ZFYVE21 promotes endothelial nitric oxide signaling and vascular barrier function in the kidney during aging.

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-/- reporter mice. These knockout mice developed accelerated aging phenotypes including reduced endothelial nitric oxide (ENOS) activity, failure to thrive, and kidney insufficiency. Kidneys from Zfyve21 EC-/- mice showed interstitial edema and glomerular EC injury. ZFYVE21-mediated phenotypes were not programmed developmentally as loss of ZFYVE21 in ECs during adulthood phenocopied its loss prenatally, and a nitric oxide donor normalized kidney function in adult hosts. Using live cell imaging and human kidney organ cultures, we found that in a GTPase Rab5- and protein kinase Akt-dependent manner, ZFYVE21 reduced vesicular levels of inhibitory caveolin-1 and promoted transfer of Golgi-derived ENOS to a perinuclear Rab5+ vesicular population to functionally sustain ENOS activity. Thus, our work defines a ZFYVE21- mediated trafficking mechanism sustaining ENOS activity and demonstrates the relevance of this pathway for maintaining kidney function with aging.

A ZFYVE21-Rubicon-RNF34 signaling complex promotes endosome-associated inflammasome activity in endothelial cells.

Internalization of complement membrane attack complexes (MACs) assembles NLRP3 inflammasomes in endothelial cells (EC) and promotes IL-ß-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.

Hedgehog costimulation during ischemia-reperfusion injury potentiates cytokine and homing responses of CD4+ T cells.

Introduction: Ischemia reperfusion injury (IRI) confers worsened outcomes and is an increasing clinical problem in solid organ transplantation. Previously, we identified a "PtchHi" 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. PtchHi 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 PtchHi T-cell population implicated in IRI.

Membrane attack complexes, endothelial cell activation, and direct allorecognition.

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.