Cryptopatches Research Articles

Intestinal fibroblastic reticular cell niches control innate lymphoid cell homeostasis and function

Innate lymphoid cells (ILCs) govern immune cell homeostasis in the intestine and protect the host against microbial pathogens. Various cell-intrinsic pathways have been identified that determine ILC development and differentiation. However, the cellular components that regulate ILC sustenance and function in the intestinal lamina propria are less known. Using single-cell transcriptomic analysis of lamina propria fibroblasts, we identify fibroblastic reticular cells (FRCs) that underpin cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Genetic ablation of lymphotoxin-β receptor expression in Ccl19-expressing FRCs blocks the maturation of CPs into mature ILFs. Interactome analysis shows the major niche factors and processes underlying FRC-ILC crosstalk. In vivo validation confirms that a sustained lymphotoxin-driven feedforward loop of FRC activation including IL-7 generation is critical for the maintenance of functional ILC populations. In sum, our study indicates critical fibroblastic niches within the intestinal lamina propria that control ILC homeostasis and functionality and thereby secure protective gut immunity.

Group 3 Innate Lymphoid Cells Program a Distinct Subset of IL-22BP-Producing Dendritic Cells Demarcating Solitary Intestinal Lymphoid Tissues

Solitary intestinal lymphoid tissues such as cryptopatches (CPs) and isolated lymphoid follicles (ILFs) constitute steady-state activation hubs containing group 3 innate lymphoid cells (ILC3) that continuously produce interleukin (IL)-22. The outer surface of CPs and ILFs is demarcated by a poorly characterized population of CD11c+ cells. Using genome-wide single-cell transcriptional profiling of intestinal mononuclear phagocytes and multidimensional flow cytometry, we found that CP- and ILF-associated CD11c+ cells were a transcriptionally distinct subset of intestinal cDCs, which we term CIA-DCs. CIA-DCs required programming by CP- and ILF-resident CCR6+ ILC3 via lymphotoxin-β receptor signaling in cDCs. CIA-DCs differentially expressed genes associated with immunoregulation and were the major cellular source of IL-22 binding protein (IL-22BP) at steady state. Mice lacking CIA-DC-derived IL-22BP exhibited diminished expression of epithelial lipid transporters, reduced lipid resorption, and changes in body fat homeostasis. Our findings provide insight into the design principles of an immunoregulatory checkpoint controlling nutrient absorption.

Differential Effects of the Absence of Nkx2-3 and MAdCAM-1 on the Distribution of Intestinal Type 3 Innate Lymphoid Cells and Postnatal SILT Formation in Mice.

Seeding of leukocytes to developing lymphoid tissues in embryonic and early postnatal age and to the mucosa throughout adulthood depends on the interaction between endothelial MAdCAM-1 addressin and its cognate ligand α4β7 integrin. Nkx2-3 as a transcriptional regulator of MAdCAM-1 controls vascular patterning in visceral lymphoid tissues in mice, and has been identified as a susceptibility factor for inflammatory bowel diseases in humans, associated with lymphoid neogenesis in the inflamed intestines. The role of Nkx2-3 in the organogenesis of the solitary intestinal lymphoid tissues (SILTs) involving type 3 innate lymphoid cells (ILC3) is still unknown. Here we investigated the effect of Nkx2-3 on the postnatal distribution of intestinal ILC3s and the development of SILTs, comparing these to mice lacking MAdCAM-1, but preserving Nkx2-3. At 1 week of age small intestines (SI) contained significantly higher number of ILC3s relative to the colon, with a substantial reduction in MAdCAM-1−/− mice compared to C57BL/6 controls. One week later SI ILC3 number decreased in all genotypes, the number of colonic ILC3 of both Nkx2-3-deficient and Nkx2-3-heterozygous mice significantly increased. On the fourth postnatal week a further reduction of SI ILC3s was observed in both Nkx2-3-deficient and Nkx2-3-heterozygous mice, while in the colon the number of ILC3s showed a significant reduction in all genotypes. At 1 week of age only sporadic SILT components were present in all genotypes. By the second week mice deficient for either Nkx2-3 or MAdCAM-1 showed absence of SILT maturation compared to their relevant controls, lacking mature isolated lymphoid follicles (ILF). By the fourth week both Nkx2-3-deficient and Nkx2-3-heterozygous mice showed a similar distribution of ILFs relative to cryptopatches (CP), whereas in MAdCAM-1−/− mice CPs and immature ILFs were present, mature ILFs were scarce. Our data demonstrate that the complete absence of MAdCAM-1 partially impairs intestinal seeding of ILC3s and causes partial blockade of SILT maturation, without affecting peripheral lymph node development. In contrast, the inactivation of Nkx2-3 permits postnatal seeding, and its blocking effect on SILT maturation prevails at later stage, thus other adhesion molecules may compensate for the intestinal homing of ILC3s in the absence of MAdCAM-1.

