René Geyeregger

A Versatile Role of Mammalian Target of Rapamycin in Human Dendritic Cell Function and Differentiation

The mammalian target of rapamycin (mTOR) regulates cell growth and survival and exists as rapamycin-sensitive mTOR complex (mTORC) 1 and as rapamycin-insensitive mTORC2. Although mTOR is a well-known regulator of diverse immune cells, its detailed role in human dendritic cell (DC) function and differentiation is only incompletely understood. In this study, we demonstrate divergent roles of mTOR during activation and differentiation of myeloid DCs (mDCs) and monocyte-derived DCs (moDCs). Inhibition of mTORC1 in mDCs activated with TLR-dependent or -independent stimuli increased proinflammatory cytokines and NF-κB, whereas IL-10 and STAT3 were blocked. Rapamycin regulated the costimulatory/surface molecules CD86, programmed death ligand-1, and CD25 on mDCs and significantly increased the T cell allostimulatory potential of mDCs. In contrast, rapamycin suppressed immunostimulatory molecules and the allostimulatory potential of LPS-stimulated moDCs by an inability to augment NF-κB signaling. In differentiating moDCs, the PI3K/Akt-dependent mTOR pathway was constitutively activated by GM-CSF to induce DC differentiation in an mTORC1-dependent manner. Inhibition of mTORC1 or mTORC1/2 during moDC differentiation decreased moDC survival and markedly hampered its immunostimulatory phenotype. Analyzing the fate of DCs in vivo, we found that kidney transplant patients treated with rapamycin displayed an increased immunostimulatory potential of mDCs compared with patients treated with calcineurin inhibitors. Furthermore, rapamycin did not interfere with mDC differentiation in these patients. Collectively, mTOR exerts divergent immunoregulatory functions during DC activation and differentiation depending on the DC type that lead to opposing T cell responses, which might be of clinical importance in transplantation, cancer, and also for novel vaccination strategies.

Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin Resistance

OBJECTIVE Obesity is associated with a state of chronic low-grade inflammation mediated by immune cells that are primarily located to adipose tissue and liver. The chronic inflammatory response appears to underlie obesity-induced metabolic deterioration including insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine, the expression of which is strongly upregulated in adipose tissue and liver upon obesity. Here, we studied OPN effects in obesity-induced inflammation and insulin resistance by targeting OPN action in vivo. RESEARCH DESIGN AND METHODS C57BL/6J mice were fed a high-fat diet to induce obesity and were then intravenously treated with an OPN-neutralizing or control antibody. Insulin sensitivity and inflammatory alterations in adipose tissue and liver were assessed. RESULTS Interference with OPN action by a neutralizing antibody for 5 days significantly improved insulin sensitivity in diet-induced obese mice. Anti-OPN treatment attenuated liver and adipose tissue macrophage infiltration and inflammatory gene expression by increasing macrophage apoptosis and significantly reducing c-Jun NH2-terminal kinase activation. Moreover, we report OPN as a novel negative regulator for the activation of hepatic signal transducer and activator of transcription 3 (STAT3), which is essential for glucose homeostasis and insulin sensitivity. Consequently, OPN neutralization decreased expression of hepatic gluconeogenic markers, which are targets of STAT3-mediated downregulation. CONCLUSIONS These findings demonstrate that antibody-mediated neutralization of OPN action significantly reduces insulin resistance in obesity. OPN neutralization partially decreases obesity-associated inflammation in adipose tissue and liver and reverses signal transduction related to insulin resistance and glucose homeostasis. Hence, targeting OPN could provide a novel approach for the treatment of obesity-related metabolic disorders.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids

Objective:Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue.Methods and procedure:Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence.Results:HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes.Conclusion:N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.

Oxytocin alleviates the neuroendocrine and cytokine response to bacterial endotoxin in healthy men.

Oxytocin is a hormone and neurotransmitter found to have anti-inflammatory functions in rodents. Here we used experimental bacterial endotoxinemia to examine the role of exogenous oxytocin administration on innate immune responses in humans. Ten healthy men received, in a randomized, placebo-controlled, crossover design, placebo, oxytocin, LPS, and LPS + oxytocin. Oxytocin treatment resulted in a transient or prolonged reduction of endotoxin-induced increases in plasma ACTH, cortisol, procalcitonin, TNF-alpha, IL-1 receptor antagonist, IL-4, IL-6, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, monocyte chemoattractant protein-1 (MCP-1), interferon-inducible protein 10, and VEGF. In vitro, oxytocin had no impact on LPS effects in releasing TNF-alpha, IL-6, and MCP-1 in monocytes and peripheral blood mononuclear cells from healthy human donors. In summary, oxytocin decreases the neuroendocrine and cytokine activation caused by bacterial endotoxin in men, possibly due to the pharmacological modulation of the cholinergic anti-inflammatory pathway. Oxytocin might be a candidate for the therapy of inflammatory diseases and conditions associated with high cytokine and VEGF levels.

