Optimization and implementation of a bimolecular fluorescence complementation (BiFC) system for the detection of plum pox potyviral protein-protein interactions in planta

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Heme Oxygenase-1 Inhibits HLA Class I Antibody-Dependent Endothelial Cell Activation

Antibody-mediated rejection (AMR) is a key limiting factor for long-term graft survival in solid organ transplantation. Human leukocyte antigen (HLA) class I (HLA I) antibodies (Abs) play a major role in the pathogenesis of AMR via their interactions with HLA molecules on vascular endothelial cells (ECs). The antioxidant enzyme heme oxygenase (HO)-1 has anti-inflammatory functions in the endothelium. As complement-independent effects of HLA I Abs can activate ECs, it was the goal of the current study to investigate the role of HO-1 on activation of human ECs by HLA I Abs. In cell cultures of various primary humanmacro- and microvascular ECs treatment with monoclonal pan-and allele-specific HLA I Abs up-regulated the expression of inducible proinflammatory adhesion molecules and chemokines (vascular cell adhesion molecule-1 [VCAM-1], intercellular cell adhesion molecule-1 [ICAM-1], interleukin-8 [IL-8] and monocyte chemotactic protein 1 [MCP-1]). Pharmacological induction of HO-1 with cobalt-protoporphyrin IX reduced, whereas inhibition of HO-1 with either zinc-protoporphyrin IX or siRNA-mediated knockdown increased HLA I Ab-dependent up-regulation of VCAM-1. Treatment with two carbon monoxide (CO)-releasing molecules, which liberate the gaseous HO product CO, blocked HLA I Ab-dependent EC activation. Finally, in an in vitro adhesion assay exposure of ECs to HLA I Abs led to increased monocyte binding, which was counteracted by up-regulation of HO-1. In conclusion, HLA I Ab-dependent EC activation is modulated by endothelial HO-1 and targeted induction of this enzyme may be a novel therapeutic approach for the treatment of AMR in solid organ transplantation

Signaling cascades of HLA I Ab-dependent VCAM-1 up-regulation in HUVECs.

<p><b>(A, B)</b> HUVECs were incubated with HLA I Ab W6/32 or isotype control (10 μg/ml) for 18 h after pre-treatment for 30 min with, <b>(A)</b> the PI3K/Akt pathway inhibitors wortmannin (1 μM) and LY294002 (4 μM), or with, <b>(B)</b> the NF-κB pathway inhibitors MG132 (100 nM) and Bay 11–7085 (10 μM). Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Data are mean ± SEM from three independent experiments. * p≤ 0.05, significant differences treatment versus control; ** p≤ 0.05, W6/32 versus W6/32 plus inhibitor. Wort, wortmannin; LY, LY294002; Bay, Bay 11–7085.</p

CORMs decrease HLA class I Ab-induced VCAM-1 expression in HUVECs.

<p>Confluent HUVECs were incubated with W6/32 or isotype control (10 μg/ml) for 18 h in the presence or absence of CORM-2 (25 μM), iCORM-2 (25 μM), CORM-3 (75 μM) or iCORM-3 (75 μM), as indicated. Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Data are represented as mean ± SEM from three independent experiments. * p≤ 0.05, significant differences CORM-2/3 versus W6/32; ** p≤ 0.05, iCORM-2/3 versus CORM2/3.</p

Pharmacological inhibition and siRNA-mediated knockdown of HO-1 reduce HLA I Ab-induced VCAM-1 expression in HUVECs.

<p><b>(A)</b> HUVECs were incubated with HLA I Ab W6/32 alone (10 μg/ml) and for 18 h in the presence of CoPPIX (5 μM) or ZnPPIX (5 μM), as indicated. Cells were lysed and subjected to RT-PCR analysis. The fold induction of VCAM-1 mRNA levels is shown. <b>(B-D)</b> HUVECs were transfected with siRNA for HO-1 or scrambled control siRNA. <b>(B)</b> mRNA expression was determined by RT-PCR analysis and relative levels of HO-1 mRNA are shown. <b>(C)</b> Protein expression was determined by Western blot analysis and probed sequentially with Abs against HO-1 and GAPDH. A representative of three independent experiments is shown. <b>(D)</b> Transfected HUVECs were treated with TNFα (15 ng/ml) or W6/32 for 18 h. Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Bar graphs represent mean ± SEM from three independent experiments. * p≤ 0.05, significant differences treatment versus control; ** p≤ 0.05, W6/32 versus W6/32 plus CoPPIX/ ZnPPIX. Con, control.</p

HO-1 modulates HLA I Ab-dependent up-regulation of THP-1 monocyte adhesion to HUVECs.

<p>(A) Confluent HUVECs were cultured for 24 h in the presence of MoAb W6/32 or isotype control Ab (10 μg/ml) in the presence or absence of a blocking antibody against VCAM-1, a control Ab (Con Ab), ZnPPIX (5 μM) and CoPPIX (5 μM), as indicated. (B) HUVECs were transfected with siRNA for HO-1 or scrambled control siRNA, as indicated. After the various treatments in (A) and (B) cell cultures were incubated with Cell Tracker green-labeled THP-1 monocytes for 30 min. After 5 washing steps firmly adherent monocytes were quantified with fluorescence microscopy in 15 preselected high-power fields by a blinded investigator. The monocyte adhesion in % of control is indicated. Data are mean ± SEM from three independent experiments. * p≤ 0.05, significant differences treatment versus control; ** p≤ 0.05, W6/32 versus W6/32 plus anti-VCAM-1/ CoPPIX/ ZnPPIX; *** p≤ 0.05, siCon plus W6/32 versus siHO-1 plus W6/32.</p

Cell-type-specific downregulation of heme oxygenase-1 by lipopolysaccharide via Bach1 in primary human mononuclear cells

Heme oxygenase (HO)-1 is the inducible isoform of the heme-degrading enzyme HO, which is upregulated by multiple stress stimuli. HO-1 has major immunomodulatory and anti-inflammatory effects via its cell-type-specific functions in mononuclear cells. Contradictory findings have been reported on HO-1 regulation by the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) in these cells. Therefore, we reinvestigated the effects of LPS on HO-1 gene expression in human and murine mononuclear cells in vitro and in vivo. Remarkably, LPS downregulated HO-1 in primary human peripheral blood mononuclear cells (PBMCs), CD14(+) monocytes, macrophages, dendritic cells, and granulocytes, but upregulated this enzyme in primary murine macrophages and human monocytic leukemia cell lines. Furthermore, experiments with human CD14(+) monocytes revealed that activation of other TLRs including TLR1, -2, -5, -6, -8, and -9 decreased HO-1 mRNA expression. LPS-dependent downregulation of HO-1 was specific, because expression of cyclooxygenase-2, NADP(H)-quinone oxidoreductase-1, and peroxiredoxin-1 was increased under the same experimental conditions. Notably, LPS upregulated expression of Bach1, a critical transcriptional repressor of HO-1. Moreover, knockdown of this nuclear factor enhanced basal and LPS-dependent HO-1 expression in mononuclear cells. Finally, downregulation of HO-1 in response to LPS was confirmed in PBMCs from human individuals subjected to experimental endotoxemia. In conclusion, LPS downregulates HO-1 expression in primary human mononuclear cells via a Bach1-mediated pathway. As LPS-dependent HO-1 regulation is cell-type- and species-specific, experimental findings in cell lines and animal models need careful interpretation. (C) 2014 Elsevier Inc. All rights reserved