A mechanism for matrikine regulation in acute inflammatory lung injury.

Proline-glycine-proline (PGP) and its acetylated form (Ac-PGP) are neutrophil chemoattractants generated by collagen degradation, and they have been shown to play a role in chronic inflammatory disease. However, the mechanism for matrikine regulation in acute inflammation has not been well established. Here, we show that these peptides are actively transported from the lung by the oligopeptide transporter, PEPT2. Following intratracheal instillation of Ac-PGP in a mouse model, there was a rapid decline in concentration of the labeled peptide in the bronchoalveolar lavage (BAL) over time and redistribution to extrapulmonary sites. In vitro knockdown of the PEPT2 transporter in airway epithelia or use of a competitive inhibitor of PEPT2, cefadroxil, significantly reduced uptake of Ac-PGP. Animals that received intratracheal Ac-PGP plus cefadroxil had higher levels of Ac-PGP in BAL and lung tissue. Utilizing an acute LPS-induced lung injury model, we demonstrate that PEPT2 blockade enhanced pulmonary Ac-PGP levels and lung inflammation. We further validated this effect using clinical samples from patients with acute lung injury in coculture with airway epithelia. This is the first study to our knowledge to determine the in vitro and in vivo significance of active matrikine transport as a mechanism of modulating acute inflammation and to demonstrate that it may serve as a potential therapeutic target.

NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis.

The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.

Exosome-mediated metabolic reprogramming: the emerging role in tumor microenvironment remodeling and its influence on cancer progression.

Metabolic reprogramming is reported to be one of the hallmarks of cancer, which is an adaptive mechanism by which fast-growing cancer cells adapt to their increasing energy demands. Recently, extracellular vesicles (EVs) known as exosomes have been recognized as crucial signaling mediators in regulating the tumor microenvironment (TME). Meanwhile, the TME is a highly heterogeneous ecosystem incorporating cancer cells, fibroblasts, adipocytes, endothelial cells, mesenchymal stem cells, and extracellular matrix. Accumulated evidence indicates that exosomes may transfer biologically functional molecules to the recipient cells, which facilitate cancer progression, angiogenesis, metastasis, drug resistance, and immunosuppression by reprogramming the metabolism of cancer cells and their surrounding stromal cells. In this review, we present the role of exosomes in the TME and the underlying mechanism of how exosomes exacerbate tumor development through metabolic reprogramming. In addition, we will also discuss the potential role of exosomes targeting metabolic process as biomarkers for tumor diagnosis and prognosis, and exosomes-mediated metabolic reprogramming as potential targets for cancer therapy. Furthermore, a better understanding of the link between exosomes and metabolic reprogramming, and their impact on cancer progression, would provide novel insights for cancer prevention and treatment in the future.

Dendritic Cells From the Cervical Mucosa Capture and Transfer HIV-1 via Siglec-1.

Antigen presenting cells from the cervical mucosa are thought to amplify incoming HIV-1 and spread infection systemically without being productively infected. Yet, the molecular mechanism at the cervical mucosa underlying this viral transmission pathway remains unknown. Here we identified a subset of HLA-DR+ CD14+ CD11c+ cervical DCs at the lamina propria of the ectocervix and the endocervix that expressed the type-I interferon inducible lectin Siglec-1 (CD169), which promoted viral uptake. In the cervical biopsy of a viremic HIV-1+ patient, Siglec-1+ cells harbored HIV-1-containing compartments, demonstrating that in vivo, these cells trap viruses. Ex vivo, a type-I interferon antiviral environment enhanced viral capture and trans-infection via Siglec-1. Nonetheless, HIV-1 transfer via cervical DCs was effectively prevented with antibodies against Siglec-1. Our findings contribute to decipher how cervical DCs may boost HIV-1 replication and promote systemic viral spread from the cervical mucosa, and highlight the importance of including inhibitors against Siglec-1 in microbicidal strategies.

Imaging Vesicular Traffic at the Immune Synapse.

Immunological synapse formation is the result of a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, as well as intracellular vesicle traffic. Endosomal vesicle traffic ensures the targeting of the T cell receptor (TCR) and various signaling molecules to the synapse, being necessary for the generation of signaling complexes downstream of the TCR. Here we describe the microscopy imaging methods that we currently use to unveil how TCR and signaling molecules are associated with endosomal compartments and deliver their cargo to the immunological synapse.