Suppression of T cell signaling by polyunsaturated fatty acids: Selectivity in inhibition of mitogen-activated protein kinase and nuclear factor activation

Polyunsaturated fatty acids (PUFAs) are known to suppress inflammatory and autoimmune responses and, therefore, clinical applications of PUFAs as immunomodulatory substances are extensively studied. PUFAs are known to inhibit T cell responses, but with respect to TCR/CD3-mediated signal transduction only a block in CD3-induced phospholipase Cγ1/calcium signaling has been shown so far. In this study, we investigated PUFA-mediated changes in downstream T cell signal transduction. We show that among the mitogen-activated protein kinase families activation of c-Jun NH2-terminal kinase, but not phosphorylation of extracellular signal-regulated kinase-1/-2 or p38 is inhibited. CD3/CD28-induced activity of NF-AT was markedly reduced by PUFA treatment, while activation of other nuclear receptors (AP-1 and NF-κB) remained unaltered. Furthermore, IL-2 promoter activity, IL-2 and IL-13 mRNA levels, IL-2 secretion, and IL-2R α-chain expression were significantly diminished by PUFA treatment, whereas the expression of IFN-γ, IL-4, IL-10, and CD69 remained essentially unaffected by PUFAs. In conclusion, PUFA treatment of T cells inhibits selectively c-Jun NH2-terminal kinase and NF-AT activation, resulting in diminished production of IL-2 and IL-13.

Liver X receptors in cardiovascular and metabolic disease.

Abstract.Liver X receptors (LXRs) α and β are nuclear oxysterol receptors and metabolic sensors initially found to regulate cholesterol metabolism and lipid biosynthesis. Recent studies have elucidated the importance of LXR in the development of cardiovascular diseases and metabolic disorders. LXR agonists prevent development of atherosclerosis by modulation of metabolic as well as inflammatory gene expression in rodent models. Moreover, LXR activation inhibits hepatic gluconeogenesis and lowers serum glucose levels, indicating possible application of LXR activation in the treatment of diabetes mellitus. However, first-generation LXR agonists elevate hepatic and serum trigylceride levels, making subtype-specific agonists and selective LXR modulators rather than unselective LXR agonists a potential pharmacological strategy. This review summarizes the multiple physiological and pathophysiological implications of LXRs and observations that identify LXRs as potential targets for therapeutic interventions in human cardiovascular and metabolic disease.

Effect of the proteasome inhibitor bortezomib on humoral immunity in two presensitized renal transplant candidates.

Background. Recipient presensitization represents a major hurdle to successful renal transplantation. Previous case series have suggested that the proteasome inhibitor bortezomib directly affects the alloantibody-secreting plasma cells in rejecting allograft recipients. However, the ability of this agent to desensitize nonimmunosuppressed transplant candidates before transplantation is currently unknown. Methods. In this analysis, two sensitized hemodialysis patients were selected to receive two subsequent bortezomib cycles. Bortezomib was given at 1.3 mg/m2 on days 1, 4, 8, and 11. Dexamethasone was added to the second cycle to enhance treatment efficiency. Serial immune monitoring included cytotoxic panel reactive antibody testing, Luminex single antigen testing for anti-human leukocyte antigen (HLA) IgG with or without C4d-fixing capability, and ABO antibody detection. Results. During a half-year follow-up period, cytotoxic panel reactive antibody decreased from 87% to 80% (patient 1) and 37% to 13% (patient 2). Patient 1 showed a 40% reduction in binding intensities of identified Luminex HLA single antigen reactivities and, in parallel, slight reductions in ABO blood group antibody and total immunoglobulin levels. In patient 2, bortezomib did not affect circulating antibody levels in a meaningful way. Both patients showed a more than 50% reduction in the levels of anti-HLA antibody-triggered C4d deposition to Luminex beads. Conclusion. Our initial experience suggests that, without additional immunosuppressive measures, bortezomib has modest effects on circulating antibodies against HLA or blood group antigens. The reduced levels of antibody-triggered complement fixation, however, imply potential clinical relevance of proteasome inhibition for recipient desensitization.

