Stefania Benatti

Circulating mitochondrial DNA increases with age and is a familiar trait: Implications for “inflamm‐aging”

Mitochondrial components, including mitochondrial DNA (mtDNA), when released extracellularly, can act as “damage‐associated molecular pattern” (DAMP) agents and cause inflammation. As many elderly people are characterized by a low‐grade, chronic inflammatory status defined “inflamm‐aging,” we evaluated if circulating mtDNA can contribute to this phenomenon. Eight hundred and thirty‐one Caucasian subjects were enrolled in the study, including 429 siblings aged 90–104 (90+ siblings). mtDNA plasma levels increased gradually after the fifth decade of life. In 90+ subjects, mtDNA values of two members of the same sibling relationship were directly correlated, suggesting a role for familiar/genetic background in controlling the levels of circulating mtDNA. The subjects with the highest mtDNA plasma levels had the highest amounts of TNF‐α, IL‐6, RANTES, and IL‐1ra; the subjects with the lowest mtDNA levels had the lowest levels of the same cytokines. In vitro stimulation of monocytes with mtDNA concentrations similar to the highest levels observed in vivo resulted in an increased production of TNF‐α, suggesting that mtDNA can modulate the production of proinflammatory cytokines. Our findings therefore show that circulating mtDNA increases with age, and can significantly contribute to the maintenance of the low‐grade, chronic inflammation observed in elderly people.

Successful treatment of HIV-1 infection increases the expression of a novel, short transcript for IL-18 receptor α chain.

Abstract:The importance of interleukin (IL)-18 in mediating immune activation during HIV infection has recently emerged. IL-18 activity is regulated by its receptor (IL-18R), formed by an &agr; and a &bgr; chain, the IL-18–binding protein, and the newly identified shorter isoforms of both IL-18R chains. We evaluated gene expression of the IL-18/IL-18R system in peripheral blood mononuclear cells from HIV+ patients. Compared with healthy donors, IL-18 expression decreased in patients with primary infection. The IL-18R&agr; short transcript expression was strongly upregulated by successful highly active antiretroviral therapy. HIV progression and its treatment can influence the expression of different components of the complex IL-18/IL-18R system.

A prokineticin-like protein responds to immune challenges in the gastropod pest Pomacea canaliculata.

Abstract The golden apple snail Pomacea canaliculata is an invasive pest originating from South America. It has already been found in Asia, the southern United States and more recently in the EU. Aiming to target the immune system of the snail as a way to control its spreading, we have developed organ‐specific transcriptomes and looked for molecules controlling replication and differentiation of snail hemocytes. The prokineticin domain‐containing protein Astakine 1 is the only cytokine known thus far capable of regulating invertebrate hematopoiesis, and we analyzed the transcriptomes looking for molecules containing a prokineticin domain. We have identified a prokineticin‐like protein (PlP), that we called Pc‐plp and we analyzed by real‐time PCR (qPCR) its expression. In control snails, highest levels of Pc‐plp were detected in the digestive gland, the ampulla (i.e., a hemocyte reservoir) and the pericardial fluid (i.e., the hematopoietic district). We tested Pc‐plp expression after triggering hematopoiesis via multiple hemolymph withdrawals, or during bacterial challenge through LPS injection. In both cases a reduction of Pc‐plp mRNA was observed. The multiple hemolymph withdrawals caused a significant decrease of Pc‐plp mRNA in pericardial fluid and circulating hemocytes, while the LPS injection promoted the Pc‐plp mRNA drop in anterior kidney, mantle and gills, organs that may act as immune barrier in molluscs. Our data indicate an important role for prokineticin domain‐containing proteins as immunomodulators also in gastropods and their dynamic expression may serve as a biosensor to gauge the effectiveness of immunological interventions aimed at curtailing the spreading of the gastropod pest P. canaliculata. HighlightsA prokineticin‐like protein (Pc‐PlP) is expressed in several organs of Pomacea canaliculata.Pc‐plp is maximally expressed in digestive gland, ampulla and hematopoietic tissue.Pc‐plp expression is specifically altered in different organs by diverse immune challenges.Pc‐plp mRNA appears to be post‐transcriptionally regulated as astakines in crustaceans.Pc‐plp may represent a biosensor to evaluate the immune system status after different challenges.

