Gudrun Strauss

A New Missense Mutation in the Leptin Gene Causes Mild Obesity and Hypogonadism without Affecting T Cell Responsiveness

OBJECTIVE Leptin, a protein product of adipocytes, plays a critical role in the regulation of body weight, immune function, pubertal development, and fertility. So far, only three homozygous mutations in the leptin gene in a total of 13 individuals have been found leading to a phenotype of extreme obesity with marked hyperphagia and impaired immune function. DESIGN Serum leptin was measured by ELISA. The leptin gene (OB) was sequenced in patient DNA. The effect of the identified novel mutation was assessed using HEK293 cells. RESULTS We describe a 14-yr-old child of nonobese Austrian parents without known consanguinity. She had a body mass index of 31.5 kg/m(2) (+2.46 SD score) and undetectable leptin serum levels. Sequencing of the leptin gene revealed a hitherto unknown homozygous transition (TTA to TCA) in exon 3 of the LEP gene resulting in a L72S replacement in the leptin protein. RT-PCR, Western blot, and immunohistochemical analysis indicated that the mutant leptin was expressed in the patients adipose tissue but retained within the cell. Using a heterologous cell system, we confirmed this finding and demonstrated that the side chain of Leu72 is crucial for intracellular leptin trafficking. Our patient showed signs of a hypogonadotropic hypogonadism. However, in contrast to the literature, she showed only mild obesity and a normal T cell responsiveness. CONCLUSIONS These findings shed a new light on the clinical consequences of leptin deficiency. Congenital leptin deficiency should be considered possible in pediatric patients with mild obesity even if parents are lean and unrelated.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While ‘classical’ apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.

TRAIL (TNF-related apoptosis-inducing ligand) regulates adipocyte metabolism by caspase-mediated cleavage of PPARgamma

Tumor necrosis factor α (TNFα) and other members of the TNF family affect adipose tissue metabolism and contribute to the obesity-related inflammation of adipose tissue. Here, we sought to identify the effects of TRAIL (TNF-related apoptosis-inducing ligand) on fat cell biology. TRAIL-receptor 2 (TRAIL-R2) and its mouse homolog DR5 were regulated upon acute and chronic energy imbalance in murine and human adipose tissue. TRAIL inhibited insulin-stimulated glucose uptake and de novo lipogenesis in human adipocytes. Interestingly, TRAIL did not interfere with the phosphorylation of insulin-stimulated kinases such as Akt or Erk and did not activate the NF-κB pathway. Instead, TRAIL activated cleavage of caspase-8 and caspase-3. The subsequent cleavage of PPARγ led to its inactivation and resulted in reduced expression of lipogenic genes, such as Glut-4, FASN, and ACC. Taken together, we discovered a so far unknown function of the death ligand TRAIL in regulating adipocyte metabolism. Our results imply that TRAIL/TRAIL-R system might provide a new target for the prevention and treatment of obesity and its co-morbidities.

In vitro-generated MDSCs prevent murine GVHD by inducing type 2 T cells without disabling antitumor cytotoxicity

Myeloid-derived suppressor cells (MDSCs) inhibit T-cell expansion and functions by versatile mechanisms such as nutrient depletion, nitrosylation, or apoptosis. Since graft-versus-host disease (GVHD) is characterized by the expansion of donor-derived T cells destroying recipient tissue, we analyzed whether MDSCs can be used for GVHD prevention in murine allogeneic bone marrow transplantation models. Transplantation of MDSCs, generated from bone marrow cells by granulocyte-macrophage colony-stimulating factor (GM-CSF)/G-CSF in vitro, inhibited GVHD-induced death and attenuated histologic GVHD, whereas antitumor cytotoxicity of alloantigen-specific T cells was maintained. MDSCs expanded in vivo and invaded lymphatic and GVHD target organs. Major histocompatibility complex class I expression on MDSCs was dispensable for their suppressive capacity. Inhibition of GVHD required the presence of MDSCs during T-cell priming, whereas allogeneic T-cell numbers and homing in lymphoid and GVHD target organs were not considerably affected in MDSC-treated mice. However, MDSCs skewed allogeneic T cells toward type 2 T cells upregulating T helper 2 (Th2)-specific cytokines. Type 2 T-cell induction was indispensable for GVHD prevention since MDSC treatment failed to prevent GVHD when allogeneic STAT6-deficient T cells, which are unable to differentiate into Th2 cells, were transplanted. MDSC-induced Th2 induction might be applicable for GVHD treatment in clinical settings.

