Abstract Background: Bone marrow (BM) derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. Methods: EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. Results: Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. Conclusions: We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs’ longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration.
Endothelial cells (ECs) form a critical immune interface regulating both the activation and trafficking of alloreactive T cells. In the setting of solid organ transplantation, donor-derived ECs represent sites where alloreactive T cells encounter major and minor tissue-derived alloantigens. During this initial encounter, ECs may formatively modulate effector responses of these T cells through expression of inflammatory mediators. Direct allorecognition is a process whereby recipient T cells recognize alloantigen in the context of donor EC-derived HLA molecules. Direct alloresponses are strongly modulated by human ECs and are galvanized by EC-derived inflammatory mediators. Complement are immune proteins that mark damaged or foreign surfaces for immune cell activation. Following labeling by natural IgM during ischemia reperfusion injury (IRI) or IgG during antibody-mediated rejection (ABMR), the complement cascade is terminally activated in the vicinity of donor-derived ECs to locally generate the solid-phase inflammatory mediator, the membrane attack complex (MAC). Via upregulation of leukocyte adhesion molecules, costimulatory molecules, and cytokine trans-presentation, MAC strengthen EC:T cell direct alloresponses and qualitatively shape the alloimmune T cell response. These processes together promote T cell-mediated inflammation during solid organ transplant rejection. In this review we describe molecular pathways downstream of IgM- and IgG-mediated MAC assembly on ECs in the setting of IRI and ABMR of tissue allografts, respectively. We describe work demonstrating that MAC deposition on ECs generates ‘signaling endosomes’ that sequester and post-translationally enhance the stability of inflammatory signaling molecules to promote EC activation, a process potentiating EC-mediated direct allorecognition. Additionally, with consideration to first-in-human xenotransplantation procedures, we describe clinical therapeutics based on inhibition of the complement pathway. The complement cascade critically mediates EC activation and improved understanding of relevant effector pathways will uncover druggable targets to obviate dysregulated alloimmune T cell infiltration into tissue allografts.
Abstract Background Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with hematological and cardiovascular pathologies are impaired and insufficient; hence, allogenic sources from adult or cord blood are considered as good options for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors. TNFR1 has ubiquitous expression and its interaction with TNFα leads to deleterious effects and apoptosis, whereas, TNFR2 is expressed on few cell types and is involved in protective and survival mechanisms. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatroy functions. Methods EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping TNFR1 stable. Then, co-cultures of human EPCs and T cells were performed to assess if EPC immunoregulatory effect is increased. Results Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, TNFα-TNFR1 interaction polarized EPCs towards pro-inflammatory and immunogenic functions. Conclusions We report for the first time the crucial role of inflammation, notably, the implication of TNFα-TNFR2 axis to boost EPC immunoregulatory functions. Priming EPCs with proper dose of TNFα prior to administration could provide a more protective immunosuppressive microenvironment and longer lasting cells which eventually lead to their better contribution to hematopoietic and vascular regeneration.
Internalized pools of membrane attack complexes (MACs) promote NF-kB and dysregulated tissue inflammation. Here, we show that C9, a MAC-associated protein, promotes loss of proteostasis to become intrinsically immunogenic. Surface-bound C9 is internalized into Rab5 + endosomes whose intraluminal acidification promotes C9 aggregates. A region within the MACPF/CDC domain of C9 stimulates aggrephagy to induce NF-kB, inflammatory genes, and EC activation. This process requires ZFYVE21, a Rab5 effector, which links LC3A/B on aggresome membranes to RNF34-P62 complexes to mediate C9 aggrephagy. C9 aggregates form in human tissues, C9-associated signaling responses occur in three mouse models, and ZFYVE21 stabilizes RNF34 to promote C9 aggrephagy
ZFYVE21 is an ancient, endosome-associated protein that is highly expressed in endothelial cells (ECs) but whose function(s) in vivo are undefined. Here, we identified ZFYVE21 as an essential regulator of vascular barrier function in the aging kidney. ZFYVE21 levels significantly decline in ECs in aged human and mouse kidneys. To investigate attendant effects, we generated EC-specific Zfyve21
