The role of IL-6 in hyperlipidemia-induced accelerated rejection.

Hyperlipidemia induces accelerated rejection of cardiac allografts and resistance to tolerance induction using costimulatory molecule blockade in mice due in part to anti-donor Th17 responses and reduced regulatory T cell function. Accelerated rejection in hyperlipidemic mice is also associated with increased serum levels of IL-6. Here, we examined the role of IL-6 in hyperlipidemia-induced accelerated rejection and resistance to tolerance. Genetic ablation of IL-6 prevented hyperlipidemia-induced accelerated cardiac allograft rejection. Using Th17-lineage fate tracking mice, we observed that IL-6 is required to promote the development of anti-donor Th17 lineage cells independently of antigen challenge. In contrast, the frequency of alloreactive T cells producing IL-2 or IFN-γ remained increased in hyperlipidemic IL-6-deficient mice. Ablation of IL-6 overcame hyperlipidemia-induced changes in Tregs, but was not sufficient to overcome resistance to costimulatory molecule blockade induced tolerance. We suggest that accelerated rejection in hyperlipidemic mice results from IL-6 driven anti-donor Th17 responses. While alterations in Tregs were overcome by ablation of IL-6, the reversal of hyperlipidemia-induced changes in Tregs was not sufficient to overcome increased Th1-type anti-donor T cell responses, suggesting that hyperlipidemia induced IL-6-independent effects on recipient immunity prevent tolerance induction.

Hyperlipidemia-induced metabolic changes in regulatory T cells result in altered function.

Regulatory T cells (Tregs) play a critical role in maintaining self-tolerance and controlling inflammation. However, physiologically relevant conditions that alter Treg function and drive disease pathogenesis are poorly understood and few have been defined. We have previously shown that induction of hyperlipidemia in mice results in changes in Tregs that reduce their function. Here, we set out to examine mechanisms by which hyperlipidemia alters Tregs. Using live-cell metabolic assays, we observed that induction of hyperlipidemia increases metabolism in Tregs but not conventional T cells. Increased metabolism resulted from preferential activation of the serine/threonine kinase Akt2 (PKB-ß). Expression of a constitutively activated form of Akt2 in CD4 T cells was sufficient to increase glycolysis in Tregs and drive changes in Treg subsets. Induction of hyperlipidemia did not alter Treg metabolism in mice lacking Akt2. Activation of Akt2 was sufficient to drive the production of inflammatory cytokines by Tregs. We suggest that hyperlipidemia alters Treg function through effects on metabolism via Akt2 activation thereby promoting plasticity and decreased function of FoxP3+ T cells.

Hyperlipidemia and Allograft Rejection.

PURPOSE OF REVIEW: Advances in the development of immunosuppressive drug regimens have led to impressive survival rates in the year following organ transplantation. However rates of long-term graft dysfunction remain undesirably high. Recently it has been shown that co-morbidities in the patient population may affect graft survival. In mouse models, hyperlipidemia, a co-morbidity present in the majority of cardiac transplant patients, can significantly alter T cell responses to cardiac and skin allografts, and accelerate graft rejection. Here we review recent advances in our understanding of how alterations in lipids affect immune function and graft survival. RECENT FINDINGS: Recent work in humans has highlighted the importance of controlling low density lipoprotein (LDL) levels in transplant recipients to reduce the development of chronic allograft vasculopathy (CAV). High serum levels of cholesterol containing particles leads to extensive immune system changes to T cell proliferation, differentiation and suppression. Changes in B cell subsets, and the ability of antigen presenting cells to stimulate T cells in hyperlipidemic animals may also contribute to increased organ allograft rejection. SUMMARY: Cholesterol metabolism is a critical cellular pathway for proper control of immune cell homeostasis and activation. Increasing evidence in both human, and in mouse models shows that elevated levels of serum cholesterol can have profound impact on the immune system. Hyperlipidemia has been shown to increase T cell activation, alter the development of T helper subsets, increase the inflammatory capacity of antigen presenting cells (APC) and significantly accelerate graft rejection in several models.

Impact of environmental factors on alloimmunity and transplant fate.

