An optimized method for IgE-mediated degranulation of human lung mast cells.

Background: Mast cells are critically involved in IgE-mediated diseases, e.g., allergies and asthma. Human mast cells are heterogeneous, and mast cells from different anatomical sites have been shown to respond differently to certain stimuli and drugs. The origin of the mast cells is therefore of importance when setting up a model system, and human lung mast cells are highly relevant cells to study in the context of asthma. We therefore set out to optimize a protocol of IgE-mediated activation of human lung mast cells. Methods: Human lung mast cells were extracted from lung tissue obtained from patients undergoing pulmonary resection by enzyme digestion and mechanical disruption followed by CD117 magnetic-activated cell sorting (MACS) enrichment. Different culturing media and conditions for the IgE-mediated degranulation were tested to obtain an optimized method. Results: IgE crosslinking of human lung mast cells cultured in serum-free media gave a stronger response compared to cells cultured with 10% serum. The addition of stem cell factor (SCF) did not enhance the degranulation. However, when the cells were put in fresh serum-free media 30 minutes prior to the addition of anti-IgE antibodies, the cells responded more vigorously. Maximum degranulation was reached 10 minutes after the addition of anti-IgE. Both CD63 and CD164 were identified as stable markers for the detection of degranulated mast cells over time, while the staining with anti-CD107a and avidin started to decline 10 minutes after activation. The levels of CD203c and CD13 did not change in activated cells and therefore cannot be used as degranulation markers of human lung mast cells. Conclusions: For an optimal degranulation response, human lung mast cells should be cultured and activated in serum-free media. With this method, a very strong and consistent degranulation response with a low donor-to-donor variation is obtained. Therefore, this model is useful for further investigations of IgE-mediated mast cell activation and exploring drugs that target human lung mast cells, for instance, in the context of asthma.

The Rac-GEF Tiam1 controls integrin-dependent neutrophil responses.

Rac GTPases are required for neutrophil adhesion and migration, and for the neutrophil effector responses that kill pathogens. These Rac-dependent functions are impaired when neutrophils lack the activators of Rac, Rac-GEFs from the Prex, Vav, and Dock families. In this study, we demonstrate that Tiam1 is also expressed in neutrophils, governing focal complexes, actin cytoskeletal dynamics, polarisation, and migration, in a manner depending on the integrin ligand to which the cells adhere. Tiam1 is dispensable for the generation of reactive oxygen species but mediates degranulation and NETs release in adherent neutrophils, as well as the killing of bacteria. In vivo, Tiam1 is required for neutrophil recruitment during aseptic peritonitis and for the clearance of Streptococcus pneumoniae during pulmonary infection. However, Tiam1 functions differently to other Rac-GEFs. Instead of promoting neutrophil adhesion to ICAM1 and stimulating ß2 integrin activity as could be expected, Tiam1 restricts these processes. In accordance with these paradoxical inhibitory roles, Tiam1 limits the fMLP-stimulated activation of Rac1 and Rac2 in adherent neutrophils, rather than activating Rac as expected. Tiam1 promotes the expression of several regulators of small GTPases and cytoskeletal dynamics, including αPix, Psd4, Rasa3, and Tiam2. It also controls the association of Rasa3, and potentially αPix, Git2, Psd4, and 14-3-3ζ/δ, with Rac. We propose these latter roles of Tiam1 underlie its effects on Rac and ß2 integrin activity and on cell responses. Hence, Tiam1 is a novel regulator of Rac-dependent neutrophil responses that functions differently to other known neutrophil Rac-GEFs.

Dock2 generates characteristic spatiotemporal patterns of Rac activity to regulate neutrophil polarisation, migration and phagocytosis.

Introduction: Rac-GTPases and their Rac-GEF activators play important roles in neutrophil-mediated host defence. These proteins control the adhesion molecules and cytoskeletal dynamics required for neutrophil recruitment to inflamed and infected organs, and the neutrophil effector responses that kill pathogens. Methods: Here, we used live cell TIRF-FRET imaging in neutrophils from Rac-FRET reporter mice with deficiencies in the Rac-GEFs Dock2, Tiam1 or Prex1/Vav1 to evaluate if these proteins activate spatiotemporally distinct pools of Rac, and to correlate patterns of Rac activity with the neutrophil responses they control. Results: All the GEFs were required for neutrophil adhesion, and Prex1/Vav1 were important during spreading and for the velocity of migration during chemotaxis. However, Dock2 emerged as the prominent regulator of neutrophil responses, as this GEF was required for neutrophil polarisation and random migration, for migration velocity during chemokinesis, for the likelihood to migrate and for the speed of migration and of turning during chemotaxis, as well as for rapid particle engulfment during phagocytosis. We identified characteristic spatiotemporal patterns of Rac activity generated by Dock2 which correlate with the importance of the Rac-GEF in these neutrophil responses. We also demonstrate a requirement for Dock2 in neutrophil recruitment during aseptic peritonitis. Discussion: Collectively, our data provide a first direct comparison of the pools of Rac activity generated by different types of Rac-GEFs, and identify Dock2 as a key regulator of polarisation, migration and phagocytosis in primary neutrophils.

