Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: A study in germ-free pigs
Introduction
There is epidemiological and experimental evidence that colonization with commensal bacteria is essential for many functional processes in the gut including the development of a balanced and regulated immune system (Hooper and Gordon, 2001, Hooper et al., 2001). The ‘hygiene hypothesis’ suggests that this regulation may be disturbed in the Western world (Wold, 1998, Liu and Murphy, 2003), accounting for the recent epidemic in atopic diseases in the Western world. However, it has proved extremely difficult to identify the detailed mechanisms involved. Microbial colonization of the gut involves a huge number of complex and interactive populations of microorganisms, most of which are still largely unknown. Microbial composition is highly variable, both with time as well as between individuals, and even varies between litter mates reared under identical environmental conditions. Therefore, the role of individual components in such a complex system is difficult to elucidate. Nevertheless, there is considerable evidence that certain subpopulations of intestinal microorganisms have desirable effects on gut health and the immune system, as so-called probiotics. Lactobacilli and bifidobacteria, certain strains of commensal Escherichia coli and other microorganisms have been used as probiotics in humans. In rodents, segmented filamentous bacteria have been shown to affect the mucosal immune system in various ways: Peyer's patch (PP) germinal centre formation is affected and mucosal epithelial cells as well as intra-epithelial lymphocytes are stimulated and expanded (Okada et al., 1994, Umesaki et al., 1995, Jiang et al., 1998, Talham et al., 1999).
The pig provides an exceptional model to study the effect of environmental factors on the development of the immune system, as piglets are born immunologically naïve, without maternal antibody or other maternally transmitted macromolecules, due to their epithelio-chorial placenta. Thus, pigs have been used extensively for studies of the effect of gut microflora on the immune system, both in conventional animals as well as in animals raised under germ-free conditions. Colonization with the probiotic bacterium Enterococcus faecium SF68 resulted in decreased colonization by certain pathogenic serovars of E. coli (Scharek et al., 2005). Pre-colonization with an avirulent Salmonella strain protected gnotobiotic piglets against subsequent colonization with the pathogenic strain Salmonella enterica serovar Infantis (Foster et al., 2003). Multiple effects on the immune system have also been reported: Pigs raised under germ-free conditions cannot make serum antibodies to T-dependent and type 2 T-independent antigens (Butler et al., 2002). Colonization diversifies the pre-immune repertoire in mucosal lymphoid tissues (Butler et al., 2000) and expands the T cell receptor (TCR) repertoire in tonsils (Wilson et al., 2005). An increase of blood monocyte oxidative burst activity (Rehakova et al., 1998b) and phenotypic changes in peripheral B cells could also be shown after colonization of germ-free piglets (Sinkora et al., 1998).
The two E. coli strains O86 and O83 used to colonize the germ-free (GF) piglets in this experiment are classified as commensals in conventional pigs, some effects on health status and/or the immune system are already known. O83 has been used successfully as an immune-modulator and ‘probiotic’ in human infants (Lodinova-Zadnikova et al., 1991, Dlabac et al., 1995, Lodinova-Zadnikova and Sonnenborn, 1997, Cukrowska et al., 2002, Vancikova et al., 2003). O86 affected the composition of peripheral T cell subsets (Rehakova et al., 1998a) in pigs and specifically and polyclonally stimulated serum and mucosal antibody (Cukrowska et al., 2001).
Peyer's patches are involved in the inductive phase of the mucosal immune response, whereas the so-called diffuse tissue is traditionally thought to be an effector site. However, there is increasing evidence that in healthy animals, the role of the diffuse lymphoid tissue in the gut is also that of immune regulation (Bailey and Haverson, 2006) and that early environmental events can affect its long-term function.
We and others have presented evidence of the highly organized and compartmentalized structure of the diffuse immune tissue of the pig jejunum in mature animals, containing large numbers of immature DC (Haverson et al., 2000, Bimczok et al., 2005, Bimczok et al., 2006, Haverson and Riffault, 2006). Also present are large numbers of T cells (Vega-Lopez et al., 1993, Rothkötter et al., 1994) prone to activation-induced apoptosis (Bailey et al., 1998, Bailey and Haverson, 2006). Immature DC are potentially involved in immune regulation as well as defence and are thought to play a major role in the decision making process between active defence and tolerance. This structure is almost totally lacking at birth and develops gradually over a period of several weeks (Rothkötter et al., 1994, Vega-Lopez et al., 1995). Previous studies with GF piglets have shown reduced T and B cell numbers (Rothkötter and Pabst, 1989, Rothkötter et al., 1991, Rothkötter et al., 1999) in PP and gut lamina propria (LP). However, no studies have been conducted during the early phase post-colonization with single microbial strains.
Therefore, we have investigated the effect of mono-association of germ-free piglets with these two commensal strains of E. coli on the immune development of the diffuse lymphoid tissue, with particularly emphasis on the antigen-presenting cells. This was done by multicolour immunohistochemistry and pixel-based image analysis, labelling molecules characteristic for DC, monocytes/macrophages and T cells.
Section snippets
Animals and bacterial association
Germ-free Minnesota minipigs reared in Novy Hradek, Czech Republic, were delivered by hysterectomy and raised colostrum-free under germ-free conditions on autoclaved bovine milk as previously described (Mandel and Travnicek, 1987). Bacterial mono-association was done when piglets were between 1 and 4 days of age. GF piglets were mono-associated orally with 107 CFU of the A0 34/86 serotype O83:K24:H31 (O83 hereafter) and the O86 E. coli strain (Sinkora et al., 1998, Cukrowska et al., 1998,
Results
Fig. 1 illustrates the events immediately post microbial colonization with O86. It is apparent that 1 day post-association (Fig. 1a and d), cells expressing CD163 (arrowhead) and cells expressing MHC II (arrow) are present, but frequently represent different cell populations. One cell co-expressing CD163 and MHC II can also be seen (**). One day later (Fig. 1b and e), many more cells have been recruited, mostly co-expressing both molecules. However, after 20 days (Fig. 1c and f), although large
Bacterial colonization
Although both O83 and O86 strains of E. coli are classified as ‘commensals’ in normal pigs, we observed significant piglet morbidity and mortality following association of germ-free animals younger than 4 days with the O83 strain, although this strain has been used successfully as a probiotic in infants. Piglets associated with O86 aged 1 or 2 days had 100% survival rates.
O83 appears to act as a pathogen in these very young germ-free colostrum-deprived animals, whereas E. coli O86 appears to be
Acknowledgements
The support of this work by funding from grant no. 523/03/0186 of the Grant Agency of the Czech Republic (GACR) and also by the European Union (Framework FP 5), as part of a project entitled ‘Defining and validating gut health criteria in young pig, based on digestive physiology, microbiology and mucosal immunology investigations for testing alternative strategies to in-feed antibiotics’, contract number QLK5-LT2000-00522, is gratefully acknowledged, as is the financial support by the British
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Address: University of Defense, Hradec Kralove, Czech Republic.
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Address: DakoCytomation AG, Brno, Czech Republic.