Gut-Innervating Nociceptor Neurons Regulate Peyer's Patch Microfold Cells and SFB Levels to Mediate Salmonella Host Defense.

Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.

Innervation and nerve-immune cell contacts in mouse Peyer's patches.

Neural regulation of the function of the gastrointestinal tract (GIT) relies on a delicate balance of the two divisions of its nervous system, namely, the intrinsic and extrinsic divisions. The intrinsic innervation is provided by the enteric nervous system (ENS), whereas the extrinsic innervation includes sympathetic/parasympathetic nerve fibers and extrinsic sensory nerve fibers. In the present study, we used immunofluorescent staining of neurofilament-heavy (NF-H) to reveal the distribution of nerve fibers and their associations with immune cells inside mouse Peyer's patches (PP), an essential part of gut-associated lymphoid tissue (GALT). Our results demonstrate (1) the presence of an extensive meshwork of NF-H-immunoreactive presumptive nerve fibers in all PP compartments including the lymphoid nodules, interfollicular region, follicle-associated epithelium, and subepithelial dome; (2) close associations/contacts of nerve fibers with blood vessels including high endothelial venules, indicating neural control of blood flow and immune cell dynamics inside the PP; (3) close contacts between nerve fibers/endings and B/T cells and various subsets of dendritic cells ( e.g., B220⁻, B220⁺, CD4⁻, CD4⁺, CD8⁻, and CD8⁺). Our novel findings concerning PP innervation and nerve-immune-cell contacts in situ should facilitate our understanding of bi-directional communications between the PNS and GALT. Since the innervation of the gut, including PP, might be important in the pathogenesis and progression of some neurological, infectious, and autoimmune diseases, e.g., prion diseases and inflammatory bowel disease, better knowledge of PNS-immune system interactions in the GALT (including PP) should benefit the development of potential treatments for these diseases via neuroimmune manipulations.

An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine.

Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer's patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiologically relevant environment that preserved normal enterocyte structure, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane-impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional interstitial cells of Cajal to the extent that nonstimulated, segmental contractions occurred for up to 48 h ex vivo. To detect changes in physiological responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respectively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model renders mechanisms of neuroimmune-microbiome interactions in a complex gut environment available to direct observation and controlled perturbation.

Zearalenone-induced changes in the lymphoid tissue and mucosal nerve fibers in the porcine ileum.

This is the first study to examine zearalenone-(ZEN) induced changes in the immune system of the ileum and substance P-(SP-) and vasoactive intestinal peptide-(VIP-) immunoreactive nerve fibers in the mucosa, which participate in the regulation of intestinal functions under physiological conditions and during pathological processes. The aim of this study was also to identify potential relationships between selected immune and neural elements in ileal Peyer's patches in pigs that were and were not exposed to ZEN. The experiment was performed on 10 prepubertal gilts divided into two groups: the experimental group (n=5) where ZEN was administered at 0.1 mg kg-1 feed day-1 for 42 days, and the control group (n=5) which was administered a placebo. The tissue levels of cytokines were determined by enzyme-linked immunosorbent assay which revealed elevated concentrations of IL-12/23 40p and IL-1 ß in animals exposed to ZEN. Flow cytometry revealed a lower percentage of CD21+ lymphocytes in pigs exposed to ZEN in comparison with control animals. The tissue levels of neuropeptides were evaluated in the dot blot procedure which demonstrated higher concentrations of VIP and SP in experimental pigs. In experimental animals, numerous VIP-like immunoreactive processes were observed, and SP-immunoreactive nerve fibers formed a very dense network. Our results demonstrate for the first time that ZEN can modify the chemical coding of nerve structures in the gastrointestinal system. Those modifications can be attributed to ZEN's impact on estrogen receptors or its pro-inflammatory properties, and they reflect changes that take place in the nervous system at the transcriptional, translational and metabolic level.

Frequency and distribution of nerves in scrapie-affected and unaffected Peyer's patches and lymph nodes.

Transmission of sheep scrapie and some other prion diseases, including variant Creutzfeldt-Jakob disease of man, probably occurs via the oral route. A disease-associated variant of the host-coded prion protein (PrP(d)) accumulates in germinal center follicles of lymphoid tissues, including Peyer's patches of the gut, where it can be detected before its accumulation in the central nervous system. To investigate the potential role of lymphoid tissue nerves in neuroinvasion, we used immunohistochemical methods to study the frequency and distribution of nerves and PrP(d) accumulation in Peyer's patches and other lymphoid tissues from scrapie-affected and unaffected sheep. Nerves were infrequently found in secondary follicles of Peyer's patches, but never in germinal centers of the other lymphoid tissues tested. No differences in the frequency or distribution of nerves were found in relation to the presence or absence of PrP(d) accumulation. PrP(d) accumulation and nerves were only infrequently present together in Peyer's patches. These results suggest that, even if amplification of infectivity in lymphoid tissues facilitates neuroinvasion, nerves within lymph nodes and germinal centers of Peyer's patches do not play a primary role in transport of infectivity to the central nervous system. However, sheep between 2 and 4 months of age had significantly more nerve fibers within follicles than older groups. It is therefore possible that a general increase in nerve density of the intestine during early phases of life may contribute to an increased susceptibility of young animals to oral prion infection.

