Inès Schmid

Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis.

Autophagy-related gene (Atg) 5 is a gene product required for the formation of autophagosomes. Here, we report that Atg5, in addition to the promotion of autophagy, enhances susceptibility towards apoptotic stimuli. Enforced expression of Atg5-sensitized tumour cells to anticancer drug treatment both in vitro and in vivo. In contrast, silencing the Atg5 gene with short interfering RNA (siRNA) resulted in partial resistance to chemotherapy. Apoptosis was associated with calpain-mediated Atg5 cleavage, resulting in an amino-terminal cleavage product with a relative molecular mass of 24,000 (Mr 24K). Atg5 cleavage was observed independent of the cell type and the apoptotic stimulus, suggesting that calpain activation and Atg5 cleavage are general phenomena in apoptotic cells. Truncated Atg5 translocated from the cytosol to mitochondria, associated with the anti-apoptotic molecule Bcl-xL and triggered cytochrome c release and caspase activation. Taken together, calpain-mediated Atg5 cleavage provokes apoptotic cell death, therefore, represents a molecular link between autophagy and apoptosis — a finding with potential importance for clinical anticancer therapies.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-γ–primed eosinophils to release mitochondrial DNA in a reactive oxygen species–dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner—in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Viable neutrophils release mitochondrial DNA to form neutrophil extracellular traps

Neutrophil extracellular traps (NETs) represent extracellular structures able to bind and kill microorganisms. It is believed that they are generated by neutrophils undergoing cell death, allowing these dying or dead cells to kill microbes. We show that, following priming with granulocyte/macrophage colony-stimulating factor (GM-CSF) and subsequent short-term toll-like receptor 4 (TLR4) or complement factor 5a (C5a) receptor stimulation, viable neutrophils are able to generate NETs. Strikingly, NETs formed by living cells contain mitochondrial, but no nuclear, DNA. Pharmacological or genetic approaches to block reactive oxygen species (ROS) production suggested that NET formation is ROS dependent. Moreover, neutrophil populations stimulated with GM-CSF and C5a showed increased survival compared with resting neutrophils, which did not generate NETs. In conclusion, mitochondrial DNA release by neutrophils and NET formation do not require neutrophil death and do also not limit the lifespan of these cells.

Apoptotic Pathways Are Inhibited by Leptin Receptor Activation in Neutrophils

Leptin regulates food intake as well as metabolic, endocrine, and immune functions. It exerts proliferative and antiapoptotic activities in a variety of cell types, including T cells. Leptin also stimulates macrophages and neutrophils, and its production is increased during inflammation. In this study, we demonstrate that human neutrophils express leptin surface receptors under in vitro and in vivo conditions, and that leptin delays apoptosis of mature neutrophils in vitro. The antiapoptotic effects of leptin were concentration dependent and blocked by an anti-leptin receptor mAb. The efficacy of leptin to block neutrophil apoptosis was similar to G-CSF. Using pharmacological inhibitors, we obtained evidence that leptin initiates a signaling cascade involving PI3K- and MAPK-dependent pathways in neutrophils. Moreover, leptin delayed the cleavage of Bid and Bax, the mitochondrial release of cytochrome c and second mitochondria-derived activator of caspase, as well as the activation of both caspase-8 and caspase-3 in these cells. Taken together, leptin is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in inflammatory diseases.