Plant viral nanoparticles-based HER2 vaccine: Immune response influenced by differential transport, localization and cellular interactions of particulate carriers.

Cancer vaccines are designed to elicit an endogenous adaptive immune response that can successfully recognize and eliminate residual or recurring tumors. Such approaches can potentially overcome shortcomings of passive immunotherapies by generating long-lived therapeutic effects and immune memory while limiting systemic toxicities. A critical determinant of vaccine efficacy is efficient transport and delivery of tumor-associated antigens to professional antigen presenting cells (APCs). Plant viral nanoparticles (VNPs) with natural tropism for APCs and a high payload carrying capacity may be particularly effective vaccine carriers. The applicability of VNP platform technologies is governed by stringent structure-function relationships. We compare two distinct VNP platforms: icosahedral cowpea mosaic virus (CPMV) and filamentous potato virus X (PVX). Specifically, we evaluate in vivo capabilities of engineered VNPs delivering human epidermal growth factor receptor 2 (HER2) epitopes for therapy and prophylaxis of HER2+ malignancies. Our results corroborate the structure-function relationship where icosahedral CPMV particles showed significantly enhanced lymph node transport and retention, and greater uptake by/activation of APCs compared to filamentous PVX particles. These enhanced immune cell interactions and transport properties resulted in elevated HER2-specific antibody titers raised by CPMV- vs. PVX-based peptide vaccine. The 'synthetic virology' field is rapidly expanding with numerous platforms undergoing development and preclinical testing; our studies highlight the need for systematic studies to define rules guiding the design and rational choice of platform, in the context of peptide-vaccine display technologies.

MST1-dependent vesicle trafficking regulates neutrophil transmigration through the vascular basement membrane.

Neutrophils need to penetrate the perivascular basement membrane for successful extravasation into inflamed tissue, but this process is incompletely understood. Recent findings have associated mammalian sterile 20-like kinase 1 (MST1) loss of function with a human primary immunodeficiency disorder, suggesting that MST1 may be involved in immune cell migration. Here, we have shown that MST1 is a critical regulator of neutrophil extravasation during inflammation. Mst1-deficient (Mst1-/-) neutrophils were unable to migrate into inflamed murine cremaster muscle venules, instead persisting between the endothelium and the basement membrane. Mst1-/- neutrophils also failed to extravasate from gastric submucosal vessels in a murine model of Helicobacter pylori infection. Mechanistically, we observed defective translocation of VLA-3, VLA-6, and neutrophil elastase from intracellular vesicles to the surface of Mst1-/- neutrophils, indicating that MST1 is required for this crucial step in neutrophil transmigration. Furthermore, we found that MST1 associates with the Rab27 effector protein synaptotagmin-like protein 1 (JFC1, encoded by Sytl1 in mice), but not Munc13-4, thereby regulating the trafficking of Rab27-positive vesicles to the cellular membrane. Together, these findings highlight a role for MST1 in vesicle trafficking and extravasation in neutrophils, providing an additional mechanistic explanation for the severe immune defect observed in patients with MST1 deficiency.

Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs.

Antigen transfer from exosomes to dendritic cells as an explanation for the immune enhancement seen by IgE immune complexes.

IgE antigen complexes induce increased specific T cell proliferation and increased specific IgG production. Immediately after immunization, CD23(+) B cells capture IgE antigen complexes, transport them to the spleen where, via unknown mechanisms, dendritic cells capture the antigen and present it to T cells. CD23, the low affinity IgE receptor, binds IgE antigen complexes and internalizes them. In this study, we show that these complexes are processed onto B-cell derived exosomes (bexosomes) in a CD23 dependent manner. The bexosomes carry CD23, IgE and MHC II and stimulate antigen specific T-cell proliferation in vitro. When IgE antigen complex stimulated bexosomes are incubated with dendritic cells, dendritic cells induce specific T-cell proliferation in vivo, similar to IgE antigen complexes. This suggests that bexosomes can provide the essential transfer mechanism for IgE antigen complexes from B cells to dendritic cells.

Reduction of proteinuria through podocyte alkalinization.

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.

