Absence Of Rapamycin Research Articles

Abstract B26: PI3K class I and mTOR regulate distinct steps in Met dependent tumorigenesis

Abstract The Receptor Tyrosine Kinase (RTK) Met, overexpressed or mutated in cancer, plays a major role in cancer progression and represents an attractive target for cancer therapy. Although several Met inhibitors are in clinical trials, resistance may occur as experienced in the clinic with other RTKs. Thus, a need for alternative / complementary therapy may be required to target Met driven tumors efficiently. The aim of this study was to investigate whether PI3K plays a role in Met oncogenic activity, in view of designing appropriate therapy. The study was performed on two cell models suitable to study Met driven NIH3T3 cells expressing Met Wt or the constitutively active mutant M1268T and the glioblastoma U87MG cell line, with Met constitutively activated due to an autocrine loop. Transwell cell migration and anchorage independent growth in soft agar were studied in presence of inhibitors targeting pan-PI3K (LY294002 and wortmannin), isoform-specific PI3K (GDC0941, pan-PI3K class I, A66, p110 alpha, TGX221, p110 beta) and mTOR (rapamycin), or following siRNA knock down of PI3K isoforms. Tumor growth of cells grafted to nude mice was assessed in presence or absence of rapamycin. LY294002 reduced the migration and the anchorage independent growth of Met mutant M1268T expressing cells to the same level as with Met Wt expressing cells, while no difference was detected in Met Wt expressing cells. Inhibitions / knock downs of PI3K class I reduced the migration of Met M1268T expressing cells and UMG87 cells. Interestingly, it was determined that p110 alpha and beta together are responsible for Met dependent cell migration. Moreover, the phosphorylation (Ser473) of Akt, the downstream effector of PI3K class I, was significantly increased in Met M1268T versus Wt Met expressing cells. It was inhibited by Met pharmacological inhibition, indicating that Met M1268T activates Akt in these cells. Moreover, the inhibition of Met M1268T internalization using the dynamin inhibitor dynasore led to a significant reduction of Akt phosphorylation in these cells, suggesting further that Met M1268T activates Akt on endosome. Interestingly, the endosomal activation of Akt seems to be Appl1 independent. Surprisingly, inhibitions / knock downs of PI3K class I did not decrease the anchorage independent growth of Met M1268T expressing cells and of U87MG cells. Wortmannin also had no effect. However, our results using the mTOR inhibitor indicate that in fact, mTOR is responsible for Met dependent anchorage independent growth in Met M1268T expressing cells and U87MG cells. Moreover, rapamycin significantly reduced Met dependent tumor growth in vivo. Our results suggest that while PI3K class I (p110 alpha and beta) regulate oncogenic Met dependent cell migration, the anchorage independent growth and tumor growth are regulated by mTOR. These results may have implication for therapeutical intervention. Citation Format: Alexia Hervieu, Stephanie Kermorgant. PI3K class I and mTOR regulate distinct steps in Met dependent tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B26.

