Project description:Transcriptional profiling shows that Peyer´s patch CD4+ T cells from mice kept on dietary antigens are skewed towards a Tfh cell programme. Continous recognition of dietary antigens does not lead to classical signature of exhaustion. Overall design: Examination of conventional and elemental diet on gene expression of PP T cells
Project description:The primary induction sites for intestinal IgA are the gut-associated lymphoid tissues (GALT), such as Peyer's patches (PPs) and isolated lymphoid follicles (ILFs). The commensal microbiota is known to contribute to IgA production in the gut; however, the role of dietary antigens in IgA production is poorly understood. To understand the effect of dietary antigens on IgA production, post-weaning mice were maintained on an elemental diet without any large immunogenic molecules. We found that dietary antigens contribute to IgA production in PPs through induction of follicular helper T cells and germinal center B cells. The role of dietary antigens in the PP responses was further confirmed by adding bovine serum albumin (BSA) into the elemental diet. Although dietary antigens are important for PP responses, they have fewer effects than the microbiota on the development and maturation of ILFs. Furthermore, we demonstrated that dietary antigens are essential for a normal antigen-specific IgA response to Salmonella typhi serovar Typhimurium infection. These results provide new insights into the role of dietary antigens in the regulation of mucosal immune responses.
Project description:To explore the requirement for M cells and the Peyer's patch (PP) in induction of oral tolerance and address the potential in vivo role of intestinal epithelial cells as nonprofessional APCs, we have attempted to induce tolerance in mice with ligated small bowel loops without M cells and Peyer's patches. A 2-centimeter section of vascularized small bowel was spliced away from the gut without disruption of the mesenteric attachments. We introduced OVA directly into the lumen of the loop prior to footpad immunization. By excising segments of bowel that contain PPs in some mice and segments without patches in others, we could study the necessity of the M cell and the underlying patch versus epithelial cells in induction of mucosal tolerance. We show that OVA-specific T cell proliferation and serum antibody responses are reduced in mice that have previously been given OVA both in PP-containing loops and in loops without patches. Furthermore, both high- and low-dose tolerance could be induced in the absence of PPs. Low-dose tolerance is associated with bystander suppression and requires IL-10, which indicates active suppression and the induction of regulatory cells. These data suggest that there is a critical role for components of the mucosal immune system other than PPs in antigen sampling and induction of oral tolerance.
Project description:Lymphocytes express integrin receptors, termed lymphocyte Peyer's patch high endothelial venule (HEV) adhesion molecules (LPAMs), that mediate their organ-specific adhesion to specialized HEVs found in mucosal lymphoid organs (Peyer's patches). LPAM-1 consists of a murine integrin alpha 4 noncovalently associated with integrin beta p. Here, we describe the cloning and expression of a mouse cDNA encoding beta p, which is an 806-amino acid transmembrane glycoprotein. The genomic Southern blot analysis indicates that beta p is the murine homologue of human beta 7. The function of alpha 4 beta 7 as a Peyer's patch-specific adhesion molecule was tested directly by expression of the murine beta 7 cDNA in an alpha 4+ beta 7-B-cell line or coexpression of the alpha 4 and beta 7 cDNAs in an alpha 4-beta 7-T-cell line. The transfected cells exhibited a new Peyer's patch-specific adhesive phenotype that could be specifically blocked by monoclonal antibodies against alpha 4 and beta 7. Moreover, an anti-beta 7 monoclonal antibody specifically blocked binding of normal lymphocytes to Peyer's patch HEV but did not inhibit their binding to peripheral lymph node HEVs, indicating that beta 7 is a unique component of the Peyer's patch-specific homing receptor.
Project description:The germinal center (GC) reaction in Peyer's patches (PP) requires continuous access to antigens, but how this is achieved is not known. Here we show that activated antigen-specific CCR6+CCR1+GL7- B cells make close contact with M cells in the subepithelial dome (SED). Using in situ photoactivation analysis of antigen-specific SED B cells, we find migration of cells towards the GC. Following antigen injection into ligated intestinal loops containing PPs, 40% of antigen-specific SED B cells bind antigen within 2 h, whereas unspecifc cells do not, indicating B cell-receptor involvment. Antigen-loading is not observed in M cell-deficient mice, but is unperturbed in mice depleted of classical dendritic cells (DC). Thus, we report a M cell-B cell antigen-specific transporting pathway in PP that is independent of DC. We propose that this antigen transporting pathway has a critical role in gut IgA responses, and should be taken into account when developing mucosal vaccines.
Project description:Sampling and immune surveillance within gut-associated lymphoid tissues such as the intestinal Peyer's patch (PP) occurs by an elegantly orchestrated effort that involves the epithelial barrier, B and T lymphocytes, and an extensive network of mononuclear phagocytes. Although we now understand more about the dynamics of antigen and microbial sampling within PPs, the gene expression changes that occur in individual cell subsets during sampling are not well characterized. This protocol describes the isolation of high-quality RNA from sorted PP, B and T-lymphocytes, and CD11c+ phagocytes for use with nCounter-NanoString technology. This method allows investigators to study gene expression changes within PPs in response to antigens, microbes, and oral vaccine delivery vehicles of interest that are sampled.