Oxysterol Sensing through the Receptor GPR183 Promotes the Lymphoid-Tissue-Inducing Function of Innate Lymphoid Cells and Colonic Inflammation

SummaryGroup 3 innate lymphoid cells (ILC3s) sense environmental signals and are critical for tissue integrity in the intestine. Yet, which signals are sensed and what receptors control ILC3 function remain poorly understood. Here, we show that ILC3s with a lymphoid-tissue-inducer (LTi) phenotype expressed G-protein-coupled receptor 183 (GPR183) and migrated to its oxysterol ligand 7α,25-hydroxycholesterol (7α,25-OHC). In mice lacking Gpr183 or 7α,25-OHC, ILC3s failed to localize to cryptopatches (CPs) and isolated lymphoid follicles (ILFs). Gpr183 deficiency in ILC3s caused a defect in CP and ILF formation in the colon, but not in the small intestine. Localized oxysterol production by fibroblastic stromal cells provided an essential signal for colonic lymphoid tissue development, and inflammation-induced increased oxysterol production caused colitis through GPR183-mediated cell recruitment. Our findings show that GPR183 promotes lymphoid organ development and indicate that oxysterol-GPR183-dependent positioning within tissues controls ILC3 activity and intestinal homeostasis.

Targeted deletion of RANKL in M cell inducer cells by the Col6a1-Cre driver

The gut-associated lymphoid tissues (GALTs), including Peyer's patches (PPs), cryptopatches (CPs) and isolated lymphoid follicles (ILFs), establish a host-microbe symbiosis by the promotion of immune reactions against gut microbes. Microfold cell inducer (MCi) cells in GALTs are the recently identified mesenchymal cells that express the cytokine RANKL and initiate bacteria-specific immunoglobulin A (IgA) production via induction of microfold (M) cell differentiation. In the previous study, the Twist2-Cre driver was utilized for gene deletion in mesenchymal cells including MCi cells. In order to investigate MCi cells more extensively, it will be necessary to develop experimental tools in addition to the Twist2-Cre driver mice and characterize such drivers in specificity and efficiency. Here we show that M cell differentiation and IgA production are impaired in the targeted deletion of RANKL by the Col6a1-Cre driver. We compared Col6a1-Cre with Twist2-Cre in terms of the specificity for mesenchymal cells in GALTs. Col6a1-Cre CAG-CAT-EGFP mice exhibited EGFP expression in podoplanin+CD31– cells including MCi cells, while Twist2-Cre mice were shown to target endothelial cells and podoplanin+CD31– cells. Tnfsf11fl/ΔCol6a1-Cre mice exhibited the absence of M cells and severe IgA reduction together with an alteration in gut microbial composition. Moreover, we analyzed germ free mice to test whether changes in the microbiota are the cause of M cell deficiency. M cell differentiation was normal in the CPs/ILFs of germ free mice, indicating that MCi cells induce M cells independently of microbial colonization. This study demonstrates that Col6a1-Cre driver mice are as useful as Twist2-Cre driver mice for functional analyses of GALT-resident mesenchymal cells, including MCi cells.

