C.R. Stokes

Environmentally-acquired bacteria influence microbial diversity and natural innate immune responses at gut surfaces

BackgroundEarly microbial colonization of the gut reduces the incidence of infectious, inflammatory and autoimmune diseases. Recent population studies reveal that childhood hygiene is a significant risk factor for development of inflammatory bowel disease, thereby reinforcing the hygiene hypothesis and the potential importance of microbial colonization during early life. The extent to which early-life environment impacts on microbial diversity of the adult gut and subsequent immune processes has not been comprehensively investigated thus far. We addressed this important question using the pig as a model to evaluate the impact of early-life environment on microbe/host gut interactions during development.ResultsGenetically-related piglets were housed in either indoor or outdoor environments or in experimental isolators. Analysis of over 3,000 16S rRNA sequences revealed major differences in mucosa-adherent microbial diversity in the ileum of adult pigs attributable to differences in early-life environment. Pigs housed in a natural outdoor environment showed a dominance of Firmicutes, in particular Lactobacillus, whereas animals housed in a hygienic indoor environment had reduced Lactobacillus and higher numbers of potentially pathogenic phylotypes. Our analysis revealed a strong negative correlation between the abundance of Firmicutes and pathogenic bacterial populations in the gut. These differences were exaggerated in animals housed in experimental isolators. Affymetrix microarray technology and Real-time Polymerase Chain Reaction revealed significant gut-specific gene responses also related to early-life environment. Significantly, indoor-housed pigs displayed increased expression of Type 1 interferon genes, Major Histocompatibility Complex class I and several chemokines. Gene Ontology and pathway analysis further confirmed these results.ConclusionEarly-life environment significantly affects both microbial composition of the adult gut and mucosal innate immune function. We observed that a microbiota dominated by lactobacilli may function to maintain mucosal immune homeostasis and limit pathogen colonization.

Restricting microbial exposure in early life negates the immune benefits associated with gut colonization in environments of high microbial diversity.

Background Acquisition of the intestinal microbiota in early life corresponds with the development of the mucosal immune system. Recent work on caesarean-delivered infants revealed that early microbial composition is influenced by birthing method and environment. Furthermore, we have confirmed that early-life environment strongly influences both the adult gut microbiota and development of the gut immune system. Here, we address the impact of limiting microbial exposure after initial colonization on the development of adult gut immunity. Methodology/Principal Findings Piglets were born in indoor or outdoor rearing units, allowing natural colonization in the immediate period after birth, prior to transfer to high-health status isolators. Strikingly, gut closure and morphological development were strongly affected by isolator-rearing, independent of indoor or outdoor origins of piglets. Isolator-reared animals showed extensive vacuolation and disorganization of the gut epithelium, inferring that normal gut closure requires maturation factors present in maternal milk. Although morphological maturation and gut closure were delayed in isolator-reared animals, these hard-wired events occurred later in development. Type I IFN, IL-22, IL-23 and Th17 pathways were increased in indoor-isolator compared to outdoor-isolator animals during early life, indicating greater immune activation in pigs originating from indoor environments reflecting differences in the early microbiota. This difference was less apparent later in development due to enhanced immune activation and convergence of the microbiota in all isolator-reared animals. This correlated with elevation of Type I IFN pathways in both groups, although T cell pathways were still more affected in indoor-reared animals. Conclusions/Significance Environmental factors, in particular microbial exposure, influence expression of a large number of immune-related genes. However, the homeostatic effects of microbial colonization in outdoor environments require sustained microbial exposure throughout development. Gut development in high-hygiene environments negatively impacts on normal succession of the gut microbiota and promotes innate immune activation which may impair immune homeostasis.

Analysis of the immunological cross reactivities of 213 well characterized monoclonal antibodies with specificities against various leucocyte surface antigens of human and 11 animal species.

213 Monoclonal antibodies (mAbs) raised against leucocyte surface antigens from human and 11 animal species were analyzed for reactivities against leucocytes from human and 15 different animal species. We found 77 mAbs (36%) to cross-react. Altogether, 217 cross reactions were registered out of 3195 possible combinations (7%). Most of the cross reacting mAbs had integrin or MHC class II specificities. This study defined cross reactions on the following markers: CD1a, 1c, 2, 4, 5, 8, 9, 11a, 11b, 14, 18, 20, 21, 23, 29, 31, 41, 43, 44, 45, 45R, 46, 49, 61, 62L, TCR gamma/delta, BCR, Thy-1, MHC class I and MHC class II, Swine-WC7 and Cattle-WC1. In order to characterize the molecular weight (MW) of the corresponding cross reacting antigens, selected mAbs were used to immunoprecipitate the antigens. The MWs of the analyzed precipitated antigens were in good agreement with the MWs of the homologous antigens. The followed strategy was found to be efficient and economical in defining new leucocyte antigen reactive mAbs.

