A population of Langerin-positive dendritic cells in murine Peyer's patches involved in sampling ?-glucan microparticles.
ABSTRACT: Glucan particles (GPs) are 2-4 ?m hollow, porous shells composed of 1,3-?-D-glucan that have been effectively used for oral targeted-delivery of a wide range of payloads, including small molecules, siRNA, DNA, and protein antigens. While it has been demonstrated that the transepithelial transport of GPs is mediated by Peyer's patch M cells, the fate of the GPs once within gut-associated lymphoid tissue (GALT) is not known. Here we report that fluorescently labeled GPs administered to mice by gavage accumulate in CD11c+ DCs situated in Peyer's patch sub-epithelial dome (SED) regions. GPs appeared in DCs within minutes after gavage and remained within the SED for days afterwards. The co-administration or sequential administration of GPs with differentially labeled GPs or poly(lactic-co-glycolic acid) nanoparticles demonstrated that the SED DC subpopulation in question was capable of internalizing particles of different sizes and material compositions. Phenotypic analysis identified the GP-containing DCs as being CD8?- and CD11blo/-, suggesting they are the so-called myeloid and/or double negative (DN) subset(s) of PP DCs. A survey of C-type lectin receptors (CLRs) known to be expressed by leukocytes within the intestinal mucosa revealed that GP-containing SED DCs were positive for Langerin (CD207), a CLR with specificity for ?-D-glucan and that has been shown to mediate the internalization of a wide range of microbial pathogens, including bacteria, viruses and fungi. The presence of Langerin+ DCs in the SED as determined by immunofluorescence was confirmed using Langerin E-GFP transgenic mice. In summary, our results demonstrate that following M cell-mediated transepithelial transport, GPs (and other micro/nanoparticles) are sampled by a population of SED DCs distinguished from other Peyer's patch DC subsets by their expression of Langerin. Future studies will be aimed at defining the role of Langerin in antigen sampling and antigen presentation within the context of the GALT.
Project description:Immunoglobulin A (IgA) induction primarily occurs in intestinal Peyer's patches (PPs). However, the cellular interactions necessary for IgA class switching are poorly defined. Here we show that in mice, activated B cells use the chemokine receptor CCR6 to access the subepithelial dome (SED) of PPs. There, B cells undergo prolonged interactions with SED dendritic cells (DCs). PP IgA class switching requires innate lymphoid cells, which promote lymphotoxin-? receptor (LT?R)-dependent maintenance of DCs. PP DCs augment IgA production by integrin ?v?8-mediated activation of transforming growth factor-? (TGF?). In mice where B cells cannot access the SED, IgA responses against oral antigen and gut commensals are impaired. These studies establish the PP SED as a niche supporting DC-B cell interactions needed for TGF? activation and induction of mucosal IgA responses.
Project description:The specialized monocyte-derived phagocytes termed LysoDC are hallmarks of Peyer’s patches where their main function is to sample pathogens. However, their differentiation pathway and migratory properties remain uncharacterized. From single-cell RNA sequencing, we built LysoDC differentiation trajectories and fate diversity in correlation with their location and functions. All LysoDC differentiation states display similar phagocytic activity. One of them is located in follicles in a CXCR5-independent manner whereas the others reside in subepithelial domes (SED) and mature as they get closer to the epithelium. Immature LysoDC proliferate. Mature LysoDC acquire a gene signature shared with SIRP alpha-expressing conventional DC and prime naïve T cells in vitro but, at steady state, do not migrate in naïve T cells-enriched interfollicular regions (IFR). However, upon stimulation, they express the chemokine receptor CCR7 and migrate from the SED to the IFR periphery where they strongly interact with proliferative T cells. Finally, we show that LysoDC populates human Peyer’s patches. Overall design: Three to five independent replicates of the 4 LysoDC differentiation states (TN, SP, DP, TP) were sorted from Peyer's patch of C57BL/6 mice at steady state. In addition, three to four independant replicates of 2 LysoDC differentiation states (DP and TP) and of TIM-4- and TIM-4+ macrophages (LysoMac) 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.
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:Bacterial flagellin activates innate immune responses by signaling through Toll-like receptor 5 and is a potential vaccine adjuvant. Mucosal lymphoid follicles, inductive sites for adaptive mucosal immune responses, are covered by a follicle-associated epithelium (FAE) specialized for the uptake of antigens. This study demonstrates that mucosal application of Salmonella dublin flagellin enhanced transepithelial transport of microparticles by the FAE of mouse Peyer's patches in vivo. Flagellin also induced rapid, matrix metalloproteinase-dependent migration of subepithelial dendritic cells (DCs) into the FAE, better positioning DCs for antigen capture. These innate responses to flagellin enhance FAE functions and may promote adaptive immune responses in the mucosa.
Project description:Skin-derived dendritic cells (DCs) are potent antigen-presenting cells with critical roles in both adaptive immunity and tolerance to self. Skin DCs carry antigens and constitutively migrate to the skin-draining lymph nodes (LNs). In mice, Langerin-CD11b- dermal DCs are a low-frequency, heterogeneous, migratory DC subset that traffics to LNs (Langerin-CD11b- migDCs). Here, we build on the observation that Langerin-CD11b- migDCs are Fms-like tyrosine kinase 3 ligand (Flt3L) dependent and strongly Flt3L responsive, which may relate them to classical DCs. Examination of DC capture of FITC from painted skin, DC isolation from skin explant culture, and from the skin of CCR7 knockout mice, which accumulate migDCs, demonstrate these cells are cutaneous residents. Langerin-CD11b- Flt3L-responsive DCs are largely CD24(+) and CX3CR1(low) and can be depleted from Zbtb46-DTR mice, suggesting classical DC lineage. Langerin-CD11b- migDCs present antigen with equal efficiency to other DC subsets ex vivo, including classical CD8? cDCs and Langerin+CD103+ dermal DCs. Finally, transcriptome analysis suggests a close relationship with other skin DCs, and a lineage relationship with other classical DCs. This work demonstrates that Langerin- CD11b- dermal DCs, a previously overlooked cell subset, may be an important contributor to the cutaneous immune environment.
Project description:Langerin is a C-type lectin receptor that recognizes glycosylated patterns on pathogens. Langerin is used to identify human and mouse epidermal Langerhans cells (LCs), as well as migratory LCs in the dermis and the skin draining lymph nodes (DLNs). Using a mouse model that allows conditional ablation of langerin(+) cells in vivo, together with congenic bone marrow chimeras and parabiotic mice as tools to differentiate LC- and blood-derived dendritic cells (DCs), we have revisited the origin of langerin(+) DCs in the skin DLNs. Our results show that in contrast to the current view, langerin(+)CD8(-) DCs in the skin DLNs do not derive exclusively from migratory LCs, but also include blood-borne langerin(+) DCs that transit through the dermis before reaching the DLN. The recruitment of circulating langerin(+) DCs to the skin is dependent on endothelial selectins and CCR2, whereas their recruitment to the skin DLNs requires CCR7 and is independent of CD62L. We also show that circulating langerin(+) DCs patrol the dermis in the steady state and migrate to the skin DLNs charged with skin antigens. We propose that this is an important and previously unappreciated element of immunosurveillance that needs to be taken into account in the design of novel vaccine strategies.
Project description:This study used an in vivo mouse model to analyze the response of dendritic cells (DCs) in Peyer's patches (PPs) within the first 48 h of infection with the wild-type murine rotavirus EDIM (EDIM(wt)). After the infection, the absolute number of DCs was increased by 2-fold in the PPs without a modification of their relative percentage of the total cell number. Also, the DCs from PPs of infected mice showed a time-dependent migration to the subepithelial dome (SED) and an increase of the surface activation markers CD40, CD80, and CD86. This response was more evident at 48 h postinfection (p.i.) and depended on viral replication, since DCs from PPs of mice inoculated with UV-treated virus did not show this phenotype. As a result of the activation, the DCs showed an increase in the expression of mRNA for the proinflammatory cytokines interleukin-12/23p40 (IL-12/23p40), tumor necrosis factor alpha (TNF-alpha), and beta interferon (IFN-beta), as well as for the regulatory cytokine IL-10. These results suggest that, a short time after rotavirus infection, the DCs from PPs play a critical role in controlling the infection and, at the same time, avoiding an excessive inflammatory immune response.
Project description:Skin dendritic cells (DCs) control the immunogenicity of cutaneously administered vaccines. Antigens targeted to DCs via the C-type lectin Langerin/CD207 are cross-presented to CD8(+) T cells in vivo. We investigated the relative roles of Langerhans cells (LCs) and Langerin(+) dermal DCs (dDCs) in different vaccination settings. Poly(I:C) and anti-CD40 agonist antibody promoted cytotoxic responses upon intradermal immunization with ovalbumin (OVA)-coupled anti-Langerin antibodies (Langerin/OVA). This correlated with CD70 upregulation in Langerin(+) dDCs, but not LCs. In chimeric mice where Langerin targeting was restricted to dDCs, CD8(+) T-cell memory was enhanced. Conversely, providing Langerin/OVA exclusively to LCs failed to prime cytotoxicity, despite initial antigen cross-presentation to CD8(+) T cells. Langerin/OVA combined with imiquimod could not prime CD8(+) T cells and resulted in poor cytotoxicity in subsequent responses. This tolerance induction required targeting and maturation of LCs. Altogether, Langerin(+) dDCs prime long-lasting cytotoxic responses, while cross-presentation by LCs negatively influences CD8(+) T-cell priming. Moreover, this highlights that DCs exposed to TLR agonists can still induce tolerance and supports the existence of qualitatively different DC maturation programs.
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.
Project description:Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)-mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin(+), skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin(+) dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin(+), skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin(+) DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin(+) DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.