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.

Defining a microRNA-mRNA interaction map for calcineurin inhibitor induced nephrotoxicity.

Calcineurin inhibitors induce nephrotoxicity through poorly understood mechanisms thereby limiting their use in transplantation and other diseases. Here we define a microRNA (miRNA)-messenger RNA (mRNA) interaction map that facilitates exploration into the role of miRNAs in cyclosporine-induced nephrotoxicity (CIN) and the gene pathways they regulate. Using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP), we isolated RNAs associated with Argonaute 2 in the RNA-induced silencing complex (RISC) of cyclosporine A (CsA) treated and control human proximal tubule cells and identified mRNAs undergoing active targeting by miRNAs. CsA causes specific changes in miRNAs and mRNAs associated with RISC, thereby altering post-transcriptional regulation of gene expression. Pathway enrichment analysis identified canonical pathways regulated by miRNAs specifically following CsA treatment. RNA-seq performed on total RNA indicated that only a fraction of total miRNAs and mRNAs are actively targeted in the RISC, indicating that PAR-CLIP more accurately defines meaningful targeting interactions. Our data also revealed a role for miRNAs in calcineurin-independent regulation of JNK and p38 MAPKs caused by targeting of MAP3K1. Together, our data provide a novel resource and unique insights into molecular pathways regulated by miRNAs in CIN. The gene pathways and miRNAs defined may represent novel targets to reduce calcineurin induced nephrotoxicity.

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.

Independent effects of sham laparotomy and anesthesia on hepatic microRNA expression in rats.

BACKGROUND: Studies on liver regeneration following partial hepatectomy (PH) have identified several microRNAs (miRNAs) that show a regulated expression pattern. These studies involve major surgery to access the liver, which is known to have intrinsic effects on hepatic gene expression and may also affect miRNA screening results. We performed two-third PH or sham laparotomy (SL) in Wistar rats to investigate the effect of both procedures on miRNA expression in liver tissue and corresponding plasma samples by microarray and qRT-PCR analyses. As control groups, non-treated rats and rats undergoing anesthesia only were used. RESULTS: We found that 49 out of 323 miRNAs (15%) were significantly deregulated after PH in liver tissue 12 to 48 hours postoperatively (>20% change), while 45 miRNAs (14%) were deregulated following SL. Out of these miRNAs, 10 miRNAs were similarly deregulated after PH and SL, while one miRNA showed opposite regulation. In plasma, miRNA upregulation was observed for miR-133a and miR-133b following PH and SL, whereas miR-100 and miR-466c were similarly downregulated following anesthesia and surgery. CONCLUSIONS: We show that miRNAs are indeed regulated by sham laparotomy and anesthesia in rats. These findings illustrate the critical need for finding appropriate control groups in experimental surgery.

The site of allergen expression in hematopoietic cells determines the degree and quality of tolerance induced through molecular chimerism.

The transplantation of allergens (e.g. Phl p 5 or Bet v 1) expressed on BM cells as membrane-anchored full-length proteins leads to permanent tolerance at the T-cell, B-cell, and effector-cell levels. Since the exposure of complete allergens bears the risk of inducing anaphylaxis, we investigated here whether expression of Phl p 5 in the cytoplasm (rather than on the cell surface) is sufficient for tolerance induction. Transplantation of BALB/c BM retrovirally transduced to express Phl p 5 in the cytoplasm led to stable and durable molecular chimerism in syngeneic recipients (∼20% chimerism at 6 months). Chimeras showed allergen-specific T-cell hyporesponsiveness. Further, Phl p 5-specific TH 1-dependent humoral responses were tolerized in several chimeras. Surprisingly, Phl p 5-specific IgE and IgG1 levels were significantly reduced but still detectable in sera of chimeric mice, indicating incomplete B-cell tolerance. No Phl p 5-specific sIgM developed in cytoplasmic chimeras, which is in marked contrast to mice transplanted with BM expressing membrane-anchored Phl p 5. Thus, the expression site of the allergen substantially influences the degree and quality of tolerance achieved with molecular chimerism in IgE-mediated allergy.

Engraftment of retrovirally transduced Bet v 1-GFP expressing bone marrow cells leads to allergen-specific tolerance.

Molecular chimerism is a promising strategy to induce tolerance to disease-causing antigens expressed on genetically modified haematopoietic stem cells. The approach was employed successfully in models of autoimmunity and organ transplantation. Recently, we demonstrated that molecular chimerism induces robust and lasting tolerance towards the major grass pollen allergen Phl p 5. Since allergens are a group of antigens differing widely in their function, origin and structure we further examined the effectiveness of molecular chimerism using the Phl p 5-unrelated major birch pollen allergen Bet v 1, co-expressed with the reporter GFP. Besides, inhibition of CD26 was used to promote engraftment of modified stem cells. Retrovirus VSV-Betv1-GFP was generated to transduce 5-FU-mobilized BALB/c hematopoietic cells to express membrane-bound Bet v 1 (VSV-GFP virus was used as control). Myeloablated BALB/c mice received Betv1-GFP or GFP expressing bone marrow cells, pre-treated with a CD26 inhibitor. Chimerism was followed by flow cytometry. Tolerance was assessed by measuring allergen-specific isotype levels in sera, RBL assays and T-cell proliferation assays. Mice transplanted with transduced BMC developed multi-lineage molecular chimerism which remained stable long-term (>8 months). After repeated immunizations with Bet v 1 and Phl p 5 serum levels of Bet v 1-specific antibodies (IgE, IgG1, IgG2a, IgG3 and IgA) remained undetectable in Betv1-GFP chimeras while high levels of Phl p 5-specific antibodies developed. Likewise, basophil degranulation was induced in response to Phl p 5 but not to Bet v 1 and specific non-responsiveness to Bet v 1 was observed in proliferation assays. These data demonstrate successful tolerization towards Bet v 1 by molecular chimerism. Stable long-term chimerism was achieved under inhibition of CD26. These results provide evidence for the broad applicability of molecular chimerism as tolerance strategy in allergy.

