Epithelial-mesenchymal transition of absorptive enterocytes and depletion of Peyer's patch M cells after PEDV infection

Abstract

This study focused on intestinal restitution including phenotype switching of absorptive enterocytes and the abundance of different enterocyte subtypes in weaned pigs after porcine epidemic diarrhea virus (PEDV) infection. At 10 days post-PEDV-inoculation, the ratio of villus height to crypt depth in both jejunum and ileum had restored, and the PEDV antigen was not detectable. However, enterocytes at the villus tips revealed epithelial-mesenchymal transition (EMT) in the jejunum in which E-cadherin expression decreased while expression of N-cadherin, vimentin, and Snail increased. Additionally, there was reduced expression of actin in microvilli and Zonula occludens-1 (ZO-1) in tight junctions. Moreover, the protein concentration of transforming growth factor β1 (TGFβ1), which mediates EMT and cytoskeleton alteration, was increased. We also found a decreased number of Peyer's patch M cells in the ileum. These results reveal incomplete restitution of enterocytes in the jejunum and potentially impaired immune surveillance in the ileum after PEDV infection.

Keywords: Enteric coronavirus; Epithelial-mesenchymal transition; Intestine; Microfold cell; Weaned pig.

Fig. 1

Diagram of the anatomical divisions in small intestinal mucosa. To investigate epithelial-mesenchymal transition, immunohistochemistry for E-cadherin, Snail, actin, and ZO-1, as well as in situ hybridization for E-cadherin and N-cadherin, were evaluated only in the villus tip area (highlighted as yellow cells in the diagram). The image analysis was performed on the HALO image analysis platform.

Fig. 2

Epithelial-mesenchymal transition at the villus tips in the jejunum after PEDV infection. (A–B) E-cadherin antigens in a control pig (A) and a PEDV-infected pig (B). Immunohistochemistry (IHC) for E-cadherin. (C) The mean values of E-cadherin immunopositive area (μm²)/μm length of epithelium. (D–E) Vimentin antigens in a control pig (D) and a PEDV-infected pig (E). Arrowheads indicate vimentin antigen in enterocytes. Insert: a higher magnification of enterocytes. IHC for vimentin. (F) The mean numbers of vimentin-positive enterocytes/μm of epithelium. (G–H) Snail antigens in a control pig (G) and a PEDV-infected pig (H). IHC for Snail. (I) The mean values of Snail immunopositive area (μm²)/μm length of epithelium. Bars show the mean values for each group, including the control (n = 6, open dots) and PEDV (n = 10, closed dots) treatments. * indicates a significant difference (P < 0.05).

Fig. 3

Increased RNA level of N-cadherin at the villus tips in the jejunum after PEDV infection. (A) RNA of E-cadherin (green) in a control pig. In situ hybridization (ISH) for E-cadherin and N-cadherin. (B) RNA of E-cadherin (green) and RNA of N-cadherin (red) in a PEDV-infected pig. Arrowheads indicate N-cadherin RNA in enterocytes. ISH for E-cadherin and N-cadherin. (C) Mean values of E-cadherin-positive area (μm²)/cell area (μm²). (D) Mean values of N-cadherin-positive area (μm²)/cell area (μm²). Bars show the mean values for each group, including the control (n = 6, open dots) and PEDV (n = 10, closed dots) treatments. * indicates a significant difference (P < 0.05).

Fig. 4

Impairment of tight junctions and microvilli in the jejunum after PEDV infection. (A–B) Actin antigens in a control pig (A) and a PEDV-infected pig (B). Immunohistochemistry (IHC) for actin. (C) The mean values of actin immunopositive area (μm²)/μm length of epithelium. (D–E) ZO-1 antigens in a control pig (D) and a PEDV pig (E). Insert: a higher magnification of enterocytes. IHC for ZO-1. (F) The mean values of ZO-1 immunopositive area (μm²)/μm length of epithelium. Bars show the mean values for each group, including the control (n = 6, open dots) and PEDV (n = 10, closed dots) treatments. * indicates a significant difference (P < 0.05).

Fig. 5

The mean values of TGFβ1 protein concentration in the jejunum of control and PEDV-infected pigs after PEDV infection. Bars show the mean values for each group, including the control (n = 6, open dots) and PEDV (n = 10, closed dots) treatments. * indicates a significant difference (P < 0.05).

Fig. 6

Intestinal restitution in the jejunum and ileum after PEDV infection. (A) Restored intestinal villi in the jejunum. PEDV-infected pig. Hematoxylin and eosin stain. (B) The mean ratios of villus length to crypt depth (VH:CD) in the jejunum and ileum in controls and PEDV-infected pigs. (C) Goblet cells in the jejunal mucosa. PEDV-infected pig. Alcian Blue/Periodic Acid–Schiff (AB/PAS) stain. (D) The mean AB/PAS-stained areas (μm²)/μm length of epithelium in the jejunum and ileum in controls and PEDV-infected pigs. (E) Villus M cells in the jejunal villi. PEDV-infected pig. Immunohistochemistry (IHC) for cytokeratin 18 (CK18). (F) The mean numbers of villus M cells/μm length of epithelium in the jejunum and ileum in controls and PEDV-infected pigs. (G) Peyer's patch M cells in the dome epithelium of the ileum. PEDV-infected pig. IHC for CK18. (H) The mean numbers of Peyer's patch M cells/μm length of epithelium in the jejunum and ileum in controls and PEDV-infected pigs. (I) Stem cell proliferation in the jejunal crypts. PEDV-infected pig. IHC for Ki67. (J) The mean Ki67-stained areas (μm²)/μm length of epithelium in jejunum and ileum in controls and PEDV-infected pigs. Bars show the mean values for each group, including the control (n = 6, open dots) and PEDV (n = 10, closed dots) treatments. * indicates a significant difference (P < 0.05).