Early Signaling Events Research Articles

FC065: Combined Proteomic Analyses of Recurrent Idiopathic Nephrotic Syndrome Plasma and Plasma-Treated Human Podocytes

Abstract BACKGROUND AND AIMS Idiopathic nephrotic syndrome (INS) comprises a group of rare glomerulopathies attributed to direct or indirect interaction of putative circulating factors with podocytes. One of the pathological manifestations of INS is the rapid recurrence of focal and segmental glomerulosclerosis (rFSGS) after renal transplant, which occurs in 30–50% of cases. To date, the nature and identity of circulating factors have not been confirmed, while the pathogenic mechanisms in the podocyte are partially understood. Direct analysis of patient plasma proteome has been scarcely addressed, mainly due to methodological difficulties associated with plasma complexity and dynamic range. To better understand the mechanisms involved in FSGS recurrence, the present study had two main objectives: (i) to perform a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from rFSGS patients and from controls; and (ii) to study the early proximal signaling events in podocytes in response to plasma from post-transplant rFSGS patients as compared to plasma from controls, by a combination of phosphoproteomics and lipid raft proteomics (raftomics). METHOD We obtained first post-transplant plasma exchange fluid from either rFSGS or non-INS patients (n = 4), the latter used as controls, as well as blood plasma from healthy individuals (n = 4). In a first study, by a combination of immunodepletion and high pH fractionation, we performed in parallel a differential proteomic analysis of soluble plasma proteins and of extracellular vesicles (EV) from the three groups. In a second study, we incubated differentiated human podocytes for 30 min with 10% of plasma exchange fluid from either rFSGS or non-INS patients. Then, we isolated lipid rafts from podocytes by a detergent-free method and performed differential proteomics. In addition, we performed differential phosphoproteomics after phosphopeptide enrichment in a TiO2 column. Proteomics by LC-MS/MS analysis was performed in a TimsTOF Pro mass spectrometer on plasma samples, and in a nanoRSLC-Q Exactive PLUS instrument on podocyte samples. RESULTS The approximate number of proteins identified was 500 in immunodepleted plasma, 1400 in EV, 2500 in podocyte rafts and 4900 podocyte phosphosites. In both soluble and EV protein sets from rFSGS patients, we found a statistically significant increase in a cluster of proteins involved in neutrophil degranulation. A group of lipid binding proteins, generally associated with lipoproteins, was found decreased in the soluble set from rFSGS patients. In addition, several aminoacid transporters involved in mTORC1 activation were found significantly increased in EV from rFSGS individuals. As regards podocyte proteome analyses, the differences found in protein expression and phosphorylation associated with rFSGS plasma incubation suggested alterations in the mTOR pathway, in autophagy, in mitochondrial metabolism and in cytoskeleton organization. In particular, validation experiments indicated an altered phosphorylation pattern of Hsp27. CONCLUSION The technological approaches used in the plasma study show the feasibility of direct proteomic analysis of this material, as well as the potential of some of the identified differential proteins as FSGS biomarkers. The observed changes in podocyte proteome associated with rFSGS plasma incubation highlight some of the potential mechanisms involved in FSGS recurrence and could be used as specific early markers of circulating factor activity on podocytes. Further research will be necessary to confirm and validate these findings.

Uncovering early thyroid hormone signalling events through temperature-mediated activation of molecular memory in the cultured bullfrog tadpole tail fin