Eosinophil-nerve interactions and neuronal plasticity in rat gut associated lymphoid tissue (GALT) in response to enteric parasitism.

Intestinal lymphoid tissues and Peyer's patches (PP) are innervated sites of immune surveillance in the gastrointestinal tract. Following infection with F. hepatica, neuronal hyperplasia and significantly increased eosinophil and mast cell trafficking to colonic PP sites were evident in rat tissues. Nerve-eosinophil associations were significantly elevated in infected colon and colonic PP, as were colonic tissue levels of the circulatory recruitment factors IL-5 and eotaxin. Increased immunoreactivity for neuronal plasticity markers GAP-43 and neural cell adhesion molecule (NCAM) was also found in infected tissues. Such neuronal alterations in the PP during enteric parasitism may have functional consequences on particular or pathogen uptake.

Anatomical evidence for ileal Peyer's patches innervation by enteric nervous system: a potential route for prion neuroinvasion?

We have examined the innervation of the gut-associated lymphoid system of the sheep ileum, with a view to identifying potential sites for neuroinvasion by pathogens, such as prions (PrP(Sc)). Special attention has been paid to the follicles of Peyer's patches (PPs), which are major sites of PrP(Sc) accumulation during infection. Evidence exists that the enteric nervous system, together with the parasympathetic and sympathetic pathways projecting to the intestine, are important for PrP(Sc) entry into the central nervous system. Thus, PrP(Sc) might move from PPs to the neurons and nerve fibres that innervate them. We investigated, by immunohistochemistry and retrograde tracing (DiI) from the follicles, the distribution and phenotype of enteric neurons innervating the follicles. Antibodies against protein gene product 9.5, tyrosine hydroxylase, dopamine beta hydroxylase, choline acetyltransferase, calbindin (CALB), calcitonin gene-related peptide (CGRP), and nitric oxide synthase were used to characterise the neurons. Immunoreactivity for each of these was observed in fibres around and inside PP follicles. CGRP-immunoreactive fibres were mainly seen at the follicular dome. Retrograde tracing revealed submucosal neurons that contributed to the innervation of PPs, including Dogiel type II neurons and neurons immunoreactive for CALB and CGRP. The major source of the adrenergic fibres are the sympathetic ganglia. Our results thus suggest that enteric and sympathetic neurons are involved during the first stage of neuroinvasion, with neurons connecting to them acting as potential carriers of PrP(Sc) to the central nervous system.

Interaction between dendritic cells and nerve fibres in lymphoid organs after oral scrapie exposure.

In transmissible spongiform encephalopathies (TSEs), the infectious agent, called PrPsc, an abnormal isoform of the cellular prion protein, accumulates and replicates in lymphoid organs before affecting the nervous system. To clarify the cellular requirements for the neuroinvasion of the scrapie agent from the lymphoid organs to the central nervous system, we have studied, by confocal microscopy, the innervations within Peyer's patches, mesenteric lymph nodes and the spleen of mice in physiological conditions and after oral exposure to prion. Contacts between nerve fibres and PrPsc-associated cells, dendritic cells (DCs) and follicular dendritic cells (FDCs), were evaluated in preclinical prion-infected mice. Using a double immunolabelling strategy, we demonstrated the lack of innervation of PrPsc-accumulating cells (FDCs). Contacts between nerve fibers and PrPsc-propagating cells (DCs) were detected in T-cell zones and cell-trafficking areas. This supports, for the first time, the possible implication of dendritic cells in the prion neuroinvasion process.

Neuroimmune connections in jejunal and ileal Peyer's patches at various bovine ages: potential sites for prion neuroinvasion.