Although gene-environment interactions have been investigated for many years to understand people's susceptibility to autoimmune diseases or cancer, a role for environmental factors in modulating alloimmune responses and transplant outcomes is only now beginning to emerge. New data suggest that diet, hyperlipidemia, pollutants, commensal microbes, and pathogenic infections can all affect T cell activation, differentiation, and the kinetics of graft rejection. These observations reveal opportunities for novel therapeutic interventions to improve graft outcomes as well as for noninvasive biomarker discovery to predict or diagnose graft deterioration before it becomes irreversible. In this Review, we will focus on the impact of these environmental factors on immune function and, when known, on alloimmune function, as well as on transplant fate.

Impact of hyperlipidemia on alloimmunity.

PURPOSE OF REVIEW: Hyperlipidemia is a comorbidity affecting a significant number of transplant patients despite treatment with cholesterol lowering drugs. Recently, it has been shown that hyperlipidemia can significantly alter T-cell responses to cardiac allografts in mice, and graft rejection is accelerated in dyslipidemic mice. Here, we review recent advances in our understanding of hyperlipidemia in graft rejection. RECENT FINDINGS: Hyperlipidemic mice have significant increases in serum levels of proinflammatory cytokines, and neutralization of interleukin 17 (IL-17) slows graft rejection, suggesting that IL-17 production by Th17 cells was necessary but not sufficient for rejection. Hyperlipidemia also causes an increase in alloreactive T-cell responses prior to antigen exposure. Analysis of peripheral tolerance mechanisms indicated that this was at least in part due to alterations in FoxP3 T cells that led to reduced Treg function and the expansion of FoxP3 CD4 T cells expressing low levels of CD25. Functionally, alterations in Treg function prevented the ability to induce operational tolerance to fully allogeneic heart transplants through costimulatory-molecule blockade, a strategy that requires Tregs. SUMMARY: These findings highlight the importance of considering the contribution of inflammatory comorbidities to cardiac allograft rejection, and point to the potential importance of managing hyperlipidemia in the transplant population.

Angiotensin II-induced TLR4 mediated abdominal aortic aneurysm in apolipoprotein E knockout mice is dependent on STAT3.

Abdominal Aortic Aneurysm (AAA) is a major cause of mortality and morbidity in men over 65 years of age. Male apolipoprotein E knockout (ApoE(-/-)) mice infused with angiotensin II (AngII) develop AAA. Although AngII stimulates both JAK/STAT and Toll-like receptor 4 (TLR4) signaling pathways, their involvement in AngII mediated AAA formation is unclear. Here we used the small molecule STAT3 inhibitor, S3I-201, the TLR4 inhibitor Eritoran and ApoE(-/-)TLR4(-/-) mice to evaluate the interaction between STAT3 and TLR4 signaling in AngII-induced AAA formation. ApoE(-/-) mice infused for 28 days with AngII developed AAAs and increased STAT3 activation and TLR4 expression. Moreover, AngII increased macrophage infiltration and the ratio of M1 (pro-inflammatory)/M2 (healing) macrophages in aneurysmal tissue as early as 7-10 days after AngII infusion. STAT3 inhibition with S3I-201 decreased the incidence and severity of AngII-induced AAA formation and decreased MMP activity and the ratio of M1/M2 macrophages. Furthermore, AngII-mediated AAA formation, MMP secretion, STAT3 phosphorylation and the ratio of M1/M2 macrophages were markedly decreased in ApoE(-/-)TLR4(-/-) mice, and in Eritoran-treated ApoE(-/-) mice. TLR4 and pSTAT3 levels were also increased in human aneurysmal tissue. These data support a role of pSTAT3 in TLR4 dependent AAA formation and possible therapeutic roles for TLR4 and/or STAT3 inhibition in AAA.

Aldosterone increases early atherosclerosis and promotes plaque inflammation through a placental growth factor-dependent mechanism.