Thyroid hormone (TH) is a critical signalling molecule for all vertebrate organisms, playing a crucial role in postembryonic development. The best-studied mechanism of TH response is through modulating gene expression, however TH’s involvement in coordinating the early steps in the TH signal transduction pathway is still poorly understood. The American bullfrog, Rana [Lithobates] catesbeiana, is a useful model to study these early responses as tadpole post-embryonic development in the form of metamorphosis of the tadpole into a frog can be experimentally induced by TH exposure. The rate of TH-induced metamorphosis can be modulated by temperature where sufficiently cold temperatures (5 °C) completely halt precocious metamorphosis. Interestingly, when premetamorphic tadpoles exposed to exogenous THs at 5 °C are shifted to permissive temperatures (24 °C), their metamorphic rate exceeds that of TH-exposed tadpoles at the permissive temperature. This suggests that a molecular memory of TH exposure is retained at 5 °C even after THs are cleared at this low temperature. However, the molecular memory machinery is poorly understood. Herein we use RNA-seq analysis to identify potential components of the molecular memory in cultured tail fin that allows for the recapitulation of the molecular memory phenomenon. Eighty-one gene transcripts were TH-responsive at 5 °C compared to matched controls indicating that the molecular memory is more complex than previously thought. Many of these transcripts encode transcription factors including thyroid hormone-induced B/Zip, thibz, and a novel krüppel-like factor family member, klfX. Actinomycin D and cycloheximide treatment had no effect on their TH induction suggesting that a change in transcription or translation is not required. Rather a change in RNA stability may be a possible mechanism contributing to the molecular memory. The ability to manipulate temperature and TH response in cultured organs provide an exciting opportunity to further elucidate the early TH signalling mechanisms during postembryonic development.

TRPM3-mediated dynamic mitochondrial activity in nerve growth factor–induced latent sensitization of chronic low back pain

The transient receptor potential ion channel TRPM3 is highly prevalent on nociceptive dorsal root ganglion (DRG) neurons, but its functions in neuronal plasticity of chronic pain remain obscure. In an animal model of nonspecific low back pain (LBP), latent spinal sensitization known as nociceptive priming is induced by nerve growth factor (NGF) injection. Here, we address the TRPM3-associated molecular basis of NGF-induced latent spinal sensitization at presynaptic level by studying TRPM3-mediated calcium transients in DRG neurons. By investigating TRPM3-expressing HEK cells, we further show the dynamic mitochondrial activity downstream of TRPM3 activation. NGF enhances TRPM3 function, attenuates TRPM3 tachyphylaxis, and slows intracellular calcium clearance; TRPM3 activation triggers more mitochondrial calcium loading than depolarization does, causing a steady-state mitochondrial calcium elevation and a delayed recovery of cytosolic calcium; mitochondrial calcium buffering accounts for approximately 40% of calcium influx subsequent to TRPM3 activation. TRPM3 activation provokes an outbreak of pulsatile superoxide production (mitoflash) that comes in the form of a surge in frequency being tunable. We suggest that mitoflash pulsations downstream of TRPM3 activation might be an early signaling event initiating pain sensitization. Tuning of mitoflash activity would be a novel bottom-up therapeutic strategy for chronic pain conditions such as LBP and beyond.

Significant impact of mTORC1 and ATF4 pathways in CHO cell recombinant protein production induced by CDK4/6 inhibitor.

The CDK4/6 inhibitor has been shown to increase recombinant protein productivity in Chinese hamster ovary (CHO) cells. Therefore, we investigated the mechanism that couples cell-cycle inhibitor (CCI) treatment with protein productivity utilizing proteomics and phosphoproteomics. We identified mTORC1 as a critical early signaling event that preceded boosted productivity. Following CCI treatment, mTOR exhibited a transient increase in phosphorylation at a novel site that is also conserved in humans and mouse. Upstream of mTORC1, increased phosphorylation of AKT1S1 and decreased phosphorylation of RB1 may provide molecular links between CDK4/6 inhibition and mTORC1. Downstream, increased EIF4EBP1 phosphorylation was observed, which can mediate cap-dependent translation. In addition, the collective effect of increased phosphorylation of RPS6, increased phosphorylation of regulators of RNA polymerase I, and increased protein expression in the transfer RNA-aminoacylation pathway may contribute to enhancing the translational apparatus for increased productivity. In concert, an elevated stress response via GCN2/EIF2AK4-ATF4 axis persisted over the treatment course, which may link mTOR to downstream responses including the unfolded protein responseand autophagy to enhance proper protein folding and secretion. Together, this comprehensive proteomics and phosphoproteomics characterization of CCI-treated CHO cells offers insights into understanding multiple aspects of signaling events resulting from CDK4/CDK6 inhibition.

Proteomic analysis in primary T cells reveals IL-7 alters T cell receptor thresholding via CYTIP/cytohesin/LFA-1 localisation and activation.

