Clusterin in human gut-associated lymphoid tissue, tonsils, and adenoids: localization to M cells and follicular dendritic cells

The follicle-associated epithelium (FAE) overlying the follicles of mucosa-associated lymphoid tissue is a key player in the initiation of mucosal immune responses. We recently reported strong clusterin expression in the FAE of murine Peyer’s patches. In this study, we examined the expression of clusterin in the human gut-associated lymphoid tissue (GALT) and Waldeyer’s ring. Immunohistochemistry for clusterin in human Peyer’s patches, appendix and colon lymphoid follicles revealed expression in M cells and in follicular dendritic cells (FDCs). Using cryo-immunogold electron microscopy in Peyer’s patches, we observed cytosolic immunoreactivity in M cells and labeling in the ER/Golgi biosynthetic pathway in FDCs. In palatine tonsils and adenoids, we demonstrated clusterin expression in germinal centers and in the lymphoepithelium in the crypts where M cells are localized. In conclusion, clusterin is expressed in M cells and follicular dendritic cells at inductive sites of human mucosa-associated lymphoid tissue suggesting a role for this protein in innate immune responses. Moreover, the use of clusterin as a human M cell marker could prove to be a valuable tool in future M cell research.     

Localization of fatty acid binding protein of epidermal type common to dendritic cells and presumptive macrophages in Peyer’s patches and epithelial M cells of mouse intestine

Fatty acid binding protein of epidermal type (E-FABP) was expressed/localized in most, if not all, populations of the dendritic cells in the subepithelial domes, follicles and interfollicular regions of Peyer’s patches and presumptive macrophages in their germinal centers, and all M cells in the follicle-associated epithelium of mouse intestine. The immunoreactivity in both of the cell populations makes it easy to recognize the accumulation of DCs in the subepithelial domes in close proximity to the base of M cells, which is essential for luminal antigens to be transported to Peyer’s patches. E-FABP may play some important roles in the mucosal immune reaction through Peyer’s patches and associated structures.     

Selective binding and transcytosis of latex microspheres by rabbit intestinal M cells

The interaction between polystyrene microspheres and the follicle-associated epithelium of rabbit Peyer's patches has been examined. Microspheres bind selectively to, and are transcytosed by, membranous or microfold (M) cells within the follicle-associated epithelium. M cells are able to transport, in 45 min, approximately 10⁵ microspheres of 0.46 μm diameter across the epithelium overlying each lymphoid follicle dome of rabbit Peyer's patches. The high capacity of M cells for particulate transcytosis and the subsequent delivery of these particulates to the mucosal immune system highlights the potential importance of this portal in the delivery of antigens and drugs. In addition, the selective binding and uptake of microspheres may be utilised as a functional marker in the identification and isolation of M cells.     

Systemic Antibodies Can Inhibit Mouse Mammary Tumor Virus-Driven Superantigen Response in Mucosa-Associated Lymphoid Tissues

Many mucosal pathogens invade the host by initially infecting the organized mucosa-associated lymphoid tissue (o-MALT) such as Peyer’s patches or nasal cavity-associated lymphoid tissue (NALT) before spreading systemically. There is no clear demonstration that serum antibodies can prevent infections in o-MALT. We have tested this possibility by using the mouse mammary tumor virus (MMTV) as a model system. In peripheral lymph nodes or in Peyer’s patches or NALT, MMTV initially infects B lymphocytes, which as a consequence express a superantigen (SAg) activity. The SAg molecule induces the local activation of a subset of T cells within 6 days after MMTV infection. We report that similar levels of anti-SAg antibody (immunoglobulin G) in serum were potent inhibitors of the SAg-induced T-cell response both in peripheral lymph nodes and in Peyer’s patches or NALT. This result clearly demonstrates that systemic antibodies can gain access to Peyer’s patches or NALT.     

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. This work was supported by the Région Wallonne and by the Leon Frederiq Foundation.     

