Intestinal M cells: Tireless samplers of enteric microbiota.

Mucosal immune responses in the inductive lymphoid tissues of the intestine begin with uptake of particulate antigens, including components of the gut microbiota by specialized antigen sampling M cells. M cells represent a distinct lineage of enterocytes that arise from crypt stem cells in response to the cytokine receptor of NF-κB ligand (RANKL). Full differentiation of M cells requires the transcription factor Spi-B to yield mature M cells that express multiple receptors for bacteria including glycoprotein 2. M cell differentiation can be recapitulated in vitro using three-dimensional enteroid cultures of primary intestinal stem cells supplemented with RANKL. This article summarizes the current knowledge about the genesis of intestinal M cells and highlights some of the remaining unanswered questions about this enigmatic cell type.

Development of intestinal M cells and follicle-associated epithelium is regulated by TRAF6-mediated NF-κB signaling.

M cells are located in the follicle-associated epithelium (FAE) that covers Peyer's patches (PPs) and are responsible for the uptake of intestinal antigens. The differentiation of M cells is initiated by receptor activator of NF-κB. However, the intracellular pathways involved in M cell differentiation are still elusive. In this study, we demonstrate that the NF-κB pathway activated by RANK is essential for M cell differentiation using in vitro organoid culture. Overexpression of NF-κB transcription factors enhances the expression of M cell-associated molecules but is not sufficient to complete M cell differentiation. Furthermore, we evaluated the requirement for tumor necrosis factor receptor-associated factor 6 (TRAF6). Conditional deletion of TRAF6 in the intestinal epithelium causes a complete loss of M cells in PPs, resulting in impaired antigen uptake into PPs. In addition, the expression of FAE-associated genes is almost silenced in TRAF6-deficient mice. This study thus demonstrates the crucial role of TRAF6-mediated NF-κB signaling in the development of M cells and FAE.

NALT M cells are important for immune induction for the common mucosal immune system.

Nasopharynx-associated lymphoid tissue (NALT) is one of the major constituents of the mucosa-associated lymphoid tissue (MALT), and has the ability to induce antigen-specific immune responses. However, the molecular mechanisms responsible for antigen uptake from the nasal cavity into the NALT remain largely unknown. Immunohistochemical analysis showed that CCL9 and CCL20 were co-localized with glycoprotein 2 (GP2) in the epithelium covering NALT, suggesting the existence of M cells in NALT. In analogy with the reduced number of Peyer's patch M cells in CCR6-deficient mice, the number of NALT M cells was drastically decreased in CCR6-deficient mice compared with the wild-type mice. Translocation of nasally administered Salmonella enterica serovar Typhimurium into NALT via NALT M cells was impaired in CCR6-deficient mice, whereas S. Typhimurium demonstrated consistent co-localization with NALT M cells in wild-type mice. When wild-type mice were nasally administered with an attenuated vaccine strain of S. Typhimurium, the mice were protected from a subsequent challenge with wild-type S. Typhimurium. Antigen-specific fecal and nasal IgA was detected after nasal immunization with the attenuated vaccine strain of S. Typhimurium only in wild-type mice but not in CCR6-deficient mice. Taken together, these observations demonstrate that NALT M cells are important as a first line of defense against infection by enabling activation of the common mucosal immune system (CMIS).

CCR6 promotes steady-state mononuclear phagocyte association with the intestinal epithelium, imprinting and immune surveillance.

The intestinal lamina propria (LP) contains antigen-presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and in vivo models to test and confirm the importance of LP-MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP-epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP-MNPs from CCR6-/- mice did not display defects in acquiring antigen and stimulating T-cell responses in ex vivo assays or in responses to antigen administered systemically. However, LP-MNPs from CCR6-deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP-MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.

IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake.

Interleukin-22 (IL-22) acts protectively and harmfully on intestinal tissue depending on the situation; therefore, IL-22 signaling needs to be tightly regulated. IL-22 binding protein (IL-22BP) binds IL-22 to inhibit IL-22 signaling. It is expressed in intestinal and lymphoid tissues, although its precise distribution and roles have remained unclear. In this study, we show that IL-22BP is highly expressed by CD11b+CD8α- dendritic cells in the subepithelial dome region of Peyer's patches (PPs). We found that IL-22BP blocks IL-22 signaling in the follicle-associated epithelium (FAE) covering PPs, indicating that IL-22BP plays a role in regulating the characteristics of the FAE. As expected, FAE of IL-22BP-deficient (Il22ra2-/-) mice exhibited altered properties such as the enhanced expression of mucus and antimicrobial proteins as well as prominent fucosylation, which are normally suppressed in FAE. Additionally, Il22ra2-/- mice exhibited the decreased uptake of bacterial antigens into PPs without affecting M cell function. Our present study thus demonstrates that IL-22BP promotes bacterial uptake into PPs by influencing FAE gene expression and function.

