Transmissible Gastroenteritis Virus Infection Promotes the Self-Renewal of Porcine Intestinal Stem Cells via Wnt/β-Catenin Pathway

Abstract

Intestinal stem cells (ISCs) play an important role in tissue repair after injury. A recent report delineates the effect of transmissible gastroenteritis virus (TGEV) infection on the small intestine of recovered pigs. However, the mechanism behind the epithelium regeneration upon TGEV infection remains unclear. To address this, we established a TGEV infection model based on the porcine intestinal organoid monolayer. The results illustrated that the porcine intestinal organoid monolayer was susceptible to TGEV. In addition, the TGEV infection initiated the interferon and inflammatory responses following the loss of absorptive enterocytes and goblet cells. However, TGEV infection did not disturb epithelial integrity but induced the proliferation of ISCs. Furthermore, TGEV infection activated the Wnt/β-catenin pathway by upregulating the accumulation and nuclear translocation of β-catenin, as well as promoting the expression of Wnt target genes, such as C-myc, Cyclin D1, Mmp7, Lgr5, and Sox9, which were associated with the self-renewal of ISCs. Collectively, these data demonstrated that the TGEV infection activated the Wnt/β-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. IMPORTANCE The intestinal epithelium is a physical barrier to enteric viruses and commensal bacteria. It plays an essential role in maintaining the balance between the host and intestinal microenvironment. In addition, intestinal stem cells (ISCs) are responsible for tissue repair after injury. Therefore, prompt self-renewal of intestinal epithelium will facilitate the rebuilding of the physical barrier and maintain gut health. In the manuscript, we found that the transmissible gastroenteritis virus (TGEV) infection did not disturb epithelial integrity but induced the proliferation of ISCs and facilitated epithelium regeneration. Detailed mechanism investigations revealed that the TGEV infection activated the Wnt/β-catenin pathway to promote the self-renewal of ISCs and resulted in intestinal epithelium regeneration. These findings will contribute to understanding the mechanism of intestinal epithelial regeneration and reparation upon viral infection.

Keywords: TGEV; Wnt/β-catenin pathway; epithelium regeneration; intestinal organoid monolayer; intestinal stem cells.

FIG 1

Establishment of a porcine intestinal organoid monolayer. (A) Graphical representation for the establishment of an organoid monolayer. (B) Single cells grow into a confluent monolayer after seeding for 3 days. (C) Transepithelial electrical resistance (TER) was measured during the development of an organoid monolayer. Values were from three independent monolayers. (D) Organoid monolayer was subjected to IFA staining for absorptive enterocytes (Villin), tight junction (ZO-1), stem cells (Sox9), enteroendocrine cells (CGA), goblet cells (Muc2), Paneth cells (LYZ), and proliferation cells (Ki67).

FIG 2

Porcine intestinal organoid monolayer is susceptible to TGEV. (A) The kinetics of TGEV replication porcine intestinal organoid monolayer at the indicated time points were measured by RT-qPCR. (B) The TGEV titers in supernatant from both the apical side and basal side of the Transwell-cultured organoid monolayer were titrated by TCID₅₀ assay on the ST cell lines. (C) Organoid monolayer infected with TGEV for 24 h was stained with TGEV N monoclonal antibody. (D) The expression of TGEV N and APN were detected by Western blotting. (E) The density ratio of APN expression was calculated with Image J and normalized against GAPDH expression. **, P ≤ 0.01; ***, P ≤ 0.001. (F) The transcription levels of IFN-λ1, IFN-λ3, IFN-γ, ISG58, ISG15, IL-8, IL-6, and TNF-α in organoid monolayer after TGEV infection were evaluated by RT-qPCR. The RT-qPCR data were calculated using the comparative threshold cycle (2^-ΔΔCT) method. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (G) Colocalization analysis of TGEV N with different epithelial cell subsets and proliferation cells was performed by confocal microscopy.

FIG 3

TGEV infection maintains epithelial integrity in intestinal organoid monolayer. (A) Transepithelial electrical resistance (TER) value in organoid monolayer infection was measured after TGEV infection. Values were from three independent monolayers. (B) TGEV-infected or mock-infected intestinal organoid monolayer at 24 hpi, 36 hpi, and 48 hpi were stained with ZO-1 and TGEV N and observed by confocal microscopy. (C) TGEV-infected or mock-infected intestinal organoid monolayers at 7 dpi were stained with ZO-1 and TGEV N and analyzed by 3D imaging of confocal microscopy.

FIG 4

TGEV infection promoted the self-renewal of ISCs. (A) The expression of Sox9, representing the number of ISCs, was detected by Western blotting. (B) The density ratio of Sox9 expression was calculated with Image J and normalized against GAPDH expression. (C) Sox9 and TGEV N were stained in intestinal organoid monolayer at indicated time points and observed under confocal microscopy. (D) The mean fluorescence intensity of Sox9⁺ cells in organoid monolayer infected with TGEV was analyzed. The data from five independent stainings. (E) The expression of Villin, LYZ, and Muc2 was detected by Western blotting, which represents absorptive enterocytes, Paneth cells, and goblet cells, respectively. (F) The density ratios of Villin, LYZ, and Muc2 were calculated with Image J and normalized against GAPDH expression. ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 5

TGEV infection activated the Wnt/β-catenin pathway. (A) The transcription levels of Wnt3a, β-catenin, TCF4, Cyclin D1, Lgr5, Sox9, C-myc, and Mmp7 in organoid monolayer were measured by RT-qPCR after TGEV infection. The RT-qPCR data were calculated using the comparative threshold cycle (2^-ΔΔCT) method. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001. (B) The expression of β-catenin, Cyclin D1, and C-myc in organoid monolayer was detected by Western blotting after TGEV infection. (C) The density ratios of β-catenin, Cyclin D1, and C-myc were calculated with Image J and normalized against GAPDH expression. ns, not significant; *, P ≤ 0.05; ***, P ≤ 0.001 (D) The nuclear translocation of β-catenin was observed in organoid monolayer by confocal microscopy after TGEV infection (bar = 20/5 μm).

FIG 6

TGEV infection promoted the self-renewal of porcine intestinal stem cells via the Wnt/β-catenin pathway. (A) The optimal concentration and treated time of ICG-0001 were screened based on the expression of β-catenin, C-myc, and Sox9. (B) The density ratios of β-catenin, C-myc, and Sox9 were calculated with Image J and normalized against GAPDH expression. ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001. (C) The expression of C-myc and Sox9 were detected in organoid monolayer treated with 10 μM ICG-001 before TGEV infection. (D) The density ratios of C-myc and Sox9 were calculated with Image J and normalized against GAPDH expression. ns, not significant; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 7

Schematic diagram of TGEV-induced intestinal epithelial regeneration via Wnt/β-catenin pathway.