Identification of a microRNA signature of renal ischemia reperfusion injury.

Renal ischemia reperfusion injury (IRI) is associated with significant morbidity and mortality. Given the importance of microRNAs (miRNAs) in regulating gene expression, we examined expression profiles of miRNAs following renal IRI. Global miRNA expression profiling on samples prepared from the kidneys of C57BL/6 mice that underwent unilateral warm ischemia revealed nine miRNAs (miR-21, miR-20a, miR-146a, miR-199a-3p, miR-214, miR-192, miR-187, miR-805, and miR-194) that are differentially expressed following IRI when compared with sham controls. These miRNAs were also differently expressed following IRI in immunodeficient RAG-2/common gamma-chain double-knockout mice, suggesting that the changes in expression observed are not significantly influenced by lymphocyte infiltration and therefore define a lymphocyte-independent signature of renal IRI. In vitro studies revealed that miR-21 is expressed in proliferating tubular epithelial cells (TEC) and up-regulated by both cell-intrinsic and -extrinsic mechanisms resulting from ischemia and TGF-beta signaling, respectively. In vitro, knockdown of miR-21 in TEC resulted in increased cell death, whereas overexpression prevented cell death. However, overexpression of miR-21 alone was not sufficient to prevent TEC death following ischemia. Our findings therefore define a molecular fingerprint of renal injury and suggest miR-21 may play a role in protecting TEC from death.

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.

Liver sinusoidal endothelial cells as possible vehicles for gene therapy: a comparison between plasmid-based and lentiviral gene transfer techniques.

UNLABELLED: Liver sinusoidal endothelial cells (LSECs) constitute an attractive target for gene therapy of several liver and systemic diseases. However, there are few reports showing an efficient plasmid-based or viral methodology to deliver recombinant genes into these cells. In the present study, the authors evaluated in vitro gene transfer efficiency of standard plasmid-based techniques (i.e., electroporation, lipofection, and calcium phosphate) and lentiviral-mediated gene transduction into primary murine LSECs, using reporter genes. The results show that electroporation is the most effective in vitro plasmid-gene transfer method to deliver GFP into LSECs (31%), as compared with lipofection and calcium phosphate transfection (6% and 4%, respectively). However, lentiviral transduction resulted in higher, efficient, and stable gene transfer (70%) as compared with plasmid-based techniques. CONCLUSIONS: The highly efficient gene expression obtained by lentiviral transduction and electroporation shows that these methodologies are highly reliable systems for gene transfer into LSECs.

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.

Ig knock-in mice producing anti-carbohydrate antibodies: breakthrough of B cells producing low affinity anti-self antibodies.

Natural Abs specific for the carbohydrate Ag Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) play an important role in providing protective host immunity to various pathogens; yet little is known about how production of these or other anti-carbohydrate natural Abs is regulated. In this study, we describe the generation of Ig knock-in mice carrying functionally rearranged H chain and L chain variable region genes isolated from a B cell hybridoma producing alphaGal-specific IgM Ab that make it possible to examine the development of B cells producing anti-carbohydrate natural Abs in the presence or absence of alphaGal as a self-Ag. Knock-in mice on a alphaGal-deficient background spontaneously developed alphaGal-specific IgM Abs of a sufficiently high titer to mediate rejection of alphaGal expressing cardiac transplants. In the spleen of these mice, B cells expressing alphaGal-specific IgM are located in the marginal zone. In knock-in mice that express alphaGal, B cells expressing the knocked in BCR undergo negative selection via receptor editing. Interestingly, production of low affinity alphaGal-specific Ab was observed in mice that express alphaGal that carry two copies of the knocked in H chain. We suggest that in these mice, receptor editing functioned to lower the affinity for self-Ag below a threshold that would result in overt pathology, while allowing development of low affinity anti-self Abs.

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.

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.

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.

High efficiency and long-term foreign gene expression in cultured liver sinusoidal endothelial cells by retroviral transduction.

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.

Induction of alloreactive CD4 T cell tolerance in molecular chimeras: a possible role for regulatory T cells.

Induction of molecular chimerism following reconstitution of mice with autologous bone marrow cells expressing a retrovirally encoded allogeneic MHC class I Ag results in donor-specific tolerance. To investigate the mechanism by which CD4 T cells that recognize allogeneic MHC class I through the indirect pathway of Ag presentation are rendered tolerant in molecular chimeras, transgenic mice expressing a TCR on CD4 T cells specific for peptides derived from K(b) were used. CD4 T cells expressing the transgenic TCR were detected in mice reconstituted with bone marrow cells transduced with retroviruses carrying the gene encoding H-2K(b), albeit detection was at lower levels than in mice receiving mock-transduced bone marrow. Despite the presence of CD4 T cells expressing an alloreactive TCR, mice receiving H-2K(b)-transduced bone marrow permanently accepted K(b) disparate skin grafts. CD4+CD25+ T cells from mice reconstituted with H-2K(b)-transduced bone marrow prevented rejection of K(b) disparate skin grafts when adoptively transferred into immunodeficient mice along with effector T cells, suggesting that induction of molecular chimerism leads to the generation of donor specific regulatory T cells, which may be involved in preventing alloreactive CD4 T cell responses that lead to rejection.