The ability of the cellular immune system to discriminate self from foreign antigens depends on the appropriate calibration of the T cell receptor (TCR) signalling threshold. The lymphocyte homeostatic cytokine interleukin 7 (IL-7) is known to affect TCR thresholding, but the molecular mechanism is not fully elucidated. A better understanding of this process is highly relevant in the context of autoimmune disease therapy and cancer immunotherapy. We sought to characterise the early signalling events attributable to IL-7 priming; in particular, the altered phosphorylation of signal transduction proteins and their molecular localisation to the TCR. By integrating high-resolution proximity- phospho-proteomic and imaging approaches using primary T cells, rather than engineered cell lines or an in vitro expanded T cell population, we uncovered transduction events previously not linked to IL-7. We show that IL-7 leads to dephosphorylation of cytohesin interacting protein (CYTIP) at a hitherto undescribed phosphorylation site (pThr280) and alters the co-localisation of cytohesin-1 with the TCR and LFA-1 integrin. These results show that IL-7, acting via CYTIP and cytohesin-1, may impact TCR activation thresholds by enhancing the co-clustering of TCR and LFA-1 integrin.

PD-L1 promotes myofibroblastic activation of hepatic stellate cells by distinct mechanisms selective for TGF-β receptor I versus II.

SUMMARYIntrahepatic cholangiocarcinoma (ICC) contains abundant myofibroblasts derived from hepatic stellate cells (HSCs) through an activation process mediated by TGF-β. To determine the role of programmed death-ligand 1 (PD-L1) in myofibroblastic activation of HSCs, we disrupted PD-L1 of HSCs by shRNA or anti-PD-L1 antibody. We find that PD-L1, produced by HSCs, is required for HSC activation by stabilizing TGF-β receptors I (TβRI) and II (TβRII). While the extracellular domain of PD-L1 (amino acids 19–238) targets TβRII protein to the plasma membrane and protects it from lysosomal degradation, a C-terminal 260-RLRKGR-265 motif on PD-L1 protects TβRI mRNA from degradation by the RNA exosome complex. PD-L1 is required for HSC expression of tumor-promoting factors, and targeting HSC PD-L1 by shRNA or Cre/loxP recombination suppresses HSC activation and ICC growth in mice. Thus, myofibroblast PD-L1 can modulate the tumor microenvironment and tumor growth by a mechanism independent of immune suppression.

Role of Skin Stretch on Local Vascular Permeability in Murine and Cell Culture Models.

Background:Excessive mechanical forces, particularly skin stretch, have been implicated in pathological cutaneous scarring. We hypothesize that this reflects, in part, stretch-induced vessel leakage that provokes prolonged wound/scar inflammation. However, this has never been observed directly. Here, a mouse model was used to examine the effect of skin flap stretching on vascular permeability. An in vitro model with pseudocapillaries grown in a stretchable chamber was also used to determine the effect of stretching on endothelial cell morphology and ion channel activity.Methods:Murine skin flaps were stretched with a biaxial stretching device, after which FITC-conjugated-dextran was injected and imaged with fluorescence stereomicroscopy. Endothelial cells were induced to form pseudocapillary networks in an elastic chamber. The chamber was stretched and differential interference contrast microscopy was used to assess cell morphology. In other experiments, markers for Ca2+ influx and K+ efflux were added before a single stretch was conducted. Histamine served as a positive-control in all experiments.Results:Cyclic stretching (20%) increased the vascular permeability of skin flaps almost as strongly as histamine. Both stimuli also partially disrupted the pseudocapillary networks, induced cell contraction, and created gaps between the cells. Both stimuli caused sustained K+ efflux; stretching had a milder effect on Ca2+ influx.Conclusions:Excessive cyclical stretching strongly increased the vascular permeability of skin vessels and in vitro pseudocapillaries. This effect associated with increased K+ efflux and some Ca2+ influx. Inhibiting such early stretch-induced signaling events may be an effective strategy for treating and preventing hypertrophic scars and keloids.

