Immune development in jejunal mucosa after colonization with selected commensal gut bacteria: A study in germ-free pigs

Abstract

The immunological structure of the porcine jejunal lamina propria in germ-free piglets was compared with that of their counterparts associated with two strains of commensal Escherichia coli, A0 34/86 serotype O83:K24:H31 and the O86 E. coli strain, up to 20 days post-colonization. In the antigen-presenting compartment, both dendritic cells (DC) and cells expressing CD163, probably macrophages were investigated. In addition we also assessed the number of CD2+/CD3+ (T) cells. In contrast to some previous reports, we show a total lack of both DC and T cells for germ-free animals in the diffuse lymphoid tissue of villi and crypts of the jejunum. Association with either strain of commensal E. coli had a profound effect on the immune structure and resulted in extensive recruitment of DC to the lamina propria and of T cells to epithelium and lamina propria. The data suggest that the earliest immigrant cells were monocytes, which soon acquired the phenotype of mucosal DC. T cells migrated in at a slightly slower rate. Nevertheless, the response could be extremely rapid: within 3 days of colonization with O83, the magnitude of this response was comparable to that observed 20 days post-colonization.

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.