Oral vaccination with recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase elicited a protective immunity in BALB/c mice

BackgroundTrichinellosis is a serious zoonotic disease distributed around the world. It is needed to develop a safe, effective and feasible anti-Trichinella vaccine for prevention and control of trichinellosis. The aim of this study was to construct a recombinant Lactobacillus plantarum encoding Trichinella spiralis inorganic pyrophosphatase (TsPPase) and investigate its immune protective effects against T. spiralis infection.Methodology/Principal findingsThe growth of recombinant L. plantarum was not affected by TsPPase/pSIP409-pgsA′ plasmid, and the recombinant plasmid was inherited stably in bacteria. Western blot and immunofluorescence assay (IFA) indicated that the rTsPPase was expressed on the surface of recombinant L. plantarum. Oral vaccination with rTsPPase induced higher levels of specific serum IgG, IgG1, IgG2a and mucosal secretory IgA (sIgA) in BALB/c mice. ELISA analysis revealed that the levels of IFN-γ and IL-4 released from spleen, mesenteric lymph nodes and Peyer’s patches were evidently increased at 2–4 weeks following vaccination, compared to MRS (De Man, Rogosa, Sharpe) medium control group (P < 0.05). Immunization of mice with rTsPPase exhibited a 67.18, 54.78 and 51.91% reduction of intestinal infective larvae, adult worms and muscle larvae at 24 hours post infection (hpi), 6 days post infection (dpi) and 35 dpi, respectively (P < 0.05), and the larval molting and development was significantly inhibited by 45.45% at 24 hpi, compared to the MRS group.ConclusionsTsPPase plays a crucial role in T. spiralis molting and development, oral vaccination with rTsPPase induced a significant local mucosal sIgA response and systemic Th1/Th2 immune response, and immune protection against T. spiralis infection in BALB/c mice.     

Recombinant expression, localization and in vitro inhibition of midgut cysteine peptidase (Sl-CathL) from sugarcane weevil, Sphenophorus levis

A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 °C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k_cat/K_m = 37.53 mM S⁻¹), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K_i = 0.196 nM), indicating that this protease is a potential target for pest control.     

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130 kDa Cry7Ab3 protoxin was degraded into the ～75 kDa proteinase-resistant fragments. In vivo analysis, 130 kDa Cry7Ab3 protoxin was also processed into ～75 kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220 kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.     

Oral scrapie infection modifies the homeostasis of Peyer’s patches’ dendritic cells

In transmitted prion diseases the immune system supports the replication and the propagation of the pathogenic agent (PrPSc). DCs, which are mobile cells present in large numbers within lymph organs, are suspected to carry prions through the lymphoid system and to transfer them towards the peripheral nervous system. In this study, C57Bl/6 mice were orally inoculated with PrPSc (scrapie strain 139A) and sacrificed at the preclinical stages of the disease. Immunolabelled cryosections of Peyer’s patches were analysed by confocal microscopy. Membrane prion protein expression was studied by flow cytometry. In Peyer’s patches (PP), dissected at day one and day 105 after oral exposure to scrapie, we observed an increased population of DCs localised in the follicular-associated epithelium. On day 105, PrPSc was found in the follicles inside the PP of prion-infected mice. A subset of Peyer’s patches DCs, which did not express cellular prion protein on their surface in non-infected mice conditions, was prion-positive in scrapie conditions. Within Peyer’s patches oral scrapie exposure thus induced modifications of the homeostasis of DCs at the preclinical stages of the disease. These results give new arguments in favour of the implication of DCs in prion diseases.     

Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells

Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer’s patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2±3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.This work was supported by the EU (5th framework programme “Healthypigut” QLK5-CT-2000-00522 and 6th framework programme “Feed for Pig Health” FOOD-CT 2004-506144).     

C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine

Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.     

Differential display analysis of gene expression during the induction of mucosal immunity

One approach to understanding the physiologically relevant events during the induction of an immune response is to identify genes that are expressed when the immune system first encounters antigen. Such an investigation requires a naive but fully functional immune system, and the fetal lamb provides these conditions during the last trimester of gestation. 'Intestinal segments,' containing a jejunal Peyer's patch, were surgically prepared in fetal lambs (>120 days gestation) and individual 'intestinal segments' were injected with either culture medium or infectious bovine rotavirus. Peyer's patch tissue was collected 18 h postinfection. Histology and virus culture confirmed that bovine rotavirus had infected the mucosal epithelium. RNA was extracted from jejunal Peyer's patch tissue and mRNA differential display was used to identify genes expressed following rotavirus infection. Ten cDNAs were identified by differential display and these cDNAs were isolated, cloned, and sequenced. One of the cDNAs sequenced, displayed homology to the gene encoding the sperm surface protein Sp17. Differential expression of this gene in antigen-exposed jejunal Peyer's patches was confirmed by Northern blot and RT-PCR. The complete sequence for sheep Sp17 mRNA was obtained from a lambda cDNA library, prepared from the jejunal Peyer's patch of a young lamb. Sp17 expression was detected by RT-PCR in a variety of mucosa-associated lymphoid tissues but not in primary or other secondary lymphoid tissues. Thus, the fetal lamb model may be appropriate for identifying genes relevant to mucosal immunity.     