Data-Independent Acquisition-Based Proteome and Phosphoproteome Profiling Reveals Early Protein Phosphorylation and Dephosphorylation Events in Arabidopsis Seedlings upon Cold Exposure.

Protein phosphorylation plays an important role in mediating signal transduction in cold response in plants. To better understand how plants sense and respond to the early temperature drop, we performed data-independent acquisition (DIA) method-based mass spectrometry analysis to profile the proteome and phosphoproteome of Arabidopsis seedlings upon cold stress in a time-course manner (10, 30 and 120 min of cold treatments). Our results showed the rapid and extensive changes at the phosphopeptide levels, but not at the protein abundance levels, indicating cold-mediated protein phosphorylation and dephosphorylation events. Alteration of over 1200 proteins at phosphopeptide levels were observed within 2 h of cold treatment, including over 140 kinases, over 40 transcriptional factors and over 40 E3 ligases, revealing the complexity of regulation of cold adaption. We summarized cold responsive phosphoproteins involved in phospholipid signaling, cytoskeleton reorganization, calcium signaling, and MAPK cascades. Cold-altered levels of 73 phosphopeptides (mostly novel cold-responsive) representing 62 proteins were validated by parallel reaction monitoring (PRM). In summary, this study furthers our understanding of the molecular mechanisms of cold adaption in plants and strongly supports that DIA coupled with PRM are valuable tools in uncovering early signaling events in plants.

SERKs regulate embryonic cuticle integrity through the TWS1-GSO1/2 signaling pathway in Arabidopsis.

The embryonic cuticle integrity is critical for the embryo to separate from the neighboring endosperm. The sulfated TWISTED SEED1 (TWS1) peptide precursor generated in the embryo diffuses through gaps of the nascent cuticle to the surrounding endosperm, where it is cleaved by ABNORMAL LEAF SHAPE1 (ALE1) and becomes an active mature form. The active TWS1 is perceived by receptor-like protein kinases GASSHO1 (GSO1) and GSO2 in the embryonic epidermal cells to start the downstream signaling and guide the formation of an intact embryonic cuticle. However, the early signaling events after TWS1 is perceived by GSO1/2 are still unknown. Here, we report that serk1/2/3 embryos show cuticle defects similar to ale1, tws1, and gso1/2. Genetic and biochemical analyses were performed to dissect the signaling pathway mediated by SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASEs (SERKs) during cuticle development. SERKs function with GSO1/2 in a common pathway to monitor the integrity of the embryonic cuticle. SERKs interact with GSO1/2, which can be enhanced dramatically by TWS1. The phosphorylation levels of SERKs and GSO1/2 rely on each other and can respond to and be elevated by TWS1. Our results demonstrate that SERKs may function as coreceptors of GSO1/2 to transduce the TWS1 signal and ultimately regulate embryonic cuticle integrity.

The Arabidopsis thaliana LysM-containing Receptor-Like Kinase 2 is required for elicitor-induced resistance to pathogens.

In Arabidopsis thaliana, perception of chitin from fungal cell walls is mediated by three LysM‐containing Receptor‐Like Kinases (LYKs): CERK1, which is absolutely required for chitin perception, and LYK4 and LYK5, which act redundantly. The role in plant innate immunity of a fourth LYK protein, LYK2, is currently not known. Here we show that CERK1, LYK2 and LYK5 are dispensable for basal susceptibility to B. cinerea but are necessary for chitin‐induced resistance to this pathogen. LYK2 is dispensable for chitin perception and early signalling events, though it contributes to callose deposition induced by this elicitor. Notably, LYK2 is also necessary for enhanced resistance to B. cinerea and Pseudomonas syringae induced by flagellin and for elicitor‐induced priming of defence gene expression during fungal infection. Consistently, overexpression of LYK2 enhances resistance to B. cinerea and P. syringae and results in increased expression of defence‐related genes during fungal infection. LYK2 appears to be required to establish a primed state in plants exposed to biotic elicitors, ensuring a robust resistance to subsequent pathogen infections.

Deciphering the Role of Ion Channels in Early Defense Signaling against Herbivorous Insects.

