David S. Donaldson

Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium

The transcytosis of antigens across the gut epithelium by microfold cells (M cells) is important for the induction of efficient immune responses to some mucosal antigens in Peyer’s patches. Recently, substantial progress has been made in our understanding of the factors that influence the development and function of M cells. This review highlights these important advances, with particular emphasis on: the host genes which control the functional maturation of M cells; how this knowledge has led to the rapid advance in our understanding of M-cell biology in the steady state and during aging; molecules expressed on M cells which appear to be used as “immunosurveillance” receptors to sample pathogenic microorganisms in the gut; how certain pathogens appear to exploit M cells to infect the host; and finally how this knowledge has been used to specifically target antigens to M cells to attempt to improve the efficacy of mucosal vaccines.

Salmonella Transforms Follicle-Associated Epithelial Cells into M Cells to Promote Intestinal Invasion

Salmonella Typhimurium specifically targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. Although M cells represent a small proportion of the specialized follicular-associated epithelium (FAE) overlying mucosa-associated lymphoid tissues, their density increases during Salmonella infection, but the underlying molecular mechanism remains unclear. Using in vitro and in vivo infection models, we demonstrate that the S. Typhimurium type III effector protein SopB induces an epithelial-mesenchymal transition (EMT) of FAE enterocytes into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/β-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, via the activity of a single secreted effector, S. Typhimurium transforms primed epithelial cells into M cells to promote host colonization and invasion.

M cell-depletion blocks oral prion disease pathogenesis

Many prion diseases are orally acquired. Our data show that after oral exposure, early prion replication upon follicular dendritic cells (FDC) in Peyers patches is obligatory for the efficient spread of disease to the brain (termed neuroinvasion). For prions to replicate on FDC within Peyers patches after ingestion of a contaminated meal, they must first cross the gut epithelium. However, the mechanism through which prions are conveyed into Peyers patches is uncertain. Within the follicle-associated epithelium overlying Peyers patches are microfold cells (M cells), unique epithelial cells specialized for the transcytosis of particles. We show that following M cell-depletion, early prion accumulation upon FDC in Peyers patches is blocked. Furthermore, in the absence of M cells at the time of oral exposure, neuroinvasion and disease development are likewise blocked. These data suggest M cells are important sites of prion uptake from the gut lumen into Peyers patches.

Expression of mesenchyme-specific gene signatures by follicular dendritic cells: insights from the meta-analysis of microarray data from multiple mouse cell populations

Follicular dendritic cells (FDC) are an important subset of stromal cells within the germinal centres of lymphoid tissues. They are specialized to trap and retain antigen‐containing immune complexes on their surfaces to promote B‐cell maturation and immunoglobulin isotype class‐switching. However, little is known of the cell types from which FDC originate. To address fundamental questions associated with the relationships between FDC and other cell populations, we took advantage of the growing body of publicly available data for transcriptome analysis. We obtained a large number of gene expression data files from a range of different primary mouse cells and cell lines and subjected these data to network‐based cluster analysis using BiolayoutExpress3D. Genes with related function clustered together in distinct regions of the graph and enabled the identification of transcriptional networks that underpin the functional activity of distinct cell populations. Several gene clusters were identified that were selectively expressed by cells of mesenchymal lineage and contained classic mesenchymal cell markers and extracellular matrix genes including various collagens, Acta2, Bgn, Fbn1 and Twist1. Our analysis showed that FDC also express highly many of these mesenchyme‐associated genes. Promoter analysis of the genes comprising the mesenchymal clusters identified several regulatory motifs that are binding sites for candidate transcription factors previously known to be candidate regulators of mesenchyme‐specific genes. Together, these data suggest FDC are a specialized mesenchymal cell population within the germinal centres of lymphoid tissues.

The functional maturation of M cells is dramatically reduced in the Peyer’s patches of aged mice

The transcytosis of antigens across the follicle-associated epithelium (FAE) of Peyer’s patches by microfold cells (M cells) is important for the induction of efficient immune responses to mucosal antigens. The mucosal immune response is compromised by ageing, but effects on M cells were unknown. We show that M-cell density in the FAE of aged mice was dramatically reduced. As a consequence, aged Peyer’s patches were significantly deficient in their ability to transcytose particulate lumenal antigen across the FAE. Ageing specifically impaired the expression of Spi-B and the downstream functional maturation of M cells. Ageing also dramatically impaired C-C motif chemokine ligand 20 expression by the FAE. As a consequence, fewer B cells were attracted towards the FAE, potentially reducing their ability to promote M-cell maturation. Our study demonstrates that ageing dramatically impedes the functional maturation of M cells, revealing an important ageing-related defect in the mucosal immune system’s ability to sample lumenal antigens.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis.

Aging and the mucosal immune system in the intestine

Bacterial and viral infections of the gastrointestinal tract are more common in the elderly and represent a major cause of morbidity and mortality. The mucosal immune system provides the first line of defence against pathogens acquired by ingestion and inhalation, but its function is adversely affected in the elderly. This aging-related decline in the immune function is termed immunosenescence and is associated with diminished abilities to generate protective immunity, reduced vaccine efficacy, increased incidence of cancer, inflammation and autoimmunity, and the impaired ability to generate tolerance to harmless antigens. In this review we describe our current understanding of the effects immunosenescence has on the innate and adaptive arms of the mucosal immune system in the intestine. Current estimates suggest that by the year 2050 up to 40xa0% of the UK population will be over 65xa0years old, bringing with it important health challenges. A thorough understanding of the mechanisms that contribute to the development of immunosenescence is therefore crucial to help identify novel approaches to improve mucosal immunity in the elderly.