Erratum to: Changes in the NMR Metabolic Profile of Live Human Neuron-Like SH-SY5Y Cells Exposed to Interferon-α2 (J Neuroimmune Pharmacol, 2015, DOI 10.1007/s11481-015-9641-x)

Interferon (IFN)-α2 is an extensively therapeutically used pro-inflammatory cytokine. Though its efficacy in controlling viral replication and tumor cells proliferation, administration of IFN-α2 is often associated with the development of central side effects. Magnetic resonance spectroscopy studies have demonstrated that IFN-α2 administration affects brain metabolism, however the exact nature of this effect is not completely known. We hypothesized that IFN-α2 can affect metabolic activity of human neuron-like SH-SY5Y cells which possess many characteristics of neurons and represent one of the most used models for studying mechanisms involved in neurotoxicity or neuroprotection. To test our hypothesis we have characterized the metabolic signature of live SH-SY5Y, and their conditioned media, after 24 and 72 h of exposure to vehicle or IFN-α2 (100 ng/ml) by using High Resolution-Magic Angle Spinning (HR-MAS) Nuclear Magnetic Resonance (NMR) spectroscopy. Our results revealed that 1) the use of HR-MAS NMR is ideally suitable for the characterization of the metabolic profile of live cells and their conditioned media without extraction procedures; and 2) a 72 h exposure to IFN-α2 increases the level of metabolites involved in maintaining energetic (including creatine and lactate) and osmotic (such asmyo-inositol, scyllo-inositol, taurine and glycerophosphorylcholine) balances in neuron-like cells and of metabolic waste products (namely lactate, ethanol and acetate), glycine and glutamine in their growth media. These results may contribute to gain more knowledge about the IFN-α2 induced effect on the brain and support the interpretation of magnetic resonance spectroscopy studies performed in humans.

Increased SHISA3 expression characterizes chronic lymphocytic leukemia patients sensitive to lenalidomide

Abstract Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change =  +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.

Angiopoietin-2 acts as a survival factor for chronic lymphocytic leukemia B cells throughout Tie-2 receptor engagement

Angiogenesis, ie, the formation of new vessels from pre‐existing one, is a complex process tightly regulated by the dynamic balance between pro‐angiogenic and anti‐angiogenic factors. Among pro‐angiogenic factors, angiopoietin‐2 (Ang2) is a 75 kDa secreted glycoprotein able to bind the receptor tyrosine kinaseTie‐2. Ang2 is a destabilizing factor able to revert vessels to a more plastic state and works in concert with vascular endothelial growth factor (VEGF) to determine angiogenic remodeling and sprouting. Chronic lymphocytic leukemia (CLL) is the commonest leukemia in the Western countries. Higher density of microvessels was detected in CLL patients with poor prognosis. Moreover, CLL cells are able to secrete several pro‐angiogenic factors, such as VEGF, Ang2, endothelin‐1 (ET‐1), and basic fibroblast growth factor (bFGF), that can be measured at high concentration in plasma samples of CLL patients, being also particularly increased in cases with unfavorable prognostic factors and worse clinical outcome. In particular, Ang2 plasma levels are able to predict progression‐free survival of CLL patients. Tie‐1 and Tie‐2 (also known as TEK) are type 1 transmembrane protein receptor tyrosine kinases (RTKs) that interact on the cellular surface through their ectodomains forming an inhibitory complex of signal transduction. Both Ang2 and Ang1 bind to the same site on Tie‐2, however Ang1 induces dissociation of Tie‐1/Tie‐2 complex, clustering of Tie‐2 and signaling initiation, whereas Ang2 inhibits Tie‐2 signaling acting as a competitive antagonist. Nevertheless, when the balance between angiopoietins is shifted to Ang2, it can also act as agonist, activating Tie‐2 receptor and downstream pathways including PI3K‐Akt prosurvival cascade. CLL cells were reported to express Tie‐1 but not Tie‐2.Here, wewonderedwhether Ang2may act in an autocrine fashion in CLL cells, investigating the expression of Tie‐2 receptor in leukemic cells and the in vitro effect of Ang2 on CLL survival. All patients included in this study (n = 38) were seen at the Hematology Division of Modena in Italy and provided informed consent in accordancewith local institutional review board requirements and the Declaration of Helsinki Principles. Clinical and biological data were provided inTable S1. First, we measured the percentage of circulating CLL cells showing expression of Tie‐2 receptor on the cellular surface in 27 untreated patients by flow cytometry, staining peripheral blood mononuclear cells (PBMCs) using APC‐conjugated anti‐CD19