BACKGROUND: Aldosterone levels correlate with the incidence of myocardial infarction and mortality in cardiovascular patients. Aldosterone promotes atherosclerosis in animal models, but the mechanisms are poorly understood. METHODS AND RESULTS: Aldosterone was infused to achieve pathologically relevant levels that did not increase blood pressure in the atherosclerosis-prone apolipoprotein E-knockout mouse (ApoE-/-). Aldosterone increased atherosclerosis in the aortic root 1.8±0.1-fold after 4 weeks and in the aortic arch 3.7±0.2-fold after 8 weeks, without significantly affecting plaque size in the abdominal aorta or traditional cardiac risk factors. Aldosterone treatment increased lipid content of plaques (2.1±0.2-fold) and inflammatory cell content (2.2±0.3-fold), induced early T-cell (2.9±0.3-fold) and monocyte (2.3±0.3-fold) infiltration into atherosclerosis-prone vascular regions, and enhanced systemic inflammation with increased spleen weight (1.52±0.06-fold) and the circulating cytokine RANTES (regulated and normal T cell secreted; 1.6±0.1-fold). To explore the mechanism, 7 genes were examined for aldosterone regulation in the ApoE-/- aorta. Further studies focused on the proinflammatory placental growth factor (PlGF), which was released from aldosterone-treated ApoE-/- vessels. Activation of the mineralocorticoid receptor by aldosterone in human coronary artery smooth muscle cells (SMCs) caused the release of factors that promote monocyte chemotaxis, which was inhibited by blocking monocyte PlGF receptors. Furthermore, PlGF-deficient ApoE-/- mice were resistant to early aldosterone-induced increases in plaque burden and inflammation. CONCLUSIONS: Aldosterone increases early atherosclerosis in regions of turbulent blood flow and promotes an inflammatory plaque phenotype that is associated with rupture in humans. The mechanism may involve SMC release of soluble factors that recruit activated leukocytes to the vessel wall via PlGF signaling. These findings identify a novel mechanism and potential treatment target for aldosterone-induced ischemia in humans.

MicroRNA expression data reveals a signature of kidney damage following ischemia reperfusion injury.

Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury, a common problem worldwide associated with significant morbidity and mortality. We have recently examined the role of microRNAs (miRs) in renal IRI using expression profiling. Here we conducted mathematical analyses to determine if differential expression of miRs can be used to define a biomarker of renal IRI. Principal component analysis (PCA) was combined with spherical geometry to determine whether samples that underwent renal injury as a result of IRI can be distinguished from controls based on alterations in miR expression using our data set consisting of time series measuring 571 miRs. Using PCA, we examined whether changes in miR expression in the kidney following IRI have a distinct direction when compared to controls based on the trajectory of the first three principal components (PCs) for our time series. We then used Monte Carlo methods and spherical geometry to assess the statistical significance of these directions. We hypothesized that if IRI and control samples exhibit distinct directions, then miR expression can be used as a biomarker of injury. Our data reveal that the pattern of miR expression in the kidney following IRI has a distinct direction based on the trajectory of the first three PCs and can be distinguished from changes observed in sham controls. Analyses of samples from immunodeficient mice indicated that the changes in miR expression observed following IRI were lymphocyte independent, and therefore represent a kidney intrinsic response to injury. Together, these data strongly support the notion that IRI results in distinct changes in miR expression that can be used as a biomarker of injury.

Costimulation-dependent expression of microRNA-214 increases the ability of T cells to proliferate by targeting Pten.

T cell activation requires signaling through the TCR and costimulatory molecules, such as CD28. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression posttranscriptionally and are also known to be involved in lymphocyte development and function. In this paper, we set out to examine potential roles of miRNAs in T cell activation, using genome-wide expression profiling to identify miRNAs differentially regulated following T cell activation. One of the miRNAs upregulated after T cell activation, miR-214, was predicted to be capable of targeting Pten based on bioinformatics and reports suggesting that it targets Pten in ovarian tumor cells. Upregulation of miR-214 in T cells inversely correlated with levels of phosphatase and tensin homolog deleted on chromosome 10. In vivo, transcripts containing the 3' untranslated region of Pten, including the miR-214 target sequence, were negatively regulated after T cell activation, and forced expression of miR-214 in T cells led to increased proliferation after stimulation. Blocking CD28 signaling in vivo prevented miR-214 upregulation in alloreactive T cells. Stimulation of T cells through the TCR alone was not sufficient to result in upregulation of miR-214. Thus, costimulation-dependent upregulation of miR-214 promotes T cell activation by targeting the negative regulator Pten. Thus, the requirement for T cell costimulation is, in part, related to its ability to regulate expression of miRNAs that control T cell activation.

