Notch2 Signaling Sensitizes Endothelial Cells to Apoptosis by Negatively Regulating the Key Protective Molecule Survivin

BACKGROUND: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively. CONCLUSIONS/SIGNIFICANCE: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity

Cross-Reactivity of Herpesvirus-Specific CD8 T Cell Lines Toward Allogeneic Class I MHC Molecules

Although association between persistent viral infection and allograft rejection is well characterized, few examples of T-cell cross-reactivity between self-MHC/viral and allogeneic HLA molecules have been documented so far. We appraised in this study the alloreactivity of CD8 T cell lines specific for immunodominant epitopes from human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV). CD8 T cell lines were generated after sorting with immunomagnetic beads coated with either pp65495–503/A*0201, BMLF1259–267/A*0201, or BZLF154–64/B*3501 multimeric complexes. Alloreactivity of the CD8 T cell lines against allogeneic class I MHC alleles was assessed by screening of (i) TNF-α production against COS-7 cells transfected with as many as 39 individual HLA class I-encoding cDNA, and (ii) cytotoxicity activity toward a large panel of HLA-typed EBV-transformed B lymphoblastoid cell lines. We identified several cross-reactive pp65/A*0201-specific T cell lines toward allogeneic HLA-A*3001, A*3101, or A*3201. Moreover, we described here cross-recognition of HLA-Cw*0602 by BZLF1/B*3501-specific T cells. It is noteworthy that these alloreactive CD8 T cell lines showed efficient recognition of endothelial cells expressing the relevant HLA class I allele, with high level TNF-α production and cytotoxicity activity. Taken together, our data support the notion that herpes virus-specific T cells recognizing allo-HLA alleles may promote solid organ rejection

The Involvement of SMILE/TMTC3 in Endoplasmic Reticulum Stress Response

The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.We first confirmed that SMILE mRNA is differentially expressed in the blood of operationally tolerant patients with drug-free long term graft function compared to stable and rejecting patients. Using a yeast two-hybrid approach and a colocalization study by confocal microscopy we furthermore report an interaction of SMILE with PDIA3, a molecule resident in the endoplasmic reticulum (ER). In accordance with this observation, SMILE silencing in HeLa cells correlated with the modulation of several transcripts involved in proteolysis and a decrease in proteasome activity. Finally, SMILE silencing increased HeLa cell sensitivity to the proteasome inhibitor Bortezomib, a drug that induces ER stress via protein overload, and increased transcript expression of a stress response protein, XBP-1, in HeLa cells and keratinocytes.In this study we showed that SMILE is involved in the endoplasmic reticulum stress response, by modulating proteasome activity and XBP-1 transcript expression. This function of SMILE may influence immune cell behavior in the context of transplantation, and the analysis of endoplasmic reticulum stress in transplantation may reveal new pathways of regulation in long-term graft acceptance thereby increasing our understanding of tolerance

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

Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells

As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information

Signaling of endothelial cytoprotection in transplantation

International audienceA better knowledge of the processes by which endothelium can resist to cell death and adapt to injury by specific intracellular signaling pathways and dedicated protein regulation is a key step to understand how vascular inflammation/injury develops and how it is regulated. This review focuses on signaling pathways and molecular effectors that trigger the balance between endothelial cell activation and dys-function. In addition to the canonical nuclear factor-jB (NF-jB), phosphatidyl inositol 3-kinase (PI-3K) and mitogen-activated protein kinases (MAPK) that orchestrated the inflammatory response and its termination we report here additive pathways such as Notch pathway and protein C/protease activated receptor (PAR) pathway that have been also reported to play a role in the control of EC activation and apoptosis. This review also provides an update of the characteristics of some established and novel protective molecules for the endothelium, identified in transplantation

Advances in Endothelial Cell Biology: From Knowledge to Control

The aim of this Special Issue is to provide an overview of recent investigations in the field of endothelial cell (EC) biology that advance our understanding of the molecular mechanisms that trigger normal EC functions and dysfunctions in pathologies and to demonstrate how improved knowledge of EC biology may lead to the discovery of novel molecular diagnostic technologies and targeted therapeutics [...