Plants and insect herbivores are in a relentless battle to outwit each other. Plants have evolved various strategies to detect herbivores and mount an effective defense system against them. These defenses include physical and structural barriers such as spines, trichomes, cuticle, or chemical compounds, including secondary metabolites such as phenolics and terpenes. Plants perceive herbivory by both mechanical and chemical means. Mechanical sensing can occur through the perception of insect biting, piercing, or chewing, while chemical signaling occurs through the perception of various herbivore-derived compounds such as oral secretions (OS) or regurgitant, insect excreta (frass), or oviposition fluids. Interestingly, ion channels or transporters are the first responders for the perception of these mechanical and chemical cues. These transmembrane pore proteins can play an important role in plant defense through the induction of early signaling components such as plasma transmembrane potential (Vm) fluctuation, intracellular calcium (Ca2+), and reactive oxygen species (ROS) generation, followed by defense gene expression, and, ultimately, plant defense responses. In recent years, studies on early plant defense signaling in response to herbivory have been gaining momentum with the application of genetically encoded GFP-based sensors for real-time monitoring of early signaling events and genetic tools to manipulate ion channels involved in plant-herbivore interactions. In this review, we provide an update on recent developments and advances on early signaling events in plant-herbivore interactions, with an emphasis on the role of ion channels in early plant defense signaling.

Protective Effect of Ciclopirox against Ovariectomy-Induced Bone Loss in Mice by Suppressing Osteoclast Formation and Function.

Postmenopausal osteoporosis is closely associated with excessive osteoclast formation and function, resulting in the loss of bone mass. Osteoclast-targeting agents have been developed to manage this disease. We examined the effects of ciclopirox on osteoclast differentiation and bone resorption in vitro and in vivo. Ciclopirox significantly inhibited osteoclast formation from primary murine bone marrow macrophages (BMMs) in response to receptor activator of nuclear factor kappa B ligand (RANKL), and the expression of genes associated with osteoclastogenesis and function was decreased. The formation of actin rings and resorption pits was suppressed by ciclopirox. Analysis of RANKL-mediated early signaling events in BMMs revealed that ciclopirox attenuates IκBα phosphorylation without affecting mitogen-activated protein kinase activation. Furthermore, the administration of ciclopirox suppressed osteoclast formation and bone loss in ovariectomy-induced osteoporosis in mice and reduced serum levels of osteocalcin and C-terminal telopeptide fragment of type I collagen C-terminus. These results indicate that ciclopirox exhibits antiosteoclastogenic activity both in vitro and in vivo and represents a new candidate compound for protection against osteoporosis and other osteoclast-related bone diseases.

The plasma membrane-associated Ca2+ -binding protein, PCaP1, is required for oligogalacturonide and flagellin-induced priming and immunity.

Early signalling events in response to elicitation include reversible protein phosphorylation and re‐localization of plasma membrane (PM) proteins. Oligogalacturonides (OGs) are a class of damage‐associated molecular patterns (DAMPs) that act as endogenous signals to activate the plant immune response. Previous data on early phosphoproteome changes in Arabidopsis thaliana upon OG perception uncovered the immune‐related phospho‐regulation of several membrane proteins, among which PCaP1, a PM‐anchored protein with actin filament–severing activity, was chosen for its potential involvement in OG‐ and flagellin‐triggered responses. Here, we demonstrate that PCaP1 is required for late, but not early, responses induced by OGs and flagellin. Moreover, pcap1 mutants, unlike the wild type, are impaired in the recovery of full responsiveness to a second treatment with OGs performed 24 h after the first one. Localization studies on PCaP1 upon OG treatment in plants expressing a functional PCaP1‐GFP fusion under the control of PCaP1 promoter revealed fluorescence on the PM, organized in densely packed punctate structures, previously reported as microdomains. Fluorescence was found to be associated also with endocytic vesicles, the number of which rapidly increased after OG treatment, suggesting both an endocytic turnover of PCaP1 for maintaining its homeostasis at the PM and an OG‐induced endocytosis.

Immunostimulatory CpG Oligodeoxynucleotides Induce Sustained ERK2 Activation and Strong Proliferative Responses in CLL B-Cells from Patients with Progressive Disease.

