Expression of IL-15 on the surface of human graft endothelial cells (ECs) bound to the IL-15Rα subunit can increase the activation of CTLs, potentiating allograft rejection. Our previous work showed that surface expression of this protein complex could be induced by alloantibody-mediated complement activation through increased IL-1β synthesis, secretion, and autocrine/paracrine IL-1-mediated activation of NF-κB. In this article, we report that cultured human ECs express eight differently spliced IL-15 transcripts. Remarkably, IL-1β does not alter the expression level of any IL-15 transcript but induces surface expression independently of RNA polymerase II-mediated transcription while requiring new protein translation. Mechanistically, IL-1β causes an NF-κB-mediated reduction in the level of microRNA Let-7c-3p, thereby relieving a block of translation of IL-15 surface protein. Let7c-3p anti-miR can induce EC surface expression of IL-15/IL-15Rα in the absence of complement activation or of IL-1, enabling IL-15 transpresentation to boost CD8 T cell activation. Because of the complexity we have uncovered in IL-15 regulation, we recommend caution in interpreting increased total IL-15 mRNA or protein levels as a surrogate for transpresentation.

Purpose ZFYVE21 is a Rab5 effector whose role(s) in cellular immunity are unknown. Here, we examined the role of ZFYVE21 as a mediator of Hedgehog signaling, specifically testing the hypothesis that Hedgehog-derived signals intended for proliferative wound repair may pathologically expand CD4+ T cells. Methods We peformed in vitro cultures using primary human endothelial cells, T cells, and B cells. We used a humanized mouse model involving surgical implantation of human artery xenografts in SCID/beige engrafted with human lymphoid cells. We validated salient findings using patient-derived biospecimens from patients with delayed graft function, a complement-mediated condition causing dysregulated tissue injury. Results Following complement-mediated injury, endothelial cells release Hh ligands to generate a highly activated population of memory CD4+ T cells (Tmem) abundantly expressing PD-1 and chemokine receptors for homing to peripheral but not lymphoid tissues. In this subset, non-canonical Hedgehog signaling upregulates ZFYVE21 to induce Akt2-dependent NLRP3 inflammasomes, a process eliciting IL-18-mediated cellular expansion. Hh-induced PD-1hi Tmem, NLRP3 inflammasomes, and lymphoid-specific ZFYVE21 regulate vascular injury in vivo. Patients with ischemia reperfusion injury show increased ZFYVE21 expression in Ki-67+PD-1hiCCR2+ Tmem, and patient-derived Hh ligands induce ZFYVE21. Conclusion ZFYVE21 links dysregulated non-canonical Hh signaling with T cell intrinsic NLRP3 inflammasome activity to expand a tissue homing subset of CD4+ T cells propagating peripheral injury.

Vascular disorders frequently have differing clinical presentations among women and men. Sex differences exist in vascular access for hemodialysis; women have reduced rates of arteriovenous fistula (AVF) maturation as well as fistula utilization compared with men. Inflammation is increasingly implicated in both clinical studies and animal models as a potent mechanism driving AVF maturation, especially in vessel dilation and wall thickening, that allows venous remodeling to the fistula environment to support hemodialysis. Sex differences have long been recognized in arterial remodeling and diseases, with men having increased cardiovascular events compared with pre-menopausal women. Many of these arterial diseases are driven by inflammation that is similar to the inflammation during AVF maturation. Improved understanding of sex differences in inflammation during vascular remodeling may suggest sex-specific vascular therapies to improve AVF success.

