5-Chloro-2-Guanidinobenzimidazole (ClGBI) Is a Non-Selective Inhibitor of the Human HV1 Channel.

5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.

The Voltage-Gated Hv1 H+ Channel Is Expressed in Tumor-Infiltrating Myeloid-Derived Suppressor Cells.

Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.

Novel STAT-3 gain-of-function variant with hypogammaglobulinemia and recurrent infection phenotype.

Signal transducer and activator of transcription 3 (STAT-3) gain-of-function (GOF) syndrome is an early-onset monogenic inborn error of immunity characterized by multi-organ autoimmune disorders, growth failure and lymphoproliferation. We describe that STAT-3 GOF syndrome may be presented with hypogammaglobulinemia and recurrent severe upper and lower respiratory tract infections. In addition, the patient had lymphoproliferation, short stature and interstitial lung disease. Chest computerized tomography examinations showed mild bronchiectasis with areas of non-fibrosing alveolar-interstitial disease and maldevelopment of bilateral first ribs. Using Sanger sequencing, we revealed a novel c.508G>C, p.D170H STAT-3 variant affecting the coiled coil domain of STAT-3. Functional studies confirmed that p.D170H was a GOF variant, as shown by increased phosphorylated STAT-3 (pSTAT-3) and STAT-3 transcriptional activity. Our observation suggests that STAT-3 GOF syndrome can manifest in early childhood with hypogammaglobulinemia and recurrent severe respiratory tract infections. We suggest that patients with lymphoproliferation, hypogammaglobulinemia and severe recurrent infections should be screened for STAT-3 variants, even if autoimmune manifestations are missing.

Intersection of TKS5 and FGD1/CDC42 signaling cascades directs the formation of invadopodia.

Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells.

Author Correction: Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced endothelial motility and multicellular sprouting is mediated by the scaffold protein TKS4.

Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.

CD84 cell surface signaling molecule: An emerging biomarker and target for cancer and autoimmune disorders.

CD84 (SLAMF5) is a member of the SLAM family of cell-surface immunoreceptors. Broadly expressed on most immune cell subsets, CD84 functions as a homophilic adhesion molecule, whose signaling can activate or inhibit leukocyte function depending on the cell type and its stage of activation or differentiation. CD84-mediated signaling regulates diverse immunological processes, including T cell cytokine secretion, natural killer cell cytotoxicity, monocyte activation, autophagy, cognate T:B interactions, and B cell tolerance at the germinal center checkpoint. Recently, alterations in CD84 have been related to autoimmune and lymphoproliferative disorders. Specific allelic variations in CD84 are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In chronic lymphocytic leukemia, CD84 mediates intrinsic and stroma-induced survival of malignant cells. In this review, we describe our current understanding of the structure and function of CD84 and its potential role as a therapeutic target and biomarker in inflammatory autoimmune disorders and cancer.

Signaling Lymphocyte Activation Molecule Family 5 Enhances Autophagy and Fine-Tunes Cytokine Response in Monocyte-Derived Dendritic Cells via Stabilization of Interferon Regulatory Factor 8.

Signaling lymphocyte activation molecule family (SLAMF) receptors are essential regulators of innate and adaptive immune responses. The function of SLAMF5/CD84, a family member with almost ubiquitous expression within the hematopoietic lineage is poorly defined. In this article, we provide evidence that in human monocyte-derived dendritic cells (moDCs) SLAMF5 increases autophagy, a degradative pathway, which is highly active in dendritic cells (DCs) and plays a critical role in orchestration of the immune response. While investigating the underlying mechanism, we found that SLAMF5 inhibited proteolytic degradation of interferon regulatory factor 8 (IRF8) a master regulator of the autophagy process by a mechanism dependent on the E3-ubiquitin ligase tripartite motif-containing protein 21 (TRIM21). Furthermore, we demonstrate that SLAMF5 influences the ratio of CD1a+ cells in differentiating DCs and partakes in the regulation of IL-1ß, IL-23, and IL-12 production in LPS/IFNγ-activated moDCs in a manner that is consistent with its effect on IRF8 stability. In summary, our experiments identified SLAMF5 as a novel cell surface receptor modulator of autophagy and revealed an unexpected link between the SLAMF and IRF8 signaling pathways, both implicated in multiple human pathologies.