Polyunsaturated fatty acids interfere with formation of the immunological synapse.

Polyunsaturated fatty acids (PUAs) exert inhibitory effects on T cell‐mediated immune responses. Activation of T cells in vivo depends on formation of an immunological synapse (IS) at the T cell/antigen‐presenting cell (APC) interface. Here, we analyzed effects of PUFA treatment on the formation of the IS and APC‐induced human T cell activation. In T cells treated with the PUFA eicosapentaenoic (EPA; 20:5,n‐3) and arachidonic acid (20:4,n‐6), stimulated by superantigen‐presenting cells or APCs, relocalization to the IS of distinct molecules [F‐actin, talin, leukocyte functional antigen‐1α, clusters of differentiation (CD)3ɛ] was inhibited markedly compared with cells treated with saturated fatty acid, whereas relocalization of protein kinase Cθ to the IS remained unaffected. CD3‐induced, sustained phosphorylation of nucleotide exchange factor Vav, which controls cytoskeletal rearrangements underlying IS formation, was significantly reduced in EPA‐treated Jurkat and peripheral blood T cells. In addition, T cell raft disruption by methyl‐β‐cyclodextrin treatment and experiments with a chimeric linker for activation of T cell proteins, which is resistant to PUFA effects on lipid rafts, revealed modifications of lipid rafts as a crucial factor for PUFA‐mediated inhibition of APC‐stimulated cytoskeletal rearrangements. Furthermore, the efficiency of T cell/APC conjugate formation was significantly reduced with EPA‐treated T cells, as was stimulation of CD69 expression, which is not altered following antibody‐mediated T cell activation. In conclusion, PUFA treatment of T cells qualitatively and quantitatively alters IS formation, thereby extending T cell signaling defects to pathways that are not intrinsically altered in PUFA‐treated T cells when stimulated by antibodies.

Liver X receptors interfere with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data indicate an additional role of LXR in immunity by controlling dendritic cell and T‐cell function and in breast and prostate cancer cells. Here, we show that LXR activation interferes with IL‐2 and IL‐7‐induced proliferation and cell cycle progression of human T‐cell blasts mainly through inhibited phosphorylation of the retinoblastoma protein and decreased expression of the cell cycle protein cyclin B. Comparable results were obtained with IL‐2‐dependent chronic lymphoblastic leukemia (CLL) T cells. Furthermore, we show for B‐CLL cells that LXR are functionally active and inhibit expression of survival genes bcl‐2 and MMP‐9, and significantly reduce cell viability, suggesting an interference of LXR with cytokine‐dependent CLL cell survival. In conclusion, our data reveal LXR as a potent modulator of cytokine‐dependent proliferation and survival of normal and malignant T and B lymphocytes. This novel LXR action could find clinical application in immunosuppressive and antileukemic therapies.

Clinical and Immunological Correction of DOCK8 Deficiency by Allogeneic Hematopoietic Stem Cell Transplantation Following a Reduced Toxicity Conditioning Regimen

Dedicator of cytokinesis 8 protein (DOCK8) deficiency is a combined immunodeficiency disorder characterized by an expanding clinical picture with typical features of recurrent respiratory or gastrointestinal tract infections, atopic eczema, food allergies, chronic viral infections of the skin, and blood eosinophilia often accompanied by elevated serum IgE levels. The only definitive treatment option is allogeneic hematopoietic stem cell transplantation (HSCT). We report a patient with early severe manifestation of DOCK8 deficiency, who underwent unrelated allogeneic HSCT at the age of 3 years following a reduced toxicity conditioning regimen. The transplant course was complicated by pulmonary aspergilloma pretransplantation, adenovirus (ADV) reactivation, and cytomegalovirus (CMV) pneumonitis 4 weeks after transplantation. With antifungal and antiviral treatment the patient recovered. Seven months after transplantation the patient is in excellent clinical condition. Eczematous rash, chronic viral skin infections, and food allergies have subsided, associated with normalization of IgE levels and absolute numbers of eosinophils. Chimerism analysis shows stable full donor chimerism. DOCK8 deficiency can be successfully cured by allogeneic HSCT. This treatment option should be considered early after diagnosis, as opportunistic infections and malignancies that occur more frequently during the natural course of the disease are associated with higher morbidity and mortality.