Research paper
Primary bovine colonic cells: A model to study strain-specific responses to Escherichia coli

https://doi.org/10.1016/j.vetimm.2010.04.010Get rights and content

Abstract

The parasitic or commensal lifestyle of bacteria in different hosts depends on specific molecular interactions with the respective host species. In vitro models to study intestinal bacteria–host interactions in cattle are not available. Bovine primary colonocyte (PC) cultures were generated from colon crypt explants. Up to day 4 of culture, the vast majority of cells were of epithelial phenotype (i.e., expressed cytokeratin but not vimentin). PCs harboured mRNA specific for Toll-like receptors (TLR) 1, TLR3, TLR4 and TLR6 but not for TLR2, TLR5, TLR7, TLR8, TLR9 and TLR10. Six hours after inoculation of PC cultures with Escherichia coli (E. coli) prototype strains representing different pathovars (enterohaemorrhagic E. coli [EHEC], enteropathogenic E. coli [EPEC], enterotoxic E. coli [ETEC]), bacteria were found attached to the cells. EPEC adhesion was accompanied by intracellular actin accumulation. An attenuated laboratory strain (E. coli K12 C600) and a bovine commensal E. coli strain (P391) both did not adhere. Bacterial or LPS challenge of PC cultures resulted in specific increases in mRNA transcripts for IL-8, GRO-α, MCP-1, RANTES, and IL-10. The level of mRNA transcripts for TGF-β stayed constant, while IL-12 mRNA was not detectable. Short-term cultures of PCs, maintaining epithelial cell properties, interacted with commensal and pathogenic bacteria in a strain-specific manner and have proven to be a useful in vitro model to study the interaction of bacteria with the bovine intestinal mucosa.

Introduction

During the pathogenesis of many enteric diseases, intestinal epithelial cells (IEC) represent the principal target cells for microbial virulence factors that specifically impair the integrity and the functionality of the epithelial layer. Notably, IEC also act as sensors for microbial infection and form an integral component of a communication network that comprises interactions between IEC, luminal microbes, and host immune cells (Kagnoff and Eckmann, 1997). To fulfil the latter function, IEC express Toll-like receptors (TLR) to sense pathogen associated molecular patterns (PAMPs) (Werling and Jungi, 2003) and are capable of secreting a variety of chemokines (Dwinell et al., 2003, Shibahara et al., 2001, Yang et al., 1997) and cytokines (Nagalakshmi et al., 2004).

The absence of pathological inflammation of the gut that is exclusively colonized by the resident microflora results from the ability to discriminate between commensal and pathogenic bacteria (Lan et al., 2005, Skjolaas et al., 2007). The maintenance of hypo-responsiveness towards the luminal microflora has led to the concept of a state of controlled or physiologic inflammation in a healthy host. The underlying mechanisms are partially understood and may comprise modulated expression of genes involved in intestinal functions (Hooper et al., 2001), inhibition of IkB-α ubiquitination (Neish et al., 2000) and acquired endotoxin tolerance in IEC (Lotz et al., 2007).

Zoonotic pathogens pose a major threat to human wellbeing worldwide causing life-threatening diseases in developing and industrial countries (Schlundt et al., 2004). The pathogenicity of these microorganisms is frequently restricted to humans while domestic animals serve as a common reservoir colonized without clinical disease (Smith et al., 2002). Although exhibiting a commensal-like lifestyle in the reservoir species, the very same factors that represent the principal virulence factors in the pathogenesis of human disease may account for the ability to persistently colonize reservoirs, as seen in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 (Smith et al., 2002). The different outcomes of infections in various hosts may result from differential abilities to colonize human and non-human epithelial cells (Sonntag et al., 2005), host-specific differences of cellular receptor distribution for bacterial virulence factors (Mundy et al., 2007, Stamm et al., 2008), and altered expression of bacterial factors in different hosts (Rashid et al., 2006). It becomes increasingly clear that a commensal or parasitic lifestyle results from specific bacteria–host interactions rather than being an inherited feature of a bacterial species. Elucidation of these specific interactions may provide new opportunities to counteract colonization and subsequent persistent shedding of zoonotic pathogens by livestock.

Host-specific in vitro systems are indispensable to achieve this goal. Cattle represent an important reservoir for food-borne pathogens, such as Campylobacter spp., Salmonella Typhimurium, and EHEC (Schlundt et al., 2004). Currently, no permanent cell line of bovine intestinal tissue origin is available for such studies, making primary cultures a good alternative. Various studies have been performed with primary colonocytes (PCs) isolated from rabbits, rats, mice and humans (for review see Kaeffer, 2002), but only few attempts have been made to culture PCs from cattle (Dibb-Fuller et al., 2001, Föllmann et al., 2000, Hoey et al., 2003, Rusu et al., 2005). Bovine PC cultures were shown to be useful tools for toxicological assays (Föllmann and Birkner, 2008), and we recently used bovine PCs to study effects of a purified bacterial virulence factor (Shiga toxin 1) on IECs in cattle (Stamm et al., 2008). In the present study, we investigated whether the bovine PC model can be used to study the complex interactions between living bacteria and bovine colonic epithelium at a single cell level.

Section snippets

Isolation of bovine colonic crypts

Intestinal specimens taken from the ascending colon were obtained from freshly slaughtered cattle (18–24 months old) of different breeds from a local abattoir (Giessen, Germany). Colonic crypts were isolated as described (Föllmann et al., 2000) with some minor modifications (Stamm et al., 2008). Briefly, mucosal tissue was separated from the lamina propria by scraping with a sterile glass slide, homogenized by a razor blade and centrifuged (5 min, 130 × g, 4 °C) in Hanks’ buffered saline solution

Assessment of epithelial cell properties in short- and long-term PC cultures

After 72–96 h post-seeding of colonic crypts, epitheloid cells formed a confluent monolayer on the surface of collagen-coated culture flasks. Immunofluorescence microscopy performed on day 4 (96 h post-seeding) showed that epitheloid, large, mono- or bi-nucleated PCs that represented the vast majority of cells expressed epithelial cell-specific cytokeratin (Fig. 1A). Extension of the cultivation time (>4 days) resulted in an increase in the number of fibroblastoid, spindle-shaped,

Discussion

Host-specific in vitro systems, cell cultures in particular, are indispensable to reveal the molecular basis of commensal and parasitic lifestyles implemented by certain bacterial strains in different hosts. In vivo, colonic crypts maintain a sterile luminal content (Sansonetti, 2004). Consequently epithelial cells physiologically exposed to luminal microbes are the highly differentiated cells situated toward the surface of the crypts. However, these cells lack proliferative capacity, have a

Acknowledgments

We thank Gabriele Köpf and Ursula Leidner for excellent technical assistance. We acknowledge Anja Taubert and Carlos Hermosilla, Institut für Parasitologie, Justus-Liebig-Universität, Giessen, for helpful discussions. This work was supported by grants from the German Research Foundation (Collaborative Research Centre 535).

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