CD95 co-stimulation blocks activation of naive T cells by inhibiting T cell receptor signaling

1. 1. Strauss, 2. et al . 2009. J. Exp. Med. doi: 10.1084/jem.20082363 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20082363%26rft_id%253Dinfo%253Apmid%252F19487421%26rft.genre%253Darticle%26rft_val_fmt%

Downregulation of FLIP by cycloheximide sensitizes human fat cells to CD95-induced apoptosis.

Adipocyte apoptosis is an important regulator of adipocyte number in fat depots. We have previously shown that an inhibition of protein synthesis sensitizes human adipocytes for apoptosis. In vivo, dramatic changes in the fat cells protein expression should be anticipated under special conditions such as calorie restriction. Here, we studied the underlying mechanism by which human preadipocytes and adipocytes are sensitized for death receptor induced apoptosis in vitro. The protein synthesis blocker cycloheximide (CHX) sensitized human fat cells for CD95-induced apoptosis in a caspase-dependent manner. Treatment with CHX differentially changed expression of pro- and anti-apoptotic proteins. Most noticeably, FLICE-like inhibitory protein (FLIP) expression rapidly decreased during CHX treatment. Reduction of FLIP levels resulted in undetectable amounts of FLIP at the CD95 death-inducing signaling complex (DISC) upon CD95 stimulation, thereby enhancing recruitment and activation at caspase-8. Down-regulation of FLIP by shRNA sensitized preadipocytes for CD95-induced apoptosis. In mice, adipose tissue mRNA levels of Flip were down-regulated upon fasting. In conclusion, we identify FLIP as an important regulator of apoptosis sensitivity in fat cells. Modulating adipocyte homeostasis by apoptosis might provide a new therapeutic concept to get rid of excess adipose tissue, and FLIP might be a possible target molecule.

Membrane-bound CD95 ligand expressed on human antigen-presenting cells prevents alloantigen-specific T cell response without impairment of viral and third-party T cell immunity.

Genetically modified antigen-presenting cells (APC) represent an attractive strategy for in vitro immunomodulation. In the human system, APC expressing HLA-A1 and a membrane-bound form of CD95L (m-CD95L) were used for selective depletion of HLA-A1-specific T cells. In short-term assays, m-CD95L-expressing APC-induced apoptosis in activated T cells and the constitutive presence of m-CD95L and HLA-A1 expressing APC in long-term T cell cultures prevented the expansion of CD4+ and CD8+ HLA-A1-specifc T cells and the development of HLA-A1-specific cytotoxicity. However, immunity towards third party, viral and bacterial antigens was maintained and T cells spared from depletion could be induced to develop cytotoxicity towards unrelated antigens. Interestingly, inhibition of HLA-A1-specific T cell response absolutely requires the coexpression of m-CD95L and HLA-A1 antigen on the same APC. Thus, m-CD95L expressing APC might be used in clinical settings to obtain tolerance induction in allogeneic transplantation systems or autoimmune diseases.