Inhibition of insulin receptor function by a human, allosteric monoclonal antibody: a potential new approach for the treatment of hyperinsulinemic hypoglycemia.

Novel therapies are needed for the treatment of hypoglycemia resulting from both endogenous and exogenous hyperinsulinema. To provide a potential new treatment option, we identified XMetD, an allosteric monoclonal antibody to the insulin receptor (INSR) that was isolated from a human antibody phage display library. To selectively obtain antibodies directed at allosteric sites, panning of the phage display library was conducted using the insulin-INSR complex. Studies indicated that XMetD bound to the INSR with nanomolar affinity. Addition of insulin reduced the affinity of XMetD to the INSR by 3-fold, and XMetD reduced the affinity of the INSR for insulin 3-fold. In addition to inhibiting INSR binding, XMetD also inhibited insulin-induced INSR signaling by 20- to 100-fold. These signaling functions included INSR autophosphorylation, Akt activation and glucose transport. These data indicated that XMetD was an allosteric antagonist of the INSR because, in addition to inhibiting the INSR via modulation of binding affinity, it also inhibited the INSR via modulation of signaling efficacy. Intraperitoneal injection of XMetD at 10 mg/kg twice weekly into normal mice induced insulin resistance. When sustained-release insulin implants were placed into normal mice, they developed fasting hypoglycemia in the range of 50 mg/dl. This hypoglycemia was reversed by XMetD treatment. These studies demonstrate that allosteric monoclonal antibodies, such as XMetD, can antagonize INSR signaling both in vitro and in vivo. They also suggest that this class of allosteric monoclonal antibodies has the potential to treat hyperinsulinemic hypoglycemia resulting from conditions such as insulinoma, congenital hyperinsulinism and insulin overdose.

Lipid abnormalities and lipid-based repair strategies in atopic dermatitis.

Prior studies have revealed the key roles played by Th1/Th2 cell dysregulation, IgE production, mast cell hyperactivity, and dendritic cell signaling in the evolution of the chronic, pruritic, inflammatory dermatosis that characterizes atopic dermatitis (AD). We review here increasing evidence that the inflammation in AD results primarily from inherited abnormalities in epidermal structural and enzymatic proteins that impact permeability barrier function. We also will show that the barrier defect can be attributed to a paracellular abnormality due to a variety of abnormalities in lipid composition, transport and extracellular organization. Accordingly, we also review the therapeutic implications of this emerging pathogenic paradigm, including several current and potentially novel, lipid-based approaches to corrective therapy. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Pathogenic mechanisms and clinical implications of congenital neutropenia syndromes.

PURPOSE OF REVIEW: The purpose of this review is to summarize pathogenic mechanisms and clinical implications of the most illustrative genetic entities of congenital neutropenia syndromes. RECENT FINDINGS: Congenital neutropenia comprise monogenetic entities with or without additional immunologic and extrahaematopoietic syndromatic features. Continuous careful explorations of known entities such as ELANE, GFI1, HAX1, G6PC3 deficiency and XLN help to define principles controlling differentiation and function of neutrophil granulocytes. Furthermore, the identification of novel genetic defects associated with congenital neutropenia, such as VPS45 deficiency, broadens our understanding of neutrophil biology. Pathogenic mechanisms imply protein and vesicle mistrafficking, endoplasmic reticulum stress, the unfolded protein response, destabilization of the mitochondrial membrane potential, disturbed energy metabolism, dysglycosylation and deregulated actin polymerization. SUMMARY: Advanced genetic and biochemical techniques have helped to expand our knowledge of congenital neutropenia syndromes. Known and novel genetic entities shed light on fundamental biological processes important for the homeostatis and functioning not only of the neutrophil granulocyte but as well of the entire haematopoietic system. Furthermore, treatment decisions become more tailored and might pave the road towards personalized molecular medicine.

Investigation of the role of FcγR and FcRn in mAb distribution to the brain.

