Early acute microvascular kidney transplant rejection in the absence of anti-HLA antibodies is associated with preformed IgG antibodies against diverse glomerular endothelial cell antigens

International audienceBACKGROUND: Although anti-HLA antibodies (Abs) cause most antibody-mediated rejections of renal allografts, non-anti-HLA Abs have also been postulated to contribute. A better understanding of such Abs in rejection is needed.METHODS: We conducted a nationwide study to identify kidney transplant recipients without anti-HLA donor-specific Abs who experienced acute graft dysfunction within 3 months after transplantation and showed evidence of microvascular injury, called acute microvascular rejection (AMVR). We developed a crossmatch assay to assess serum reactivity to human microvascular endothelial cells, and used a combination of transcriptomic and proteomic approaches to identify non-HLA Abs.RESULTS: We identified a highly selected cohort of 38 patients with early acute AMVR. Biopsy specimens revealed intense microvascular inflammation and the presence of vasculitis (in 60.5%), interstitial hemorrhages (31.6%), or thrombotic microangiopathy (15.8%). Serum samples collected at the time of transplant showed that previously proposed anti-endothelial cell Abs-angiotensin type 1 receptor (AT1R), endothelin-1 type A and natural polyreactive Abs-did not increase significantly among patients with AMVR compared with a control group of stable kidney transplant recipients. However, 26% of the tested AMVR samples were positive for AT1R Abs when a threshold of 10 IU/ml was used. The crossmatch assay identified a common IgG response that was specifically directed against constitutively expressed antigens of microvascular glomerular cells in patients with AMVR. Transcriptomic and proteomic analyses identified new targets of non-HLA Abs, with little redundancy among individuals.CONCLUSIONS: Our findings indicate that preformed IgG Abs targeting non-HLA antigens expressed on glomerular endothelial cells are associated with early AMVR, and that cell-based assays are needed to improve risk assessments before transplant

Effector mechanisms of acute humoral rejection : contribution of the Notch signaling pathway and of DLL4 to the endothelium/macrophages crosstalk and to macrophages polarization

Le rejet aigu humoral (RAH) est une complication post-transplantation qui peut conduire à la dysfonction puis la perte du greffon. Le RAH est caractérisé, au niveau histologique, par la présence de lésions endothéliales et d’un infiltrat de macrophages intravasculaires. Cette étude avait pour objectif d’identifier les mécanismes et voies de signalisation impliqués dans l’altération des cellules endothéliales (CE) au cours du RAH et de nouvelles molécules régulatrices. Nous montrons que l’induction du ligand de la voie Notch Dll4 dans les CE et les macrophages est caractéristique du RAH dans les greffons cardiaques. L’expression endothéliale de Dll4et la sécrétion de l’IL-6 par les CE induit la polarisation des macrophages vers un profil pro-inflammatoire de type M1. Nous identifions aussi Dll4 comme un régulateur négatif et pro-apoptotique de la différentiation vers un phénotype suppressif de typeM2. Nous montrons que les cellules microvasculaires sont les cibles cellulaires privilégiées des anticorps non HLA préformés associés à certains RAH en transplantation rénale. Enfin, nous avons mis en évidence l’activité d’une famille de coumarines issues de végétaux sur l’expression endothéliale de molécules de l’inflammation et de l’immunité. Pour conclure,notre étude montre l’importance de la voie Notch dansl’inflammation liée au RAH et identifie DLL4 et IL-6comme de nouveaux médiateurs de l’inflammation et d’une interaction spatiale et fonctionnelle des CE et des macrophages. Ces travaux proposent donc DLL4 etl’IL-6 comme des cibles moléculaires et les coumarines comme nouvelles molécules bioactives pour le contrôle de l’inflammation et du RAH en transplantation.Acute humoral rejection (RAH) is a post-transplantcomplication that can lead to dysfunction and graftloss. RAH is characterized histologically by thepresence of endothelial lesions and of an infiltrate ofintravascular macrophages. This study aimed toidentify the mechanisms, the signaling pathwaysinvolved in the alteration of endothelial cells (EC) upon RAH as well as new regulatory molecules fortherapeutic approaches. Here, we show that theinduction of the Notch ligand DLL4 in both the EC andmacrophages is a feature of RAH in cardiac allografts.The expression of Dll4 and secretion of IL-6 inducedpolarization of macrophages into proinflammatory M1-type. We further identify Dll4 as a negative and pro-apoptoticregulator of macrophage differentiation towards a suppressive phenotype M2-type. We foundthat glomerular microvascular cells are the cell targetsof preformed non HLA antibodies causing RAH inkidney transplantation. Finally, we have demonstratedthe inhibitory activity of a series of coumarins issued from plants on the endothelial expression of a panel of inflammation and immunity molecules. In conclusion,our study shows the importance of the Notch pathwayin inflammation-related RAH and identifies DLL4 and IL-6 as new mediators of inflammation and spatial andfunctional interaction of the EC and macrophages. Thiswork therefore propose DLL4 and IL-6 as moleculartargets and coumarins as new bioactive molecules forthe control of inflammation and RAH

Notch signaling triggered via the ligand DLL4 impedes M2 macrophage differentiation and promotes their apoptosis

International audienc

Additional Insights into Hypericum perforatum Content: Isolation, Total Synthesis, and Absolute Configuration of Hyperbiphenyls A and B from Immunomodulatory Root Extracts