Immunostimulatory DNA sequences that are present in microbial DNA, DNA-containing immune complexes and synthetic CpG-oligodeoxynucleotides (ODNs) can activate cells of the immune system via Toll-like receptor 9 (TLR9). Normal B-cells respond to such stimulation by upregulation of co-stimulatory molecules, proliferation and prolonged survival. The response of chronic lymphocytic leukemia B-cells to CpG ODN stimulation appears to be more variable, since both proliferation and induction of apoptosis have been reported. Because CLL B-cells with mutated and unmutated Ig VH genes show distinct clinical and biological behaviors, we compared their responsiveness to CpG ODN stimulation in terms of proliferation, survival, activation of TLR9 signaling pathways and expression of cell-cycle regulatory and anti-apoptotic proteins. A significant proliferative response was induced in most of the VH unmutated CLL samples (14/23), whereas 3H thymidine incorporation was low or absent in the majority of the VH mutated cases (22/27, P = 0.005). More strikingly, stratification of the CLL cases based on the clinical course showed strong proliferative responses in 14/21 cases with progressive disease and in only 5/29 cases with stable disease (P=0.001). The absence of a proliferative response to CpG ODN stimulation in the stable/VH mutated cases was not caused by different early signaling events, since phosphorylation of JNK, ERK1/2 and Akt, as well as degradation of IkB, was detected in almost all investigated samples (7/7 proliferating and 5/6 nonproliferating). However, a notable difference was observed in the duration of ERK2 activation, which was transient in the nonproliferating CLL B-cells, but lasted more than 24 hours in the proliferating cases. This difference was associated with dimished phosphorylation of the retinoblastoma protein (pRb) and less pronounced or absent induction of cyclin E and cyclin A in the nonproliferating cases, suggesting a block in the G1/S phase transition. On the other hand, cyclin D3 was induced and p27KIP1 was downregulated in all samples, indicating that CLL B-cells from both subgroups can enter the G1 phase of the cell cycle. Analysis of CLL B-cell survival by Annexin V/PI staining and PARP cleavage after 48 hours stimulation with CpG ODN showed significant levels of apoptosis in most of the nonproliferating cases. In contrast, proliferating CLL B-cells remained viable after CpG ODN stimulation, which was associated with upregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL. These data indicate that immunostimulatory CpG oligodeoxynucleotides can induce distinct responses in progressive/VH unmutated and stable/VH mutated CLL B-cells, which are characterized by proliferation and inhibition of spontaneous apoptosis in the former and induction of apoptosis in the latter group of cases. Although both subgroups show similar early activation of TLR9 signaling pathways and both enter into the G1 phase of the cell cycle, the absence of sustained ERK2 activation precludes cell cycle progression in most stable/VH mutated CLL B-cells. The greater capacity to traverse the cell cycle and proliferate in response to immunostimulatory CpG DNA may therefore be related to the more aggressive clinical course that is associated with CLL B-cells that express unmutated VH genes.

Phloem exudate metabolic content reflects the response to water-deficit stress in pea plants (Pisum sativum L.).

SummaryDrought stress impacts the quality and yield of Pisum sativum. Here, we show how short periods of limited water availability during the vegetative stage of pea alters phloem sap content and how these changes are connected to strategies used by plants to cope with water deficit. We have investigated the metabolic content of phloem sap exudates and explored how this reflects P. sativum physiological and developmental responses to drought. Our data show that drought is accompanied by phloem‐mediated redirection of the components that are necessary for cellular respiration and the proper maintenance of carbon/nitrogen balance during stress. The metabolic content of phloem sap reveals a shift from anabolic to catabolic processes as well as the developmental plasticity of P. sativum plants subjected to drought. Our study underlines the importance of phloem‐mediated transport for plant adaptation to unfavourable environmental conditions. We also show that phloem exudate analysis can be used as a useful proxy to study stress responses in plants. We propose that the decrease in oleic acid content within phloem sap could be considered as a potential marker of early signalling events mediating drought response.

Quantitative Phosphoproteomic Analysis Reveals Dendritic Cell- Specific STAT Signaling After α2-3-Linked Sialic Acid Ligand Binding.