293. Optimal In Vivo Treg Induction and Suppression of Immune Responses By Synergistic Use of Rapamycin and FLT3 Ligand

Immune-mediated rejection of therapeutic proteins is a major hurdle for gene and protein replacement therapies for genetic disease. However, optimal induction and expansion of CD4+CD25+FoxP3+ regulatory T cells (Treg) counteract effector responses. We have previously shown that co-administration of antigen with rapamycin simultaneously promotes deletion of conventional CD4+ T cells and induction of Treg, so that we were able to induce tolerance to factors VIII and IX in treatment of the X-linked bleeding disorders hemophilia A and B. Here, we found in DO11.10 Rag−/− mice (homozygous for ovalbumin-peptide specific TCR, deficient in endogenous mature B, T or Treg cells) that the cytokine FMS-like receptor tyrosine kinase ligand (Flt3L) enhances the in vivo effect of rapamycin via selective expansion of plasmacytoid dendritic cells (pDC), resulting in substantially improved induction of OVA-specific Treg. This regimen, consisting of co-administration of rapamycin/Flt3L/OVA 3x/week for 1 month, also prevented OVA-specific CD8+ T cell responses in immune competent mice after intrasmuscular OVA gene transfer with an AAV1 vector. Flt3L signaling is critical to the generation and steady state expansion of both the conventional (CD11c+, CD8+CD11c+) and plasmacytoid (CD11cmid-loPDCA-1+) subsets of DC, which indirectly expands naturally occurring Treg. Interestingly, Flt3L-induced signaling in DC and DC precursors occurs through the mTOR pathway and can therefore be blocked by rapamycin, posing the question of why we are able to use these drugs synergistically for Treg induction. We find that limited doses of rapamycin selectively block Flt3L-induced expansion of conventional DC (cDC) but not pDC. Induced mTOR signaling is effectively blocked in cDC, while pDC have increased mTOR activity and are thus more resistant to inhibition by rapamycin. Consequently, Flt3L and rapamycin synergistically promote induction of antigen-specific Treg via selective expansion of pDC. During Flt3L/rapamycin/antigen co-administration, Treg induction is abrogated upon pDC depletion, thus confirming that Treg induction is pDC dependent. Similarly, Flt3L/antigen by itself fails to induce Treg, indicating that both pDC and rapamycin are required for Treg induction in this regimen.Finally, we are able to demonstrate in a therapeutic model for FVIII protein replacement therapy that combined Flt3L/rapamycin/FVIII treatment is superior in prevention of inhibitory antibody responses in hemophilia A mice (BALB/c F8e16−/−) when compared to rapamycin/FVIII only. In contrast, Flt3L/antigen administration in the absence of rapamycin, while enhancing activation induced cell death (AICD) of conventional CD4+ T cells, fails to induce Treg and also fails to induce tolerance to OVA or to FVIII. Thus, the differential effect of rapamycin on DC subsets can be exploited to improve tolerance induction via enhanced Treg induction, creating new opportunities for development of immune tolerance therapies with wide applicability.

Expansion and generation of Tregs cells with potential clinical application in kidney transplantation

Expansion and generation of Tregs cells with potential clinical application in kidney transplantation

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Objectives Preeclampsia (PE) is a clinical complication of pregnancy associated with high levels of inflammatory cytokines, leukocyte activation and oxidative stress. Autophagy, an intracellular catabolic process, which removes damaged organelles and cytoplasmic proteins seems to be able to maintain cellular homeostasis. Moreover, it also has a potent anti-inflammatory mechanism by destroying inflammasomes, an intracellular multi-protein structure required for processing and releasing bioactive Interleukin-1 beta (IL-1beta). The present study compared the ability of sera from pregnant women with or without preeclampsia to induce autophagy in peripheral blood mononuclear cells (PBMC), and the relationship between this activity and serum concentrations of uric acid, IL-1beta, IL-10 and TNF-alpha. Methods PBMC from 20 healthy non-pregnant women were incubated with sera from 45 pregnant women, being 23 normotensive (NT) and 22 with PE in the presence or absence of rapamycin, an autophagy inducer. After 48h, the cells were assayed for p62, a cytoplasmic protein associated with autophagy inhibition. Sera from both groups of pregnant women were tested for IL-1beta, IL-10 and TNF-alpha by enzyme-linked immunosorbent assay. Results Autophagy was induced to a greater extent by sera from women with PE than by NT pregnant women. The relationship between inflammatory cytokines in sera of preeclamptic pregnant women showed association between autophagy induction (decrease in cytoplasmic p62) and higher concentrations of IL-1beta ( r = −0.4949; p = 0.0192), and TNF-alpha ( r = − 0.5302; p = 0.0111). This relationship was absent in sera from NT women. There was no association between IL-10 or uric acid levels and autophagy both in preeclamptic and NT group. Conclusions Association between autophagy induction and higher levels of inflammatory cytokines in sera from patients with preeclampsia suggests that the enhanced pro-inflammatory state characteristic of preeclampsia may result in the up-regulation of autophagy induction in these women. Financial support: Fapesp 2014/00693-9. Disclosures M.L. Matias: None. M.T. Peracoli: None. T.T. Kanninen: None. G. Sisti: None. M. Romao: None. I.C. Weel: None. J.C. Peracoli: None. A.M. Bongiovanni: None. S.S. Witkin: None.

The relative merits of cord blood as a cell source for autologous T regulatory cell therapy in type 1 diabetes.