During preclinical stages of cattle orally infected with bovine spongiform encephalopathy (BSE), the responsible agent is confined to ileal Peyer's patches (IPP), namely in nerve fibers and in lymph follicles, before reaching the peripheral and central nervous systems. No infectivity has been reported in other bovine lymphoid organs, including jejunal Peyer's patches (JPP). To determine the potential sites for prion neuroinvasion in IPP, we analyzed the mucosal innervation and the interface between nerve fibers and follicular dendritic cells (FDC), two dramatic influences on neuroinvasion. Bovine IPP were studied at three ages, viz., newborn calves, calves less than 12 months old, and bovines older than 24 months, and the parameters obtained were compared with those of JPP. No differences in innervation patterns between IPP and JPP were found. The major difference observed was that, in calves of less than 12 months, IPP were the major mucosal-associated lymphoid organ that possessed a large number of follicles with extended FDC networks. Using a panel of antibodies, we showed that PP in 24-month-old bovines were highly innervated at various strategic sites assumed to be involved in the invasion and replication of the BSE pathogen: the suprafollicular dome, T cell area, and germinal centers. In PP in calves of less than 12 months old, no nerve fibers positive for the neurofilament markers NF-L (70 kDa) and NF-H (200 kDa) were observed in contact with FDC. Thus, in view of the proportion of these protein subunits present in neurofilaments, the innervation of the germinal centers can be said to be an age-dependent dynamic process. This variation in innervation might influence the path of neuroinvasion and, thus, the susceptibility of bovines to the BSE agent.

Anatomical evidence for enteric neuroimmune interactions in Peyer's patches.

Peyer's patches (PP), a key component of the gut-associated lymphoid tissue, serve as the primary inductive sites for intestinal immunity. In the present study, we addressed the hypothesis that the morphological features of PP innervation are consistent with an immunomodulatory role for the enteric nervous system. Laser scanning confocal microscopy was used to collect images through large tissue volumes, yielding a three-dimensional perspective of the neuronal network superimposed on PP follicles from porcine jejunum and human ileum. Peptidergic nerve fibers were found in close apposition to immunocytes within PP subepithelial domes and the adjacent villi. The results suggest that nerve fibers in PP may participate in neuroimmune cross-talk within individual antigen-sampling sites as well as integrate information across multiple antigen-sampling sites.

Immmunohistochemical study of the blood and lymphatic vasculature and the innervation of mouse gut and gut-associated lymphoid tissue.

The blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut-associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double-positive cells. Dendritic cells, the most important antigen-presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane-membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron-immune regulation/cross-talk and probably neuroinvasion of prion pathogens in the gut and GALT.

Neuromodulation of enteropathogen internalization in Peyer's patches from porcine jejunum.

Jejunal Peyer's patches (JPP) are innervated sites of immune induction and enteropathogen infection. We investigated the role of enteric nerves in modulating pathogen entry into porcine JPP. Presumptive norepinephrine (NE)-containing nerve fibers were localized in JPP domes and follicle-associated villi by secondary immunofluorescence histochemistry. NE or the neuronal conduction blocker saxitoxin increased intracellular internalization of pathogenic Salmonella choleraesuis and Escherichia coli O157:H7, but not nonpathogenic E. coli, into isolated JPP mucosa. NE action was prevented by the alpha-adrenergic antagonist phentolamine. Withdrawal of enteric neural activity or NE administration appears to modulate JPP interactions with pathogenic bacteria.

The topography, architecture and structure of the enteric nervous system in the jejunum and ileum of cattle.

To date, there appear to have been no detailed and clear descriptions of the nerve plexuses and their subdivisions in the intestine of cattle. In this study, the enteric nervous system in the jejunum and ileum of 12 1-y-old calves was examined using neurofilament protein and vasoactive intestinal peptide immunohistochemistry in wholemounts and paraffin sections combined with staining of paraffin and historesin sections with haematoxylin and eosin. The main organisation of the plexuses was similar to that of the pig, horse and man with external and internal submucous plexuses being morphologically distinct, with further subdivisions of the internal submucous plexus into the external and internal subplexuses. However, in contrast to pig, horse and man, the submucous layer was firmly attached to the inner circular muscle layer. The myenteric plexus was well developed with large ganglia, and primary and secondary nerve strands. Its main axis was oriented parallel to the outer longitudinal smooth muscle; large ganglia and primary nerve strands fused to form complex ganglia, and 2 types of tertiary nerve strands were observed. Antibodies to neurofilament proteins and vasoactive intestinal peptide revealed adendritic, pseudouniaxonal or multiaxonal type II neurons only in the myenteric and submucous plexuses. This appears to be the first report of the identification of isolated uniaxonal, multidendritic type IV neurons in the mucous pericryptal plexus. The new information presented here provides further evidence for the existence of anatomical and functional differences between the external and internal submucous plexuses and for supporting the nomenclature proposed earlier.

[Innervation of grouped lymphoid nodules (Peyer's patches) by the enteric nervous system and the topography of their interior neural elements in the rat]. / Dlitel'naia morfofunktsional'naia sokhrannost' srezov gippokampa morskoi svinki posle kratkoi obrabotki ingibitorami tsiklooksigenaz.