Project description:OBJECTIVE:Exposure to lethal doses of radiation has severe effects on normal tissues. Exposed individuals experience a plethora of symptoms in different organ systems including the gastrointestinal (GI) tract, summarized as Acute Radiation Syndrome (ARS). There are currently no approved drugs for mitigating GI-ARS. A recent high-throughput screen performed at the UCLA Center for Medical Countermeasures against Radiation identified compounds containing sulfonylpiperazine groups with radiation mitigation properties to the hematopoietic system and the gut. Among these 1-[(4-Nitrophenyl)sulfonyl]-4-phenylpiperazine (Compound #5) efficiently mitigated gastrointestinal ARS. However, the mechanism of action and target cells of this drug is still unknown. In this study we examined if Compound #5 affects gut-associated lymphoid tissue (GALT) with its subepithelial domes called Peyer's patches. METHODS:C3H mice were irradiated with 0 or 12?Gy total body irradiation (TBI). A single dose of Compound #5 or solvent was administered subcutaneously 24?h later. 48?h after irradiation the mice were sacrificed, and the guts examined for changes in the number of visible Peyer's patches. In some experiments the mice received 4 daily injections of treatment and were sacrificed 96?h after TBI. For immune histochemistry gut tissues were fixed in formalin and embedded in paraffin blocks. Sections were stained with H&E, anti-Ki67 or a TUNEL assay to assess the number of regenerating crypts, mitotic and apoptotic indices. Cells isolated from Peyer's patches were subjected to immune profiling using flow cytometry. RESULTS:Compound #5 significantly increased the number of visible Peyer's patches when compared to its control in non-irradiated and irradiated mice. Additionally, assessment of total cells per Peyer's patch isolated from these mice demonstrated an overall increase in the total number of Peyer's patch cells per mouse in Compound #5-treated mice. In non-irradiated animals the number of CD11bhigh in Peyer's patches increased significantly. These Compound #5-driven increases did not coincide with a decrease in apoptosis or an increase in proliferation in the germinal centers inside Peyer's patches 24?h after drug treatment. A single dose of Compound #5 significantly increased the number of CD45+ cells after 12?Gy TBI. Importantly, 96?h after 12?Gy TBI Compound #5 induced a significant rise in the number of visible Peyer's patches and the number of Peyer's patch-associated regenerating crypts. CONCLUSION:In summary, our study provides evidence that Compound #5 leads to an influx of immune cells into GALT, thereby supporting crypt regeneration preferentially in the proximity of Peyer's patches.
Project description:To define gene expression profiling of microfold (M) cells in follicle-associated epithelium (FAE), we isolated M cells and other epithelial cells from EpCAM+CD45-CD24- cells of Peyer's patches. The gene expression profilings were clearly different between GP2+ M cells and GP2- epithelial cells. Overall design: GP2+ M cells and GP2- epithelial cells were sorted from EpCAM+CD45-CD24- fraction of C57BL/6J mouse Peyer's patch
Project description:Here, we set out to delineate the effect of fluid flow on stromal populations in the peyer's patch. Overall design: Mice were unperturbed or treated with PEG to diminish fluid flow for three days. Then fibroblastic reticular cells and high endothelial venules were FACS sorted for RNAseq.
Project description:The initiation of the mucosal immune response in Peyer’s patch (PP) relies on the sampling, processing and efficient presentation of foreign antigens by dendritic cells (DC). PP DC encompass five subsets, among which CD11b+ conventional DC (cDC) and LysoDC have distinct progenitors and functions but share many cell surface markers. This has previously led to confusion between these two subsets. In addition, another PP DC subset, termed double-negative (DN), remains poorly characterized. Here, we have studied the genetic relatedness of the different subsets of PP cDC at steady state and under TLR7 ligand stimulation. We also provide the transcriptional profiles of subepithelial TIM-4- and interfollicular TIM-4+ macrophages. Overall design: Three independent replicates of TIM-4- and TIM-4+ LysoMac, CD8a+ and DN dome cDC and five independent replicates of CD11b+ dome cDC were sorted from Peyer's patch of C57BL/6 mice at steady state. In addition, three and five independant replicates of CD11b+ and CD8a+ dome cDC, respectively were sorted from Peyer's patch of C57BL/6 mice 9 hours after R848 gavage. Total RNA of Peyer's patch sorted cells was extracted with a Qiagen RNeasy Plus Micro Kit. Quantity, quality and absence of genomic DNA contamination were assessed with a Bioanalyser (Agilent). Microarray experiments were performed by the Plateforme Biopuces of Strasbourg (France) using the Affymetrix GeneChip® Mouse Gene 1.0 ST Array.