Development and Function of Secondary and Tertiary Lymphoid Organs in the Small Intestine and the Colon.

The immune system of the gut has evolved a number of specific lymphoid structures that contribute to homeostasis in the face of microbial colonization and food-derived antigenic challenge. These lymphoid organs encompass Peyer’s patches (PP) in the small intestine and their colonic counterparts that develop in a programed fashion before birth. In addition, the gut harbors a network of lymphoid tissues that is commonly designated as solitary intestinal lymphoid tissues (SILT). In contrast to PP, SILT develop strictly after birth and consist of a dynamic continuum of structures ranging from small cryptopatches (CP) to large, mature isolated lymphoid follicles (ILF). Although the development of PP and SILT follow similar principles, such as an early clustering of lymphoid tissue inducer (LTi) cells and the requirement for lymphotoxin beta (LTβ) receptor-mediated signaling, the formation of CP and their further maturation into ILF is associated with additional intrinsic and environmental signals. Moreover, recent data also indicate that specific differences exist in the regulation of ILF formation between the small intestine and the colon. Importantly, intestinal inflammation in both mice and humans is associated with a strong expansion of the lymphoid network in the gut. Recent experiments in mice suggest that these structures, although they resemble large, mature ILF in appearance, may represent de novo-induced tertiary lymphoid organs (TLO). While, so far, it is not clear whether intestinal TLO contribute to the exacerbation of inflammatory pathology, it has been shown that ILF provide the critical microenvironment necessary for the induction of an effective host response upon infection with enteric bacterial pathogens. Regarding the importance of ILF for intestinal immunity, interfering with the development and maturation of these lymphoid tissues may offer novel means for manipulating the immune response during intestinal infection or inflammation.

Absence of organized lymphoid tissues in the intestine of the freshwater fish, C. commersonii (VET2P.1042)

Abstract Microscopic organized lymphoid tissues in the small intestine, such as cryptopatches (CP) and isolated lymphoid follicles (ILF), have been well characterized in the laboratory mouse. Descriptions of these structures have also been reported in rat and human, but the presence or absence of CP and ILF in other animals has not been explored. Given the evolutionary development of the adaptive immune system beginning in jawed fish, we hypothesized that CP and ILF would be present in the intestines of fish. We examined the intestines of 10 wild-caught Catostomus commersonii, a bottom-feeding freshwater fish found in local streams in Western Maryland and throughout the United States. The intestines of these fish were opened longitudinally and arranged in flattened layers for horizontal sectioning. Samples of mouse small intestine were prepared simultaneously as a positive control. After quick-freezing and sectioning, the tissue was stained with hematoxylin and eosin. Interestingly, a complete absence of organized lymphoid tissue was noted, suggesting that CP and ILF, in addition to the well-known Peyer’s patches, are limited to higher-order animals and potentially only mammals. Plans to study other fish species, especially carnivorous varieties (since the composition of the intestinal immune system can change in response to diet) are currently underway to further support this finding.

The Aryl Hydrocarbon Receptor: A Sentinel Safeguarding the Survival of Immune Cells in the Gut

Innate lymphoid cells (ILCs) regulate the epithelial barrier function and immunity in the gut. How ILC numbers are maintained is unknown. In this issue of Immunity, Qiu et al. (2012) report that the transcription factor aryl hydrocarbon receptor controls survival and function of gut-residing ILCs.

