunostained cells (cell(max)) in at least 50 hemi-crypts of 6 histoanatomically distinct intestinal mucosal regions were numbered and extracted for statistical procedures. For the mice cohort (n = 28), the walking distance of enterocytes was evaluated from cell(max) within crypt (n = 57), plateau (n = 19), and villus (n = 172) positions, resulting in a total of 6838 observations. Data analysis was performed by fitting a three-level mixed effects model to the data.

Results: In cell culture experiments with Caco2 cells, Casp8 knockdown efficiency mediated by RNA interference on Casp8 transcripts was 80% controlled as determined by Western blotting. In the scratch assay, migration of Casp8-deleted Caco2 cells was significantly diminished when compared with controls (Casp8(∆scramble) and Caco2). In BrdU-labeled Casp8(∆int) mice, cell(max) locations were found along the hemi-crypts in a lower position than it was for Casp8(+/∆int) or control (cre-negative) animals. Statistical data analysis with a three-level mixed effects model revealed that in the six different intestinal locations (distinct segments of the small and large intestine), cell movement between the three mice groups differed widely. Especially in duodenal hemi-crypts, enterocyte movement was different between the groups. At 20 h, duodenal cell(max) location was significantly lower in Casp8(∆int) (25.67 ± 2.49) than in Casp8(+/∆int) (35.67 ± 4.78; P < 0.05) or control littermates (44.33 ± 0.94; P < 0.01).

Conclusion: Casp8-dependent migration of enterocytes is likely involved in intestinal physiology and inflammation-related pathophysiology.

Keywords: Barrier function; Caspase 8; Cell migration; Inflammatory bowel disease; Intestinal morphogenesis.

Figure 1

Migration of caspase-8 (Casp8)-deleted Caco2 cells is diminished. A: Western blots Casp8 protein and densitometric analysis after treatment of Caco2 cells with small interfering RNAs (siRNAs) interfering with Casp8 transcripts or with negative control siRNA (∆scramble); B: Measurement of cellular proliferation by proliferating cell nuclear antigen (PCNA) expression in Caco2 cells with siRNA interfering with Casp8 transcripts or with negative control siRNA; C: Migration of Casp8 siRNA-treated Caco2 cells and scramble controls at 6 h and 24 h after scratching; D-F: Control siRNA-treated Caco2 cells at 0 h (D), 6 h (E), or 24 h (F) after scratching; G-I: Casp8 siRNA-treated Caco2 cells at 0 h (G), 6 h (H) or 24 h (I) after scratching.

Figure 2

Intestinal phenotype of Casp8^∆int mice. Morphologic pattern of A: Small intestinal; B: Large intestinal architecture in Casp8^f/f mice; C: Small intestinal; D: Large intestinal architecture in Casp8^+/∆int mice; E: Small intestinal; F: Large intestinal architecture in Casp8^∆int mice. Paneth cells are marked by arrows (hematoxylin and eosin,100 × magnification). Casp8: Caspase-8.

Figure 3

Morphometry examples of small and large intestinal tissues. Small or large intestinal tissue sections of Casp8^f/f, Casp8^+/∆int, and Casp8^∆int mice were anti-Ki67 (left) or anti-BrdU (right) immunostained. BrdU was injected 2 h, 20 h, or 40 h before sacrificing mice. Examples of cell^max locations in the small or large intestine are highlighted with a red dot (200 × magnification). Casp8: Caspase-8.

Figure 4

Tissue-specific analysis of enterocyte migration. Cell^max locations (enterocyte migration) in six histoanatomically distinct intestinal mucosal regions in cre-negative Casp8^f/f (f/f), heterozygous Casp8^+/∆int (+/∆int), and homozygous-knockout mice Casp8^∆int (∆int) at 2 h, 20 h, or 40 h after BrdU injection.