Objective: To investigate the association of β₂‐glycoprotein I (β₂GPI) with lipid rafts in monocytic cells and to evaluate the proinflammatory and procoagulant effects of anti‐β₂GPI binding to its target antigen on the monocyte plasma membrane.Methods: Human monocytes were fractionated by sucrose density‐gradient centrifugation and analyzed by Western blotting. Immunoprecipitation experiments were performed to analyze the association of β₂GPI with lipid rafts and the possible interaction of β₂GPI with annexin A2 and Toll‐like receptor 4 (TLR‐4). Monocytes were then stimulated with affinity‐purified anti‐β₂GPI antibodies from patients with the antiphospholipid syndrome (APS). Interleukin‐1 receptor–associated kinase (IRAK) phosphorylation and NF‐κB activation were evaluated by immunoprecipitation and transcription factor assay, respectively. Supernatants from monocytes were tested for tumor necrosis factor α (TNFα) and tissue factor (TF) levels by enzyme‐linked immunosorbent assay.Results: We found β₂GPI and its putative receptor annexin A2 in lipid raft fractions of human monocytes. Moreover, there was an association between β₂GPI and TLR‐4, suggesting that it was partially dependent on raft integrity. Triggering with anti‐β₂GPI antibodies induced IRAK phosphorylation and consequent NF‐κB activation, which led to the release of TNFα and TF.Conclusion: Anti‐β₂GPI antibodies react with their target antigen, likely in association with annexin A2 and TLR‐4, in lipid rafts in the monocyte plasma membrane. Anti‐β₂GPI binding triggers IRAK phosphorylation and NF‐κB translocation, leading to a proinflammatory and procoagulant monocyte phenotype characterized by the release of TNFα and TF, respectively. These findings provide new insight into the pathogenesis of APS, improving our knowledge of valuable therapeutic targets.

Autophagy and apoptosis represent two fundamental pathophysiological mechanisms of cell fate regulation. However, the signaling pathways of these processes are significantly interconnected through various mechanisms of crosstalk. Indeed, autophagy/apoptosis crosstalk is still an emerging field, in which an increasing number of molecules are involved, including, for example, PINK1 and ERLINs. On the other hand, this crosstalk involves signal transduction pathways which are strongly dependent on Ca²⁺. Interestingly, crosstalk between autophagy and apoptosis impacts several pathologies, including multiple rheumatic diseases. The purpose of this Special Issue is also to investigate the bioactive properties of drugs with antitumor activity, focusing particularly on the role of anthraquinone derivatives in the regulation of cell death and autophagy crosstalk. This Special Issue of Cells brings together the most recent advances in understanding the various aspects of crosstalk between autophagy and apoptosis and the interconnected signaling pathways, implying therapeutic perspectives for the utility of its modulation in an anti-cancer setting.

Despite inducing a strong host cellular and humoral immune response, the helminth Echinococcus granulosus is a highly successful parasite that develops, progresses, and ultimately causes chronic disease. Although surgery remains the preferred therapeutic option, pharmacological research now envisages antihelminthic strategies. To understand the mechanisms that E. granulosus uses to escape host immunosurveillance and promote chronic infection, we investigated how two hydatid cyst components, purified antigen B (AgB) and sheep hydatid fluid (SHF), act on host dendritic cell (DC) differentiation from monocyte precursors and how they influence maturation of DC that have already differentiated. We evaluated the immunomodulatory potential of these antigens by performing immunochemical and cytofluorimetric analyses of monocyte-derived DCs from healthy human donors. During monocyte differentiation, AgB and SHF downmodulated CD1a expression and upregulated CD86 expression. Compared with immature DCs differentiated in medium alone (iDCs), AgB- and SHF-differentiated cells stimulated with lipopolysaccharide included a significantly lower percentage of CD83 ⁺ cells ( P < 10 ⁻⁴ ) and had weaker costimulatory molecule expression. When stimulated with AgB and SHF, iDCs matured and primed lymphocytes towards the Th2 response typical of E. granulosus infection. SHF and particularly AgB reduced the production of interleukin-12p70 (IL-12p70) and tumor necrosis factor alpha in lipopolysaccharide-stimulated iDCs. Anti-IL-10 antibodies increased the levels of IL-12p70 secretion in AgB- and SHF-matured DCs. AgB and SHF induced interleukin-1 receptor-associated kinase phosphorylation and activated nuclear factor-κB, suggesting that Toll-like receptors could participate in E. granulosus -stimulated DC maturation. These results suggest that E. granulosus escapes host immunosurveillance in two ways: by interfering with monocyte differentiation and by modulating DC maturation.

Mitochondria-associated membranes (MAMs) are subdomains of the endoplasmic reticulum (ER) that interact with mitochondria. This membrane scrambling between ER and mitochondria appears to play a critical role in the earliest steps of autophagy. Recently, lipid microdomains, i.e. lipid rafts, have been identified as further actors of the autophagic process. In the present work, a series of biochemical and molecular analyses has been carried out in human fibroblasts with the specific aim of characterizing lipid rafts in MAMs and to decipher their possible implication in the autophagosome formation. In fact, the presence of lipid microdomains in MAMs has been detected and, in these structures, a molecular interaction of the ganglioside GD3, a paradigmatic “brick” of lipid rafts, with core-initiator proteins of autophagy, such as AMBRA1 and WIPI1, was revealed. This association seems thus to take place in the early phases of autophagic process in which MAMs have been hypothesized to play a key role. The functional activity of GD3 was suggested by the experiments carried out by knocking down ST8SIA1 gene expression, i.e., the synthase that leads to the ganglioside formation. This experimental condition results in fact in the impairment of the ER-mitochondria crosstalk and the subsequent hindering of autophagosome nucleation. We thus hypothesize that MAM raft-like microdomains could be pivotal in the initial organelle scrambling activity that finally leads to the formation of autophagosome.

We previously reported that during death receptor-mediated apoptosis, cardiolipin (CL) relocates to the cell surface, where it reacts with autoantibodies from antiphospholipid syndrome sera. Here, we analysed the intracellular distribution of CL and its metabolites during the early phase of cell death signalling triggered by Fas stimulation in U937 cells and mouse liver. We found a redistribution of mitochondrial CL to the cell surface by using confocal microscopy and flow cytometry. Mass spectrometry revealed that CL and its metabolites relocated from mitochondria to other intracellular organelles during apoptosis, with a conversion into non-mitochondrial lipids. Concomitantly, cytosolic Bid relocated to the light membranes comprised in fraction P100, including the plasma membrane and associated vesicular systems. A direct Bid–CL interaction was demonstrated by the observation that CL and monolysoCL coimmunoprecipitated with Bid especially after Fas stimulation, suggesting a dynamic interaction of the protein with CL and its metabolites.