Cord blood has been used as a cell source for therapeutic purposes in children with type 1 diabetes and other disorders. Here, we explore the benefits of cord blood as an autologous source of T regulatory cells for immune cell therapy in patients. CD4(+)CD25(+) T regulatory cells were isolated from cord blood and adult peripheral blood of healthy donors and compared during and after expansion in a 14-day protocol incorporating anti-CD3/anti-CD28 beads, and IL-2 with or without rapamycin. Cord blood T regulatory cells were largely naïve (89±7 vs. 31±10% in young adults, p<0.0001), and had higher expansion yields (median 5,968-fold) than adult T regulatory cells (median 516-fold, p=0.001) and adult naïve T regulatory cells (median 820-fold, p=0.003). Rapamycin reduced expansion yields, but was not necessary to obtain pure expanded cord blood T regulatory cells as judged by FOXP3 staining (94±3%), methylation status of FOXP3 (97%), and intracellular effector cytokine staining (< 6%). Expanded adult T regulatory cells were much less pure in the absence of rapamycin (72±19% FOXP3; 76% by methylation status, <13% INF-γ, <16% IL-4, <5% IL-17 positive), but purity was achieved by inclusion of rapamycin during expansion. Despite differences in purity, all preparations of expanded T regulatory from all sources were able to strongly suppress proliferation of T effector cells in vitro. Our findings suggest that cord blood is an excellent source of T regulatory cells for expansion and autologous cell therapy that may be considered as a strategy to prevent immune-mediated destruction of beta cells in type 1 diabetes.

Inhibitor-induced conformational stabilization and structural alteration of a mip-like peptidyl prolyl cis-trans isomerase and its C-terminal domain.

FKBP22, an Escherichia coli-encoded PPIase (peptidyl-prolyl cis-trans isomerase) enzyme, shares substantial identity with the Mip-like pathogenic factors, caries two domains, exists as a dimer in solution and binds some immunosuppressive drugs (such as FK506 and rapamycin) using its C-terminal domain (CTD). To understand the effects of these drugs on the structure and stability of the Mip-like proteins, rFKBP22 (a chimeric FKBP22) and CTD+ (a CTD variant) have been studied in the presence and absence of rapamycin using different probes. We demonstrated that rapamycin binding causes minor structural alterations of rFKBP22 and CTD+. Both the proteins (equilibrated with rapamycin) were unfolded via the formation of intermediates in the presence of urea. Further study revealed that thermal unfolding of both rFKBP22 and rapamycin-saturated rFKBP22 occurred by a three-state mechanism with the synthesis of intermediates. Intermediate from the rapamycin-equilibrated rFKBP22 was formed at a comparatively higher temperature. All intermediates carried substantial extents of secondary and tertiary structures. Intermediate resulted from the thermal unfolding of rFKBP22 existed as the dimers in solution, carried an increased extent of hydrophobic surface and possessed relatively higher rapamycin binding activity. Despite the formation of intermediates, both the thermal and urea-induced unfolding reactions were reversible in nature. Unfolding studies also indicated the considerable stabilization of both proteins by rapamycin binding. The data suggest that rFKBP22 or CTD+ could be exploited to screen the rapamycin-like inhibitors in the future.

Requirement of B7-H1 in mesenchymal stem cells for immune tolerance to cardiac allografts in combination therapy with rapamycin

BackgroundThe potential of mesenchymal stem cells (MSCs) for immunosuppression has been tested in transplantation, but its mechanisms are not fully understood. This study investigated the role of MSC-expressing B7-H1 in the induction of immune tolerance to cardiac allografts by the combination therapy of MSCs and rapamycin (RAPA). MethodsThe anti-alloimmunity of donor MSCs in the presence or absence of RAPA was examined in both mouse cardiac allograft model (C57BL/6 to BALB/c mice) and a variety of cultured immune cells. Immunohistochemical staining was used for the measurement of intragraft antibody deposition, and fluorescence-activated cell sorting (FACS) for the determination of serum alloantibodies and leukocyte phenotypes. ResultsB7-H1 expression in cultured MSCs was up-regulated following IFN-γ stimulation. In transplant recipients, combination therapy of MSCs and RAPA induced immune tolerance to allografts, but blockade of B7-H1 on MSCs with monoclonal antibody abrogated the combination therapy-induced immune tolerance as heart allografts were rejected. The negative effect of MSC-expressing B7-H1 neutralization on graft survival was correlated with a reduction of regulatory immune cells (CD4+CD25+Foxp3+ T cells, tolerogenic dendritic cells and IL-4highIL-10HighCD83low B cells), and also with an increase in alloantibody (IgG and IgM) levels both inside the grafts and in the circulation as compared with un-neutralized controls. In vitro MSC-mediated suppression of antibody production and B cell proliferation depended on B7-H1 function and cell contact between CD19+ B cells and MSCs. ConclusionThese data suggest that MSC-expressing B7-H1 mediates the immune tolerance to cardiac allografts in recipients receiving MSC and RAPA combination therapy.