Dendritic cells (DCs) are key initiators of the adaptive immunity, and upon recognition of pathogens are able to skew T cell differentiation to elicit appropriate responses. DCs possess this extraordinary capacity to discern external signals using receptors that recognize pathogen-associated molecular patterns. These can be glycan-binding receptors that recognize carbohydrate structures on pathogens or pathogen-associated patterns that additionally bind receptors, such as Toll-like receptors (TLRs). This study explores the early signaling events in DCs upon binding of α2-3 sialic acid (α2-3sia) that are recognized by Immune inhibitory Sialic acid binding immunoglobulin type lectins. α2-3sias are commonly found on bacteria, e.g. Group B Streptococcus, but can also be expressed by tumor cells. We investigated whether α2-3sia conjugated to a dendrimeric core alters DC signaling properties. Through phosphoproteomic analysis, we found differential signaling profiles in DCs after α2-3sia binding alone or in combination with LPS/TLR4 co-stimulation. α2-3sia was able to modulate the TLR4 signaling cascade, resulting in 109 altered phosphoproteins. These phosphoproteins were annotated to seven biological processes, including the regulation of the IL-12 cytokine pathway. Secretion of IL-10, the inhibitory regulator of IL-12 production, by DCs was found upregulated after overnight stimulation with the α2-3sia dendrimer. Analysis of kinase activity revealed altered signatures in the JAK-STAT signaling pathway. PhosphoSTAT3 (Ser727) and phosphoSTAT5A (Ser780), involved in the regulation of the IL-12 pathway, were both downregulated. Flow cytometric quantification indeed revealed de- phosphorylation over time upon stimulation with α2-3sia, but no α2-6sia. Inhibition of both STAT3 and -5A in moDCs resulted in a similar cytokine secretion profile as α-3sia triggered DCs. Conclusively, this study revealed a specific alteration of the JAK-STAT pathway in DCs upon simultaneous α2-3sia and LPS stimulation, altering the IL10:IL-12 cytokine secretion profile associated with reduction of inflammation. Targeted control of the STAT phosphorylation status is therefore an interesting lead for the abrogation of immune escape that bacteria or tumors impose on the host.

Pattern-recognition receptors are required for NLR-mediated plant immunity.

The plant immune system is fundamental for plant survival in natural ecosystems and for productivity in crop fields. Substantial evidence supports the prevailing notion that plants possess a two-tiered innate immune system, called pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered by microbial patterns via cell surface-localized pattern-recognition receptors (PRRs), whereas ETI is activated by pathogen effector proteins via predominantly intracellularly localized receptors called nucleotide-binding, leucine-rich repeat receptors (NLRs)1-4. PTI and ETI are initiated by distinct activation mechanisms and involve different early signalling cascades5,6. Here we show that Arabidopsis PRR and PRR co-receptor mutants-fls2 efr cerk1 and bak1 bkk1 cerk1 triple mutants-are markedly impaired in ETI responses when challenged with incompatible Pseudomonas syrinage bacteria. We further show that the production of reactive oxygen species by the NADPH oxidase RBOHD is a critical early signalling event connecting PRR- and NLR-mediated immunity, and that the receptor-like cytoplasmic kinase BIK1 is necessary for full activation of RBOHD, gene expression and bacterial resistance during ETI. Moreover, NLR signalling rapidly augments the transcript and/or protein levels of key PTI components. Our study supports a revised model in which potentiation of PTI is an indispensable component of ETI during bacterial infection. This revised model conceptually unites two major immune signalling cascades in plants and mechanistically explains some of the long-observed similarities in downstream defence outputs between PTI and ETI.