Purpose Complement promotes vascular inflammation in transplant organ rejection, a process leading to cardiac allograft vasculopathy. In this study, we identify a new Rab5 effector protein, ZFYVE21, that mediates this process. We elucidate a ZFYVE21-SMURF2-pAkt signaling axis which activates non-canonical NF-kB, and we define the clinical relevance of this sequence. Methods We developed FACS-assisted protocols enabling proteomic analysis of MAC+Rab5+ endosomes and semi-quantitative analyses of the phosphoinositide content of these structures. A humanized mouse model incorporating human coronary artery segments and human lymphoid cells was employed to define a role for ZFYVE21-associated signaling in cardiac allograft vasculopathy. Software-assisted analyses of human transplant biopsies analyzing ZFYVE21 colocalization was performed using a computer algorithm we helped to developed in-house. Results In response to deposition of membrane attack complexes (MAC) on endothelial cells (EC), ZFYVE21 is post-translationally stabilized via recruitment to MAC+Rab5+ endosomes in a Rab5- and PI(3)P-dependent manner. ZFYVE21 promotes SMURF2-mediated polyubiquitinylation and proteasome-dependent degradation of endosome-associated PTEN to induce vesicular enrichment of PI(3,4,5)P3 and sequential recruitment of activated Akt and NF-κB-inducing kinase (NIK). Pharmacologic alteration of the phosphoinositide content of MAC+Rab5+ endosomes with miltefosine reduces ZFYVE21 induction, EC activation, and MAC-induced allograft vasculopathy in a humanized mouse model. ZFYVE21 induction distinctly occurs in response to MAC- but not ligand-induced non-canonical NF-κB and is a biomarker for complement-mediated endothelial signaling in transplant biopsies with antibody-mediated rejection. Conclusion Our data identifies ZFYVE21 as a novel Rab5 effector, defines a Rab5-ZFYVE21-SMURF2-pAkt axis by which it mediates EC activation, and demonstrates a role for this pathway as a drug target and biomarker for allograft vasculopathy.

PURPOSE OF REVIEW:Despite considerable advances in controlling acute rejection, the longevity of cardiac and renal allografts remains significantly limited by chronic rejection in the form of allograft vasculopathy. This review discusses recently reported mechanistic insights of allograft vasculopathy pathogenesis as well as recent clinical evaluations of new therapeutic approaches.RECENT FINDINGS:Although adaptive immunity is the major driver of allograft vasculopathy, natural killer cells mediate vasculopathic changes in a transplanted mouse heart following treatment with donor-specific antibody (DSA). However, natural killer cells may also dampen chronic inflammatory responses by killing donor-derived tissue-resident CD4 T cells that provide help to host B cells, the source of DSA. DSA may directly contribute to vascular inflammation by inducing intracellular signaling cascades that upregulate leukocyte adhesion molecules, facilitating recruitment of neutrophils and monocytes. DSA-mediated complement activation additionally enhances endothelial alloimmunogenicity through activation of noncanonical NF-κB signaling. New clinical studies evaluating mammalian target of rapamycin and proteasome inhibitors to target these pathways have been reported.SUMMARY:Allograft vasculopathy is a disorder resulting from several innate and adaptive alloimmune responses. Mechanistic insights from preclinical studies have identified agents that are currently being investigated in clinical trials.

Allograft vasculopathy (AV) is characterized by diffuse stenoses in the vasculature of solid organ transplants. Previously, we developed two humanized models showing that alloantibody and ischemia reperfusion injury (IRI) exacerbated T cell-mediated AV in human arterial xenografts in vivo. Herein we examined a causal role for terminal complement activation in both settings. IRI, in contrast to alloantibody, elicited widespread membrane attack complex (MAC) assembly throughout the vessel wall. Both alloantibody and IRI caused early (24 h) and robust endothelial cell (EC) activation localized to regions of intimal MAC deposition, indicated by increases in nuclear factor kappa B (NF-κB)-inducing kinase, an MAC-dependent activator of noncanonical NF-kB, VCAM-1 expression and Gr-1+ neutrophil infiltration. Endothelial cell activation by alloantibody was inhibited by antimouse C5 mAb, but not by anti-C5a mAb or by control mAb, implicating MAC as the primary target of anti-C5 mAb. Antimouse C5 mAb significantly reduced alloantibody- and IRI-enhanced T cell infiltration and AV-like changes, including neointimal hyperplasia as well as intraluminal thrombosis in a subset of IRI-treated arterial grafts. These results indicate that increased AV lesion formation in response to either alloantibody or IRI is dependent on complement C5 activation and, accordingly, inhibition of this pathway may attenuate AV.

