A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Mechanisms of internalization of apoptotic and necrotic L929 cells by a macrophage cell line studied by electron microscopy

Rapid and efficient phagocytic removal of dying cells is a key feature of apoptosis. In necrotic caspase-independent modes of death, the role and extent of phagocytosis is not well documented. To address this issue, we studied at the ultrastructural level the phagocytic response to dying cells in an in vitro phagocytosis assay with a mouse macrophage cell line (Mf4/4). As target cells, murine L929sAhFas cells were induced to die by TNFR1-mediated necrosis or by Fas-mediated apoptosis. Apoptotic L929sAhFas cells are taken up by complete engulfment of apoptotic bodies as single entities forming a tight-fitting phagosome, thus resembling the "zipper"-like mechanism of internalization. In contrast, primary and secondary necrotic cells were internalized by a macropinocytotic mechanism with formation of multiple ruffles by the ingesting macrophage. Ingestion of necrotic cellular material was invariably taking place after the integrity of the cell membrane was lost and did not occur as discrete particles, in contrast to apoptotic material that is surrounded by an intact membrane. Although nuclei of necrotic cells have been observed in the vicinity of macrophages, no uptake of necrotic nuclei was observed. The present report provides a basis for future studies aimed at discovering molecular pathways that precede these diverse mechanisms of uptake.     

Derivatives of tetrahydroisoquinoline: Synthesis and initial evaluation of novel non-peptide antagonists of the αIIbβ3-integrin

The novel RGDF mimetics were synthesized with the use of 4-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-4-oxobutyric or 5-(1,2,3,4-tetrahydroisoquinoline-7-yl)amino-5-oxopentanoic acids as a surrogate of Arg-Gly motif. The synthesized compounds have demonstrated a high potency to inhibit platelet aggregation in vitro and to block FITC-Fg binding to α_IIbβ₃ on washed human platelets.     

In situ adaptation and ecological release facilitate the occupied niche expansion of a non‐native Madagascan day gecko in Florida

AimTo investigate whether the frequently advocated climate‐matching species distribution modeling approach could predict the well‐characterized colonization of Florida by the Madagascar giant day gecko Phelsuma grandis.LocationMadagascar and Florida, USA.MethodsTo determine the climatic conditions associated with the native range of P. grandis, we used native‐range presence‐only records and Bioclim climatic data to build a Maxent species distribution model and projected the climatic thresholds of the native range onto Florida. We then built an analogous model using Florida presence‐only data and projected it onto Madagascar. We constructed a third model using native‐range presences for both P. grandis and the closely related parapatric species P. kochi.ResultsDespite performing well within the native range, our Madagascar Bioclim model failed to identify suitable climatic habitat currently occupied by P. grandis in Florida. The model constructed using Florida presences also failed to reflect the distribution in Madagascar by overpredicting distribution, especially in western areas occupied by P. kochi. The model built using the combined P. kochi/P. grandis dataset modestly improved the prediction of the range of P. grandis in Florida, thereby implying competitive exclusion of P. grandis by P. kochi from habitat within the former''s fundamental niche. These findings thus suggest ecological release of P. grandis in Florida. However, because ecological release cannot fully explain the divergent occupied niches of P. grandis in Madagascar versus Florida, our findings also demonstrate some degree of in situ adaptation in Florida.Main conclusionsOur models suggest that the discrepancy between the predicted and observed range of P. grandis in Florida is attributable to either in situ adaptation by P. grandis within Florida, or a combination of such in situ adaptation and competition with P. kochi in Madagascar. Our study demonstrates that climate‐matching species distribution models can severely underpredict the establishment risk posed by non‐native herpetofauna.     

ATP release from dying autophagic cells and their phagocytosis are crucial for inflammasome activation in macrophages

Pathogen-activated and damage-associated molecular patterns activate the inflammasome in macrophages. We report that mouse macrophages release IL-1β while co-incubated with pro-B (Ba/F3) cells dying, as a result of IL-3 withdrawal, by apoptosis with autophagy, but not when they are co-incubated with living, apoptotic, necrotic or necrostatin-1 treated cells. NALP3-deficient macrophages display reduced IL-1β secretion, which is also inhibited in macrophages deficient in caspase-1 or pre-treated with its inhibitor. This finding demonstrates that the inflammasome is activated during phagocytosis of dying autophagic cells. We show that activation of NALP3 depends on phagocytosis of dying cells, ATP release through pannexin-1 channels of dying autophagic cells, P(2)X(7) purinergic receptor activation, and on consequent potassium efflux. Dying autophagic Ba/F3 cells injected intraperitoneally in mice recruit neutrophils and thereby induce acute inflammation. These findings demonstrate that NALP3 performs key upstream functions in inflammasome activation in mouse macrophages engulfing dying autophagic cells, and that these functions lead to pro-inflammatory responses.     