Cry Protein Crystals: A Novel Platform for Protein Delivery

Protein delivery platforms are important tools in the development of novel protein therapeutics and biotechnologies. We have developed a new class of protein delivery agent based on sub-micrometer-sized Cry3Aa protein crystals that naturally form within the bacterium Bacillus thuringiensis. We demonstrate that fusion of the cry3Aa gene to that of various reporter proteins allows for the facile production of Cry3Aa fusion protein crystals for use in subsequent applications. These Cry3Aa fusion protein crystals are efficiently taken up and retained by macrophages and other cell lines in vitro, and can be delivered to mice in vivo via multiple modes of administration. Oral delivery of Cry3Aa fusion protein crystals to C57BL/6 mice leads to their uptake by MHC class II cells, including macrophages in the Peyer’s patches, supporting the notion that the Cry3Aa framework can be used to stabilize cargo protein against degradation for delivery to gastrointestinal lymphoid tissues.     

Immunoaffinity-isolated antigens induce protective immunity against larval Strongyloides stercoralis in mice

The objective of this study was to identify soluble protein antigens that would induce protective immunity against infective-stage larvae (L-3) of Strongyloides stercoralis in mice. Deoxycholate (DOC)-soluble proteins derived from L-3, adsorbed to aluminum hydroxide, induced protective immunity in BALB/c mice. The immunized mice generated parasite-specific IgG that could transfer passive immunity to na?ve animals. The protective antibodies bound to parasite antigens found in the muscles and nerve cords of the L-3. An IgG affinity chromatography column generated with IgG from the sera of DOC-immunized mice was used to purify specific larval antigens. Proteins were eluted from the affinity column with sizes of 80, 75, 61, 57, 43, and 32 kDa. This antigen pool stimulated both proliferation and IL-5 production by splenocytes recovered from mice immunized with live L-3. Vaccination of mice with the immunoaffinity-isolated antigens led to significant protective immunity, with 83% of challenge larvae killed. This study demonstrates that IgG-isolated proteins are candidate antigens for a vaccine against larval S. stercoralis.     

Toxocara canis: Larval migration dynamics, detection of antibody reactivity to larval excretory-secretory antigens and clinical findings during experimental infection of gerbils (Meriones unguiculatus)

In this study, Mongolian gerbils were used to analyse features of Toxocara infection that included larval migration, humoral immune responses to Toxocara canis excretory-secretory antigens (TES) and aspects of host physiology. At day 10 post-infection (p.i.) most larvae were in the intestine and the lungs while later the total number of larvae was higher in the carcass tissue; the number of larvae per gram of tissue was lower elsewhere other than in the brain. Infected animals showed several neurological abnormalities, an early increase in leukocyte and neutrophil levels, two peaks of peripheral eosinophilia (5 and 40 d.p.i.) and high antibody levels against TES in the circulation and in the vitreous humor. A sequential recognition of eight T.canis larval antigens with MW from 24 to 200 kDa was detected by Western blot. The results obtained in this study further support the use of gerbils as an experimental model for systemic, ocular and cerebral toxocariasis.     

Echinostoma caproni (Trematoda): Differential in vivo cytokine responses in high and low compatible hosts

In order to investigate the factors determining the expulsion of intestinal trematodes, we have analyzed the in vivo cytokine responses at several levels and the local responses against Echinostoma caproni (Trematoda) in two host species displaying different compatibility with the parasite. The response of the high compatible host (mice) is characterized by a mixed Th1/Th2 phenotype in the spleen, Peyer’s patches and mesenteric lymph nodes. At the intestine, a marked Th1 response with a marked increase of IFN-γ together with elevated number of mucosal neutrophils and expression of induced nitric oxide synthase were observed. The responses in the host of low compatibility (rats) with the parasite at the spleen, Peyer’s patches and mesenteric lymph node did not show clear differences with regard to the mice. However, the response in the intestine was markedly different. In rats, a Th2 response with increase in the levels of IL-5, IL-6 and IL-13 expression was detected. According to these results, the local production of IFN-γ and the local inflammatory responses with neutrophilic infiltration are associated with the development of chronic infections, whereas the worm expulsion is related with the development of Th2 responses and appears to be based on effects on non-bone narrow-derived cells.     

