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Neutrophils play a central role in immunity and inflammation via their intrinsic ability to migrate into inflamed tissue, to phagocytose pathogens, and to kill bacterial and fungi by releasing large quantities of superoxide anions and lytic enzymes. The molecular pathways controlling neutrophil microbicidal functions are still unclear, because neutrophils have a short half-life and are resistant to genetic manipulation. Neutrophil-like cells (NLC) can be generated from myeloid progenitors conditionally immortalized with the ER-HoxB8 oncoprotein, but whether these cells can replace neutrophils in high-throughput functional assays is unclear. Here, we assess the ability of NLC derived from ER-HoxB8 progenitors to produce ROS and to perform chemotaxis and phagocytosis. We compare the Ca 2+ responses and effector functions of NLC to primary murine neutrophils and document the molecular basis of their functional differences by mRNA profiling. Pro-inflammatory cytokines enhanced the expression by NLC of neutrophil surface markers and transcription factors. Ca 2+ elevations evoked in NLC by agonists, adhesion receptors, and store depletion resembled the physiological responses recorded in primary neutrophils, but NLC expressed reduced amounts of Ca 2+ signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H 2 O 2 when adhered to fibronectin, migrated toward chemotactic peptides, phagocytosed opsonized particles, and generated intracellular ROS. NLC phagocytosed as efficiently as primary neutrophils but produced 50 times less ROS and migrated less efficiently toward chemoattractant. Our data indicate that NLC can replace neutrophils to study Ca 2+ signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays., (© 2019 The Authors. Journal of Leukocyte Biology published by Wiley Periodicals, Inc. on behalf of Society for Leukocyte Biology.)

Antiphospholipid antibody syndrome is an autoimmune disease characterized by the presence of so-called antiphospholipid antibodies and clinical manifestations such as recurrent thromboembolic or pregnancy complications. Although the main antigenic determinant for antiphospholipid antibodies has been identified as the β-2-glycoprotein 1 (β2GP1), the precise epitope recognized by antiphospholipid antibodies still remains largely unknown. In the study herein, we wanted to identify a sequence in domain I of β2GP1 able to induce the proliferation of CD4 + T cells isolated from antiphospholipid antibody syndrome patients, but not from healthy donors, and to interact with antiphospholipid antibodies. We have characterized a sequence in domain I of β2GP1 that triggers CD4 + T-cell proliferation. A comparison of this sequence with the previously reported binding of antiphospholipid antibodies to discontinuous epitope R39-R43 reveals the presence of an indeterminate motif in β2GP1, in which the polarity determines the characteristics and specificity of antiphospholipid antibodies-interacting motifs. Using point mutations, we characterized the main antiphospholipid antibodies-interacting motif as ϕϕϕζζFxC, but also established ϕϕϕζζFxϕ-related motifs as potential antiphospholipid antibodies epitopes, in which ϕ represents nonpolar residues and ζ polar residues, with charges of the residues not being involved. Of specific importance, these different motifs are present at least once in all antiphospholipid antibodies-related receptors described so far. We have further demonstrated, in vitro , that peptides and domains of β2GP1 containing these motifs were able to interact with antiphospholipid antibodies and inhibit their monocyte activating activity. These results established that the antiphospholipid antibodies-interacting motifs are determined by the polarity, but not by the sequence or charge, of amino acids. These data could also contribute to the future development of more sensitive and specific diagnostic tools for antiphospholipid antibody syndrome determination and potential peptide- or β2GP1 domain-based clinical therapies., (Copyright© 2017 Ferrata Storti Foundation.)

NLRP3 and ASC are able to form a large multimeric complex called inflammasome in response to a number danger signals. The NLRP3 inflammasome is required for the activation of caspase-1 and subsequent maturation of pro-IL-1β into active IL-1β. Although the mechanisms regulating the formation and activity of NLRP3 inflammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 inflammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that microfilaments (F-actin) inhibit NLRP3 inflammasome activity and interact with NLRP3 and ASC. We demonstrate that the inhibition depends on the actin polymerization state but not on the active polymerization process. In ATP- or nigericin-activated macrophages, our data further indicate that Flightless-I (FliI) and leucine-rich repeat FliI-interaction protein 2 (LRRFIP2) are required for the co-localization of NLRP3, ASC and F-actin. We also established that the ability of Ca(2+) to accentuate the activity of NLRP3 inflammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca(2+) signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca(2+)/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 inflammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 inflammasome activity strengthening the importance of cytoskeleton in the regulation of inflammation.