Regulation of type I interferon responses by mitochondria-derived reactive oxygen species in plasmacytoid dendritic cells.

Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.

The scaffold protein Tks4 is required for the differentiation of mesenchymal stromal cells (MSCs) into adipogenic and osteogenic lineages.

The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.

RIG-I inhibits the MAPK-dependent proliferation of BRAF mutant melanoma cells via MKP-1.

BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.

Reactive oxygen species-mediated bacterial killing by B lymphocytes.

Regulated production of ROS is mainly attributed to Nox family enzymes. In neutrophil granulocytes and macrophages, Nox2 has a crucial role in bacterial killing, and the absence of phagocytic ROS production leads to the development of CGD. Expression of Nox2 was also described in B lymphocytes, where the role of the enzyme is still poorly understood. Here, we show that peritoneal B cells, which were shown recently to possess phagocytic activity, have a high capacity to produce ROS in a Nox2-dependent manner. In phagocytosing B cells, intense intraphagosomal ROS production is detected. Finally, by studying 2 animal models of CGD, we demonstrate that phagocyte oxidase-deficient B cells have a reduced capacity to kill bacteria. Our observations extend the number of immune cell types that produce ROS to kill pathogens.

Oxidative modification enhances the immunostimulatory effects of extracellular mitochondrial DNA on plasmacytoid dendritic cells.

Inflammation is associated with oxidative stress and characterized by elevated levels of damage-associated molecular pattern (DAMP) molecules released from injured or even living cells into the surrounding microenvironment. One of these endogenous danger signals is the extracellular mitochondrial DNA (mtDNA) containing evolutionary conserved unmethylated CpG repeats. Increased levels of reactive oxygen species (ROS) generated by recruited inflammatory cells modify mtDNA oxidatively, resulting primarily in accumulation of 8-oxo-7,8-dihydroguanine (8-oxoG) lesions. In this study, we examined the impact of native and oxidatively modified mtDNAs on the phenotypic and functional properties of plasmacytoid dendritic cells (pDCs), which possess a fundamental role in the regulation of inflammation and T cell immunity. Treatment of human primary pDCs with native mtDNA up-regulated the expression of a costimulatory molecule (CD86), a specific maturation marker (CD83), and a main antigen-presenting molecule (HLA-DQ) on the cell surface, as well as increased TNF-α and IL-8 production from the cells. These effects were more apparent when pDCs were exposed to oxidatively modified mtDNA. Neither native nor oxidized mtDNA molecules were able to induce interferon (IFN)-α secretion from pDCs unless they formed a complex with human cathelicidin LL-37, an antimicrobial peptide. Interestingly, simultaneous administration of a Toll-like receptor (TLR)9 antagonist abrogated the effects of both native and oxidized mtDNAs on human pDCs. In a murine model, oxidized mtDNA also proved a more potent activator of pDCs compared to the native form, except for induction of IFN-α production. Collectively, we demonstrate here for the first time that elevated levels of 8-oxoG bases in the extracellular mtDNA induced by oxidative stress increase the immunostimulatory capacity of mtDNA on pDCs.

Ragweed subpollen particles of respirable size activate human dendritic cells.

Ragweed (Ambrosia artemisiifolia) pollen grains, which are generally considered too large to reach the lower respiratory tract, release subpollen particles (SPPs) of respirable size upon hydration. These SPPs contain allergenic proteins and functional NAD(P)H oxidases. In this study, we examined whether exposure to SPPs initiates the activation of human monocyte-derived dendritic cells (moDCs). We found that treatment with freshly isolated ragweed SPPs increased the intracellular levels of reactive oxygen species (ROS) in moDCs. Phagocytosis of SPPs by moDCs, as demonstrated by confocal laser-scanning microscopy, led to an up-regulation of the cell surface expression of CD40, CD80, CD86, and HLA-DQ and an increase in the production of IL-6, TNF-α, IL-8, and IL-10. Furthermore, SPP-treated moDCs had an increased capacity to stimulate the proliferation of naïve T cells. Co-culture of SPP-treated moDCs with allogeneic CD3(+) pan-T cells resulted in increased secretion of IFN-γ and IL-17 by T cells of both allergic and non-allergic subjects, but induced the production of IL-4 exclusively from the T cells of allergic individuals. Addition of exogenous NADPH further increased, while heat-inactivation or pre-treatment with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, strongly diminished, the ability of SPPs to induce phenotypic and functional changes in moDCs, indicating that these processes were mediated, at least partly, by the intrinsic NAD(P)H oxidase activity of SPPs. Collectively, our data suggest that inhaled ragweed SPPs are fully capable of activating dendritic cells (DCs) in the airways and SPPs' NAD(P)H oxidase activity is involved in initiation of adaptive immune responses against innocuous pollen proteins.