The MacBlue Binary Transgene (csf1r-gal4VP16/UAS-ECFP) Provides a Novel Marker for Visualisation of Subsets of Monocytes, Macrophages and Dendritic Cells and Responsiveness to CSF1 Administration

The MacBlue transgenic mouse uses the Csf1r promoter and first intron to drive expression of gal4-VP16, which in turn drives a cointegrated gal4-responsive UAS-ECFP cassette. The Csf1r promoter region used contains a deletion of a 150 bp conserved region covering trophoblast and osteoclast-specific transcription start sites. In this study, we examined expression of the transgene in embryos and adult mice. In embryos, ECFP was expressed in the large majority of macrophages derived from the yolk sac, and as the liver became a major site of monocytopoiesis. In adults, ECFP was detected at high levels in both Ly6C+ and Ly6C- monocytes and distinguished them from Ly6C+, F4/80+, CSF1R+ immature myeloid cells in peripheral blood. ECFP was also detected in the large majority of microglia and Langerhans cells. However, expression was lost from the majority of tissue macrophages, including Kupffer cells in the liver and F4/80+ macrophages of the lung, kidney, spleen and intestine. The small numbers of positive cells isolated from the liver resembled blood monocytes. In the gut, ECFP+ cells were identified primarily as classical dendritic cells or blood monocytes in disaggregated cell preparations. Immunohistochemistry showed large numbers of ECFP+ cells in the Peyers patch and isolated lymphoid follicles. The MacBlue transgene was used to investigate the effect of treatment with CSF1-Fc, a form of the growth factor with longer half-life and efficacy. CSF1-Fc massively expanded both the immature myeloid cell (ECFP−) and Ly6C+ monocyte populations, but had a smaller effect on Ly6C− monocytes. There were proportional increases in ECFP+ cells detected in lung and liver, consistent with monocyte infiltration, but no generation of ECFP+ Kupffer cells. In the gut, there was selective infiltration of large numbers of cells into the lamina propria and Peyers patches. We discuss the use of the MacBlue transgene as a marker of monocyte/macrophage/dendritic cell differentiation.

Reciprocal regulation of lymphoid tissue development in the large intestine by IL-25 and IL-23

Isolated lymphoid follicles (ILFs) develop after birth in the small and large intestines (SI and LI) and represent a dynamic response of the gut immune system to the microbiota. Despite their similarities, ILF development in the SI and LI differs on a number of levels. We show that unlike ILF in the SI, the microbiota inhibits ILF development in the colon as conventionalization of germ-free mice reduced colonic ILFs. From this, we identified a novel mechanism regulating colonic ILF development through the action of interleukin (IL)-25 on IL-23 and its ability to modulate T regulatory cell (Treg) differentiation. Colonic ILF develop in the absence of a number of factors required for the development of their SI counterparts and can be specifically suppressed by factors other than IL-25. However, IL-23 is the only factor identified that specifically promotes colonic ILFs without affecting SI-ILF development. Both IL-23 and ILFs are associated with inflammatory bowel disease, suggesting that disruption to this pathway may have an important role in the breakdown of microbiota-immune homeostasis.

The Gut-Associated Lymphoid Tissues in the Small Intestine, Not the Large Intestine, Play a Major Role in Oral Prion Disease Pathogenesis

ABSTRACT Prion diseases are infectious neurodegenerative disorders characterized by accumulations of abnormally folded cellular prion protein in affected tissues. Many natural prion diseases are acquired orally, and following exposure, the early replication of some prion isolates upon follicular dendritic cells (FDC) within gut-associated lymphoid tissues (GALT) is important for the efficient spread of disease to the brain (neuroinvasion). Prion detection within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, the relative contributions of the small and large intestinal GALT to oral prion pathogenesis were unknown. To address this issue, we created mice that specifically lacked FDC-containing GALT only in the small intestine. Our data show that oral prion disease susceptibility was dramatically reduced in mice lacking small intestinal GALT. Although these mice had FDC-containing GALT throughout their large intestines, these tissues were not early sites of prion accumulation or neuroinvasion. We also determined whether pathology specifically within the large intestine might influence prion pathogenesis. Congruent infection with the nematode parasite Trichuris muris in the large intestine around the time of oral prion exposure did not affect disease pathogenesis. Together, these data demonstrate that the small intestinal GALT are the major early sites of prion accumulation and neuroinvasion after oral exposure. This has important implications for our understanding of the factors that influence the risk of infection and the preclinical diagnosis of disease. IMPORTANCE Many natural prion diseases are acquired orally. After exposure, the accumulation of some prion diseases in the gut-associated lymphoid tissues (GALT) is important for efficient spread of disease to the brain. However, the relative contributions of GALT in the small and large intestines to oral prion pathogenesis were unknown. We show that the small intestinal GALT are the essential early sites of prion accumulation. Furthermore, congruent infection with a large intestinal helminth (worm) around the time of oral prion exposure did not affect disease pathogenesis. This is important for our understanding of the factors that influence the risk of prion infection and the preclinical diagnosis of disease. The detection of prions within large intestinal GALT biopsy specimens has been used to estimate human and animal disease prevalence. However, our data suggest that using these biopsy specimens may miss individuals in the early stages of oral prion infection and significantly underestimate the disease prevalence.