Insights from yeast into whether the inhibition of heat shock transcription factor (Hsf1) by rapamycin can prevent the Hsf1 activation that results from treatment with an Hsp90 inhibitor.

In human cells TORC1 mTOR (target of rapamycin) protein kinase complex renders heat shock transcription factor 1 (Hsf1) competent for stress activation. In such cells, as well as in yeast, the selective TORC1 inhibitor rapamycin blocks this activation in contrast to Hsp90 inhibitors which potently activate Hsf1. Potentially therefore rapamycin could prevent the Hsf1 activation that frequently compromises the efficiency of Hsp90 inhibitor cancer drugs. Little synergy was found between the effects of rapamycin and the Hsp90 inhibitor radicicol on yeast growth. However certain rapamycin resistance mutations sensitised yeast to Hsp90 inhibitor treatment and an Hsp90 mutation that overactivates Hsf1 sensitised cells to rapamycin. Rapamycin inhibition of the yeast Hsf1 was abolished by this Hsp90 mutation, as well as with the loss of Ppt1, the Hsp90-interacting protein phosphatase that is the ortholog of mammalian PP5. Unexpectedly Hsf1 activation was found to have a requirement for the rapamycin binding immunophilin FKBP12 even in the absence of rapamycin, while TORC1 "bypass" strains revealed that the rapamycin inhibition of yeast Hsf1 is not exerted through two of the major downstream targets of TORC1, the protein phosphatase regulator Tap42 and the protein kinase Sch9--the latter the ortholog of human S6 protein kinase 1. A problem with most of the Hsp90 inhibitor drugs now in cancer clinic trials is that they potently activate Hsf1. This leads to an induction of heat shock proteins, many of which have a "pro-survival" role in that they help to protect cells from apopotosis. As the activation of Hsf1 requires TORC1, inhibitors of mTOR kinase could potentially block this activation of Hsf1 and be of value when used in combination drug therapies with Hsp90 inhibitors. However many of the mechanistic details of the TORC1 regulation of Hsf1, as well as the interplay between cellular resistances to rapamycin and to Hsp90 inhibitors, still remain to be resolved.

Role of mammalian target of rapamycin in ischemia‐reperfusion induced kidney injury (692.11)

mTOR has anti‐inflammatory effect and inhibition of mTOR initiates IL‐12 and IL‐6 production leading to inflammatory response. However; it is not fully known whether mTOR attenuates the inflammation in renal ischemia ‐reperfusion (I/R). We hypothesized that activation of mTOR suppresses the inflammatory response induced by I/R in the kidney. In rats subjected to I/R injury (25 min renal artery occlusion) in the presence or absence of rapamycin, mTOR inhibitor, or Clenbuterol, mTOR activator, markers of renal function, injury and inflammation were measured. In euvolemic anesthetized rats, Rapamycin increased blood pressure (142 ± 5 vs. 120 ± 3mmHg; p&lt;0.05), decreased glomerular filtration rate (2 ± 0.3 to 0.6 ± 0.4 ml/min; p&lt;0.05) and increased urinary sodium excretion (15 ± 2 vs. 107 ± 42 mM/hr; P&lt;0.05).In IR rats (vehicle‐treated), serum creatinine tended to increase (0.5+0.17 vs.1.3+0.1; p&gt;0.05). Rapamycin increased serum creatinine (1.3 ± 0.1 vs. 5 ± 1.6 mg/dl; p&lt;0.05) with no significant difference in serum IL‐6. Clenbuterol tended to antagonize the effect of rapamycin on serum creatinine (3.2 ± 1.6 vs. 5 ± 1.6 mg/dl; p&gt;0.05). In conclusion, mTOR does not appear to play a role in the inflammatory response in renal I/R. However, mTOR plays a role in renal injury via an IL‐6 independent pathway.