Ontogeny of murine T lymphocytes: II. Postnatal appearance and organ distribution of lymphocytes bearing Qa-4 and Qa-5 surface antigens

Cells from the lymphoid organs of C57BL/6 mice (from birth to 20 weeks) were monitored by the cytotoxicity assay for the presence of Qa-4 and Qa-5 surface antigens. Qa-4- and Qa-5-bearing cells are detectable in spleen, lymph nodes, and Peyer's patches, but not in thymus, liver, or bone marrow. Both antigens are present on small fractions of cells in each of these organs during the first week after birth. At 4–6 weeks of age, the fractions of Qa-4- and Qa-5-bearing cells rise to maximal levels which are then maintained throughout the ages studied (4–20 weeks). The relative proportion of these cell populations is greatest in the lymph nodes and smallest in the Peyer's patches, and in all three organs, more Qa-4- than Qa-5-positive cells are detected. The majority of Qa-4- and Qa-5-positive cells are Thy-1 positive, however, not all Thy-1- positive cells are Qa-4, Qa-5 positive. During postnatal development the ratio of Qa-4 or Qa-5-positive cells to Thy-1-positive cells increases in spleen, lymph nodes, and Peyer's patches indicating that cells bearing these antigens become a larger fraction of the T-cell population with age.     

Application of a Mouse Ligated Peyer’s Patch Intestinal Loop Assay to Evaluate Bacterial Uptake by M cells

The inside of our gut is inhabited with enormous number of commensal bacteria. The mucosal surface of the gastrointestinal tract is continuously exposed to them and occasionally to pathogens. The gut-associated lymphoid tissue (GALT) play a key role for induction of the mucosal immune response to these microbes^{1, 2}. To initiate the mucosal immune response, the mucosal antigens must be transported from the gut lumen across the epithelial barrier into organized lymphoid follicles such as Peyer''s patches. This antigen transcytosis is mediated by specialized epithelial M cells^{3, 4}. M cells are atypical epithelial cells that actively phagocytose macromolecules and microbes. Unlike dendritic cells (DCs) and macrophages, which target antigens to lysosomes for degradation, M cells mainly transcytose the internalized antigens. This vigorous macromolecular transcytosis through M cells delivers antigen to the underlying organized lymphoid follicles and is believed to be essential for initiating antigen-specific mucosal immune responses. However, the molecular mechanisms promoting this antigen uptake by M cells are largely unknown. We have previously reported that glycoprotein 2 (Gp2), specifically expressed on the apical plasma membrane of M cells among enterocytes, serves as a transcytotic receptor for a subset of commensal and pathogenic enterobacteria, including Escherichia coli and Salmonella enterica serovar Typhimurium (S. Typhimurium), by recognizing FimH, a component of type I pili on the bacterial outer membrane ⁵. Here, we present a method for the application of a mouse Peyer''s patch intestinal loop assay to evaluate bacterial uptake by M cells. This method is an improved version of the mouse intestinal loop assay previously described ^{6, 7}. The improved points are as follows: 1. Isoflurane was used as an anesthetic agent. 2. Approximately 1 cm ligated intestinal loop including Peyer''s patch was set up. 3. Bacteria taken up by M cells were fluorescently labeled by fluorescence labeling reagent or by overexpressing fluorescent protein such as green fluorescent protein (GFP). 4. M cells in the follicle-associated epithelium covering Peyer''s patch were detected by whole-mount immunostainig with anti Gp2 antibody. 5. Fluorescent bacterial transcytosis by M cells were observed by confocal microscopic analysis. The mouse Peyer''s patch intestinal loop assay could supply the answer what kind of commensal or pathogenic bacteria transcytosed by M cells, and may lead us to understand the molecular mechanism of how to stimulate mucosal immune system through M cells.