PCTP contributes to human platelet activation by enhancing dense granule secretion

PCTP (phosphatidylcholine transfer protein) was discovered recently to regulate aggregation of human platelets stimulated with PAR4 activating peptide (PAR4AP). However, the role of PCTP following thrombin stimulation, the mechanisms by which PCTP contributes to platelet activation, and the role of PCTP with other receptors remained unknown. As mouse platelets do not express PCTP, we treated human platelets with various agonists in the presence of the specific PCTP inhibitor A1. We observed that PCTP inhibition significantly reduced dense granule secretion in response to thrombin, PAR1AP, PAR4AP, convulxin (GPVI agonist) and FcγRIIA crosslinking. In contrast, among these agonists, PCTP inhibition reduced aggregation only to low dose thrombin and PAR4AP. Unlike its effects on dense granule secretion, PCTP inhibition did not reduce alpha granule secretion in response to thrombin or PAR4AP. PCTP inhibition reduced both the increase in cytoplasmic Ca2+ as well as PKC activity downstream of thrombin. These data are consistent with PCTP contributing to secretion of dense granules, and to being particularly important to human PAR4 early signaling events. Future studies will address further these molecular mechanisms and consequences for hemostasis and thrombosis.

Unique molecular characteristics and microglial origin of Kv1.3 channel–positive brain myeloid cells in Alzheimer’s disease

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aβ) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b+CD45+ CNS-MPs acutely isolated from an Aβ mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b+Kv1.3+ CNS-MPs, CD11b+CD45int Kv1.3neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers (Tmem119, P2ry12) and are distinct from peripheral Ly6chigh/Ly6clow monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c, and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3+ CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aβ burden, increased CD11c+ CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes (IL1b). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype.

Inhibition of the HEG1-KRIT1 interaction increases KLF4 and KLF2 expression in endothelial cells.

ABSTRACTThe transmembrane protein heart of glass1 (HEG1) directly binds to and recruits Krev interaction trapped protein 1 (KRIT1) to endothelial junctions to form the HEG1–KRIT1 protein complex that establishes and maintains junctional integrity. Genetic inactivation or knockdown of endothelial HEG1 or KRIT1 leads to the upregulation of transcription factors Krüppel‐like factors 4 and 2 (KLF4 and KLF2), which are implicated in endothelial vascular homeostasis; however, the effect of acute inhibition of the HEG1–KRIT1 interaction remains incompletely understood. Here, we report a high‐throughput screening assay and molecular design of a small‐molecule HEG1–KRIT1 inhibitor to uncover acute changes in signaling pathways downstream of the HEG1–KRIT1 protein complex disruption. The small‐molecule HEG1–KRIT1 inhibitor 2 (HKi2) was demonstrated to be a bona fide inhibitor of the interaction between HEG1 and KRIT1 proteins, by competing orthosterically with HEG1 through covalent reversible interactions with the FERM (4.1, ezrin, radixin, and moesin) domain of KRIT1. The crystal structure of HKi2 bound to KRIT1 FERM revealed that it occupies the same binding pocket on KRIT1 as the HEG1 cytoplasmic tail. In human endothelial cells (ECs), acute inhibition of the HEG1–KRIT1 interaction by HKi2 increased KLF4 and KLF2 mRNA and protein levels, whereas a structurally similar inactive compound failed to do so. In zebrafish, HKi2 induced expression of klf2a in arterial and venous endothelium. Furthermore, genome‐wide RNA transcriptome analysis of HKi2‐treated ECs under static conditions revealed that, in addition to elevating KLF4 and KLF2 expression, inhibition of the HEG1–KRIT1 interaction mimics many of the transcriptional effects of laminar blood flow. Furthermore, HKi2‐treated ECs also triggered Akt signaling in a phosphoinositide 3‐kinase (PI3K)‐dependent manner, as blocking PI3K activity blunted the Akt phosphorylation induced by HKi2. Finally, using an in vitro colocalization assay, we show that HKi6, an improved derivative of HKi2 with higher affinity for KRIT1, significantly impedes recruitment of KRIT1 to mitochondria‐localized HEG1 in CHO cells, indicating a direct inhibition of the HEG1–KRIT1 interaction. Thus, our results demonstrate that early events of the acute inhibition of HEG1–KRIT1 interaction with HKi small‐molecule inhibitors lead to: (i) elevated KLF4 and KLF2 gene expression; and (ii) increased Akt phosphorylation. Thus, HKi’s provide new pharmacologic tools to study acute inhibition of the HEG1–KRIT1 protein complex and may provide insights to dissect early signaling events that regulate vascular homeostasis.