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.

BACKGROUND: 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 in vitro cell-based assays are needed to improve risk assessments before transplant.

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

BACKGROUND: Notch signaling controls many cellular processes, including cell fate determination, cell differentiation, proliferation and apoptosis. In mammals, four Notch receptors (Notch 1-4) can interact with five distinct ligands [Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4]. We previously reported that Notch activation is modulated in endothelial cells and monocytes during inflammation and showed that inflammation upregulates DLL4 on endothelial cells. DLL4 promotes differentiation of blood monocytes into proinflammatory M1 macrophages. Here, we further investigated the ability of DLL4 to interfere with the polarization of blood monocytes into immunosuppressive M2 macrophages. METHODS: Human blood monocytes were differentiated in vitro into M0 macrophages and then polarized into M1 or M2 macrophages with LPS/IFNγ and IL-4, respectively. Polarization steps were performed in the presence of immobilized recombinant DLL4. Immune phenotype and apoptosis of macrophage subsets were analyzed and quantified by flow cytometry. Regulatory effects of DLL4 on gene expression, cell signaling and apoptotic pathways were investigated by QPCR and western blots. RESULTS: The phenotype of M2 macrophages was subject to specific alterations in the presence of recombinant DLL4. DLL4 inhibits the upregulation of IL-4 induced M2 markers such as CD11b, CD206, and CD200R. Survival of macrophages upon M2 polarization was also strongly reduced in the presence of DLL4. DLL4 induces a caspase3/7-dependent apoptosis during M2 but not M1 macrophage polarization. The Notch ligand DLL1 has no apoptotic effect. Both DLL4 signaling via Notch1 as well as DLL4-mediated apoptosis are Notch-dependent. Fully differentiated M2 macrophages became resistant to DLL4 action. Mechanistically, DLL4 selectively upregulates gene expression in macrophages upon M2 polarization, thereby affecting the Notch pattern (Notch1, 3, Jag1), activity (HES1), and transcription (IRF5, STAT1). The pro-apoptotic effectors Bax and Bak and the BH3-only proteins Bid and Bim seem to convey DLL4 apoptotic signal. CONCLUSION: Interplay between the DLL4/Notch and IL-4/IL-4R signaling pathways impairs M2 differentiation. Thus, DLL4 may drive a Notch-dependent selection process not only by promoting M1 macrophage differentiation but also by preventing M2 macrophage differentiation through inhibition of M2-specific gene expression and apoptotic cell death.

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

Phytochemical 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 hyperbiphenyl A followed by 1H and 19F 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.

Prenylated Polyphenols from Clusiaceae and Calophyllaceae with Immunomodulatory Activity on Endothelial Cells.

Endothelial cells (ECs) are key players in inflammation and immune responses involved in numerous pathologies. Although attempts were experimentally undertaken to prevent and control EC activation, drug leads and probes still remain necessary. Natural products (NPs) from Clusiaceous and Calophyllaceous plants were previously reported as potential candidates to prevent endothelial dysfunction. The present study aimed to identify more precisely the molecular scaffolds that could limit EC activation. Here, 13 polyphenols belonging to 5 different chemical types of secondary metabolites (i.e., mammea coumarins, a biflavonoid, a pyranochromanone acid, a polyprenylated polycyclic acylphloroglucinol (PPAP) and two xanthones) were tested on resting and cytokine-activated EC cultures. Quantitative and qualitative changes in the expression of both adhesion molecules (VCAM-1, ICAM-1, E-selectin) and major histocompatibility complex (MHC) molecules have been used to measure their pharmaceutical potential. As a result, we identified 3 mammea coumarins that efficiently reduce (up to >90% at 10 µM) both basal and cytokine-regulated levels of MHC class I, class II, MICA and HLA-E on EC surface. They also prevented VCAM-1 induction upon inflammation. From a structural point of view, our results associate the loss of the free prenyl group substituting mammea coumarins with a reduced cellular cytotoxicity but also an abrogation of their anti-inflammatory potential and a reduction of their immunosuppressive effects. A PPAP, guttiferone J, also triggers a strong immunomodulation but restricted to HLA-E and MHC class II molecules. In conclusion, mammea coumarins with a free prenyl group and the PPAP guttiferone J emerge as NPs able to drastically decrease both VCAM-1 and a set of MHC molecules and to potentially reduce the immunogenicity of the endothelium.

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

Although 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.