Dynamics of SIV-specific CXCR5+ CD8 T cells during chronic SIV infection.

A significant challenge to HIV eradication is the elimination of viral reservoirs in germinal center (GC) T follicular helper (Tfh) cells. However, GCs are considered to be immune privileged for antiviral CD8 T cells. Here, we show a population of simian immunodeficiency virus (SIV)-specific CD8 T cells express CXCR5 (C-X-C chemokine receptor type 5, a chemokine receptor required for homing to GCs) and expand in lymph nodes (LNs) following pathogenic SIV infection in a cohort of vaccinated macaques. This expansion was greater in animals that exhibited superior control of SIV. The CXCR5+ SIV-specific CD8 T cells demonstrated enhanced polyfunctionality, restricted expansion of antigen-pulsed Tfh cells in vitro, and possessed a unique gene expression pattern related to Tfh and Th2 cells. The increase in CXCR5+ CD8 T cells was associated with the presence of higher frequencies of SIV-specific CD8 T cells in the GC. Following TCR-driven stimulation in vitro, CXCR5+ but not CXCR5- CD8 T cells generated both CXCR5+ as well as CXCR5- cells. However, the addition of TGF-ß to CXCR5- CD8 T cells induced a population of CXCR5+ CD8 T cells, suggesting that this cytokine may be important in modulating these CXCR5+ CD8 T cells in vivo. Thus, CXCR5+ CD8 T cells represent a unique subset of antiviral CD8 T cells that expand in LNs during chronic SIV infection and may play a significant role in the control of pathogenic SIV infection.

Increased Abundance of M Cells in the Gut Epithelium Dramatically Enhances Oral Prion Disease Susceptibility.

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases.

TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures.

Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1 (myristoylated alanine-rich protein kinase C substrate) in 1 day. The mature M cell marker glycoprotein 2 (Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 (Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.

Characterization and application of two RANK-specific antibodies with different biological activities.

Antibodies play an important role in therapy and investigative biomedical research. The TNF-family member Receptor Activator of NF-κB (RANK) is known for its role in bone homeostasis and is increasingly recognized as a central player in immune regulation and epithelial cell activation. However, the study of RANK biology has been hampered by missing or insufficient characterization of high affinity tools that recognize RANK. Here, we present a careful description and comparison of two antibodies, RANK-02 obtained by phage display (Newa, 2014 [1]) and R12-31 generated by immunization (Kamijo, 2006 [2]). We found that both antibodies recognized mouse RANK with high affinity, while RANK-02 and R12-31 recognized human RANK with high and lower affinities, respectively. Using a cell apoptosis assay based on stimulation of a RANK:Fas fusion protein, and a cellular NF-κB signaling assay, we showed that R12-31 was agonist for both species. R12-31 interfered little or not at all with the binding of RANKL to RANK, in contrast to RANK-02 that efficiently prevented this interaction. Depending on the assay and species, RANK-02 was either a weak agonist or a partial antagonist of RANK. Both antibodies recognized human Langerhans cells, previously shown to express RANK, while dermal dendritic cells were poorly labeled. In vivo R12-31 agonist activity was demonstrated by its ability to induce the formation of intestinal villous microfold cells in mice. This characterization of two monoclonal antibodies should now allow better evaluation of their application as therapeutic reagents and investigative tools.

Role of T-cell reconstitution in HIV-1 antiretroviral therapy-induced bone loss.

HIV infection causes bone loss. We previously reported that immunosuppression-mediated B-cell production of receptor activator of NF-κB ligand (RANKL) coupled with decline in osteoprotegerin correlate with decreased bone mineral density (BMD) in untreated HIV infection. Paradoxically, antiretroviral therapy (ART) worsens bone loss although existing data suggest that such loss is largely independent of specific antiretroviral regimen. This led us to hypothesize that skeletal deterioration following HIV disease reversal with ART may be related to T-cell repopulation and/or immune reconstitution. Here we transplant T cells into immunocompromised mice to mimic ART-induced T-cell expansion. T-cell reconstitution elicits RANKL and TNFα production by B cells and/or T cells, accompanied by enhanced bone resorption and BMD loss. Reconstitution of TNFα- or RANKL-null T-cells and pharmacological TNFα antagonist all protect cortical, but not trabecular bone, revealing complex effects of T-cell reconstitution on bone turnover. These findings suggest T-cell repopulation and/or immune reconstitution as putative mechanisms for bone loss following ART initiation.

Efficient norovirus and reovirus replication in the mouse intestine requires microfold (M) cells.