Using sera from transplant patients with high-titer panel reactive antibody (PRA), we previously described a model for studying the effects of alloantibody binding and complement activation on human endothelial cells (EC). With this model we found that complement membrane attack complexes (MAC) induced rapid post-translational stabilization of NF-kB-inducing kinase (NIK), the activator of non-canonical NF-kB signaling, which was required for inflammatory changes in cultured human EC and for vasculopathic changes in human coronary artery segments implanted into immunodeficient mouse hosts. To elucidate the mechanism of NIK stabilization, we performed a genome-wide siRNA screen and discovered a novel, TRAF3-independent signaling mechanism in which MAC induces NIK stabilization on endosomes. MAC is internalized by EC in a clathrin-, AP2-, and dynamin-dependent mechanism into Rab5+ endosomes. Activated Akt is recruited in a Rab5-dependent manner to the same endosomes which then bind and stabilize NIK and subsequently recruit and activate IKK-alpha, the catalytic activator of p52/RelB complexes, the non-canonical form of NF-kB. Pharmacological inhibition of MAC endocytosis blocked this pathway both in cultured EC and in human coronary artery xenografts. These data reveal a novel endosome-based signaling cascade for activating non-canonical NF-kB and identify new targets for inhibiting complement-mediated tissue injury such as chronic antibody-mediated rejection and cardiac allograft vasculopathy.

Age-dependent changes in pulmonary endothelium contribute to worsened clinical outcomes in elderly individuals. Due to altered pulmonary endothelial responses, older participants have increased vulnerability to infection-related sequelae, higher prevalence of pulmonary hypertension, mitigated DNA repair mechanisms, and attenuated parenchymal healing. Aberrant signaling in pulmonary endothelium undergird these clinical processes. In this review, we provide an overview of the work that has elucidated age-related molecular derangements in pulmonary endothelial cells. In particular, we summarize studies describing mishandling of intracellular reactive oxygen species, pathological nitric oxide signaling, and deficient recruitment of endothelial stem cell precursors. We conclude with a summary of potential future avenues of investigation. The signaling pathways associated with pulmonary endothelial senescence reviewed herein suggest a number of putative therapeutic drug targets. Further elucidation of the cellular processes associated with aging in the pulmonary endothelium may provide critical insights into the rational design of therapies that may subvert or even reverse the effects of aging on a molecular level.

Objectives. An immune response sufficient to induce organ failure may provide protection and therapy against tumors derived from the targeted organ particularly when removal or ablation of the organ is part of the standard therapy and does not threaten survival. We have previously shown that a targeted immune response directed against the ovarian-specific protein, inhibin-a, causes ovarian failure. Here we determined whether inhibin-a autoimmunity is effective in both prevention and treatment of ovarian tumors.Methods. A transgene consisting of the SV40 large tumor transformation antigen under the regulation of an anti-Mullerian hormone promoter (AMH-SV40Tag) was transferred by backcrossing for 12 generations to SJL/J mice producing SJLAMH-SV40Tag (H-2(s)) females that develop a high incidence of autochthonous granulosa cell tumors. We determined whether immunization of SJLAMH-SV40Tag female mice with the IA(s)-restricted p215-234 peptide of mouse inhibin-a was capable of preventing and treating these ovarian tumors.Results. The growth of autochthonous ovarian granulosa cell tumors in SJL.AMH-SV40Tag transgenic mice was significantly inhibited in mice immunized with In alpha 215-234. In addition, significant inhibition of tumor growth occurred when mice with established ovarian granulosa cell tumors were therapeutically vaccinated with Ina 215-234.Conclusions. Our results indicate that induction of ovarian-specific autoimmunity may serve as an effective way to prevent the emergence of autochthonous ovarian tumors and control the growth of established ovarian malignancies. (C) 2011 Elsevier Inc. All rights reserved.

Objective: Randomized controlled trials (RCTs) are considered the highest grade of research evidence, yet properly conducted trials investigating the same association often yield conflicting, results. Our objective was to assess whether variability in treatment protocols of RCTs investigating the same topic could explain distinct patterns of outcomes.Study Design and Setting: A review of meta-analyses identified clinical topics involving RCTs with variable pharmacologic dosing and disparate outcomes. Topics were retained if at least two pairs of trials had results suggesting contradictory yet strong exposure-outcome associations.Results: The search yielded 6 clinical topics and 58 RCTs, and individual RCTs were classified into two groups, based on low and high dosages of the intervention. Aggregate odds ratios for studies in the low- and high-dose groups were often substantially discordant. For example, odds ratios were 1.76 (95% confidence interval [CI] = 1.02-3.03) for low-dose and 0.56 (95% CI = 0.31-1.03) for high-dose trials evaluating low-molecular weight heparin and pulmonary embolism. In an exploratory analysis, Outcomes for low- and high-dose groups in the comparison arms of trials (including patients assigned to placebo) had statistically significant differences in four of five analyzable topics, suggesting differences in patient characteristics across trials.Conclusion: Conflicting results from RCTs can represent a spectrum of "real" outcomes for specific treatments. Such trials are best evaluated by considering concurrently both the validity of study design as well as the generalizability of patients and interventions involved. (C) 2010 Elsevier Inc. All rights reserved.

Immunologically targeting α-lactalbumin, a breast-specific protein highly expressed in breast carcinomas but absent from nonlactating mammary cells, provides protection against breast cancer in mice. This strategy may protect women against breast cancer in their post–child-bearing years, when lactation is readily avoidable and risk for developing breast cancer is high. Although vaccination is most effective when used to prevent disease, cancer vaccine development has focused predominantly on providing therapy against established growing tumors1. The difficulty in developing prophylactic cancer vaccines is primarily due to the fact that tumor antigens are variations of self proteins and would probably mediate profound autoimmune complications if used in a preventive vaccine setting2. Here we use several mouse breast cancer models to define a prototypic strategy for prophylactic cancer vaccination. We selected α-lactalbumin as our target vaccine autoantigen because it is a breast-specific differentiation protein expressed in high amounts in the majority of human breast carcinomas3,4,5 and in mammary epithelial cells only during lactation6,7,8,9. We found that immunoreactivity against α-lactalbumin provides substantial protection and therapy against growth of autochthonous tumors in transgenic mouse models of breast cancer and against 4T1 transplantable breast tumors in BALB/c mice. Because α-lactalbumin is conditionally expressed only during lactation, vaccination-induced prophylaxis occurs without any detectable inflammation in normal nonlactating breast tissue. Thus, α-lactalbumin vaccination may provide safe and effective protection against the development of breast cancer for women in their post–child-bearing, premenopausal years, when lactation is readily avoidable and risk for developing breast cancer is high10.

Adenosine-responsive ventricular tachycardias (VTs) typically occur in patients without structural heart disease; and thus, its association with myocardial ischemia is rare. In this case report, we describe a patient who had demonstrable ischemia along the anterolateral wall of the left ventricle and who developed a VT that was clinically terminated with adenosine. Surface electrocardiogram demonstrated a monomorphic VT with a right bundle-branch block morphology and a rightward axis configuration, and electrophysiologic testing showed atrioventricular dissociation upon atrial pacing and retrograde His waves following induction of VT. These findings localized the patient's VT to the left anterior fascicle, an anatomical region that coincided with the patient's territory of ischemia. We describe the electrophysiologic testing involved in elucidating the patient's tachyarrhythmia, and we provide a brief discussion of the pathogenesis and clinical features of adenosine-sensitive VT. Our case demonstrates that heterogeneous mechanisms of VT are operative in patients with ischemic heart disease.

T helper cells that produce interleukin 17 (IL-17) are associated with inflammation and the control of certain bacteria. We report here the essential involvement of the adaptor protein Act1 in IL-17 receptor (IL-17R) signaling and IL-17-dependent immune responses. After stimulation with IL-17, recruitment of Act1 to IL-17R required the IL-17R conserved cytoplasmic 'SEFIR' domain, followed by recruitment of the kinase TAK1 and E3 ubiquitin ligase TRAF6, which mediate 'downstream' activation of transcription factor NF-κB. IL-17-induced expression of inflammation-related genes was abolished in Act1-deficient primary astroglial and gut epithelial cells. This reduction was associated with much less inflammatory disease in vivo in both autoimmune encephalomyelitis and dextran sodium sulfate–induced colitis. Our data show that Act1 is essential in IL-17-dependent signaling in autoimmune and inflammatory disease.