Persistent CD8 T Cell Marks Caused by the HCMV Infection in Seropositive Adults: Prevalence of HLA-E-Reactive CD8 T Cells

International audienceThis study investigated the frequency and peptide specificity of long-lasting HCMV-specific CD8 T cells in a cohort of 120 cytomegalovirus seropositive (HCMV+) healthy carriers with the aim of deciphering the relative contribution of unconventional HLA-E- versus conventional HLA-A2-specific CD8 T cells to long-term T cell memory expansion in HCMV immunity. The presence of HCMV-specific CD8 T cells was investigated by flow cytometry using five MHC/peptide tetramer complexes (HLA-A2/pp65, HLA-A2/IE1 and three different HLA-E/UL40). Here, we report that 50% of HCMV+ healthy individuals possess HCMV-specific CD8 T cells, representing ≥0.1% of total blood CD8 T cells years post-infection. Around a third (30.8%) of individuals possess HLA-A2-restricted (A2pp65 or A2IE1) and an equal proportion (27.5%) possess an HLA-E/UL40 CD8 T response. Concomitant HLA-E- and HLA-A2-reactive CD8 T cells were frequently found, and VMAPRTLIL peptide was the major target. The frequency of HLA-E/VMAPRTLIL among total blood CD8 T cells was significantly higher than the frequency of HLA-A2pp65 T cells (mean values: 5.9% versus 2.3%, p = 0.0354). HLA-EUL40 CD8 T cells display lower TCR avidity but similar levels of CD3 and CD8 coreceptors. In conclusion, HLA-E-restricted CD8 T cells against the VMAPRTLIL UL40 peptide constitute a predominant subset among long-lasting anti-HCMV CD8 T cells

Rôle du HLA dans la signalisation des cellules endothéliales en transplantation et contribution de HLA-E aux fonctions immunorégulatrices de l'endothélium

Mieux comprendre le rôle de l'endothélium dans la réponse immune et identifier de nouvelles cibles moléculaires pour le contrôle des dysfonctions endothéliales sont des enjeux majeurs pour combattre de nombreuses pathologies vasculaires et inflammatoires et le rejet de greffe en Transplantation. Les travaux réalisés au cours de ma thèse ont mis en évidence l'activation de GTPase RhoA dans les cellules endothéliales (CE) vasculaires consécutive à la liaison du HLA de classe I par les anticorps allospécifiques. Nous montrons le rôle central de RhoA dans un mécanisme à médiation immune de la prolifération endothéliale qui requiert la géranylgéranylation de RhoA et peut être inhibé par une statine. De façon complémentaire, nous montrons que la fixation d anticorps anti-HLA de classe II induit l apoptose des lymphocytes B mais pas des CE issues du même donneur et suggèrent que les CE échappent à l apoptose par l activation d une voie de survie impliquant la PI3-kinase. Enfin, par la mise en évidence de l expression des formes membranaire et soluble de HLA-E par les CE, nos travaux apportent la première description in vivo et in vitro de l expression et de la régulation de HLA-E par l endothélium. L ensemble de ces travaux montrent que, dans un contexte allogénique, l expression des molécules du HLA de classe I et de classe II par l endothélium contribue, via des voies de signalisation propres, à moduler les fonctions endothéliales telles que la prolifération et l apoptose. De plus, l expression de HLA-E procure de nouvelles fonctions immunorégulatrices aux CE et HLA-E soluble est un nouvel outil pour le diagnostic et l immunothérapie.The identification of new molecular targets to prevent endothelium dysfunction as well as a better understanding of the role of endothelium in the immune response are required to improve vascular injury and transplant outcome. The present study indicates that ligation of HLA class I and class II molecules on graft endothelial cells (EC) mediated by alloreactive anti-donor antibodies selectively activates signaling pathways affecting EC functions such as cell proliferation and apoptosis. Firstly, we showed that RhoA is a key mediator of signaling pathways leading to cytoskeleton reorganization and EC proliferation in response to HLA class I ligation and demonstrated the potent inhibitory effect of simvastatin on allostimulated EC growth. We also found that, in contrast to B cells, graft ECs escape from apoptosis mediated by HLA-DR ligation not as a result of moderate HLA-DR expression but rather as a result of specific signaling pathway involving the PI3-kinase. This study also demonstrated that EC express the non classical class I HLA-E molecules at the cell surface and release soluble HLA-E upon inflammation providing new immunoregulatory functions to EC and tools for diagnosis and immunotherapy.NANTES-BU Médecine pharmacie (441092101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Etude de l'expression constitutive et régulée de la molécule de CMH de classe I non classique MICA par les cellules endothéliales vasculaires humaines (conséquences fonctionnelles et mécanismes de contrôle )

