Regulation of intestinal epithelial cell cytoskeletal remodeling by cellular immunity following gut infection

Abstract

Gut infections often lead to epithelial cell damage followed by a healing response. We examined changes in the epithelial cell cytoskeleton and the involvement of host adaptive immunity in these events using an in vivo model of parasitic infection. We found that both ezrin and villin, key components of the actin cytoskeleton comprising the brush border (BB) of intestinal epithelial cells (IECs), underwent significant post-translational changes following gut infection and during the recovery phase of gut infection. Intriguingly, using mice lacking either CD4(+) or CD8(+) T-cell responses, we demonstrated that the mechanisms by which ezrin and villin are regulated in response to infection are different. Both ezrin and villin undergo proteolysis during the recovery phase of infection. Cleavage of ezrin requires CD4(+) but not CD8(+) T cells, whereas cleavage of villin requires both CD4(+) and CD8(+) T-cell responses. Both proteins were also regulated by phosphorylation; reduced levels of phosphorylated ezrin and increased levels of villin phosphorylation were observed at the peak of infection and correlated with reduced BB enzyme activity. Finally, we show that infection also leads to enhanced proliferation of IECs in this model. Cytoskeletal remodeling in IECs can have critical roles in the immunopathology and healing responses observed during many infectious and non-infectious intestinal conditions. These data indicate that cellular immune responses can be significant drivers of these processes.

Figure 1

Post-translational regulation of ezrin following gut infection. The experimental protocol for infecting mice with both strains of G. duodenalis is depicted in (a). Mice were maintained on neomycin, vancomycin and ampicillin throughout the experiment to facilitate infection with either strain of parasite. WT (b), SCID (c), β₂m^−/− (d), and CD4^−/− (e) mice were infected with the GS strain of G. duodenalis and jejunal homogenates were examined for post-translational modifications in ezrin using Western blots. GAPDH was used as a loading control. Each figure is representative of 4 mice / time point. Ezrin was also analyzed following infection of WT C57BL/6 mice with the WB strain of the parasite (f). Using paraffin-embedded tissue and IF, the localization of total ezrin (upper panel) and phosphorylated ezrin (p-ezrin, lower panel) was also determined in the intestines of mice infected with the GS strain of the parasite for 0, 5 or 18 days (g). Each panel represents 4 mice/time point.

Figure 2

Activation of µ-calpain following infection. WT (a) and SCID (b) mice were infected with the GS strain of G. duodenalis and jejunal homogenates were tested for activated µ-calpain by Western blot using an antibody capable of detecting both active and inactive forms of µ-calpain. GAPDH was used as a loading control. Each figure is representative of 4 mice/time point.

Figure 3

Villin proteolysis following gut infection. WT (a), SCID (b), β₂m^−/− (c), and CD4^−/− mice (d) were infected with the GS strain of G. duodenalis. Jejunal homogenates were analyzed by Western blot for villin post-translational modifications using a polyclonal antibody against a peptide in the COOH-terminal portion of the protein. Villin proteolysis was also examined in WT mice infected with the WB strain of the parasite (e). Renal villin in WT mice infected with the GS strain was also analyzed (f). Each figure is representative of four animals / time point. GAPDH was used as a loading control.

Figure 4

Villin localization following gut infection. WT mice were infected with the GS strain and villin distribution and localization were determined by IF in jejunal tissue at day 5 (middle panel) and day 18 (lower panel) compared with uninfected mice (upper panel). Each panel is representative of 4 animals / time point.

Figure 5

Tyrosine-phosphorylation of villin following gut infection. Villin was immunoprecipitated from jejunal homogenates of WT mice infected with the GS strain (a) or the WB strain (b). Increased tyrosine-phosphorylation of villin was not seen in SCID mice following infection with the GS strain of G. duodenalis (c). Immunoprecipitated proteins were analyzed for tyrosine phosphorylation by Western blot and levels of phosphorylated protein (upper panels) were normalized to levels of total villin (lower panels) by densitometry. Each figure represents 4 animals / time point.

Figure 6

Analysis of NH₂-terminal-containing villin fragments following G. duodenalis infection. WT (a), SCID (b), β₂m^−/− (c) and CD4^−/− (d) mice were infected with the GS strain of the parasite, and jejunal homogenates were analyzed using an antibody against a peptide in the NH₂-terminal portion of villin by Western blot. GAPDH was used as a loading control. Each figure is representative of four animals / time point.

Figure 7

Intestinal epithelial cell proliferation and positional distribution following gut infection. WT mice were infected with the GS strain of the parasite and IEC proliferation was measured by BrdU incorporation during a two-hour pulse of BrdU. BrdU⁺ cells were visualized using IHC and light microscopy and the percentage of BrdU⁺ cells/total cells counted in each crypt that were labeled was determined (A). The positional distribution of BrdU⁺ cells IECs was then determined (B). A schematic diagram of an intestinal villus indicating the positioning of cells along the crypt-villus axis (C). Each figure represents 4 animals / time point. Data represent means ± SEM for four mice per group. *p < 0.05 by Mann–Whitney U test versus uninfected mice.

Figure 8

Model of immune-dependent changes in epithelial cell function following gut infection. Infection leads to an immune response that is required for increased villin phosphorylation at day 5. This correlates with enhanced proliferation of intestinal epithelial cells and reduced levels of disaccharidase enzymes.