Distinct Developmental Requirements for Isolated Lymphoid Follicle Formation in the Small and Large Intestine: RANKL Is Essential Only in the Small Intestine

Cryptopatches (CPs) and isolated lymphoid follicles (ILFs) are organized intestinal lymphoid tissues that develop postnatally in mice and include stromal cells expressing the receptor activator of nuclear factor kappa-B ligand (RANKL). We investigated how stromal RANKL influences the development and differentiation of CPs and ILFs by analyzing the development of these lymphoid structures in knockout mice lacking RANKL. We found that RANKL(-/-) mice had a fourfold reduction in the overall density of CPs in the small intestine compared to control mice, with the largest decrease in the proximal small intestine. No B cells were present in CPs from the small intestine of RANKL(-/-) mice and ILF formation was completely blocked. In sharp contrast, colonic ILFs containing B cells were present in RANKL(-/-) mice. Stromal cells within CPs in the small intestine of RANKL(-/-) mice did not express CXCL13 (originally called B lymphocyte chemoattractant) and often lacked other normally expressed stromal cell antigens, whereas colonic lymphoid aggregates in RANKL(-/-) mice retained stromal CXCL13 expression. The CXCL13-dependent maturation of precursor CPs into ILFs is differentially regulated in the small intestine and colon, with an absolute requirement for RANKL only in the small intestine.

Lymphotoxin-β receptor-independent development of intestinal IL-22-producing NKp46+ innate lymphoid cells

The natural cytotoxicity receptor NKp46 is an activating receptor expressed by several distinct innate lymphoid cell (ILC) subsets, including NK cells, some γδ T cells and intestinal RORγt(+) IL-22(+) cells (NCR22 cells, IL-22-producing NKp46(+) cell). NCR22 cells may play a role in mucosal barrier function through IL-22-mediated production of anti-bacterial peptides from intestinal epithelial cells. Previous studies identified a predominant proportion of NCR22 cells in gut cryptopatches (CP), lymphoid structures that are strategically positioned to collect and integrate signals from luminal microbes; however, whether CP or other lymphoid structures condition NCR22 cell differentiation is not known. Programmed and inducible lymphoid tissue development requires cell-surface-expressed lymphotoxin (LT)α(1) β(2) heterotrimers (provided by lymphoid tissue inducer (LTi) cells) to signal lymphotoxin-β receptor (LTR)(+) stromal cells. Here, we analyzed NCR22 cells in LTβR-deficient Ncr1(GFP/+) mice that lack organized secondary lymphoid tissues. We found that NCR22 cells develop in the absence of LTβR, become functionally competent and localize to the lamina propria under steady-state conditions. Following infection of LTβR(-/-) mice with the Gram-negative pathogen Citrobacter rodentium, IL-22 production from NCR22 cells was not affected. These results indicate that organized lymphoid tissue structures are not critical for the generation of an intact and fully functional intestinal NCR22 cell compartment.

Aging impacts isolated lymphoid follicle development and function

BackgroundImmunosenescence is the age-related decline and dysfunction of protective immunity leading to a marked increase in the risk of infections, autoimmune disease, and cancer. The majority of studies have focused on immunosenescence in the systemic immune system; information concerning the effect of aging on intestinal immunity is limited. Isolated lymphoid follicles (ILFs) are newly appreciated dynamic intestinal lymphoid structures that arise from nascent lymphoid tissues, or cryptopatches (CP), in response to local inflammatory stimuli. ILFs promote "homeostatic" responses including the production of antigen-specific IgA, thus playing a key role in mucosal immune protection. ILF dysfunction with aging could contribute to immunosenescence of the mucosal system, and accordingly we examined phenotypic and functional aspects of ILFs from young (2 month old) and aged (2 year old) mice.ResultsWe observed that aged mice have increased numbers of ILFs and increased numbers of structures corresponding to an early stage of CPs transforming into ILFs. The cellular composition of ILFs in aged mice is altered with a smaller B-lymphocyte population and an increased T-lymphocyte population. The ILF T-lymphocyte population is notable by the presence of CD4+ CD8αα+ T-lymphocytes, which are absent from the systemic compartment. The smaller B-lymphocyte population in ILFs from aged mice is directly correlated with decreased mRNA and protein expression of CCL20 and CXCL13, two chemokines that play crucial roles in recruiting B-lymphocytes into ILFs. Aged mice had elevated levels of serum and fecal immunoglobulins and despite the decreased B-lymphocyte population, ILFs from aged mice displayed increased IgA production. The immunoglobulin repertoire was skewed in aged mice, and ILFs demonstrated a repertoire usage similar to that of the systemic pool in both young and aged mice.ConclusionsHere we observed that ILF development, cellular composition, and immunoglobulin production are altered with aging suggesting that ILF dysfunction contributes to mucosal immunosenescence.