UNLABELLED: Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. M cells have also been suggested as a portal for pathogen entry into the host. While virus particles have been observed in M cells, it is not known whether viruses use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect host organisms via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells and provides a tractable experimental system for understanding how an enteric virus overcomes the intestinal epithelial barrier to infect underlying target cells. We found that replication of two divergent MNV strains was reduced in mice depleted of M cells. Reoviruses are double-stranded RNA viruses that infect hosts via respiratory or enteric routes. In contrast to MNV, reovirus infects enterocytes in the intestine. Despite differences in cell tropism, reovirus infection was also reduced in M cell-depleted mice. These data demonstrate that M cells are required for the pathogenesis of two unrelated enteric viruses that replicate in different cell types within the intestine. IMPORTANCE: To successfully infect their hosts, pathogens that infect via the gastrointestinal tract must overcome the multilayered system of host defenses. Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. Virus particles have been observed within M cells. However, it is not known whether viruses use M cells to initiate a productive infection. To address this question, we use MNV and reovirus, two enteric viruses that replicate in different cell types in the intestine, intestinal epithelial cells for reovirus and intestinal mononuclear phagocytes for MNV. Interestingly, MNV- and reovirus-infected mice depleted of M cells showed reduced viral loads in the intestine. Thus, our work demonstrates the importance of M cells in the pathogenesis of enteric viruses irrespective of the target cell type in which the virus replicates.

Ectopic lymphoid tissue alters the chemokine gradient, increases lymphocyte retention and exacerbates murine ileitis.

BACKGROUND: The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood. DESIGN: Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed. RESULTS: Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis. CONCLUSIONS: Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.

The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.

Intestinal microfold cells (M cells) are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. The mechanisms of M-cell differentiation are poorly understood, as the rarity of these cells has hampered analysis. Exogenous administration of the cytokine RANKL can synchronously activate M-cell differentiation in mice. Here we show the Ets transcription factor Spi-B was induced early during M-cell differentiation. Absence of Spi-B silenced the expression of various M-cell markers and prevented the differentiation of M cells in mice. The activation of T cells via an oral route was substantially impaired in the intestine of Spi-B-deficient (Spib(-/-)) mice. Our study demonstrates that commitment to the intestinal M-cell lineage requires Spi-B as a candidate master regulator.

CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice.

The two most common forms of inflammatory bowel disease (IBD), Crohn's disease and ulcerative colitis, affect approximately 1 million people in the United States. Uncontrolled APC reactivity toward commensal bacteria is implicated in the pathogenesis of the disease. A number of functionally distinct APC populations exist in the mucosal lamina propria (LP) below the intestinal epithelium, but their relative contributions to inflammation remain unclear. Here, we demonstrate in mice important roles for the chemokine receptor CX3CR1 in maintaining LP macrophage populations, preventing translocation of commensal bacteria to mesenteric lymph nodes (mLNs), and limiting colitogenic Th17 responses. CX3CR1 was found to be expressed in resident LP macrophages (defined as CD11b(+)F4/80(+)) but not DCs (defined as CD11c(+)CD103(+)). LP macrophage frequency and number were decreased in two strains of CX3CR1-knockout mice and in mice deficient in the CX3CR1 ligand CX3CL1. All these knockout strains displayed markedly increased translocation of commensal bacteria to mLNs. Additionally, the severity of DSS-induced colitis was dramatically enhanced in the knockout mice as compared with controls. Disease severity could be limited by either administration of neutralizing IL-17A antibodies or transfer of CX3CR1-sufficient macrophages. Our data thus suggest key roles for the CX3CR1/CX3CL1 axis in the intestinal mucosa; further clarification of CX3CR1 function will likely direct efforts toward therapeutic intervention for mucosal inflammatory disorders such as IBD.

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.

p90RSK2 is essential for FLT3-ITD- but dispensable for BCR-ABL-induced myeloid leukemia.

p90 ribosomal S6 kinase 2 (p90RSK2) is important in diverse cellular processes including gene expression, cell proliferation, and survival. We found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including BCR-ABL and FMS-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD). Interestingly, in a murine BM transplantation (BMT) model, genetic deficiency of RSK2 did not affect the pathogenesis or disease progression of BCR-ABL-induced myeloproliferative neoplasm (PN). In contrast, FLT3-ITD induced a T-cell acute lymphoblastic leukemia in BMT mice receiving RSK2 knockout (KO) BM cells, phenotypically distinct from the myeloproliferative neoplasm induced by FLT3-ITD using wild-type BM cells. In consonance with these results, inhibition of RSK2 by an RSK inhibitor, fmk, did not effectively induce apoptosis in BCR-ABL-expressing murine Ba/F3 cells, human K562 cells or primary tissue samples from CML patients, whereas fmk treatment induced significant apoptotic cell death not only in FLT3-ITD-positive Ba/F3 cells, human Molm14 and Mv(4;11) leukemia cells, but also in primary tissue samples from AML patients. These results suggest that RSK2 is dispensable for BCR-ABL-induced myeloid leukemia, but may be required for pathogenesis and lineage determination in FLT3-ITD-induced hematopoietic transformation. RSK2 may thus represent an alternative therapeutic target in the treatment of FLT3-ITD-positive leukemia.