Induction of transplantation tolerance to fully mismatched cardiac allografts by T cell mediated delivery of alloantigen.

Induction of transplantation tolerance has the potential to allow for allograft acceptance without the need for life-long immunosuppression. Here we describe a novel approach that uses delivery of alloantigen by mature T cells to induce tolerance to fully allogeneic cardiac grafts. Adoptive transfer of mature alloantigen-expressing T cells into myeloablatively conditioned mice results in long-term acceptance of fully allogeneic heart transplants without evidence of chronic rejection. Since myeloablative conditioning is clinically undesirable we further demonstrated that adoptive transfer of mature alloantigen-expressing T cells alone into mice receiving non-myeloablative conditioning resulted in long-term acceptance of fully allogeneic heart allografts with minimal evidence of chronic rejection. Mechanistically, tolerance induction involved both deletion of donor-reactive host T cells and the development of regulatory T cells. Thus, delivery of alloantigen by mature T cells induces tolerance to fully allogeneic organ allografts in non-myeloablatively conditioned recipients, representing a novel approach for tolerance induction in transplantation.

Prevention of type 1 diabetes in NOD mice by genetic engineering of hematopoietic stem cells.

Type 1 diabetes is caused by autoimmune destruction of insulin-producing cells in the pancreas. Type 1 diabetes could potentially be treated by islet transplantation; however, the recurrence of autoimmunity leads to the destruction of islet grafts in a relatively short time frame. Therefore, a major goal of diabetes research is the induction of tolerance in diabetic patients to prevent recurrence of diabetes. Diabetes is a polygenic disease, and not all the determinants responsible for disease susceptibility have been identified. However, in both humans and mouse models of this disease, one of the principle determining genetic factors in diabetes incidence is the inheritance of mutant MHC class II alleles that are associated with increased occurrence of disease. We have shown that in the NOD mouse model, the introduction of protective MHC class II alleles through retroviral gene therapy can prevent the onset of autoimmune diabetes. Prevention of diabetes appears to be mediated, at least in part, by the deletion of autoreactive T cells in the presence of protective MHC class II. Here, we outline the procedures involved in the modification of murine hematopoietic cells through retroviral transduction, the reconstitution of recipients with modified bone marrow, and the monitoring of gene therapy recipients after reconstitution.

Tolerization of a type I allergic immune response through transplantation of genetically modified hematopoietic stem cells.

Allergy represents a hypersensitivity disease that affects >25% of the population in industrialized countries. The underlying type I allergic immune reaction occurs in predisposed atopic individuals in response to otherwise harmless Ags (i.e., allergens) and is characterized by the production of allergen-specific IgE, an allergen-specific T cell response, and the release of biologically active mediators such as histamine from mast cells and basophils. Regimens permanently tolerizing an allergic immune response still need to be developed. We therefore retrovirally transduced murine hematopoietic stem cells to express the major grass pollen allergen Phl p 5 on their cell membrane. Transplantation of these genetically modified hematopoietic stem cells led to durable multilineage molecular chimerism and permanent immunological tolerance toward the introduced allergen at the B cell, T cell, and effector cell levels. Notably, Phl p 5-specific serum IgE and IgG remained undetectable, and T cell nonresponsiveness persisted throughout follow-up (40 wk). Besides, mediator release was specifically absent in in vitro and in vivo assays. B cell, T cell, and effector cell responses to an unrelated control allergen (Bet v 1) were unperturbed, demonstrating specificity of this tolerance protocol. We thus describe a novel cell-based strategy for the prevention of allergy.

Induction of transplantation tolerance by combining non-myeloablative conditioning with delivery of alloantigen by T cells.