MICA est une molécule du CMH, dite de classe I non classique. Fortement polymorphique, MICA est le ligand du récepteur activateur NKG2D. L'objectif de cette étude était de caractériser l'expression et les fonctions des molécules MICA exprimées par l'endothélium et d'évaluer leur importance dans un contexte de Transplantation d organes. Nos résultats confirment l'expression basale de MICA par les cellules endothéliales (CE) vasculaires et montrent que, bien que modérée, l expression de MICA est suffisante pour activer des cellules NK allogéniques via le récepteur NKG2D. La co-culture de CE et de NK induit l internalisation de NKG2D et réduit l activité cytotoxique Les cytokines modulent l expression de MICA selon un profil spécifique puisque le TNFa augmente son expression alors que l IFNg diminue MICA à la surface des CE activées. L expression de MICA est fortement contrôlée par la glycosylation et par l activité des métalloprotéases de la matrice (MMP). Nos travaux montrent aussi pour la première fois que la régulation de la protéine MICA par les CE humaines dans un contexte inflammatoire est associée à la production de MICA soluble. Enfin, des inhibiteurs pharmacologiques des principales voies de signalisation intracellulaires nous ont aidés pour montrer l importance des voies NF B et MAPKinases dans la régulation de MICA par les cytokines TNFa et IFNg. En conclusion, nos résultats soulignent l impact fonctionnel de l expression de MICA par les CE ; la régulation sélective de MICA à la membrane et la production de sMICA affectent l activation des effecteurs (cellules NK et CTL) via NKG2D et procurent de nouvelles fonctions immunorégulatrices à l endothélium.MICA are highly polymorphic MHC class I related molecules shown to be expressed by endothelial cells (ECs). Consequently, MICA may be target for specific alloantibodies and NKG2D-expressing NK and T effector cells in organ allografts. In this study, we show that basal levels of MICA expressed on human vascular ECs, isolated from transplant donors, is sufficient to functionally modulate the immunoreceptor NKG2D expression and activity in allogeneic NK cells. We found that MICA expression is differentially regulated at the EC surface in response to cytokines. TNFa, up-regulated MICA while IFNg significantly decreased MICA at the EC surface. Both cytokines induced the release of soluble MICA in culture supernatant from activated vascular ECs. Allogeneic NK-mediated lysis of ECs correlated with MICA level on the EC cell surface. We identified several molecular mechanisms, including glycosylation and metalloproteinase activities that control MICA regulation and function at posttranscriptional level in ECs. Our results indicate that, in addition to the NF B pathway, the MAPKs JNK, ERK1/2 and p38 are key signaling pathways in the control of MICA by the cytokines. Together, the fine tune modulation of MICA expression and release provides further evidence for an immunomodulatory role for graft s ECs.NANTES-BU Médecine pharmacie (441092101) / SudocSudocFranceF