Dendritic Cells Produce CXCL13 and Participate in the Development of Murine Small Intestine Lymphoid Tissues

In the adult intestine, luminal microbiota induce cryptopatches to transform into isolated lymphoid follicles (ILFs), which subsequently act as sites for the generation of IgA responses. The events leading to this conversion are incompletely understood. Dendritic cells (DCs) are components of cryptopatches (CPs) and ILFs and were therefore evaluated in this process. We observed that the adult murine intestine contains clusters of DCs restricted to the CP/ILF continuum. A numerical and cell associative hierarchy in the adult intestine and a chronologic hierarchy in the neonatal intestine demonstrated that these clusters form after the coalescence of CD90+ cells to form CPs and before the influx of B220+ B lymphocytes to form ILFs. Cluster formation was dependent on lymphotoxin and the lymphotoxin beta receptor and independent of lymphocytes. The ILF DC population was distinguished from that of the lamina propria by the absence of CD4+CD11c+ cells and an increased proportion of CD11c+B220+ cells. The formation of clusters was not limited by DC numbers but was induced by luminal microbiota. Moreover, in the absence of the chemokine CXCL13, CP transformation into ILF was arrested. Furthermore, ILF DCs express CXCL13, and depletion of DCs resulted in regression of ILFs and disorganization of CPs. These results reveal DC participation in ILF transformation and maintenance and suggest that in part this may be due to CXCL13 production by these cells.

CCR6 identifies lymphoid tissue inducer cells within cryptopatches

The chemokine receptor CCR6 is expressed by dendritic cells, B and T cells predominantly within the organized structures of the gut-associated lymphatic tissue. Its ligand CCL20 is synthesized by the follicle-associated epithelium and is crucial for the development of M cells within Peyer's patches. In addition, lineage-negative c-kit positive lymphocytes within cryptopatches (CP) express CCR6. CCR6-deficient mice exhibit an altered intestinal immune system containing increased amounts of intraepithelial lymphocytes and show smaller Peyer's patches, while progression of cryptopatches to mature isolated lymphoid follicles (ILF) is inhibited. In this report, we show that lin(-) c-kit(+) lymphocytes express a variety of different chemokine receptors and that CCR6 identifies those cells located within CP. In contrast, cells found outside CP are positive for CXCR3 and exhibit a different surface marker profile, suggesting that at least two different populations of lin(-) c-kit(+) cells are present. The presence of CCR6 does not influence the expression of Notch molecules on lin(-) c-kit(+) cells, nor does it influence Notch ligand expression on bone marrow-derived dendritic cells. In the human gut, CCR6 identifies clusters of lymphocytes resembling murine CP. CCR6 seems to have an important role for lin(-) c-kit(+) cells inside CP, is expressed in a regulated manner and identifies potential human CP.

Lymphotoxin-Independent Expression of TNF-Related Activation-Induced Cytokine by Stromal Cells in Cryptopatches, Isolated Lymphoid Follicles, and Peyer’s Patches

Stromal cells play a crucial role in the organogenesis of lymphoid tissues. We previously identified VCAM-1(+) stromal cells in cryptopatches (CP) and isolated lymphoid follicles (ILF) in the small intestine of C57BL/6 mice. Nonhemopoietic stromal cell networks in CP and ILF of adult mice also expressed FDC-M1, CD157 (BP-3), and TNF-related activation-induced cytokine (TRANCE). Individual stromal cells were heterogeneous in their expression of these markers, with not all stromal cells expressing the entire set of stromal cell markers. Expression of VCAM-1, FDC-M1, and CD157 on CP stromal cells was absent in alymphoplasia mice deficient in NF-kappaB-inducing kinase (NIK) and NIK knockout mice. Administration of lymphotoxin beta receptor (LTbetaR)-Ig to wild-type mice on day 13 resulted in the absence of CP on day 20; delaying administration of LTbetaR-Ig until day 18 resulted in an 80% decrease in the number of CP on day 22 and diminished expression of VCAM-1, FDC-M1, and CD157 on the remaining CP. In sharp contrast, TRANCE expression by stromal cells was completely independent of NIK and LTbetaR. In addition, expression of TRANCE in ILF was concentrated just beneath the follicle-associated epithelium, a pattern of polarization that was also observed in Peyer's patches. These findings suggest that TRANCE on stromal cells contributes to the differentiation and maintenance of organized lymphoid aggregates in the small intestine.