Idelalisib impairs T-cell-mediated immunity in chronic lymphocytic leukemia

In the last few years, new drugs able to inhibit the B-cell receptor (BCR) pathway have been approved by the US and European drug control agencies for the treatment of chronic lymphocytic leukemia (CLL) and some indolent B lymphomas. Among them, ibrutinib and idelalisib, respectively, target the

Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia

The crosstalk between chronic lymphocytic leukemia (CLL) cells and tumor microenvironment is essential for leukemic clone maintenance, supporting CLL cells survival, proliferation and protection from drug-induced apoptosis. Over the past years, the role of several soluble factors involved in these processes has been studied. CLL cells express higher levels of endothelin 1 (ET-1) and ETA receptor as compared to normal B cells. Upon ET-1 stimulation, CLL cells improve their survival and proliferation and reduce their sensitivity to the phosphoinositide-3-kinase δ inhibitor idelalisib and to fludarabine. Here, we demonstrate that CLL cells express not only ETA receptor but also ETB receptor. ET-1 acts as a homing factor supporting CLL cells migration and adhesion to microenvironmental cells. In addition, ET-1 stimulates a pro-angiogenic profile of CLL cells increasing VEGF expression through hypoxia-inducible factor-1 (HIF-1α) accumulation in CLL cells. Macitentan, a specific dual inhibitor of ETA and ETB receptors, targets CLL cells affecting leukemic cells migration and adhesion and overcoming the pro-survival and proliferation signals mediated by microenvironment. Furthermore, macitentan cooperates with ibrutinib inhibiting the BCR pathway and with ABT-199 disrupting BCL2 pathway. Our data describe the biological effects of a new drug, macitentan, able to counteract essential processes in CLL pathobiology as survival, migration, trafficking and drug resistance. These findings envision the possibility to interfere with ET receptors activity using macitentan as a possible novel therapeutic strategy for CLL patients.

The expression of endothelin-1 in chronic lymphocytic leukemia is controlled by epigenetic mechanisms and extracellular stimuli

Endothelin-1 (ET-1) is a hormone peptide widely expressed and is involved in several biological processes, important not only for normal cell function but also for tumor development, including cell proliferation, invasion, metastasis, angiogenesis and osteogenesis. In accordance, ET-1 was already shown to contribute to the growth and progression of many different solid cancers. We recently demonstrated that ET-1 has a role in the pathogenesis of chronic lymphocytic leukemia (CLL) where it is abnormally expressed. In the context of this malignancy, ET-1 is able to mediate survival, drug-resistance and growth signals in leukemic cells. Previous studies, not conducted in CLL, have shown that ET-1 regulatory mechanisms are numerous and cell specific. Here, we valued the expression of ET-1 in CLL, in relation to DNA methylation but also in response to stimulation of some important pathways for the dialogue between CLL and microenvironment. We found that a high methylation of ET-1 first intron affects the basal expression of ET-1 in CLL. Moreover, we showed that the activation of CD40 or Toll-like receptor (TLR) by extracellular stimuli produces an augment of ET-1 level in CLL cells. Finally, we demonstrated the fundamental role of NF-kB signalling pathway in promoting and maintaining ET-1 expression in CLL cells, both in basal conditions and after CD40 activation.