F-actin dampens NLRP3 inflammasome activity via Flightless-I and LRRFIP2

NLRP3 and ASC are able to form a large multimeric complex called in ammasome in response to a number danger signals. The NLRP3 in ammasome 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 in ammasome are yet not fully elucidated, data suggest that the assembly of NLRP3 in ammasome requires microtubules to induce the proximity of ASC and NLRP3. In this study we show that micro laments (F-actin) inhibit NLRP3 in ammasome 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 Ca2+ to accentuate the activity of NLRP3 in ammasome is abrogated in FliI- and LRRFIP2-knockdown macrophages, suggesting that Ca2+ signaling requires the presence of FliI and LRRFIP2. Accordingly, we observed that Ca2+/FliI-dependent severing of F-actin suppresses F-actin/FliI/LRRFIP2-dependent NLRP3 in ammasome inhibition leading to increase IL-1β production. Altogether, our results unveil a new function of F-actin in the regulation of NLRP3 in ammasome activity strengthening the importance of cytoskeleton in the regulation of in ammation

Signaling and functional competency of neutrophils derived from bone-marrow cells expressing the ER-HOXB8 oncoprotein

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 Ca2+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. Ca2+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 Ca2+signaling proteins and of chemotactic receptors. Unlike their myeloid progenitors, NLC produced H2O2when 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 Ca2+signaling and phagocytosis, but that their incomplete granulocytic differentiation limits their use for chemotaxis and ROS production assays

TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes

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

Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae

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

Patient-derived anti-β2GP1 antibodies recognize a peptide motif pattern and not a specific sequence of residues

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

Effects of clathrin knockdown on NF-κB responses to LTA and Pam₃CSK₄.

<p>(A) The right panel shows quantification by western blot of the heavy chain of clathrin in HEK-Blue2™ and representative experience obtains with HEK-Blue2™ and HEK-Blue4™cells treated with stealth siRNA for the heavy chain of clathrin or Stealth RNAi™ negative control duplex. The left panel shows NF-κB activity of HEK-Blue2™ and HEK-Blue4™cells siRNA-treated for 72h and then activated with Pam3CSK4, LTA or LPS (100ng/ml, 1μg/ml, 100ng/ml, respectively) for 24h. Cells were tested for NF-κB activity by measuring SEAP activity in cell supernatants. NF-κB activity is presented as percentage of production in mock-siRNA transfected HEK-Blue2™or HEK-Blue4™ cells. Data are represented as mean +/- SD of at least 3 independent experiments. </p

CD14 controls LTA and Pam₃CSK₄ internalization and NF-κB activation.

<div><p>HEK-Blue2™ cells are TLR2⁺ and CD14⁺ (CD14) while HEK-TLR2 cells are TLR2⁺ and CD14^- cells (no CD14).</p> <p>(<b>A</b> and <b>C</b>) HEK-Blue2™ cells ((red dot) and HEK-TLR2 cells (■ were treated with LTA-biotin 1μg/ml or Pam₃CSK₄-biotin 100ng/ml and TLR2 ligands endocytosis was measured by flow cytometry at the times indicated. Displayed are the mean +/- SD of the percentage of corrected fluorescence index (MFI) of specific extracellular TLR2 ligands staining at each time point.</p> <p>(<b>B</b> and <b>D</b>) HEK-Blue2™ cells ((red dot) and HEK-TLR2 cells (■ were treated with LTA-biotin 1μg/ml or Pam₃CSK₄-biotin 100ng/ml and intracellular accumulation of TLR2 ligands was measured by flow cytometry at the times indicated. Displayed are the mean +/- SD of the percentage of corrected fluorescence index (MFI) of specific intracellular TLR2 ligands staining at each time point. </p> <p>(<b>E</b>) HEK-Blue2™ cells and HEK-TLR2 cells were activated by indicated TLR2 ligands (LTA 1μg/ml or Pam₃CSK₄ 100ng/ml) and IL-8 production was assayed by ELISA. Data are represented as mean +/- SD of at least 5 independent experiments.</p> <p>(<b>F</b>) Monocytes were treated with blocking antibody against CD14 (10μg/ml) during 45min prior to be activated with Pam₃CSK₄ 100ng/ml or LTA 1μg/ml. After 24h, TNF secretion was assayed by ELISA. CD14 blocking antibody decreases significantly TNF production. Data are represented as mean +/- SD of at least 4 independent experiments. </p> <p>(<b>G</b>) HEK-Blue2™ cells (CD14⁺ cells) and HEK-TLR2 cells (no CD14 cells) were activated with LTA 1μg/ml or Pam₃CSK₄ 100ng/ml. The presence of CD14 and activation of IκB (NF-κB) were analyzed by Western blot. CD14 controls NF-κB activation in TLR2 ligands-activated cells. Data are representative of 3 independent experiments. </p></div

Toll-like receptor 2 mediates the activation of human monocytes and endothelial cells by antiphospholipid antibodies

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