The observation that bone marrow derived hematopoietic cells are potent inducers of tolerance has generated interest in trying to establish transplantation tolerance by inducing a state of hematopoietic chimerism through allogeneic bone marrow transplantation. However, this approach is associated with serious complications that limit its utility for tolerance induction. Here we describe the development of a novel approach that allows for tolerance induction without the need for an allogeneic bone marrow transplant by combining non-myeloablative host conditioning with delivery of donor alloantigen by adoptively transferred T cells. CBA/Ca mice were administered 2.5 Gy whole body irradiation (WBI). The following day the mice received K(b) disparate T cells from MHC class I transgenic CBK donor mice, as well as rapamycin on days 0-13 and anti-CD40L monoclonal antibody on days 0-5, 8, 11 and 14 relative to T cell transfer. Mice treated using this approach were rendered specifically tolerant to CBK skin allografts through a mechanism involving central and peripheral deletion of alloreactive T cells. These data suggest robust tolerance can be established without the need for bone marrow transplantation using clinically relevant non-myeloablative conditioning combined with antigen delivery by T cells.

Gene therapy in type 1 diabetes.

Type 1 diabetes (T1D) is caused by the autoimmune-mediated destruction of insulin-producing beta cells in the pancreas. T1D affects as many as 3 million patients in the United States alone, with 15,000 new cases developing every year (Juvenile Diabetes Research Foundation), and presently there is no cure for T1D. In recent years, there has been a great deal of interest in developing gene therapy approaches to treat T1D. Gene therapy approaches tend to fall into three broad categoriesthose aimed at preventing or curing autoimmunity, those aimed at restoring insulin production through islet transplant or genetically engineered insulin production, and approaches that aim to prevent the morbidity and mortality associated with this complex disease. We review these studies here.

Induction of robust diabetes resistance and prevention of recurrent type 1 diabetes following islet transplantation by gene therapy.

We have previously shown that the development of type 1 diabetes (T1D) can be prevented in nonobese diabetic (NOD) mice by reconstitution with autologous hemopoietic stem cells retrovirally transduced with viruses encoding MHC class II I-A beta-chain molecules associated with protection from the disease. In this study we examined whether a blockade of the programmed death-1 (PD-1)-programmed death ligand-1 (PD-L1) pathway, a major pathway known to control diabetes occurrence, could precipitate T1D in young NOD mice following reconstitution with autologous bone marrow retrovirally transduced with viruses encoding protective MHC class II I-A beta-chain molecules. In addition, we examined whether the expression of protective MHC class II alleles in hemopoietic cells could be used to prevent the recurrence of diabetes in mice with pre-existing disease following islet transplantation. Protection from the occurrence of T1D diabetes in young NOD mice by the expression of protective MHC class II I-A beta-chain molecules in bone marrow-derived hemopoietic cells was resistant to induction by PD-1-PD-L1 blockade. Moreover, reconstitution of NOD mice with pre-existing T1D autologous hemopoietic stem cells transduced with viruses encoding protective MHC class II I-A beta-chains allowed for the successful transplantation of syngeneic islets, resulting in the long-term reversal of T1D. Reversal of diabetes was resistant to induction by PD-1-PDL-1 blockade and depletion of CD25(+) T cells. These data suggest that expression of protective MHC class II alleles in bone marrow-derived cells establishes robust self-tolerance to islet autoantigens and is sufficient to prevent the recurrence of autoimmune diabetes following islet transplantation.

New approaches to the prevention of organ allograft rejection and tolerance induction.

The therapeutic use of organ allograft transplantation is dependent on the discovery and clinical application of immunologic strategies to blunt the immune response and prevent graft rejection. It was the discovery of powerful immunotherapeutics such as cyclosporine A and rapamycin that has allowed for the widespread use of organ transplantation to treat organ failure. However, despite the attainment of impressive survival rates 1 year after organ transplantation, a significant number of organ allografts are lost to immune-mediated chronic rejection. Furthermore, significant morbidity and mortality can be associated with the use of currently available immunosuppressive regimens. Thus, the development of novel approaches to prevent of organ allograft rejection remains extremely important. Here we discuss two promising and novel avenues of research. First, the discovery and characterization of naturally occurring immune inhibitory signals have led to recent research aimed at exploiting these pathways to induce peripheral tolerance to alloantigen. Furthermore, we discuss new approaches to the induction of donor-specific tolerance by induction of molecular chimerism and the transfer of alloantigen-expressing mature T cells.

