Structure and function of mucosal immun function. Innate and adaptive immunity Andras Arato Structure of GALT The epithelial barrier of the gut separates luminal antigens and microbes from underlying lymphoid elements and acts as a first responder in the mucosal immune system. This system has to answer to pathogens and remain relatively unresponsive to food antigens and the commensal intestinal flora (1). The surface epithelial monolayer on the intestinal mucosa as a tight barrier prevents movement of various molecules from the lumen to the interstitium (2). The surface area of epithelium is about 400 m2, because it is formed into millions of fingerlike villi in the small bowel. In the upper bowel, the majority of antigen exposure comes from diet, whereas in the ileum and colon, the complex commensal microflora give additional antigenic load. Excessive entry of antigens is also prevented by nonspecific factors such as mucus and glycocalyx. However, the epithelial barrier does not completely prevent lumenal antigens going through it. Antigens can cross the epithelial surface through breaks of tight junctions, probably at the villus tips, where epithelial cells are shed. Antigens can also cross the epithelim through the follicle-associated epithelium (FAE), that overlies the Peyer’s patches, which are organized lymphoid tissues of the intestinal mucosa and their number is about 200 in adult. The immune cells at this mucosal sites, intraepithelial lymphocytes (IEL), and isolated lymphocytes or aggregates dispersed throughout the lamina propria constitute the GALT (3). FAE contains specialized epithelial cells called M cells. These cells have no lysosomes and are capable of invaginating upon attachment of microorganisms and large proteint. The M cells are a pecialized lineage of epithelium dedicated to antigen sampling. Unlike the other enterocytes, M cells have fewer and shorter microvilli on their apical surface and the basolateral surface display numerous invaginations in which mucosal lymphocytes reside (4). M cells act as a conduit to underlying antigen-presenting cells, e.g. macrophages and dendritic cells that lie beneath the epithelium directly in contact with M cells. Dendritic cells in the lamina propria reach through epithelial cells and also sample gut bacteria. The epithelium contain mostly CD8 positive intraepithelia T-cells. These IELs are juxtaposed between the epithelium at the basolateral surface. The IELs recognize bacterial antigens and immune-activated epithelial surface molecules so that they could respond rapidly upon stimulation (5). The two thirds of lamina propria lymphocytes are CD4 positive cells. The lamina propria also contains mast cells, and various lymphocytes, paricularly IgAproducing B cells. IgA acts on luminal bacteria to prevent their adherence to the enterocyte surface. The schematic structure of gut associated lymphoid structure is presented on figure 5.

Figure 1. Structure of GALT (taken from reference 1.)

A deficiency in secretory intestinal antibody may impair mucosal barrier function, resulting in increased uptake of macromolecular antigens that could then contribute to the pathogenesis of intestinal or systemic disease states. The interaction of intestinal antibodies with antigens, enterotoxins, or bacteria can prevent their attachment to epithelial cell membranes, inhibiting antigen uptake or penetration by pathogens. Activation of lymphocytes results in the proliferation of T and B cells and then migration to

the lymphatic vessels to reach the systemic circulation. Subsequently these cells migrate back through the intestinal lamina propria, where they remain after interacting with homing receptors. These cells can also disperse to other mucosal sites such as the lung, the genitourinary tract, and the breast during lactation (common mucosal immune system). Returning again the the intestinal mucosa, B cells further differentiate into antibody producing cells. On subsequent reexposure to antigen, IgA is produced in the lamina propria. A secretory component is attached to the IgA which aids in the transport of IgA across the epithelium and also prevents adherence of microbes to the mucosa. It also prevents proteolytic digestion of the IgA molecule in the intestinal lumen. Innate and adaptive immunity The innate immune system is the first line of defense against pathogenic microorganisms. It has an important protective function in the GALT and among its main functions are the recognition of microbial organisms as foreign, the incapacitation of pathogens and also the important regulation of adaptive immune response. The innate immune system can recognize conserved PAMPs on microorganisms through the Toll-like receptors (TLRs) which are crucial for the activation of the antigen-presenting cells. TLR-mediated pathogen recognition on dendritic cells leads to a TH1 type lymphocyte response (6, 7). These molecules are called Toll-like receptors because of their homolgy to receptors first discovered and named in Drosophilia. The TLRs are usually associated withe cell membranes and have an external leucine-reach repeat (LRR) recognition domain. Among the pattern recognition receptor it has to be mentioned the CARD15 molecules which are in the cytosol and have similar structure to TLRs (1). There is abundant evidence that signaling through CARD15 and TLR activates transcription factor NF-.B, leading to proinflammatory gene expression. The interaction of PAMPs and TLRs causes the appearance of B7 costimulatory molecules at the cell surface of antigen-presenting cells. The T cells have the CD28 receptor for B7. For the activation of T cells it is necessary the simultaneous binding of CD28 to B7 and the antigen/class II complex to T cell receptor (TCR) specific for it. Tolerance Oral tolerance means a systemic hyporesponsiveness or nonresponsiveness of mature T and B cells to antigen challenge after prior oral exposure to antigen. This phenomenon is a very important field of mucosal immunology, and its disturbance has a major role in the pathogenesis of several immune mediated gastrointestinal disorders (e.g. inflammatory bowel disease). Oral tolerance counter-regulate both arms of polarized T cell response, the Th1 mediated autoimmune disease and the Th2 type atopic disease. These tolerogenic responses are mediated by a coordinated interaction between the innate and adaptive immune systems. This regulation is done by the regulatory T cell subsets (Th3 and Tr1 cells) (3, 8). In these

down regulatory effect TGF-. and IL-10 are the most important cytokines (9) (Figure 3). TGF-. has been proved to have a suppressive effect on both Th1 and Th2 cells, and so this cytokine has a protection against Th1 and Th2 mediated disorders.