Rapamycin Resistant Murine Th9 Cells Have a Stable In Vivo Phenotype and Inhibit Graft-Versus-Host Reactivity

The cytokine micro-environment can direct murine CD4+ T cells towards various differentiation lineages such as Th1, Th2 and Tregs even in the presence of rapamycin, which results in T cells that mediate increased in vivo effects. Recently, a new lineage of T cells known as Th9 cells that secrete increased IL-9 have been described. However, it is not known whether Th9 differentiation occurs in the presence of rapamycin or whether adoptively transferred donor Th9 cells would augment or restrict alloreactivity after experimental bone marrow transplantation. We found that CD4+ T cells that were co-stimulated and polarized with TGF-β and IL-4 in the presence or absence of rapamycin each yielded effector cells of Th9 phenotype that secreted increased IL-9 and expressed a transcription factor profile characteristic of both Th9 and Th2 cells (high GATA-3/low T-bet). Augmentation of T cell replete allografts with manufactured rapamycin resistant Th9 cells markedly reduced both CD4+ and CD8+ T cell engraftment and strongly inhibited allo-specific T cell secretion of IFN-γ. The potency of Th9 cell inhibition of alloreactivity was similar to that of rapamycin resistant Th2 cells. Importantly, rapamycin resistant Th9 cells persisted and maintained their cytokine phenotype, thereby indicating limited differentiation plasticity of the Th9 subset. As such, Th9 differentiation proceeds in the presence of rapamycin to generate a cell therapy product that maintains high IL-9 expression in vivo while inhibiting IFN-γ driven alloreactivity.

Comparison of Regulatory T Cells in Hemodialysis Patients and Healthy Controls

Cell-based therapy with natural (CD4(+)CD25(hi)CD127(lo)) regulatory T cells to induce transplant tolerance is now technically feasible. However, regulatory T cells from hemodialysis patients awaiting transplantation may be functionally/numerically defective. Human regulatory T cells are also heterogeneous, and some are able to convert to proinflammatory Th17 cells. This study addresses the suitability of regulatory T cells from hemodialysis patients for cell-based therapy in preparation for the first clinical trials in renal transplant recipients (the ONE Study). Healthy controls and age- and sex-matched hemodialysis patients without recent illness/autoimmune disease on established, complication-free hemodialysis for a minimum of 6 months were recruited. Circulating regulatory T cells were studied by flow cytometry to compare the regulatory T cell subpopulations. Regulatory T cells from members of each group were compared for suppressive function and plasticity (IL-17-producing capacity) before and after in vitro expansion with and without Rapamycin, using standard assays. Both groups had similar total regulatory T cells and subpopulations I and III. In each subpopulation, regulatory T cells expressed similar levels of the function-associated markers CD27, CD39, HLA-DR, and FOXP3. Hemodialysis regulatory T cells were less suppressive, expanded poorly compared with healthy control regulatory T cells, and produced IL-17 in the absence of Rapamycin. However, Rapamycin efficiently expanded hemodialysis regulatory T cells to a functional and stable cell product. Rapamycin-based expansion protocols should enable clinical trials of cell-based immunotherapy for the induction of tolerance to renal allografts using hemodialysis regulatory T cells.

Molecular cloning and expression analyses of porcine MAP1LC3A in the granulosa cells of normal and miniature pig