Temporally designed treatment of melanoma cells by ATRA and polyI: C results in enhanced chemokine and IFNß secretion controlled differently by TLR3 and MDA5.

In the last three decades, the incidence of melanoma has increased worldwide and no effective treatment modalities have been developed yet. All-trans retinoic acid (ATRA) and polyinosinic:polycytidylic acid (polyI:C) are strong inducers of toll-like receptor 3 (TLR3) and MDA5 expression, and polyI:C-induced TLR3 and MDA5 signaling specifically causes cell death in melanoma cells in vitro. We addressed the question of whether ATRA pretreatment could enhance the efficacy of polyI:C and, if so, would ATRA have any additional effects on this process. We found that the combined treatment of human melanoma cells with ATRA and polyI:C strongly increased the expression of TLR3 and MDA5 in both WM35 and WM983A cells associated with significantly higher mRNA and secreted levels of interferon ß (IFNß), CXCL1, CXCL8/IL-8, CXCL9, and CXCL10 than cells treated with either ATRA or polyI:C. Silencing of MDA5 by siRNA moderately affected IFNß secretion, whereas TLR3 knockdown interfered with both CXCL chemokine and IFNß production. Furthermore, the supernatants of ATRA+polyI:C-activated cultures increased the migration of both human monocyte-derived macrophages and CD1a dendritic cells significantly as compared with the supernatants of cells treated with either ATRA or polyI:C, and this effect occurred in a TLR3-dependent manner. In conclusion, consecutive treatment with ATRA and polyI:C results in strong, TLR3/MDA5-mediated chemokine and IFN responses in cultured human melanoma cells, which triggers a functional migratory response in professional antigen-presenting cells. This novel mode of concomitant activation may represent a more efficient treatment option for future melanoma therapy.

Frank-ter Haar syndrome protein Tks4 regulates epidermal growth factor-dependent cell migration.

Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.

RLR-mediated production of interferon-ß by a human dendritic cell subset and its role in virus-specific immunity.

Cytosolic RIG-I-like helicases (RLR) are PRRs involved in type I IFN production and antiviral immunity. This study focuses to the comparison of the expression, function, and signaling cascades associated to RLR in the previously identified CD14(-)DC-SIGN(+)PPARγ(low)CD1a(+) and CD14(low)DC-SIGN(+)PPARγ(high)CD1a(-) human moDC subsets. Our results revealed that the expression of RLR genes and proteins as well as the activity of the coupled signaling pathways are significantly higher in the CD1a(+) subset than in its phenotypically and functionally distinct counterpart. Specific activation of RLR in moDCs by poly(I:C) or influenza virus was shown to induce the secretion of IFN-ß via IRF3, whereas induction of proinflammatory cytokine responses were predominantly controlled by TLR3. The requirement of RLR-mediated signaling in CD1a(+) moDCs for priming naïve CD8(+) T lymphocytes and inducing influenza virus-specific cellular immune responses was confirmed by RIG-I/MDA5 silencing, which abrogated these functions. Our results demonstrate the subset-specific activation of RLR and the underlying mechanisms behind its cytokine secretion profile and identify CD1a(+) moDCs as an inflammatory subset with specialized functional activities. We also provide evidence that this migratory DC subset can be detected in human tonsil and reactive LNs.

Constraints for monocyte-derived dendritic cell functions under inflammatory conditions.

The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.