To analyze the glycosylation of anti-β2GP1, we investigated purified IgG from healthy children, patients with APS, and asymptomatic adult carriers of antiphospholipid antibodies. We observed that in the sera of healthy children and of patients with APS, IgG3 and IgG2 were predominant, respectively. The potentially protective anti-β2GP1-IgM was lower in the sera of healthy children. Although anti-β2GP1-associated C1q did not differ between children and patients with antiphospholipid syndrome, the associated C3c was significantly higher in the sera of healthy children. This indicates a more efficient clearance of anti-β2GP1 immune complexes in the healthy children. This clearance is not accompanied by inflammation or coagulatory events. It is likely that the most important pathogenic factor of the anti-β2GP1-IgG is related to the different glycosylation observed in healthy and diseased individuals. We detected a significantly higher sialylation of anti-β2GP1-IgG isolated from the sera of healthy children and asymptomatic adults when compared with that of patients with clinically apparent antiphospholipid syndrome. Low sialylated IgG reportedly ameliorates inflammation and inflammation promotes hyposialylation. Thus, both reactions create a vicious circle that precipitates the pathology of the antiphospholipid syndrome including thrombus-formation. We conclude that the increased sialylation of anti-β2GP1-IgG of sera of healthy individuals limits their pathogenicity.

Background: The antiphospholipid antibody syndrome (APS) is an autoimmune disease associated with arterial or venous thrombosis and/or recurrent fetal loss and is caused by pathogenic antiphospholipid antibodies (aPLA). We recently demonstrated that Toll-like receptor 2 (TLR2) and CD14 contribute to monocyte activation of aPLA., Objective: To study the mechanisms of cell activation by aPLA, leading to pro-coagulant and pro-inflammatory responses., Methods and Results: For this study, we used purified antibodies from the plasmas of 10 different patients with APS and healthy donors. We demonstrate that aPLA, but not control IgG, co-localizes with TLR2 and TLR1 or TLR6 on human monocytes. Blocking antibodies to TLR2, TLR1 or TLR6, but not to TLR4, decreased TNF and tissue factor (TF) responses to aPLA. Pharmacological and siRNA approaches revealed the importance of the clathrin/dynamin-dependent endocytic pathway in cell activation by aPLA. In addition, soluble aPLA induced NF-κB activation, while bead-immobilized aPLA beads, which cannot be internalized, were unable to activate NF-κB. Internalization of aPLA in monocytes and NF-κB activation were dependent on the presence of CD14., Conclusion: We show that TLR2 and its co-receptors, TLR1 and TLR6, contribute to the pathogenicity of aPLA, that aPLA are internalized via clathrin- and CD14-dependent endocytosis and that endocytosis is required for NF-κB activation. Our results contribute to a better understanding of the APS and provide a possible therapeutic approach., (© 2014 International Society on Thrombosis and Haemostasis.)

Localization of Toll-like receptors (TLR) in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

The presence of antiphospholipid antibodies (aPLAs) is associated with arterial or venous thrombosis and/or recurrent fetal loss. The proposed pathogenic mechanisms for aPLA effects include the inflammatory activation of monocytes and endothelial cells. Toll-like receptors (TLRs) are candidate signaling intermediates. The aim of this study was to investigate the relative contribution of TLR2 and TLR4 in cell activation by aPLAs. Of 32 patient-derived aPLAs, 19 induced an inflammatory activation of human monocytes and umbilical vein endothelial cells (HUVECs). In HUVECs, inflammatory responses to these aPLAs were increased by TNF pretreatment, which increases the expression of TLR2 but not TLR4. Anti-TLR2 but not anti-TLR4 antibodies reduced the aPLA-induced activation of monocytes and HUVECs. aPLAs activated TLR2-expressing human embryonic kidney 293 (HEK293) cells but not TLR4-expressing cells. Binding studies demonstrated an interaction between aPLAs and TLR2 but not TLR4. A role for CD14, a coreceptor for TLR2 and TLR4, can be inferred by observations that anti-CD14 antibodies reduced responses to aPLAs in monocytes, and that responses in HEK293 cells expressing TLR2 and CD14 were greater than in HEK293 cells expressing TLR2 alone. Our results demonstrate a role for TLR2 and CD14 in human endothelial cell and monocyte activation by aPLAs.

Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications.