Next-Generation Porcine Intestinal Organoids: an Apical-Out Organoid Model for Swine Enteric Virus Infection and Immune Response Investigations

Abstract

The intestinal organoid culture system is a pathbreaking working model for investigating pathogen-host interactions in the intestines. However, due to the limitations of the first generation of intestinal organoids, basal-out structure and growth in Matrigel, most pathogens can rarely attach to the apical membrane directly and hardly initiate infection. In this study, we first developed a next-generation porcine intestinal organoid culture system, characterized by an apical membrane on the surface, called apical-out. To investigate the infectivity and antiviral immune responses of this apical-out porcine intestinal organoid, a swine enteric virus, transmissible gastroenteritis virus (TGEV), was employed to inoculate the culture system. Both RT–qPCR and IFA analysis demonstrated that TGEV replicated in the apical-out porcine intestinal organoid culture system. Additionally, our results illustrated that TGEV infection significantly upregulated the expression levels of IFN-α, IFN-λ1, ISG15, ISG58, TNF-α, and IL-6 in this culture system. Hence, we successfully developed a porcine intestinal apical-out organoid culture system, which will facilitate the investigation of pathogen-host interactions in pig intestines. IMPORTANCE Intestinal organoids are a newly developed culture system for investigating pathogen-host interactions. Intestinal organoid models have been widely used since their development because the results obtained from this type of culture model better represent physiological conditions than those from well-established cell lines. The 3D porcine intestinal organoid model was reported in 2018 and 2019 for the investigation of intestinal pathogens. However, those organoid culture models were basal-out intestinal organoids, which are not suitable for porcine enteric virus research because they invade the intestines via the apical side of epithelial cells on villi. In this study, we developed a porcine apical-out intestinal organoid culture system and verified its infectivity, type I, and type III IFN antiviral responses, and inflammatory responses following infection by a swine enteric virus. Our results imply that this apical-out porcine intestinal organoid culture system is an ideal model for the investigation of interactions between swine enteric viruses and the intestines.

Intestinal organoids in farm animals

“…A recent study showed that human enteroid cell polarity can be reversed by removal of the extracellular matrix [ 33 ]. The method developed by Co et al was applied to reverse epithelial polarity in pig jejunum enteroids in suspension cultures [ 34 ]. However, in this study, the polarity failed to be reversed in ~20% organoids after 3 days, which indicates phenotypic variability in these culture conditions.…”

“…Livestock intestinal organoids were successfully infected by a variety of enteric pathogens. Pig intestinal organoids were infected by several swine coronaviruses (porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV)) [ 21 , 29 , 34 , 38 ], by the bacteria Salmonella Typhimurium [ 20 ] and Lawsonia intracellularis [ 36 ] and by the protozoan parasite Toxoplasma gondii [ 20 ]. Bovine intestinal organoids were infected by Salmonella Typhimurium , Toxoplasma gondii and by group A rotaviruses [ 20 , 24 ].…”

“…To study the infection process, it is essential to replicate the physiological route of entry of enteric pathogen. Apical-out organoids and 2D organoid cell monolayers can be used to study enteric pathogens that penetrate epithelial cells through the apical side, as performed for the study of swine coronaviruses or Lawsonia intracellularis in pig intestinal organoids [ 21 , 29 , 34 , 36 , 38 ]. Alternatively, the apical surface of organoid epithelial cells can be exposed to luminal components when organoids are mechanically disrupted during the subculture process, as performed for Toxoplasma gondii or Salmonella Typhimurium infection in pig and bovine intestinal organoids [ 20 ].…”

Farm Animal-derived Models of the Intestinal Epithelium: Recent Advances and Future Applications of Intestinal Organoids

“…Intestinal organoids can also be maintained in suspension culture, where the cells switch their polarity so that the apical membrane faces outwards; this has already been achieved with crypts isolated from the porcine colon and jejunum. 71,78 This technique permits easier access to the apical surface of the epithelium and increases the available surface area, while keeping the cells in a proliferative state. Figure 3 illustrates a range of different methods used for the propagation of intestinal organoids.…”

“…Successful infection of this ‘next-generation’ model with a swine enteric virus was confirmed, as well as the elicitation of antiviral inflammatory responses. 71…”