A monoclonal antibody recognising an epitope associated with pig interleukin-2 receptors.

A monoclonal antibody is described which recognises an epitope associated with a receptor for interleukin-2 (IL-2) on pig lymphocytes. The monoclonal antibody inhibits high affinity binding of radiolabelled recombinant human IL-2 (rhIL-2) by pig lymphoblasts and also non-competitively inhibits both pig-TCGF and rhIL-2 maintained proliferation. By flow cytometry the antigen is apparently not present on freshly isolated blood lymphocytes but is detectable on small cells between 6 and 12 h after activation and on large cells by 24-h. These findings are comparable with those obtained using monoclonal antibodies recognising the 55 kDa alpha chain of the human and mouse IL-2 receptor (p55, TAC) expressed on activated cells in vivo and in vitro. However, the molecular weight of the porcine antigen is between 65 and 70 kDa.

Characterization of monoclonal antibodies specific for monocytes, macrophages and granulocytes from porcine peripheral blood and mucosal tissues.

A panel of four monoclonal antibodies produced in our laboratory, MIL1, MIL2, MIL3, MIL4, and the type-specific monocyte/granulocyte marker 74-22-15 were used to isolate and to discriminate between monocytes, macrophages and granulocytes derived from porcine peripheral blood, lung and gut lamina propria. Two-colour flow cytometry and cell sorting showed that while no monoclonal antibody was specific for just a single cell population, each cell type had a unique and characteristic combination of surface antigens. These differences could be used to identify and purify monocytes, macrophages, neutrophils, eosinophils and basophils from the three different sites. The study also demonstrated similarities and differences within cell types from the same site and from different sites: polymorphonuclear neutrophils (PMN) from peripheral blood were subdivided into two subpopulations by the presence or absence of the surface antigen recognized by MIL4, while PMN from alveolar lavage did not express this antigen. Peripheral blood eosinophils were also divided into subpopulations by the presence or absence of the same surface antigen. Lamina propria eosinophils strongly expressed the MIL4 marker and differed morphologically from blood eosinophils. Peripheral blood basophils and lamina propria mast cells were morphologically similar and expressed similar antigens. Monocytes and alveolar macrophages also expressed the same surface antigens.

Establishment of normal gut microbiota is compromised under excessive hygiene conditions

Background Early gut colonization events are purported to have a major impact on the incidence of infectious, inflammatory and autoimmune diseases in later life. Hence, factors which influence this process may have important implications for both human and animal health. Previously, we demonstrated strong influences of early-life environment on gut microbiota composition in adult pigs. Here, we sought to further investigate the impact of limiting microbial exposure during early life on the development of the pig gut microbiota. Methodology/Principal Findings Outdoor- and indoor-reared animals, exposed to the microbiota in their natural rearing environment for the first two days of life, were transferred to an isolator facility and adult gut microbial diversity was analyzed by 16S rRNA gene sequencing. From a total of 2,196 high-quality 16S rRNA gene sequences, 440 phylotypes were identified in the outdoor group and 431 phylotypes in the indoor group. The majority of clones were assigned to the four phyla Firmicutes (67.5% of all sequences), Proteobacteria (17.7%), Bacteroidetes (13.5%) and to a lesser extent, Actinobacteria (0.1%). Although the initial maternal and environmental microbial inoculum of isolator-reared animals was identical to that of their naturally-reared littermates, the microbial succession and stabilization events reported previously in naturally-reared outdoor animals did not occur. In contrast, the gut microbiota of isolator-reared animals remained highly diverse containing a large number of distinct phylotypes. Conclusions/Significance The results documented here indicate that establishment and development of the normal gut microbiota requires continuous microbial exposure during the early stages of life and this process is compromised under conditions of excessive hygiene.