Apoptosis and necrosis: detection, discrimination and phagocytosis.

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.     

Life and death of female gametes during oogenesis and folliculogenesis

The vertebrate ovary is an extremely dynamic organ in which excessive or defective follicles are rapidly and effectively eliminated early in ontogeny and thereafter continuously throughout reproductive life. More than 99% of follicles disappear, primarily due to apoptosis of granulosa cells, and only a minute fraction of the surviving follicles successfully complete the path to ovulation. The balance between signals for cell death and survival determines the destiny of the follicles. An abnormally high rate of cell death followed by atresia can negatively affect fertility and eventually lead irreversibly to premature ovarian failure. In this review we provide a short overview of the role of programmed cell death in prenatal differentiation of the primordial germ cells and in postnatal folliculogenesis. We also discuss the issue of neo-oogenesis. Next, we highlight molecules involved in regulation of granulosa cell apoptosis. We further discuss the potential use of scores for apoptosis in granulosa cells and characteristics of follicular fluid as prognostic markers for predicting the outcome of assisted reproduction. Potential therapeutic strategies for combating premature ovarian failure are also addressed.     

Impairment of phagocytosis of apoptotic cells and its role in chronic airway diseases

Phagocytosis of dying cells is a complex and dynamic process coordinated by the interaction of many surface molecules, adaptors, and chemotactic molecules, and it is controlled at multiple levels. This well regulated clearance process is of utmost importance for the development and homeostasis of organisms because defective or inefficient phagocytosis may contribute to human pathologies. In this review we discuss recent advances in the knowledge of the molecular interactions involved in recognition and clearance of apoptotic cells and how derangement of these processes can contribute to the pathogenesis of chronic airway diseases such as chronic obstructive pulmonary disease, cystic fibrosis and asthma. We will briefly consider how different types of macrophages are implicated in chronic airway diseases. Finally, we will address possible therapeutic strategies, such as the use of macrolide antibiotics and statins, for modulating apoptotic cell clearance.     

BNIP3 supports melanoma cell migration and vasculogenic mimicry by orchestrating the actin cytoskeleton

BNIP3 is an atypical BH3-only member of the BCL-2 family of proteins with reported pro-death as well as pro-autophagic and cytoprotective functions, depending on the type of stress and cellular context. In line with this, the role of BNIP3 in cancer is highly controversial and increased BNIP3 levels in cancer patients have been linked with both good as well as poor prognosis. In this study, using small hairpin RNA (shRNA) lentiviral transduction to stably knockdown BNIP3 (BNIP3-shRNA) expression levels in melanoma cells, we show that BNIP3 supports cancer cell survival and long-term clonogenic growth. Although BNIP3-shRNA increased mitochondrial mass and baseline levels of reactive oxygen species production, which are features associated with aggressive cancer cell behavior, it also prevented cell migration and completely abolished the ability to form a tubular-like network on matrigel, a hallmark of vasculogenic mimicry (VM). We found that this attenuated aggressive behavior of these melanoma cells was underscored by severe changes in cell morphology and remodeling of the actin cytoskeleton associated with loss of BNIP3. Indeed, BNIP3-silenced melanoma cells displayed enhanced formation of actin stress fibers and membrane ruffles, while lamellopodial protrusions and filopodia, tight junctions and adherens junctions were reduced. Moreover, loss of BNIP3 resulted in re-organization of focal adhesion sites associated with increased levels of phosphorylated focal adhesion kinase. Remarkably, BNIP3 silencing led to a drop of the protein levels of the integrin-associated protein CD47 and its downstream signaling effectors Rac1 and Cdc42. These observations underscore that BNIP3 is required to maintain steady-state levels of intracellular complexes orchestrating the plasticity of the actin cytoskeleton, which is integral to cell migration and other vital processes stimulating cancer progression. All together these results unveil an unprecedented pro-tumorigenic role of BNIP3 driving melanoma cell''s aggressive features, like migration and VM.