Regulation of oxidative stress responses by ataxia-telangiectasia mutated is required for T cell proliferation.

Mutations in the gene encoding ataxia-telangiectasia (A-T) mutated (Atm) cause the disease A-T, characterized by immunodeficiency, the molecular basis of which is not known. Following stimulation through the TCR, Atm-deficient T cells and normal T cells in which Atm is inhibited undergo apoptosis rather than proliferation. Apoptosis is prevented by scavenging reactive oxygen species (ROS) during activation. Atm therefore plays a critical role in T cell proliferation by regulating responses to ROS generated following T cell activation. The inability of Atm-deficient T cells to control responses to ROS is therefore the molecular basis of immunodeficiency associated with A-T.

Long-term survival of transplanted allogeneic cells engineered to express a T cell chemorepellent.

BACKGROUND: Alloantigen specific T cells have been shown to be required for allograft rejection. The chemokine, stromal cell derived factor-1 (SDF-1) at high concentration, has been shown to act as a T-cell chemorepellent and abrogate T-cell infiltration into a site of antigen challenge in vivo via a mechanism termed fugetaxis or chemorepulsion. We postulated that this mechanism could be exploited therapeutically and that allogeneic cells engineered to express a chemorepellent protein would not be rejected. METHODS: Allogeneic murine insulinoma beta-TC3 cells and primary islets from BALB/C mice were engineered to constitutively secrete differential levels of SDF-1 and transplanted into allogeneic diabetic C57BL/6 mice. Rejection was defined as the permanent return of hyperglycemia and was correlated with the level of T-cell infiltration. The migratory response of T-cells to SDF-1 was also analyzed by transwell migration assay and time-lapse videomicroscopy. The cytotoxicity of cytotoxic T cell (CTLs) against beta-TC3 cells expressing high levels of SDF-1 was measured in standard and modified chromium-release assays in order to determine the effect of CTL migration on killing efficacy. RESULTS: Control animals rejected allogeneic cells and remained diabetic. In contrast, high level SDF-1 production by transplanted cells resulted in increased survival of the allograft and a significant reduction in blood glucose levels and T-cell infiltration into the transplanted tissue. CONCLUSIONS: This is the first demonstration of a novel approach that exploits T-cell chemorepulsion to induce site specific immune isolation and thereby overcomes allograft rejection without the use of systemic immunosuppression.

Oocyte generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood.

It has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries. Here we show that adult mouse ovaries rapidly generate hundreds of oocytes, despite a small premeiotic germ cell pool. In considering the possibility of an extragonadal source of germ cells, we show expression of germline markers in bone marrow (BM). Further, BM transplantation restores oocyte production in wild-type mice sterilized by chemotherapy, as well as in ataxia telangiectasia-mutated gene-deficient mice, which are otherwise incapable of making oocytes. Donor-derived oocytes are also observed in female mice following peripheral blood transplantation. Although the fertilizability and developmental competency of the BM and peripheral blood-derived oocytes remain to be established, their morphology, enclosure within follicles, and expression of germ-cell- and oocyte-specific markers collectively support that these cells are bona fide oocytes. These results identify BM as a potential source of germ cells that could sustain oocyte production in adulthood.

Preventing autoimmune diabetes through gene therapy.

Extract: Type 1 diabetes is an autoimmune disease in which an individual develops T cells that are able to destroy insulin-producing beta cells in the pancreas. Type 1 diabetics require life-long treatment with exogenous insulin for survival. Susceptibility to type 1 diabetes is influenced by both genetic and environmental factors. The first diabetes-susceptibility genes to be identified were the human leukocyte antigen (HLA) genes. Subsequent studies demonstrated an association of these genes with the insulin gene region. High throughput screening of the human genome in families with two or more affected siblings led to the identification of additional chromosomal regions that may contain susceptibility genes for type 1 diabetes. However, linkage between the HLA gene region and susceptibility to disease suggested that the principal genetic component leading to development of diabetes is the inheritance of mutant HLA class II alleles. These are so-called "at-risk" alleles which lack an aspartic acid, a positively amino acid, at position 57 of the major histocompatibility complex (MHC) class II beta chain.