Inflammation-associated Cell Cycle–independent Block of Apoptosis by Survivin in Terminally Differentiated Neutrophils

Survivin has received great attention due to its expression in many human tumors and its potential as a therapeutic target in cancer. Survivin expression has been described to be cell cycle–dependent and restricted to the G2-M checkpoint, where it inhibits apoptosis in proliferating cells. In agreement with this current view, we found that survivin expression was high in immature neutrophils, which proliferate during differentiation. In contrast with immature cells, mature neutrophils contained only little or no survivin protein. Strikingly, these cells reexpressed survivin upon granulocyte/macrophage colony-stimulating factor (CSF) or granulocyte CSF stimulation in vitro and under inflammatory conditions in vivo. Moreover, survivin-deficient mature neutrophils were unable to increase their lifespan after survival factor exposure. Together, our findings demonstrate the following: (a) overexpression of survivin occurs in primary, even terminally differentiated cells and is not restricted to proliferating cells; and (b) survivin acts as an inhibitor of apoptosis protein in a cell cycle–independent manner. Therefore, survivin plays distinct and independent roles in the maintenance of the G2-M checkpoint and in apoptosis control, and its overexpression is not restricted to proliferating cells. These data provide new insights into the regulation and function of survivin and have important implications for the pathogenesis, diagnosis, and treatment of inflammatory diseases and cancer.

ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy

Anticancer drug therapy activates both molecular cell death and autophagy pathways. Here we show that even sublethal concentrations of DNA-damaging drugs, such as etoposide and cisplatin, induce the expression of autophagy-related protein 5 (ATG5), which is both necessary and sufficient for the subsequent induction of mitotic catastrophe. We demonstrate that ATG5 translocates to the nucleus, where it physically interacts with survivin in response to DNA-damaging agents both in vitro and in carcinoma tissues obtained from patients who had undergone radiotherapy and/or chemotherapy. As a consequence, elements of the chromosomal passenger complex are displaced during mitosis, resulting in chromosome misalignment and segregation defects. Pharmacological inhibition of autophagy does not prevent ATG5-dependent mitotic catastrophe, but shifts the balance to an early caspase-dependent cell death. Our data suggest a dual role for ATG5 in response to drug-induced DNA damage, where it acts in two signalling pathways in two distinct cellular compartments, the cytosol and the nucleus.

A novel FIP1L1‐PDGFRA mutant destabilizing the inactive conformation of the kinase domain in chronic eosinophilic leukemia/hypereosinophilic syndrome

Background:  The Fip1‐like‐1–platelet‐derived growth factor receptor alpha (FIP1L1‐PDGFRA) gene fusion is a common cause of chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome (HES), and patients suffering from this particular subgroup of CEL/HES respond to low‐dose imatinib therapy. However, some patients may develop imatinib resistance because of an acquired T674I mutation, which is believed to prevent drug binding through steric hindrance.

ATG5 can regulate p53 expression and activation.