BackgroundThe members of the microtubule-associated protein 1 light chain (MAP1LC) family, especially those of the LC3 family (MAP1LC3A, B, C), are known to induce autophagy upon localization onto the autophagosomal membrane. In this regard, LC3 can be utilized as a marker for the formation of autophagosomes during the process of autophagy. The aims of this study are to clone porcine MAP1LC3A, and analyze the pattern of its expression in the ovarian tissues of normal and miniature pig ovary in an attempt to understand the distinct mode of apoptosis between two strains.MethodsRapid amplification of cDNA ends (RACE) were used to obtain the 5′ and 3′ ends of the porcine MAP1LC3A full length cDNA. Reverse-transcriptase-PCR (RT-PCR), real-time PCR, and western blot analysis were performed to examine the expression of porcine MAP1LC3A. The localization of MAP1LC3A in the ovary was determined by In situ Hybridization and Immunohistochemical staining.ResultsWe cloned the full-length cDNA of porcine MAP1LC3A and identified an open reading frame of 980 bp encoding 121 amino acids. Based on its homology to known mammalian proteins (98%) this novel cDNA was designated as porcine MAP1LC3A and registered to the GenBank (Accession No. GU272221). We compared the expression of MAP1LC3A in the Graafian follicles of normal and miniature pigs by in situ hybridization at day 15 of the estrus cycle. While normal pigs showed a stronger expression of MAP1LC3A mRNA than miniature pigs in the theca cell area, the expression was lower in the granulosa cells. Immunofluorescence analysis of the MAP1LC3A fusion reporter protein showed the subcellular localization of porcine MAP1LC3A and ATG5 as a punctate pattern in the cytoplasm of porcine granulosa cells under stress conditions. In addition, the expressions of MAP1LC3A and ATG5 were higher in normal pigs than in miniature pigs both in the presence and absence of rapamycin.ConclusionsThe newly cloned porcine MAP1LC3A provides a novel autophagosomal marker in both normal and miniature pig. We demonstrated that the expression of MAP1LC3A in graafian follicle is distinct in normal and miniature pig, which may explain the unique folliculogenesis of miniature pigs.

Regulation of the glutamate transporter EAAT3 by mammalian target of rapamycin mTOR

The serine/threonine kinase mammalian target of rapamycin (mTOR) is stimulated by insulin, growth factors and nutrients and confers survival of several cell types. The kinase has previously been shown to stimulate amino acid uptake. In neurons, the cellular uptake of glutamate by the excitatory amino-acid transporters (EAATs) decreases excitation and thus confers protection against excitotoxicity. In epithelia, EAAT3 accomplishes transepithelial glutamate and aspartate transport. The present study explored, whether mTOR regulates EAAT3 (SLC1A1). To this end, cRNA encoding EAAT3 was injected into Xenopus oocytes with or without cRNA encoding mTOR and the glutamate induced current (Iglu), a measure of glutamate transport, determined by dual electrode voltage clamp. Moreover, EAAT3 protein abundance was determined utilizing chemiluminescence. As a result, Iglu was observed in Xenopus oocytes expressing EAAT3 but not in water injected oocytes. Coexpression of mTOR significantly increased Iglu, an effect reversed by rapamycin (100nM). mTOR coexpression increased EAAT3 protein abundance in the cell membrane. The decay of Iglu following inhibition of carrier insertion with brefeldin A in oocytes coexpressing EAAT3 with mTOR was similar in the presence and absence of rapamycin (100nM). In conclusion, mTOR is a novel powerful regulator of EAAT3 and may thus contribute to protection against neuroexcitotoxicity.

Regulatory T cells expanded by rapamycin in vitro suppress colitis in an experimental mouse model

To provide rapid immunosuppression without side effects, we analyzed whether rapamycin alone, and regulatory T cells (Tregs) expanded ex vivo by rapamycin, suppressed colitis in a mouse model. Severe combined immunodeficiency (SCID) mice reconstituted with naive CD4(+) T cells were treated with or without intraperitoneal rapamycin. Body weight was evaluated. CD4(+) T cells were cultured in the presence of rapamycin for three 7-day rounds of stimulation. The ratio of Tregs to CD4(+) T cells was analyzed by flow cytometry. Naive CD4(+) T cells were transferred into SCID mice with CD4(+) T cells expanded in the presence or absence of rapamycin. Clinical symptoms of colitis, histological changes, and cytokine expression were investigated. Systemic rapamycin partially prevented the development of colonic inflammation in a transfer model of colitis, but decreased body weight in control mice. With rapamycin, stimulated CD4(+) T cells expanded eightfold in 3 weeks in vitro, and the proportion of Tregs increased to about 40%. Without rapamycin, CD4(+) T cells expanded 20-fold in 3 weeks, but the proportion of Tregs remained at about 15%. CD4(+) T cells expanded with rapamycin prevented the development of colitis in a naïve CD4(+) T-cell transfer model, in association with the downregulation of Th1 and Th17 responses. We demonstrated, for the first time, that CD4(+) T cells expanded with rapamycin in vitro suppressed colitis. Therefore, rapamycin-expanded Treg transfer therapy is expected to be efficacious for inflammatory bowel disease.

