Interferon gamma and interleukin 1, but not interferon alfa, inhibit rotavirus entry into human intestinal cell lines

We describe a method for long-term culture of primary small intestinal epithelial cells (IEC) from suckling mice. IEC were digested from intestinal fragments as small intact units of epithelium (organoids) by using collagenase and dispase. IEC proliferated from organoids on a basement-membrane-coated culture surface and remained viable for 3 weeks. Cultured IEC had the morphologic and functional characteristics of immature enterocytes, notably sustained expression of cytokeratin and alkaline phosphatase. Few mesenchymal cells were present in the IEC cultures. IEC were also cultured from adult BALB/c mice and expressed major histocompatibility complex (MHC) class II antigens for at least 48 h in vitro. Primary IEC supported the growth of rhesus rotavirus (RRV) to a greater extent than a murine small intestinal cell line, m-IC cl2 . Cell-culture-adapted murine rotavirus strain EDIM infected primary IEC and m-IC cl2 cells to a lesser extent than RRV. Wild-type EDIM did not infect either cell type. Long-term culture of primary murine small intestinal epithelial cells provides a method to study (i) virus-cell interactions, (ii) the capacity of IEC to act as antigen-presenting cells using a wide variety of MHC haplotypes, and (iii) IEC biology.Rotavirus causes acute diarrheal disease by infecting villous epithelial cells of the small intestine. Unfortunately, in vitro studies of interactions between rotavirus and intestinal epithelial cells (IEC) have been limited by the lack of established small intestinal cell lines. Studies of rotavirus using MA104 cells (monkey kidney epithelial cells), Madin-Darby canine kidney cells (MDCK cells) (13), and human colonic carcinoma cell lines, particularly Caco-2 and HT-29 cells (12,13,16,17,30), have been carried out to investigate rotavirus-epithelial cell interactions in vitro. However, the utility of these cell lines is limited by the fact that they (i) are not derived from the small intestine, (ii) lack major histocompatibility complex (MHC) compatibility with a variety of genetically defined strains of experimental animals, and (iii) are malignantly transformed (colonic carcinoma cell lines). Although immortalized murine small intestinal cell lines have been established (e.g., m-IC cl2 ), they have not previously been studied for susceptibility to rotavirus (4, 31, 33).We developed a method to cultivate primary murine small IEC in vitro. We found that primary IEC proliferated in culture, remained viable for 3 weeks, and maintained expression of cytokeratin, alkaline phosphatase, and class II antigens, characteristic of IEC. Primary cultured murine small IEC supported the growth of rotavirus to a greater extent than the murine small intestinal cell line, m-IC cl2 . MATERIALS AND METHODS Animals.Male and female 5-to 8-week-old BALB/c mice were obtained from Taconic Breeding Laboratories (Germantown, N.Y.) and were mated. At 7 days, BALB/c mice were sacrificed by cervical dislocation, and their small intestines were removed.Epithelial cell isolation and culture. IEC were isolate...

Section: Discussionmentioning

confidence: 60%

Primary Murine Small Intestinal Epithelial Cells, Maintained in Long-Term Culture, Are Susceptible to Rotavirus Infection

Macartney

Baumgart

Carding

et al. 2000

“…Yet one dose of VirHRV induces almost complete protection against rotavirus shedding, whereas one dose of AttHRV induces protection at low rates (49,51). Studies of mice have shown the lack of a role for IFN-␥ in the resolution of rotavirus-induced diarrhea and infection of IFN-␥ Ϫ/Ϫ mice (1, 41), although an in vitro study has shown that IFN-␥ inhibited rotavirus entry into epithelial cells (7).…”

Section: Discussionmentioning

confidence: 99%

Cytokine Responses in Gnotobiotic Pigs after Infection with Virulent or Attenuated Human Rotavirus

Azevedo

Yuan

Pouly

et al. 2006

102

100

“…Fresh medium was replaced approximately every 10 days. The rhesus monkey epithelial cell line MA104 was grown in Medium 199 with Earle's balanced salt solution, 2.2 g of NaHCO 3 /liter, and 100 mg of L-glutamine (BioWhittaker)/liter, supplemented with 100 IU of penicillin and streptomycin/ml and 10% FBS. Cell cultures and viral infections were kept in a 5% CO 2 incubator at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Gene Expression Pattern in Caco-2 Cells following Rotavirus Infection

Cuadras¹,

Feigelstock²,

An³

et al. 2002

Rotaviruses are recognized as the leading cause of severe dehydrating diarrhea in infants and young children worldwide. Preventive and therapeutic strategies are urgently needed to fight this pathogen. In tissue culture and in vivo, rotavirus induces structural and functional alterations in the host cell. In order to better understand the molecular mechanisms involved in the events after rotavirus infection, we identified host cellular genes whose mRNA levels changed after infection. For this analysis, we used microarrays containing more than 38,000 human cDNAs to study the transcriptional response of the human intestinal cell line Caco-2 to rotavirus infection. We found that 508 genes were differentially regulated >2-fold at 16 h after rotavirus infection, and only one gene was similarly regulated at 1 h postinfection. Of these transcriptional changes, 73% corresponded to the upregulation of genes, with the majority of them occurring late, at 12 or more hours postinfection. Some of the regulated genes were classified according to known biological function and included genes encoding integral membrane proteins, interferon-regulated genes, transcriptional and translational regulators, and calcium metabolism-related genes. A new picture of global transcriptional regulation in the infected cell is presented and families of genes which may be involved in viral pathogenesis are discussed.