Dear Editor, DNA-damaging anti-cancer drugs cause cell death by apoptosis, but they also activate macroautophagy1 (hereafter just autophagy), a fundamental survival pathway under stress where cells enclose cytosol or organelles in double-membrane autophagosomes, then fuse them with lysosomes for recovery of metabolic precursors.2 This process depends upon autophagy-related (ATG) proteins.2 Activation of this survival pathway is unwanted in cancer therapy, because even a few surviving tumor cells can accumulate mutations, gain genetic diversity, and, potentially, resume proliferation.3 We recently reported that expression of ATG5 was upregulated by treatment with low concentrations of etoposide.4 Few cells entered apoptosis, but almost all showed autophagy. Surprisingly, much of the induced ATG5 was found in the cell nucleus, binding to BIRC5/survivin and causing cell cycle arrest at G2/M followed by mitotic catastrophe.4, 5 We then asked whether just ectopic ATG5 expression, without etoposide, would lead to mitotic catastrophe. Indeed, a large part of the expressed ATG5 was again found in the nucleus and rapid cell cycle arrest together with mitotic catastrophe was observed.4 DNA damage, however, was not in evidence; neither ATM nor ATR phosphorylation was detected and foci of H2AX phosphorylation were absent.4 This has led us to ask: how can ATG5 induce the same kind of stress response as DNA-damaging drugs? This report documents increased p53 expression after lentivirus-mediated, ectopic ATG5 expression (Figure 1a). Upregulated p21 demonstrated p53 transactivation of a target gene. Vector alone elicited only a slightly elevated p53. As expected, ATG5 expression also caused autophagy as measured by lipidated LC3 (LC3-II). Figure 1 ATG5 expression induces and activates p53. (a) Jurkat T cells were transduced with lentivirus constructs of vector alone or ATG5. Here shown is the ATG5 33-kDa monomer band. At the indicated intervals in hours, lysates were prepared, electrophoresed and ... One wonders whether the observed p53 upregulation/activation is necessary to initiate autophagy?6 Using p53 null Saos-2 cells made DOX inducible for p53 with a Tet-on construct, we showed that p53 was not required for autophagy (Figure 1b). Ectopically expressed ATG5 is shown as both 33-kDa monomer and 57-kDa conjugate with ATG12. Autophagy was apparent from LC3-II. An additional stimulus with nutrient starvation was followed within 1 h by further increased LC3-II, but without any requirement for p53 induction (Figure 1b). As lentivirus-mediated gene transfer might have produced some local anomalies in DNA, we studied p53 expression/activation in an ATG5 knockout mouse embryo fibroblast (mEF) line that had been subsequently transfected with a Tet-off ATG5 expression system (clone M5-7).7 As these cells had been maintained since 2006 without DOX stimulation, a DNA damage response seems unlikely; however, we examined ATM phospho-Ser1981 as confirmation (Figure 1c). After DOX treatment, ATG5 expression in these cells was rapidly suppressed. Note: please ignore the unspecific band seen in mEF cells at 36 kDa. In mEF cells, ATG5 exists almost entirely as a conjugate with ATG12. Corresponding to the downregulation of ATG5 after DOX, both p53 expression and autophagy, monitored as LC3-II levels, declined sharply (Figure 1c). Also in Figure 1d, using the same Tet-off ATG5 expression system, these differences between ATG5-non-expressing and ATG5-expressing cells were apparent. Noteworthy is that, ATG5-expressing cells showed not only elevated p53 levels, but also increased p53 activation as evidenced by p53 Ser18 phosphorylation (Figure 1d). Interestingly, starvation also induced p53 in DOX-treated cells not expressing ATG5, not, however, LC3-II or autophagy. Our findings thus indicate that increased ATG5 expression represents for the cell something like a stress response. In consequence, p53 upregulation and transactivation of p21 are followed by cell cycle arrest. Here p53 is not acting in its generally accepted role as a regulator of autophagy and apoptosis,6 but itself exhibits a secondary response to upregulation of ATG5 expression. Similar findings have been reported recently with ATG7 knockout mEF cells,8 showing that ATG7 also can impose a reciprocal regulation on p53. Surprisingly too, activation of the NF-κB pathway, an important stress response, is blocked in cells lacking either ATG5 or ATG7.9 All these observations suggest a network of interactive responses available to the cell, initiated by different homeostatic imbalances, but integrated in the same overall program.

RhoH/TTF negatively regulates leukotriene production in neutrophils.

Leukotriene B4 (LTB4) is an important proinflammatory lipid mediator generated by neutrophils upon activation. GM-CSF stimulation is known to enhance agonist-mediated LTB4 production of neutrophils within minutes, a process called “priming”. In this study, we demonstrate that GM-CSF also limits the production of LTB4 by neutrophils via a transcriptional mechanism at later time points. We identified hemopoietic-specific Ras homologous (RhoH)/translocation three four (TTF), which was induced following GM-CSF stimulation in neutrophils, as a key regulator in this process. Neutrophils derived from RhoH/TTF-deficient (Rhoh−/−) mice demonstrated increased LTB4 production upon activation compared with normal mouse neutrophils. Moreover, neutrophils from cystic fibrosis patients expressed enhanced levels of RhoH/TTF and generated less LTB4 upon activation compared with normal human neutrophils. Taken together, these data suggest that RhoH/TTF represents an inducible feedback inhibitor in neutrophils that is involved in the limitation of innate immune responses.

Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.