Abstract Background Bone marrow derived endothelial progenitor cells (EPCs) are immature endothelial cells (ECs) involved in neo-angiogenesis and endothelial homeostasis and are considered as a circulating reservoir for endothelial repair. Many studies showed that EPCs from patients with cardiovascular pathologies are impaired and insufficient; hence, allogenic sources of EPCs from adult or cord blood are considered as good choices for cell therapy applications. However, allogenic condition increases the chance of immune rejection, especially by T cells, before exerting the desired regenerative functions. TNFα is one of the main mediators of EPC activation that recognizes two distinct receptors, TNFR1 and TNFR2. We have recently reported that human EPCs are immunosuppressive and this effect was TNFα-TNFR2 dependent. Here, we aimed to investigate if an adequate TNFα pre-conditioning could increase TNFR2 expression and prime EPCs towards more immunoregulatory functions. Methods EPCs were pre-treated with several doses of TNFα to find the proper dose to up-regulate TNFR2 while keeping the TNFR1 expression stable. Then, co-cultures of human EPCs and human T cells were performed to assess whether TNFα priming would increase EPC immunosuppressive and immunomodulatory effect. Results Treating EPCs with 1 ng/ml TNFα significantly up-regulated TNFR2 expression without unrestrained increase of TNFR1 and other endothelial injury markers. Moreover, TNFα priming through its interaction with TNFR2 remarkably enhanced EPC immunosuppressive and anti-inflammatory effects. Conversely, blocking TNFR2 using anti-TNFR2 mAb followed by 1 ng/ml of TNFα treatment led to the TNFα-TNFR1 interaction and polarized EPCs towards pro-inflammatory and immunogenic functions. Conclusions We report for the first time the crucial impact of inflammation notably the TNFα-TNFR signaling pathway on EPC immunological function. Our work unveils the pro-inflammatory role of the TNFα-TNFR1 axis and, inversely the anti-inflammatory implication of the TNFα-TNFR2 axis in EPC immunoregulatory functions. Priming EPCs with 1 ng/ml of TNFα prior to their administration could boost them toward a more immunosuppressive phenotype. This could potentially lead to EPCs’ longer presence in vivo after their allogenic administration resulting in their better contribution to angiogenesis and vascular regeneration.
Introduction Ischemia reperfusion injury (IRI) confers worsened outcomes and is an increasing clinical problem in solid organ transplantation. Previously, we identified a “Ptch Hi ” T-cell subset that selectively received costimulatory signals from endothelial cell-derived Hedgehog (Hh) morphogens to mediate IRI-induced vascular inflammation. Methods Here, we used multi-omics approaches and developed a humanized mouse model to resolve functional and migratory heterogeneity within the PtchHi population. Results Hh-mediated costimulation induced oligoclonal and polyclonal expansion of clones within the PtchHi population, and we visualized three distinct subsets within inflamed, IRI-treated human skin xenografts exhibiting polyfunctional cytokine responses. One of these PtchHi subsets displayed features resembling recently described T peripheral helper cells, including elaboration of IFN-y and IL-21, expression of ICOS and PD-1, and upregulation of positioning molecules conferring recruitment and retention within peripheral but not lymphoid tissues. Ptch Hi T cells selectively homed to IRI-treated human skin xenografts to cause accelerated allograft loss, and Hh signaling was sufficient for this process to occur. Discussion Our studies define functional heterogeneity among a Ptch Hi T-cell population implicated in IRI.
Internalization of complement membrane attack complexes (MACs) assembles NLRP3 inflammasomes in endothelial cells (EC) and promotes IL-β-mediated tissue inflammation. Informed by proteomics analyses of FACS-sorted inflammasomes, we identify a protein complex modulating inflammasome activity on endosomes. ZFVYE21, a Rab5 effector, partners with Rubicon and RNF34, forming a "ZRR" complex that is stabilized in a Rab5- and ZFYVE21-dependent manner on early endosomes. There, Rubicon competitively disrupts inhibitory associations between caspase-1 and its pseudosubstrate, Flightless I (FliI), while RNF34 ubiquitinylates and degradatively removes FliI from the signaling endosome. The concerted actions of the ZRR complex increase pools of endosome-associated caspase-1 available for activation. The ZRR complex is assembled in human tissues, its associated signaling responses occur in three mouse models in vivo, and the ZRR complex promotes inflammation in a skin model of chronic rejection. The ZRR signaling complex reflects a potential therapeutic target for attenuating inflammasome-mediated tissue injury.
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