S35 Rapamycin inhibits IL-33-induced, nuocyte-driven airway inflammation

Introduction IL-33 is an innate cytokine that promotes Th2 responses in both the innate and the adaptive immune systems, with an established role in allergic airway inflammation.1 The signalling pathway...

Liver-specific Inducible Nitric-oxide Synthase Expression Is Sufficient to Cause Hepatic Insulin Resistance and Mild Hyperglycemia in Mice

Inducible nitric-oxide synthase (iNOS), a major mediator of inflammation, plays an important role in obesity-induced insulin resistance. Inhibition of iNOS by gene disruption or pharmacological inhibitors reverses or ameliorates obesity-induced insulin resistance in skeletal muscle and liver in mice. It is unknown, however, whether increased expression of iNOS is sufficient to cause insulin resistance in vivo. To address this issue, we generated liver-specific iNOS transgenic (L-iNOS-Tg) mice, where expression of the transgene, iNOS, is regulated under mouse albumin promoter. L-iNOS-Tg mice exhibited mild hyperglycemia, hyperinsulinemia, insulin resistance, and impaired insulin-induced suppression of hepatic glucose output, as compared with wild type (WT) littermates. Insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1) and -2, and Akt was significantly attenuated in liver, but not in skeletal muscle, of L-iNOS-Tg mice relative to WT mice without changes in insulin receptor phosphorylation. Moreover, liver-specific iNOS expression abrogated insulin-stimulated phosphorylation of glycogen synthase kinase-3β, forkhead box O1, and mTOR (mammalian target of rapamycin), endogenous substrates of Akt, along with increased S-nitrosylation of Akt relative to WT mice. However, the expression of insulin receptor, IRS-1, IRS-2, Akt, glycogen synthase kinase-3β, forkhead box O1, protein-tyrosine phosphatase-1B, PTEN (phosphatase and tensin homolog), and p85 phosphatidylinositol 3-kinase was not altered by iNOS transgene. Hyperglycemia was associated with elevated glycogen phosphorylase activity and decreased glycogen synthase activity in the liver of L-iNOS-Tg mice, whereas phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and proliferator-activated receptor γ coactivator-1α expression were not altered. These results clearly indicate that selective expression of iNOS in liver causes hepatic insulin resistance along with deranged insulin signaling, leading to hyperglycemia and hyperinsulinemia. Our data highlight a critical role for iNOS in the development of hepatic insulin resistance and hyperglycemia.

Characterization of the Metabolic Phenotype of Rapamycin-Treated CD8+ T Cells with Augmented Ability to Generate Long-Lasting Memory Cells

BackgroundCellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized.Design and MethodsUsing lymphocytic choriomeningitis virus (LCMV) peptide gp33-specific CD8+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo.ResultsAntigen-activated CD8+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells.ConclusionsOur findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

Stability of human rapamycin-expanded CD4+CD25+ T regulatory cells

The clinical use of ex vivo-expanded T-regulatory cells for the treatment of T-cell-mediated diseases has gained increasing momentum. However, the recent demonstration that FOXP3(+) T-regulatory cells may contain interleukin-17-producing cells and that they can convert into effector cells once transferred in vivo raises significant doubts about their safety. We previously showed that rapamycin permits the ex vivo expansion of FOXP3(+) T-regulatory cells while impairing the proliferation of non-T-regulatory cells. Here we investigated the Th17-cell content and the in vivo stability of rapamycin-expanded T-regulatory cells as pertinent aspects of cell-based therapy. T-regulatory-enriched cells were isolated from healthy volunteers and were expanded ex vivo with rapamycin with a pre-clinical applicable protocol. T-regulatory cells cultured with and without rapamycin were compared for their regulatory activity, content of pro-inflammatory cells and stability. We found that CD4(+)CCR6(+)CD161(+) T cells (i.e., precursor/committed Th17 cells) contaminate the T-regulatory cells cultured ex vivo in the absence of rapamycin. In addition, Th17 cells do not expand when rapamycin-treated T-regulatory cells are exposed to a "Th17-favorable" environment. Rapamycin-expanded T-regulatory cells maintain their in vitro regulatory phenotype even after in vivo transfer into immunodeficient NOD-SCID mice despite being exposed to the irradiation-induced pro-inflammatory environment. Importantly, no additional rapamycin treatment, either in vitro or in vivo, is required to keep their phenotype fixed. These data demonstrate that rapamycin secures ex vivo-expanded human T-regulatory cells and provide additional justification for their clinical use in future cell therapy-based trials.