To evaluate the role of Fc receptors (FcR) on IgG distribution to the brain, the disposition of 8C2, a murine monoclonal IgG1 antibody, was investigated after intravenous administration in FcRn α-chain knockout mice, FcγRIIb knockout mice, FcγRI/RIII knockout mice, and C57BL/6 control mice. (125)I-8C2 was co-administered with (51)Cr-labeled red blood cells to allow accurate assessment of residual blood content in brain samples. Blood and brain tissues were harvested from subgroups of three mice at several time-points up to 10 days, and radioactivity was counted. The blood and brain areas under 8C2 concentration vs time curves (AUCs) were calculated using the linear trapezoidal rule, and the associated standard deviations (SD) were assessed using a modified Bailer method. Concentration data were also analyzed with a semiphysiological population pharmacokinetic model. The brain/blood AUC ratios were comparable across all strains of mice (ratios ± SD): 0.00774 ± 0.000452, 0.00841 ± 0.000535, 0.00636 ± 0.000548, and 0.00917 ± 0.000478 for C57BL/6 control mice, FcγRI/RIII knockouts, FcγRIIb knockouts, and FcRn α-chain knockout mice (p > 0.05). Statistically significant improvement in model fitting of the data was shown with incorporation of a strain-specific parameter for antibody clearance for FcRn knockout mice; however, no significant improvements in model fitting were found for strain effects on any other parameter, including the brain uptake clearance or efflux clearances for 8C2. The predicted 8C2 brain efflux clearance was found to be ∼135-fold faster than the brain uptake clearance, consistent with the observed low ratio of brain-blood exposure. The experimental results and modeling results indicate that, in mice, FcRn and FcγR do not contribute to the "blood-brain barrier" that limits mAb uptake into the brain.

Toll-like receptor 11 (TLR11) prevents Salmonella penetration into the murine Peyer patches.

Toll-like receptors (TLRs) are key molecular sensors used by the mammalian innate immune system to detect microorganisms. Although TLR functions in colonic immune homeostasis and tolerance to commensal bacteria have been intensively researched, the precise roles of different TLRs in response to pathogen infection in the gut remain elusive. Peyer patches are the major entrance of Salmonella infection and antigen transportation in intestine. Here, we report that, in contrast to TLR5 as a "carrier of Salmonella," TLR11 works as a "blocker of Salmonella" to prevent highly invasive Salmonella from penetrating into the murine Peyer patches and spreading systemically. TLR11 plays an important role in mediating TNF-α induction and systemic inflammation in response to Salmonella infection. Remarkably, in mice lacking TLR11, apparent hemorrhages at Peyer patches are induced by highly invasive Salmonella, a phenotype resembling human Salmonella infection. Therefore, our results indicate a potentially important role for TLR11 in preventing murine intestinal infection and modulating antigen transportation in the gut and imply an important role for various TLRs in cooperation with tight control of pathogens penetrating into Peyer patches. The TLR11 knock-out mouse can serve as a good animal model to study Salmonella infection.

Protective role of commensals against Clostridium difficile infection via an IL-1ß-mediated positive-feedback loop.

Clostridium difficile is a Gram-positive obligate anaerobic pathogen that causes pseudomembranous colitis in antibiotic-treated individuals. Commensal bacteria are known to have a significant role in the intestinal accumulation of C. difficile after antibiotic treatment, but little is known about how they affect host immunity during C. difficile infection. In this article, we report that C. difficile infection results in translocation of commensals across the intestinal epithelial barrier that is critical for neutrophil recruitment through the induction of an IL-1ß-mediated positive-feedback loop. Mice lacking ASC, an essential mediator of IL-1ß and IL-18 processing and secretion, were highly susceptible to C. difficile infection. ASC(-/-) mice exhibited enhanced translocation of commensals to multiple organs after C. difficile infection. Notably, ASC(-/-) mice exhibited impaired CXCL1 production and neutrophil influx into intestinal tissues in response to C. difficile infection. The impairment in neutrophil recruitment resulted in reduced production of IL-1ß and CXCL1 but not IL-18. Importantly, translocated commensals were required for ASC/Nlrp3-dependent IL-1ß secretion by neutrophils. Mice lacking IL-1ß were deficient in inducing CXCL1 secretion, suggesting that IL-1ß is the dominant inducer of ASC-mediated CXCL1 production during C. difficile infection. These results indicate that translocated commensals play a crucial role in CXCL1-dependent recruitment of neutrophils to the intestine through an IL-1ß/NLRP3/ASC-mediated positive-feedback mechanism that is important for host survival and clearance of translocated commensals during C. difficile infection.