International audiencePhytochemical investigation of the root extracts of Hypericum perforatum led to the isolation of two biphenyl derivatives named hyperbiphenyls A and B (1 and 2) and four known xanthones (3−6). These structures were elucidated by spectroscopic and spectrometric methods including UV, NMR, and HRMS. The absolute configuration of the biphenyl derivatives was defined by two different approaches: biomimetic total synthesis of racemic hyper-biphenyl A followed [1]H and [19]F NMR Mosher's esters analysis and stereoselective total synthesis of hyperbiphenyl B, permitting assignment of the S absolute configuration for both compounds. The bioactivity of compounds 1−6 toward a set of biomolecules, including major histocompatibility complex (MHC) molecules expressed on vascular endothelial cells, was measured. The results showed that the major xanthone, i.e., 5-O-methyl-2-deprenylrheediaxanthone B (3), is a potent inhibitor of MHC that efficiently reduces HLA-E, MHC-II, and MICA biomolecules on cell surfaces

Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation

International audienceAlthough short-term outcomes have improved with modern era immunosuppression, little progress has been made in long-term graft survival in cardiac transplantation. Antibody-mediated rejection (AMR) is one of the leading causes of graft failure and contributes significantly to poor long-term outcomes. Endothelial cell (EC) injury, intravascular macrophage infiltrate and microvascular inflammation are the histological features of AMR. Nevertheless, mechanisms of AMR remain unclear and treatment is still limited. Here, we investigated the mechanisms underlying vascular and inflammatory cell network involved in AMR at endothelial and macrophage levels, using endomyocardial transplant biopsies and EC/monocyte cocultures. First, we found that AMR associates with changes in Notch signaling at endothelium/monocyte interface including loss of endothelial Notch4 and the acquisition of the Notch ligand Dll4 in both cell types. We showed that endothelial Dll4 induces macrophage polarization into a pro-inflammatory fate (CD40 high CD64 high CD200R low HLA-DR low CD11b low) eliciting the production of IL-6. Dll4 and IL-6 are both Notch-dependent and are required for macrophage polarization through selective down and upregulation of M2-and M1-type markers, respectively. Overall, these findings highlight the impact of the graft's endothelium on macrophage recruitment and differentiation upon AMR via Notch signaling. We identified Dll4 and IL-6 as coregulators of vascular inflammation in cardiac transplantation and as potential targets for immunotherapy

Prenylated polyphenols from <i>Clusiaceae</i> and <i>Calophyllaceae</i> as regulators of inflammation and immunity: Overview and hypothesis.

<p>(1) NPs efficiently inhibit the induction of VCAM-1, an adhesion molecule involved in the firm adhesion of leukocytes on inflamed endothelium and a prerequisite step for leukocyte activation (2) and extravasation in the tissues. (3) NPs strongly impair the expression of MHC molecules expressed on endothelium and involved in innate and adaptive immunity via the activation of NK and CD4T and CD8T cells, respectively.</p

Effect of compounds 1–6 and 8–11 on TNF-induced expression of inflammatory molecules in ECs.

<p><b>(A) Basal <i>versus</i> TNF-induced expression of VCAM-1, ICAM-1 and E-selectin after 6h of incubation.</b> Data shown are representative histograms from fluorescence-activated cell sorting (Facs) analysis showing constitutive <i>versus</i> induced expression of inflammatory molecules (VCAM-1, ICAM-1, E-selectin) at the cell surface of ECs. Histograms show the intensity of fluorescence (log, x-axis) versus cell number (y-axis) for untreated (purple line) and cytokine-treated (green line) ECs analyzed by flow cytometry. Immunostaining using an irrelevant isotype-matched IgG (dark line) was used as a negative control. (<b>B</b>) <b>Inhibitory effects of NPs.</b> Expression of VCAM-1, ICAM-1 and E-selectin was measured by Facs. Each bar represents geometric mean ± SD of fluorescence intensity calculated from three to five independent experiments. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s post-test; *<i>p</i> < 0.05, **<i>p</i> < 0.01, ***<i>p</i> < 0.001 compared with TNF control group. PDTC (200 μM) was used as a positive control for inhibition.</p

Effect of NPs 1–6 and 8–11 on endothelial cell viability.

<p>Cell viability was assessed on confluent EC monolayers using MTT assay. Cells were incubated with NPs (10 μM) for 48h before analysis. Diluted DMSO (1/1000 –dark dashed line) was used as control for diluent (Dil), as well as a non-treated (NT) control—grey dashed line—and the cytotoxicity positive control glyoxal (Gly) at 4 mM. The cytotoxic activity of the immunosuppressive drug zoledronic acid (ZA, 10 μM) was also assessed. Statistical analysis of values (O.D.) obtained for treated versus non-treated cells were performed using non-parametric ANOVA test.</p

Basal <i>versus</i> IFNγ-modulated expression of MHC class I, MHC class II, HLA-E and MICA on EC cultures.

<p>ECs were treated with or without IFNγ for 48h before analysis. Data shown are representative histograms from Facs analysis showing constitutive <i>versus</i> IFNγ-regulated expression of inflammatory molecules (MHC class I, MHC class II, HLA-E and MICA) at the EC surface. Histograms show the intensity of fluorescence (log, x-axis) <i>versus</i> cell number (y-axis) for untreated (purple line) and cytokine-treated (green line) ECs analyzed by flow cytometry. Immunostaining using an irrelevant isotype-matched IgG (dark line) was used as a negative control.</p