Resistin Promotes Cardiac Hypertrophy via the AMP-activated Protein Kinase/Mammalian Target of Rapamycin (AMPK/mTOR) and c-Jun N-terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathways

Resistin has been suggested to be involved in the development of diabetes and insulin resistance. We recently reported that resistin is expressed in diabetic hearts and promotes cardiac hypertrophy; however, the mechanisms underlying this process are currently unknown. Therefore, we wanted to elucidate the mechanisms associated with resistin-induced cardiac hypertrophy and myocardial insulin resistance. Overexpression of resistin using adenoviral vector in neonatal rat ventricular myocytes was associated with inhibition of AMP-activated protein kinase (AMPK) activity, activation of tuberous sclerosis complex 2/mammalian target of rapamycin (mTOR) pathway, and increased cell size, [(3)H]leucine incorporation (i.e. protein synthesis) and mRNA expression of the hypertrophic marker genes, atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain. Activation of AMPK with 5-aminoimidazole-4-carbozamide-1-β-D-ribifuranoside or inhibition of mTOR with rapamycin or mTOR siRNA attenuated these resistin-induced changes. Furthermore, resistin increased serine phosphorylation of insulin receptor substrate (IRS1) through the activation of the apoptosis signal-regulating kinase 1/c-Jun N-terminal Kinase (JNK) pathway, a module known to stimulate insulin resistance. Inhibition of JNK (with JNK inhibitor SP600125 or using dominant-negative JNK) reduced serine 307 phosphorylation of IRS1. Resistin also stimulated the activation of p70(S6K), a downstream kinase target of mTOR, and increased phosphorylation of the IRS1 serine 636/639 residues, whereas treatment with rapamycin reduced the phosphorylation of these residues. Interestingly, these in vitro signaling pathways were also operative in vivo in ventricular tissues from adult rat hearts overexpressing resistin. These data demonstrate that resistin induces cardiac hypertrophy and myocardial insulin resistance, possibly via the AMPK/mTOR/p70(S6K) and apoptosis signal-regulating kinase 1/JNK/IRS1 pathways.

Regulation of alternative splicing of skeletal muscle Troponin T in C2C212 myotubes in response to cyclic strain and manipulation of mTORC1 signaling

Experimental manipulation of body weight in rats causes precise adjustments to the pattern of fast skeletal muscle troponin T (TNNT3) alternative splicing, so that it matches that of animals with actual body weight equivalent to that of the weight-manipulated rats. In this study, we examined muscle cell-autonomous TNNT3 alternative splicing responses to increased strain, and the potential role of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway in mediating these responses. To this end, C2C12 myotubes were exposed to intermittent cyclic strain (0.5Hz, 15%) for 24–48 hours, in the presence or absence of rapamycin. After treatment, we measured the relative abundance of individual TNNT3 splice forms in the total TNNT3 mRNA mixture, and quantified phosphorylation of S6K1, a downstream effector of mTORC1. Experimentally imposed strain caused significant alterations to the relative abundance of TNNT3 splice forms that correlated with observed changes in mTORC1 signaling. Rapamycin treatment caused effects that either were independent of, or attenuated specific TNNT3 splice form responses to imposed strain regimes. To our knowledge, these findings are the first to demonstrate effects of cyclic strain on TNNT3 alternative splicing in muscle cell culture, and suggest a modulatory role of mTORC1 signaling in the regulation of TNNT3 alternative splicing in vivo. (Supported by NIH grant DK-15658)