CC Chemokine Receptor 6 Expression by B Lymphocytes Is Essential for the Development of Isolated Lymphoid Follicles

Isolated lymphoid follicles (ILFs) are organized lymphoid structures that facilitate the efficient interaction of antigen, antigen-presenting cells, and lymphocytes to generate controlled adaptive immune responses within the intestine. Because CC chemokine receptor 6 (CCR6) deficiency affects the generation of mucosal immune responses, we evaluated a potential role for CCR6 in the development of ILFs. We observed that CCR6 and its ligand CCL20 are highly expressed within ILFs and that B lymphocytes are the largest CCR6-expressing population within ILFs. ILF development was profoundly arrested in the absence of CCR6. Concordant with a block in ILF development at a stage corresponding to the influx of B lymphocytes, we observed that CCR6-deficient mice had a diminished population of intestinal B lymphocytes. Bone marrow reconstitution studies demonstrated that ILF development is dependent on CCR6-sufficient B lymphocytes, and adoptive transfers demonstrated that CCR6(-/-) B lymphocytes were inefficient at localizing to intestinal lymphoid structures. Paralleling these findings, we observed that CCR6-deficient mice had a reduced proportion of Peyer's patch B lymphocytes and an associated re-duction in the number and size of Peyer's patch follicular domes. These observations define an essential role for CCR6 expression by B lymphocytes in localizing to intestinal lymphoid structures and in ILF development.

Selective expression of TRANCE by subepithelial dome stromal cells in Peyer's patches regulates local induction of intestinal immune responses

The TNF superfamily member TRANCE and its receptor RANK are essential for lymph node development. TRANCE has been previously shown to promote development of oral tolerance and regulate cytokine production by dendritic cells (DC). To investigate the relevance of the TRANCE-RANK axis for gut-associated lymphoid tissue (GALT), we localized TRANCE-expressing cells in small intestine from C57BL/6 mice by immunofluorescence microscopy. A network of TRANCE+ stromal cells was found throughout cryptopatches (CP). In sharp contrast, TRANCE expression in both isolated lymphoid follicles (ILF) and Peyer's patches (PP) was polarized to the subepithelial dome (SED). A subset of the TRANCE+ stromal cells in the SED co-expressed PDGFRα (CD140a). In contrast to other GALT stromal cell markers (e.g. VCAM-1), expression of TRANCE was independent of lymphotoxin signaling. In addition, PP in TRANCE-deficient mice were substantially smaller than those of wild type littermates. Our data collectively demonstrate an important role for TRANCE-RANK signaling in the development and maintenance of multiple GALT structures. Given the importance of PP as a site of oral tolerance induction, we hypothesize that the TRANCE+ stromal cells in the SED area have immunoregulatory properties that influence the function of local DC to favor induction of tolerogenic responses to lumenal antigens. Supported by the NIH (DK-64730, DK-64399).