Using the complex of histological methods (staining with toluidine blue, silver nitrate impregnation and application of retrograde fluorescent dye primulin) the data on the neural elements spatial localization within Peyer patches of the small intestine and their connections with the rest of enteric metasympathetic nervous system in rat was obtained. Submucosal plexus that is significantly developed within this lymphoid organ and is divided into internal and external plexuses was found to be most essential to the innervation of rat Peyers patches. These plexuses innervate all Peyers patch areas:nodules, cupula and internodular zones and the nodule-associated epithelium. Moreover, it was shown that within Peyers patch the plexuses form an integral part with the rest of the enteric nervous system and possess close connections with ganglia that are distant from the patch and are related both to submucosal and myenteric nervous plexuses. Direct inputs into Peyers patch nervous plexuses from extramural ganglia are present as well. These data was considered as a morphological basis for functional interaction of nervous and immune systems within the enteric immune organ and for possible enteric nervous system regulation of immune functions.

Immunohistochemical localization of peptidergic nerve fibers and neuropeptide receptors in Peyer's patches of the cat ileum.

This study examined the distribution of peptidergic nerve fibers in Peyer's patches to determine whether appropriate receptors were present. Vasoactive intestinal peptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP) and receptors for VIP and SP were localized in lymphoid follicles of the cat ileum using a combined indirect horseradish peroxidase and streptavidin-biotin method. The margins of follicles were innervated by nerve fibers containing VIP, SP and CGRP. Nerve fibers were predominantly around lymphatics and high endothelial venules at the edges of follicles. Specific receptors for VIP and SP were present at the margins of follicles and in the lamina propria around crypts. VIP receptors were numerous on T cells within and around high endothelial venules and lymphatic vessels and at the margins of follicles. SP receptors were identified on a small number of T and B cells, granulocytes and macrophages, restricted to the margins of follicles. The defined distribution in ileal lymphoid tissue of nerve fibers containing VIP and SP and the corresponding localization of their appropriate receptors support immunoregulatory roles for neuropeptides in mucosal immunity.

The enteric nervous and immune systems: interactions for mucosal immunity and inflammation.

A number of anatomical studies have demonstrated the presence of peptidergic nerve fibers infiltrating mucosal lymphoid tissues. The exact mechanisms of how neuropeptides are released to affect these lymphoid sites are unclear, but radiolabeled binding studies have shown that mucosal leukocytes bear a number of neuropeptide receptors on their cell surfaces capable of responding to neural signals. The presence of neuropeptide-containing fibers and the ability to receive neural signals suggest that mucosal lymphocytes can be influenced by neurogenic mediators. The objectives set forth in this review are to provide what is currently known about the ability of substance P and vasoactive intestinal peptide to promote mucosal IgA responses in the gastrointestinal tract via Th2 mechanisms and to discuss how these neuropeptides contribute to the exacerbation of the inflammatory diseases of the gastrointestinal tract. We describe how immune responses develop in the gastrointestinal immune system and emphasize how neuropeptides may influence the differentiation of lymphocytes in mucosal inductive tissues and their subsequent expression in mucosal effector sites. Finally, we discuss new techniques developed by the Mucosal Immunization Research Group that have enabled the study of mucosal immune responses.

Topography of the enteric nervous system in Peyer's patches of the porcine small intestine.

The mechanisms of intercommunication between the immune and nervous systems are not fully understood. In the case of the intestine, the enteric nervous system is involved in the regulation of immune responses. It was therefore decided to employ immuno-histochemical techniques to investigate the structural organization of the enteric nervous system in Peyer's patches of the porcine small intestine. Using antibodies against various nervous system-specific markers (protein gene product 9.5, neuron-specific enolase, neurofilament 200, S-100 protein and the glial fibrillary acidic protein), an intimate and specific structural association could be demonstrated between enteric nerves and the compartments of Peyer's patches: follicles, interfollicular regions and domes. Peyer's patches have a close topographical relationship to the two submucosal plexuses. Enteric nerves are located around the follicle in the interfollicular area--the so-called "traffic area"--and in the dome area, which plays an important role in the uptake and presentation of antigens.

Distribution of somatostatin-immunoreactive nerve fibres in Peyer's patches.

The distribution of somatostatin-immunoreactive nerve fibres in Peyer's patches of the cat was demonstrated by immunocytochemical techniques. A large number of immunoreactive nerve fibres was observed in the tela submucosa very close to the Peyer's patches. Some immunoreactive nerve cell bodies were also found in this layer. The immunoreactive nerve terminals ran around the margin of the follicles and only a few nerve fibres were observed in the centre of follicles. Electron-microscopic investigation showed that these immunoreactive nerve terminals were in very close contact with lymphocytes and plasma cells, where no Schwann cell sheath was interposed. The gap between the nerve processes and the lymphocytes and plasma cells was about 20-200 nm, and occasionally less. These results provide morphological evidence consistent with the view that somatostatin has a neuroimmunomodulatory action.