Distinct Effects of IL-6 Classic and Trans-Signaling in Bone Fracture Healing

Bone healing is a complex process with closely linked phases of inflammation, regeneration, and remodeling. IL-6 may crucially regulate this process; however, the underlying mechanisms are unclear. IL-6 signals are transmitted via the transmembrane glycoprotein 130 by two distinct mechanisms: classic signaling using the membrane-anchored IL-6 receptor and trans-signaling using its soluble form. Herein, we investigated the hypothesis that IL-6 classic and trans-signaling have different functions during bone healing. To investigate fracture healing, 12-week-old C57BL/6J mice underwent a femur osteotomy. To study the function of IL-6 during the inflammatory phase, either an anti-IL-6 antibody, which inhibits IL-6 classic and trans-signaling, or soluble glycoprotein 130 fusion protein, which selectively blocks trans-signaling, was injected after 30 minutes and 48 hours. To analyze IL-6 effects in the repair phase, compounds were injected from day 7 onwards. Global IL-6 inhibition in the early phase after fracture reduced systemic inflammation, the recruitment of immune cells, and bone regeneration, resulting in delayed fracture healing. Global IL-6 inhibition during the repair phase disturbed bone formation and remodeling. In contrast, inhibition of IL-6 trans-signaling exerted minor effects on the immune response and did not influence bone repair, suggesting that the classic pathway accounts for most of the effects observed after global IL-6 inhibition. Our results reveal that IL-6 classic signaling, but not IL-6 trans-signaling, is essential for bone repair.

A paired comparison between glioblastoma "stem cells" and differentiated cells.

Cancer stem cells (CSC) have been postulated to be responsible for the key features of a malignancy and its maintenances, as well as therapy resistance, while differentiated cells are believed to make up the rapidly growing tumour bulk. It is therefore important to understand the characteristics of those two distinct cell populations in order to devise treatment strategies which effectively target both cohorts, in particular with respect to cancers, such as glioblastoma. Glioblastoma is the most common primary brain tumour in adults, with a mean patient survival of 12–15 months. Importantly, therapeutic improvements have not been forthcoming in the last decade. In this study we compare key features of three pairs of glioblastoma cell populations, each pair consisting of stem cell‐like and differentiated cells derived from an individual patient. Our data suggest that while growth rates and expression of key survival‐ and apoptosis‐mediating proteins are more similar according to differentiation status than genetic similarity, we found no intrinsic differences in response to standard therapeutic interventions, namely exposure to radiation or the alkylating agent temozolomide. Interestingly, we could demonstrate that both stem cell‐like and differentiated cells possess the ability to form stem cell‐containing tumours in immunocompromised mice and that differentiated cells could potentially be dedifferentiated to potential stem cells. Taken together our data suggest that the differences between tumour stem cell and differentiated cell are particular fluent in glioblastoma.

TRAIL–Receptor Costimulation Inhibits Proximal TCR Signaling and Suppresses Human T Cell Activation and Proliferation

The TRAIL–receptor/TRAIL system originally described to induce apoptosis preferentially in malignant cells is also known to be involved in T cell homeostasis and the response to viral infections and autoimmune diseases. Whereas the expression of TRAIL on activated NK and T cells increases their cytotoxicity, induction of TRAIL on APCs can turn them into apoptosis inducers but might also change their immunostimulatory capacity. Therefore, we analyzed how TRAIL–receptor (TRAIL–R) costimulation is modulating TCR-mediated activation of human T cells. T cells triggered by rTRAIL in combination with anti-CD3 and -CD28 Abs exhibited a strong decrease in the expression of activation markers and Th1 and Th2 cytokines compared with CD3/CD28-activated T cells. Most importantly, proliferation of TRAIL–R costimulated T cells was strongly impaired, but no apoptosis was induced. Addition of exogenous IL-2 could not rescue T cells silenced by TRAIL–R costimulation, and TRAIL-mediated inhibition of T cell proliferation only prevented TCR-triggered proliferation but was ineffective if T cells were activated downstream of the TCR. Inhibition of T cell proliferation was associated with abrogation of proximal TCR signaling by inhibiting recruitment of TCR-associated signaling molecules to lipid rafts, followed by abrogation of protein tyrosine phosphorylation of ZAP70, phospholipase C-γ1, and protein kinase C-θ, and impaired nuclear translocation of NFAT, AP-1, and NF-κB. Most importantly, TRAIL–R costimulation efficiently inhibited alloantigen-induced T cell proliferation and CD3/28-induced activation and proliferation of autoreactive T cells derived from patients with Omenn syndrome, indicating that coactivation of TRAIL–R and TCR represents a mechanism to downmodulate T cell immune responses.