CCR6hiCD11c(int) B cells promote M-cell differentiation in Peyer's patch.

M cells are responsible for uptake of mucosal antigens in Peyer's patches (PPs). Differentiation of M cells is thought to be induced by interactions between follicle-associated epithelium and PP cells; however, it remains elusive what types of immune cells function as M-cell inducers. Here, we attempted to identify the cells that serve as an M-cell inducer in PP. We found that a unique B-cell subset characterized by CCR6(hi)CD11c(int) resided in the subepithelial dome (SED) in mouse PP. CCR6(hi)CD11c(int) B cells showed chemotactic migration in response to CCL20. Furthermore, this unique B-cell subset substantially decreased in PP of CCR6-deficient mice, indicating that the SED localization of CCR6(hi)CD11c(int) B cells is most likely regulated by the CCL20-CCR6 system. Concomitantly, CCR6 deficiency caused remarkable decrement of M cells. Moreover, adoptive transfer of CCR6(hi)CD11c(int) B cells from wild-type mice restored the M-cell decrement in CCR6-deficient mice. Collectively, the spatial regulation of CCR6(hi)CD11c(int) B cells via the CCL20-CCR6 system may play a vital role in M-cell differentiation in mice.

Receptor activator of NF-kappaB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit.

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK-RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities.

RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.

Microfold cells (M cells) are specialized epithelial cells situated over Peyer's patches (PP) and other organized mucosal lymphoid tissues that transport commensal bacteria and other particulate Ags into intraepithelial pockets accessed by APCs. The TNF superfamily member receptor activator of NF-kappaB ligand (RANKL) is selectively expressed by subepithelial stromal cells in PP domes. We found that RANKL null mice have <2% of wild-type levels of PP M cells and markedly diminished uptake of 200 nm diameter fluorescent beads. Ab-mediated neutralization of RANKL in adult wild-type mice also eliminated most PP M cells. The M cell deficit in RANKL null mice was corrected by systemic administration of exogenous RANKL. Treatment with RANKL also induced the differentiation of villous M cells on all small intestinal villi with the capacity for avid uptake of Salmonella and Yersinia organisms and fluorescent beads. The RANK receptor for RANKL is expressed by epithelial cells throughout the small intestine. We conclude that availability of RANKL is the critical factor controlling the differentiation of M cells from RANK-expressing intestinal epithelial precursor cells.

Toll-like receptor 5-dependent regulation of inflammation in systemic Salmonella enterica Serovar typhimurium infection.

Salmonella enterica, a gram-negative pathogen, causes a spectrum of human infections including enterocolitis and typhoid fever. We previously showed that Salmonella flagellin played a role in suppressing intestinal mucosal inflammation in a murine model of acute enterocolitis. In this study, we examined the role of flagellin in the typhoid-like systemic murine Salmonella infection by measuring bacterial proliferation, inflammation, leukocyte recruitment, and cellular apoptosis in Peyer's patches (PPs), mesenteric lymph node (MLN), and spleen. We found that relative to an isogenic wild-type (WT) strain, aflagellate Salmonella exhibited increased proliferation at 4 days postinfection in PPs and MLN but not spleen. The aflagellate mutant also elicited increased local and systemic secretion of inflammatory cytokines such as interleukin-1beta, gamma interferon, and tumor necrosis factor alpha and enhanced surface expression of ICAM-1 on macrophages and dendritic cells (DCs). Furthermore, the recruitment of macrophages and DCs in PPs and MLN, but not spleen, was enhanced upon infection with aflagellate Salmonella. The relative differences between WT and aflagellate Salmonella were highly attenuated in Toll-like receptor 5-deficient (TLR5(-/-)) mice, indicating involvement of TLR5-dependent signaling. Interestingly, infection with the aflagellate mutant also resulted in decreased levels of T-cell apoptosis in PPs relative to infection with WT Salmonella. We postulate that the initial lack of detection of the aflagellate mutant in the mucosa permits increased proliferation within the host and enhances inflammatory signaling in nonepithelial cell types, which subsequently promotes leukocyte recruitment. In contrast, lack of difference in any disease parameter measured in the spleen likely reflects that Salmonella expression of flagellin is downregulated in this organ. Thus, the characteristic inflammatory pathology of Salmonella infection occurs only in PPs and to a lesser extent in MLN during the initial phases of infection and these early responses are dependent on TLR5.