Regulation of mucosal immune responses in effector sites

In human disease and rodent models, immune responses in the intestinal mucosa can be damaging. Damage is characterised by villus atrophy, crypt hyperplasia and reduced ability to digest and absorb nutrients. In normal individuals active responses to harmless environmental antigens associated with food and commensal bacteria are controlled by the development of immunological tolerance. Similar pathological changes occur in piglets weaned early from their mothers. Active immune responses to food antigens are observed in these piglets, and we and others have hypothesised that the changes occur as a result of transient allergic immune responses to novel food or bacteria antigens. The normal mechanism for producing tolerance to food antigens may operate at induction (Peyers patches and mesenteric lymph nodes) or at the effector stage (intestinal lamina propria). In our piglet studies immunological tolerance occurs despite the initial active response. Together with evidence from rodents, this observation suggests that active responses are likely to be controlled at the effector stage, within the intestinal lamina propria. Support for this mechanism comes from the observation that human and pig intestinal T-cells are susceptible to apoptosis, and that this process is accelerated by antigen. We suggest that the role of the normal mature intestinal lamina propria is a balance between immunological effector and regulatory function. In neonatal animals this balance develops slowly and is dependant on contact with antigen. Immunological insults such as weaning may tip the balance of the developing mucosal immune system into excessive effector or regulatory function resulting in transient or chronic allergy or disease susceptibility.

Whole Cholera Toxin and B Subunit Act Synergistically as an Adjuvant for the Mucosal Immune Response of Mice to Keyhole Limpet Haemocyanin

Cholera toxin (CT) is a potent stimulator of IgA responses when adtninistered orally and has been shown to promote IgA responses to a second protein such as keyhole limpet haemocyanin (KLH) if this is fed simultaneously. ln this paper we show that whilst feeding 5 mg KLH with either 0.5 μg CT or 10 μg B subunit fails to stimulate a mucosal IgA response to KLH, feeding 0.5μg CT and 10 μg B subunit together with 5 mg KLH produces a local IgA anti‐KLH response as greal as that produced by 10 μg of whole CT. ln addition to stimulating IgA responses in the lamina propria, preliminary results indicate that cellular responses are also stimulated, as we have demonstrated KLH antigen‐driven proliferation of cells isolated from groups of mice fed either 10 μg CT + 5 mg KLH or O.5 μg CT + 10 μg CTB + 5 mg KLH but not mice fed KLH alone or with either 10 μg CTB or O.5 μg CT.

Immune cell distribution in the small intestine of the pig: immunohistological evidence for an organized compartmentalization in the lamina propria.

Using monoclonal antibodies in immunohistochemistry, the distribution of the cells with the following surface antigens was studied in samples of proximal and distal small intestine of five 6-month-old pigs: CD2, CD4 (helper/inducer T-cells), CD8 (suppressor/cytotoxic T cells), accessory cell marker (monocyte/granulocyte), MHC Class II (DRw), and interleukin 2 (IL-2) receptor. CD2+ cells were found in high numbers in both the epithelium and the lamina propria. More cells were demonstrated in villis than in crypts (proportion approximately 4:1). At least two subpopulations of intraepithelial lymphocytes were identified: apically in the epithelium there were CD2+CD4-CD8- (double negative) cells, whereas cells expressing CD8 marker were concentrated around the basement membrane. CD4+ cells were localized in the lamina propria towards the villus core. Accessory cells were distributed in crypts and the villus base and more cells were found in ileum than in duodenum. In contrast, MHC Class II+ cells were located predominantly in villi, just underneath the basement membrane, forming a sheath of cells between the CD8+ and the CD4+ cells. Cells expressing IL-2 receptor were sparse but widely distributed in both the lamina propria and the epithelium. This organized cell distribution may be related to the physiology of the mucosal immune system in the gut.

Effect of early weaning on the development of immune cells in the pig small intestine

The controlled effects of age and weaning on the numbers of CD2+ T cells, subsets (CD4+, CD8+), accessory cells (macrophage/granulocyte) and cells expressing MHC class II (DQw) and IL-2R in the piglet intestine was investigated. At birth low numbers of CD2+CD4-CD8- cells were the only demonstrable T cells in the intestine. Monocyte/granulocyte and MHC class II+ cells were also detected in low numbers and IL-2R+ cells were proportionally quite numerous. All those cell populations, except the IL-2R+ cells, increased thereafter and peaked at Week 7 when the numbers of cells were comparable with those of adult animals. CD4+ cells increased dramatically after Week 1. In contrast, CD8+ remained scarce until after 5-7 weeks of age in unweaned animals. Four days after weaning at 3 weeks old, there were increases in CD2+ (P < 0.001) and macrophage/granulocyte (P < 0.01) cells in proximal small intestinal villi and in CD2+ cells only (P < 0.01) in crypts. No significant changes in cell numbers were demonstrated in the distal small intestine.