idiopathic dilated cardiomyopathy (DCM) is a disease of putative autoimmune origin that kills males at a twofold to threefold greater frequency than females. The reasons underlying these differential outcomes may be related to sex-divergent self-recognition. Here we examined sex-specific autoimmune responses to cardiac self and their impact on DCM development. We found that males immunized to the p406-425 peptide derived from mouse cardiac a-myosin heavy chain preferentially develop a predominant Th17 lineage response that provides sustained T-cell memory and a high DCM incidence whereas females preferentially develop a predominant Th17 lineage response that becomes anergized to cardiac self resulting in compensatory protection against DCM. The distinct sex-defined disease phenotypes are interchangeable after in vivo manipulation of Th1 (interleukin-2) and Th17 (interleukin-17) cytokines. Our study shows that male and female SWXJ mice differentially respond to cardiac self in ways that lead to distinct autoimmune outcomes and implies that optimized therapy for autoinmmunity may require consideration of the qualitatively different ways that males and females engage self.

Background - Antibodies to the beta(1)-adrenergic receptor (beta(1)AR) are detected in a substantial number of patients with idiopathic dilated cardiomyopathy (DCM). The mechanism whereby these autoantibodies exert their pathogenic effect is unknown. Here, we define a causal mechanism whereby beta(1)AR-specific autoantibodies mediate noninflammatory cardiomyocyte cell death during murine DCM.Methods and Results - We used the beta(1)AR protein as an immunogen in SWXJ mice and generated a polyclonal battery of autoantibodies that showed selective binding to the beta(1)AR. After transfer into naive male hosts, beta(1)AR antibodies elicited fulminant DCM at high frequency. DCM was attenuated after immunoadsorption of beta(1)AR IgG before transfer and by selective pharmacological antagonism of host beta(1)AR but not beta(2)AR. We found that beta(1)AR autoantibodies shifted the beta(1)AR into the agonist-coupled high-affinity state and activated the canonical cAMP-dependent protein kinase A signaling pathway in cardiomyocytes. These events led to functional alterations in intracellular calcium handling and contractile function. Sustained agonism by beta(1)AR autoantibodies elicited caspase-3 activation, cardiomyocyte apoptosis, and DCM in vivo, and these processes were prevented by in vivo treatment with the pan-caspase inhibitor Z-VAD-FMK.Conclusions - Our data show how beta(1)AR-specific autoantibodies elicit DCM by agonistically inducing cardiomyocyte apoptosis.

Dilated cardiomyopathy (DCM) is a common cause of congestive heart failure and commonly occurs in the setting of autoimmune cardio-inflammatory processes. Consistent with this notion myocardial damage and dilatory remodeling consistent with DCM occurs upon adoptive transfer of T cell subsets reactive to self-antigens abundantly expressed in cardiac tissue. In this review we discuss etiologic mechanisms by which cardio-destructive T cells are generated during T cell ontogeny, and we review accumulated work identifying myocardial-derived T cell epitopes. Additionally, we describe several possible mechanisms whereby autoimmune T cell responses are sustained during chronic DCM. Epitope spreading, intra-cardiac persistence of pathogen-derived proteinaceous determinants, and generation of long-lived memory T cells are discussed as putative processes operative in the chronic maintenance of DCM.

Autoimmune sensorineural hearing loss (ASNHL) is the most common cause of sudden hearing loss in adults. Although autoimmune etiopathogenic events have long been suspected in ASNHL, inner ear-specific Ags capable of targeting T cell autoreactivity have not been identified in ASNHL. In this study, we show by ELISPOT analysis that compared with normal hearing age- and sex-matched control subjects, ASNHL patients have significantly higher frequencies of circulating T cells producing either IFN-gamma (p = 0.0001) or IL-5 (p = 0.03) in response to recombinant human cochlin, the most abundant inner ear protein. In some patients, cochlin responsiveness involved both CD4(+) and CD8(+) T cells whereas other patients showed cochlin responsiveness confined to CD8(+) T cells. ASNHL patients also showed significantly elevated cochlin-specific serum Ab titers compared with both normal hearing age- and sex-matched control subjects and patients with noise- and/or age-related hearing loss (p < 0.05 at all dilutions tested through 1/2048). Our study is the first to show T cell responsiveness to an inner ear-specific protein in ASNHL patients, and implicates cochlin as a prominent target Ag for mediating autoimmune inner ear inflammation and hearing loss.