B7-H1, which represses EBV-immortalized B cell killing by autologous T and NK cells, is oppositely regulated by c-Myc and EBV latency III program at both mRNA and secretory lysosome levels.

EBV-immortalized B cells induce a complex immune response such that the virus persists as a clinically silent infection for the lifetime of the infected host. B7-H1, also called PD-L1, is a cosignaling molecule of the B7 family that can inhibit activated T cell effectors by interaction with its receptor PD-1. In this work, we have studied the dependence of B7-H1 on NF-κB and c-Myc, the two main transcription factors in EBV latency III proliferating B cells, on various lymphoblastoid and Burkitt lymphoma cell lines, some of them being inducible or not for the EBV latency III program and/or for c-Myc. We found that B7-H1 repressed killing of EBV-immortalized B cells by their autologous T and NK cells. At the mRNA level, NF-κB was a weak inducer whereas c-Myc was a strong repressor of B7-H1 expression, an effect mediated by STAT1 inhibition. At the protein level, B7-H1 molecules were stored in both degradative and unconventional secretory lysosomes. Surface membrane B7-H1 molecules were constitutively internalized and proteolyzed in lysosomes. The EBV latency III program increased the amounts of B7-H1-containing secretory lysosomes and their export to the surface membrane. By repressing actin polymerization, c-Myc blocked secretory lysosome migration and B7-H1 surface membrane export. In addition to B7-H1, various immunoregulatory molecules participating in the immunological synapse are stored in secretory lysosomes. By playing on actin polymerization, c-Myc could thus globally regulate the immunogenicity of transformed B cells, acting on export of secretory lysosomes to plasma membrane.

Proinflammatory mediators disrupt glucose homeostasis in airway surface liquid.

The glucose concentration of the airway surface liquid (ASL) is much lower than that in blood and is tightly regulated by the airway epithelium. ASL glucose is elevated in patients with viral colds, cystic fibrosis, chronic obstructive pulmonary disease, and asthma. Elevated ASL glucose is also associated with increased incidence of respiratory infection. However, the mechanism by which ASL glucose increases under inflammatory conditions is unknown. The aim of this study was to investigate the effect of proinflammatory mediators (PIMs) on the mechanisms governing airway glucose homeostasis in polarized monolayers of human airway (H441) and primary human bronchial epithelial (HBE) cells. Monolayers were treated with TNF-α, IFN-γ, and LPS during 72 h. PIM treatment led to increase in ASL glucose concentration and significantly reduced H441 and HBE transepithelial resistance. This decline in transepithelial resistance was associated with an increase in paracellular permeability of glucose. Similar enhanced rates of paracellular glucose flux were also observed across excised trachea from LPS-treated mice. Interestingly, PIMs enhanced glucose uptake across the apical, but not the basolateral, membrane of H441 and HBE monolayers. This increase was predominantly via phloretin-sensitive glucose transporter (GLUT)-mediated uptake, which coincided with an increase in GLUT-2 and GLUT-10 abundance. In conclusion, exposure of airway epithelial monolayers to PIMs results in increased paracellular glucose flux, as well as apical GLUT-mediated glucose uptake. However, uptake was insufficient to limit glucose accumulation in ASL. To our knowledge, these data provide for the first time a mechanism to support clinical findings that ASL glucose concentration is increased in patients with airway inflammation.

Differential intestinal M-cell gene expression response to gut commensals.

Different rates of bacterial translocation across the gut mucosa have been reported but few studies have examined translocation of commensals at the level of the gut epithelial microfold (M) cell. We used an in vitro M-cell model to quantify translocation and determine the transcriptional response of M cells to various commensal bacteria. The transport kinetics and gene expression profile of M cells in response to different bacterial strains, namely Lactobacillus salivarius, Escherichia coli and Bacteroides fragilis, was assessed. Bacterial strains translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency whereas L. salivarius translocated with less efficiency. Microarray analysis of the M cell response showed both common and differential gene expression changes between the bacterial strains. In the presence of bacteria, but not control beads, up-regulated genes were mainly involved in transcription regulation whereas pro-inflammatory and stress response genes were primarily up-regulated by E. coli and B. fragilis, but not L. salivarius nor beads. Translocation of bacteria and M-cell gene expression responses were confirmed in murine M cells following bacterial challenge in vivo. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses.