Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC−/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-γ in HDC−/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC−/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response

Histidine Decarboxylase Deficiency Prevents Autoimmune Diabetes in NOD Mice

Recent evidence has highlighted the role of histamine in inflammation. Since this monoamine has also been strongly implicated in the pathogenesis of type-1 diabetes, we assessed its effect in the nonobese diabetic (NOD) mouse model. To this end, we used mice (inactivated) knocked out for the gene encoding histidine decarboxylase, the unique histamine-forming enzyme, backcrossed on a NOD genetic background. We found that the lack of endogenous histamine in NOD HDC −/− mice decreased the incidence of diabetes in relation to their wild-type counterpart. Whereas the proportion of regulatory T and myeloid-derived suppressive cells was similar in both strains, histamine deficiency was associated with increased levels of immature macrophages, as compared with wild-type NOD mice. Concerning the cytokine pattern, we found a decrease in circulating IL-12 and IFN-in HDC −/− mice, while IL-6 or leptin remained unchanged, suggesting that histamine primarily modulates the inflammatory environment. Paradoxically, exogenous histamine given to NOD HDC −/− mice provided also protection against T1D. Our study supports the notion that histamine is involved in the pathogenesis of diabetes, thus providing additional evidence for its role in the regulation of the immune response

Mouse Basophils Reside in Extracellular Matrix-Enriched Bone Marrow Niches Which Control Their Motility

Made available in DSpace on 2015-06-08T14:01:47Z (GMT). No. of bitstreams: 2 license.txt: 1914 bytes, checksum: 7d48279ffeed55da8dfe2f8e81f3b81f (MD5) wilson_sovino2etal_IOC_2014.pdf: 3263108 bytes, checksum: 9132ef912d83ec3454d9e1b5b492795b (MD5) Previous issue date: 2013Universidade Federal de Alagoas. Instituto de Ciências Biológicas e da Saúde. Laboratório de Biologia Celular.. Maceió, AL, Brasil.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. Cell Imaging Platform. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Université Paris Descartes. Hôpital Necker. CNRS UMR-8147. Paris, France.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ, Brasil.Basophils co-express FceRIa and CD49b, the a-2 chain of integrin-type receptor VLA-2 (a2b1), which recognizes type-1 collagen as a major natural ligand. The physiological relevance of this integrin for interactions with extracellular bone marrow matrix remains unknown. Herein, we examined the expression of several receptors of this family by bone marrowderived basophils sorted either ex-vivo or after culture with IL-3. Having established that both populations display CD49d, CD49e and CD49f (a-4, a-5 and a-6 integrins subunits, respectively), we addressed receptor functions by measuring migration, adhesion, proliferation and survival after interacting with matched natural ligands. Type I collagen, laminin and fibronectin promoted basophil migration/adhesion, the former being the most effective. None of these ligands affected basophil viability and expansion. Interactions between basophils and extracellular matrix are likely to play a role in situ, as supported by confocal 3D cell imaging of femoral bone marrow sections, which revealed basophils exclusively in type-1 collagen-enriched niches that contained likewise laminin and fibronectin. This is the first evidence for a structure/function relationship between basophils and extracellular matrix proteins inside the mouse bone marrow

Urea-Peptide Hybrids as VEGF-A165/NRP-1 Complex Inhibitors with Improved Receptor Affinity and Biological Properties

Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A165 to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea–peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF165/NRP-1 inhibitors

Mast cell-mediated inflammation relies on insulin-regulated aminopeptidase controlling cytokine export from the Golgi

International audienceBackground: Upon activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules via regulated exocytosis. This acute degranulation is followed by a late activation phase involving synthesis and secretion of cytokines, growth factors and other inflammatory molecules via the constitutive pathway that remains ill-defined.Objective: Here we investigate the role for an insulin-responsive vesicle (IRV)-like endosomal compartment, marked by insulin-regulated aminopeptidase (IRAP), in the secretion of TNF-α- and IL-6 in mast cells and macrophages.Methods: Murine ko mouse models (IRAPko and kit-Wsh/sh) were used to study inflammatory disease models and to measure and mechanistically investigate cytokine secretion and degranulation in bone-marrow-derived mast cells in vitro.Results: We show that IRAPko mice are protected from TNF-α-dependent kidney injury and inflammatory arthritis. In the absence of IRAP, TNF-α and IL-6 but not IL-10 fail to be efficiently secreted. Moreover, chemical targeting of IRAP+ endosomes reduced pro-inflammatory cytokine secretion. Mechanistically, impaired TNF-α export from the Golgi and reduced co-localization of VAMP3+ TNF-α transport vesicles with Stx4 was observed in IRAPko mast cells, while VAMP8-dependent exocytosis of secretory granules was facilitated.Conclusion: Our study describes a novel role for IRAP in mast cell-mediated inflammation through the regulation of exocytic trafficking of cytokines

Innate-like T cells in children with sickle cell disease

International audienceBACKGROUND: The implication of lymphocytes in sickle cell disease pathogenesis is supported by a number of recent reports. These studies provided evidence for the activation of invariant natural killer T (iNKT) cells in adult patients, but did not investigate the involvement of other innate-like T cell subsets so far.METHODS: Here we present a monocentric prospective observational study evaluating the number and functional properties of both circulating conventional and innate-like T cells, namely iNKT, Mucosal-Associated Invariant T (MAIT) and gammadelta (γδ) T cells in a cohort of 39 children with sickle cell disease.RESULTS: Relative to age-matched healthy controls, we found that patients had a higher frequency of IL-13- and IL-17-producing CD4+ T cells, as well as higher MAIT cell counts with an increased frequency of IL-17-producing MAIT cells. Patients also presented increased Vδ2 γδ T cell counts, especially during vaso-occlusive crisis, and a lower frequency of IFNγ-producing Vδ2 γδ T cells, except during crisis. iNKT cell counts and the frequency of IFNγ-producing iNKT cells were unchanged compared to controls. Our study revealed positive correlations between 1) the frequency of IFNγ-producing CD4+, CD8+ and Vδ2 γδ T cells and the number of hospitalizations for vaso-occlusive crisis in the previous year; 2) the frequency of IFNγ-producing iNKT cells and patients' age and 3) the frequency of IL-17-producing Vδ2 γδ T cells and hemoglobin S level.CONCLUSION: These results strongly suggest a role of innate-like T cells in sickle cell disease pathophysiology, especially that of IL-17-producing MAIT and γδ T cells

Downregulation of basophil-derived IL-4 and in vivo TH2 IgE responses by serotonin and other organic cation transporter 3 ligands

International audienceBACKGROUND:Murine basophils can contribute to the T(H)2 polarization of the immune response by providing rapidly large amounts of IL-4, which suggests that pharmacologic downregulation of this cytokine might provide a strategy to attenuate pathologies associated with excessive production.OBJECTIVE:We examined a number of physiological and pharmacologic ligands of the organic cation transporter 3 (OCT3), a membrane carrier of biogenic amines, for their inhibitory effect on IL-4 production by basophils, selecting the most efficient compounds for in vivo evaluation in basophil-dependent experimental models.METHODS:IL-4 production by basophils isolated ex vivo or from bone marrow cultures was assessed in response to various stimuli with or without biogenic monoamines or pharmacologic analogs. Selected compounds were administered in vivo to examine their effect on levels of circulating IgE generated during a basophil-dependent T(H)2 response and on basophil activation in mice receiving IL-33.RESULTS:We found a drastic decrease in IL-4 production by stimulated basophils on exposure to serotonin (5-hydroxytryptamine [5-HT]) that is taken up by basophils through the specific high-affinity transporters serotonin transporter and the polyspecific, high-capacity organic cation transporter 3 (OCT3; or Slc22a3) but inhibits their function exclusively through the latter. This downregulation is likewise observed in vivo in response to 5-HT and other OCT3 ligands, as well as in human basophils sorted from PMBCs of nonatopic donors.CONCLUSIONS:We provide evidence for a new means of downregulating IL-4 production by basophils, both in vitro and in vivo, through OCT3 targeted by 5-HT and pharmacologic ligands

Extracellular matrix ligands do not modulate expansion or survival of basophils derived from bone marrow cultures.

<p>Percentages of basophils (phenotypically defined by CD49b-FcεRIα co-expression) expanded from bone marrow cells cultured with IL-3 remain unchanged whatever the ECM ligand added during culture: type I collagen (T-I col), fibronectin (FN) or laminin (LM) (panel <b>a)</b>. Lack of ECM ligand effect on survival of mature basophils as assessed by means of XTT staining of viable cells, quantified by absorbance (panels <b>b</b> and <b>c</b>). Results represented in panel b were obtained by positive selection based on CD49b expression and confirmed with one experiment using depletion of CD117⁺ cells (panel <b>c</b>). Cells were incubated during 24 hours in culture medium alone or supplemented with IL-3 and no significant differences were induced by the addition of integrin ligands. Data are expressed as means ± SE from three distinct experiments except for Fig. 3c that represents one experiment.</p

Bone marrow-derived basophils adhere onto purified extracellular matrix through integrin-type receptors.

<p>The panel shows that basophils obtained from IL-3 stimulated bone marrow cultures are able to adhere onto selected ECM proteins, namely type-1 collagen, laminin and fibronectin. Freshly isolated basophils behave similarly. Adhesion is abrogated by monoclonal antibodies that recognize matching integrin-type receptors. Experiments were performed with 2–3-month-old mice, with at least 5 animals being evaluated <i>per</i> group, and the adhesion assay was performed in 24-well culture plates coated with ECM proteins or BSA. Total numbers of adhered basophils correspond to specific adhesion after subtracting the background values from plates coated with BSA. Results are expressed as mean ± SE*** <i>p</i><0.001.</p

Antibodies applied in cell enrichment, cytofluorometry, Immunofluorescence, cell migration and cell adhesion assays.

*<p>MAb: monoclonal antibody; ^** This polyclonal antibody recognizes various laminin isoforms; FITC: fluoresceinisothyocyanate;</p><p>PE: phycoerythin.</p