Fif.3 So release of TGF-. is a sine qua non step in the induction of oral tolerance. In this process, immune reactivity against orally ingested antigens is selectively suppressed. TGF-.-triggered

signals are transduced by Smads, a family of proteins that serve as substrates for TG-F receptor type I and type II. The type I recptor recognizes Smad2 and -3 which moving toward the nucleus, associate with Smad4, forming complexes that participate in DNA binding and recruitment of transcription factors. In addition to these agonistic Smads, antagonist or inhibitory Smads also exist, like Smad7, which blocks activated receptors and interferes with phosphorylation of Smad2 and -3. The overproduction of Smad7 in IBD explains that despite the observed upregulation of TGF-., the proinflammatory processes are dominated in disorder (10) Bacterial-epithelial crosstalk The intestinal lumen contain a complex mixture of non-pathogenic microorganisms, which often called the indigenous or normal microflora. They are composed of more than 400 species. The host evolved various mechanims of tolerance to these organisms while it is remain responsive to enteropahtogenic bacterial species (12) The newborn infant resides in a sterile environment in utero and its mucosal immune system, athough poised ready to respond, is immature because of the lack of microbial stimulation. Accordingly, the exposure to initial bacterial colonization of the gut is important in the proper transition from a protective intrauterine environment to the rigors of extrauterine life. The nature of bacteria colonizing the gut is in part dependent on nenatal nutrition (breast milk vs. formula fedding), nature of birth (natural vaginal deivery vs. Caesarean section) and whether perinatal antibiotics are used. The immature neonatal T helper cell response is imbalanced at birth, favoring a skewed Th2 predominance. The colonizing bacteria help to balance the Th response between Th1 and Th2 cells and stimulate the induction of tolerance, presumably via the stimulation of Th3 cells producing TGF-. and IL-10. Therefore nonpathogenic bacteria may have the ability to directly influence the inflammatory response elicited by pathogens by downregulating specific signaling pathways. One goal would obviously be to use non-pathogenic microorganisms to treat inflammatory bowel disease. It has been shown that nonpathogenic bacteria may directly influence the intestinal epithelial cells to limit immune activation by inhibiting the ubiquination and degradation of I.B (13). As a result, the nuclear penetration of NF-.B becomes arrested, leading to a significant reduction in the amount of IL-8 that is secreted from the intestinal epithelial cells (13, 14) (Figure 5). References 1. MacDonald TT, Monteleone TT. Immunity, inflammation, and allergy int he gut. Science, 2005, 307, 1920-1925. 2. Mitic LL, Anderson JM. Molecular architecture of thight junctions. Annu Rev Physiol 1998, 60, 121-142. 3. Spiekermann GM, Walker WA. Oral tolerance and its role in clinical disease. J Pediatr Gastroenterol Nutr 2001, 32, 237-255. 4. Kraehenbuhl JP, Neutra MR. Epithelial M cells: Differentiation and function. Annu

Rev Cell Dev Biol 2000, 16, 301-332. 5. Hayday A, Theodoridis E, Ramsburg E, Shires J. Intraepithelial lymphocytes: Exploring the third way in immunology. Nat Immunol 2001, 2, 997-1003. 6. Yuan Q, Walker WA. Innate immunity of the gut: mucosal defense in health and disease. J Pediatr Gastroenterol Nutr 2004, 38, 463-473. 7. Schnare M, Barton GM, Holt AC, Takeda K, Akira S, Medzhitov R. Toll-like receptors control activation on adaptive immune responses. Nat Immunol 2001, 2, 947-950. 8. Rautava S, Ruuskanen O, Ouwehand A, Salminen S, Isolauri E. The hygiene hypothesis of atopic disease – An extended version. J Pediatr Gastroenterol Nutr 2004, 38, 378-388. 9. Nagler-Anderson C. Tolerance and immunity in the intestinal immune system. Crit Rev Immunol 2000, 20, 103-120. 10. Monteleone G. Blocking Smad7 restores TGF-.1 signaling in chronic inflammatory bowel disease. J Clin Invest 2001, 108, 601-609. 11. Fiocchi C. TGF-./Smad signaling defects in inflammatory bowel disease: mechanisms and possible novel therapies for chronic inflammation. J Clin Invest 2001, 108, 523526. 12. Haller D, Jobin C. Interaction between resident luminal bacteria and the host: can a healthy relationship turn sour. J Pediatr Gastroenterol Nutr 2004, 38, 121-136. 13. Neish AS, Gewirtz AT, Zeng H. Prokaryotic regulation of epithelial responses by inhibition of I.B-. ubiquination. Science 2000; 289:1560-63. 14. Resta-Lenert S, Barrett K. Probiotics and commensals reverse TNF-.- and INF-.induced dysfunction in human intestinal epithelial cells. Gastroenterology 2006, 130, 731-746.