The extrathymic T‐cell differentiation in the murine gut

The gut epithelial border is in continuous contact with exogenous antigens and harbors a distinctive and very abundant CD8 alpha alpha intraepithelial T-lymphocyte effector population. We describe here the characteristics of these cells that distinguish them from all other T-cell types in the body as well as their functions in local protection. We also describe how these cells differentiate from local precursors present in the gut cryptopatches (CPs) following a pathway of T-cell differentiation unique to the gut wall. Finally, we describe the origin of the precursors of CD8 alpha alpha T cells, which come from the bone marrow in athymic mice but are first imprinted in the thymus in euthymic mice. Indeed, CD3(-)CD4(-)CD8(-) T-cell-committed precursors can leave the thymus before T-cell receptor rearrangements and then colonize the gut CPs, proceeding with their differentiation within the gut wall.

Induction of Intestinal Lymphoid Tissue

The intestinal immune system includes several organized structures, such as Peyer's patches, isolated lymphoid follicles (ILFs), cryptopatches (CPs) as well as mesenteric lymph nodes (MLNs) that constitute an extensive network with other nonorganized parts, such as intraepithelial and lamina propria lymphocytes. CPs are small clusters of lymphoid cells with an immature lymphocyte phenotype and dendritic cells. Initial observations in transfer experiments suggested that the immature lymphocytes are T cell precursors and CPs are a potential site of extrathymic intraepithelial lymphocyte (IEL) differentiation. This feature has recently been challenged particularly by the observation that CP cells express the orphan receptor RORgammat and are phenotypically indistinguishable from lymphoid tissue-inducer (LTi) cells, suggesting that CP cells are the adult counterpart of fetal LTi cells. In addition, the chemokine receptor CCR6 is specifically expressed by precursor cells within CPs and its deletion inhibits the development of ILFs. Therefore, it is likely that ILFs derive from CPs under the control of CCR6 under inflammatory conditions and might constitute a valuable target for anti-inflammatory therapies.

CCR6 and CCL20

The CCR6 chemokine receptor is expressed by most B cells and subsets of T cells and dendritic cells (DCs) found in the gut mucosal immune system. CCL20, the single chemokine ligand for CCR6, is selectively made by the follicle-associated epithelium (FAE) overlying Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). CCL20 contributes to the recruitment of CCR6-expressing B cells to these structures. CCL20 expression by the intestinal epithelium is also highly inducible in response to inflammatory stimuli. Thus, CCL20 functions as both an inflammatory and homeostatic chemokine. Interactions between CCR6 and CCL20 play a role at several stages in the development of intestinal lymphoid structures. A subset of the c-kit(+) lymphoid precursors found in cryptopatches (CPs) expresses CCR6. Recruitment of B cells to CPs and the subsequent expansion and organization of these B cells allows differentiation of some of these structures into ILFs. In CCR6 knockout mice, PPs are smaller with fewer follicles and the development of ILFs is compromised. These defects in the development of mucosal inductive sites in CCR6-deficient mice are responsible for decreased IgA production to oral antigens. CCR6 can be included with CXCR5 and CCR7 in a list of chemokine receptors that participate in shaping the organized lymphoid structures that are part of the intestinal immune system.

Organizing a mucosal defense

Gastrointestinal associated lymphoid tissue can be divided into loosely organized effector sites, which include the lamina propria and intraepithelial lymphocytes, and more organized structures, such as mesenteric lymph nodes (LNs), Peyer's patches (PPs), isolated lymphoid follicles, and cryptopatches (CPs). These organized structures in the gastrointestinal tract have been hypothesized to play the role of primary lymphoid organ, supporting the extrathymic development of T lymphocytes (CPs), secondary lymphoid organs involved in the induction of the mucosal immune response (PPs), and tertiary lymphoid structures whose function is still under debate (isolated lymphoid follicles). The most widely studied lymphoid structure found in the small intestine is the PP. PPs are secondary lymphoid structures, and their development and function have been extensively investigated. However, single lymphoid aggregates resembling PPs have been also described in humans and in the murine small intestines. These isolated lymphoid follicles have both germinal centers and an overlying follicle-associated epithelium, suggesting that they also can function as inductive sites for the mucosal immune response. This review compares and contrasts the development and function of the four main organized gastrointestinal lymphoid tissues: CPs, isolated lymphoid follicles, PPs, and mesenteric LNs.