Modulating T-cell activation with antisense oligonucleotides targeting lymphocyte cytosolic protein 2.

Dysregulated T-cell activation is a hallmark of several autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS). The lymphocyte cytosolic protein 2 (LCP2), also known as SLP-76, is essential for the development and activation of T cells. Despite the critical role of LCP2 in T-cell activation and the need for developing drugs that modify T-cell activation, no LCP2 inhibitors have been developed. This can be explained by the "undruggable" nature of LCP2, lacking a structure permissive to standard small molecule inhibitor modalities. Here, we explored an alternative drug modality, developing antisense oligonucleotides (ASOs) targeting LCP2 mRNAs, and evaluated its activity in modulating T-cell activation. We identified a set of 3' UTR targeting LCP2 ASOs, which knocked down LCP2 in a human T-cell line and primary human T cells and found that these suppressed T-cell receptor mediated activation. We also found that the ASOs suppressed FcεR1-mediated mast cell activation, in line with the role of LCP2 in mast cells. Taken together, our data provide examples of how immunomodulatory ASOs that interfere with undruggable targets can be developed and propose that such drug modalities can be used to treat autoimmune diseases.

Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging.

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Förster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival.

Selective inhibition of prostaglandin D2 biosynthesis in human mast cells to overcome need for multiple receptor antagonists: Biochemical consequences.

BACKGROUND: The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. OBJECTIVES: To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. METHODS: Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. RESULTS: All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. CONCLUSIONS: Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

COX-1 dependent biosynthesis of 15-hydroxyeicosatetraenoic acid in human mast cells.

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Eosinophils synthesize trihydroxyoctadecenoic acids (TriHOMEs) via a 15-lipoxygenase dependent process.

Trihydroxyoctadecenoic acids (TriHOMEs) are linoleic acid-derived lipid mediators reported to be dysregulated in obstructive lung disease. In contrast to many other oxylipins, TriHOME biosynthesis in humans is still poorly understood. The association of TriHOMEs with inflammation prompted the current investigation into the ability of human granulocytes to synthesize the 16 different 9,10,13-TriHOME and 9,12,13-TriHOME isomers and of the TriHOME biosynthetic pathway. Following incubation with linoleic acid, eosinophils and (to a lesser extent) the mast cell line LAD2, but not neutrophils, formed TriHOMEs. Stereochemical analysis revealed that TriHOMEs produced by eosinophils predominantly evidenced the 13(S) configuration, suggesting 15-lipoxygenase (15-LOX)-mediated synthesis. TriHOME formation was blocked following incubation with the 15-LOX inhibitor BLX-3887 and was shown to be largely independent of soluble epoxide hydrolase and cytochrome P450 activities. TriHOME synthesis was abolished when linoleic acid was replaced with 13-HODE, but increased in incubations with 13-HpODE, indicating the intermediary role of epoxy alcohols in TriHOME formation. In contrast to eosinophils, LAD2 cells formed TriHOMEs having predominantly the 13(R) configuration, demonstrating that there are multiple synthetic routes for TriHOME formation. These findings provide for the first-time insight into the synthetic route of TriHOMEs in humans and expand our understanding of their formation in inflammatory diseases.

IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.

BACKGROUND: Specific inflammatory pathways are indicated to contribute to severe asthma, but their individual involvement in the development of airway hyperresponsiveness remains unexplored. OBJECTIVE: This experimental study in human small bronchi aimed to provide insight into which of the type 2 and type 17 cytokines cause hyperresponsiveness of airway smooth muscle. METHODS: Explanted small bronchi isolated from human lung tissue and human airway smooth muscle cells were treated for 2 and 1 day(s), respectively, with 100 ng/mL of IL-4, IL-5, IL-13, or IL-17A, and contractile responses, Ca2+ mobilization, and receptor expression were assessed. RESULTS: Treatment with IL-13 increased the potency of histamine, carbachol, and leukotriene D4 as contractile agonists. IL-4, but not IL-5 or IL-17A, also increased the potency of histamine. In human airway smooth muscle cells, IL-13 and IL-4, but not IL-5 and IL-17A, enhanced the histamine-induced Ca2+ mobilization that was accompanied with increased mRNA expression of histamine H1 and cysteinyl leukotriene CysLT1 receptors. RNA sequencing of isolated bronchi confirmed the IL-13-mediated upregulation of H1 and CysLT1 receptors, without showing an alteration of muscarinic M3 receptors. Dexamethasone had no effects on IL-13-induced hyperresponsiveness in human bronchi, the increased Ca2+ mobilization, or the enhanced receptor expression. In contrast, antagonism of the common receptor for IL-13 and IL-4 by the biologic dupilumab prevented the effects of both IL-13 and IL-4 in human bronchi and human airway smooth muscle cells. CONCLUSIONS: The glucocorticoid-insensitive hyperrresponsiveness in isolated human airways induced by IL-13 and IL-4 provides further evidence that the IL-4Rα pathway should be targeted as a new strategy for the treatment of airway hyperresponsiveness in asthma.

A RhoA-FRET Biosensor Mouse for Intravital Imaging in Normal Tissue Homeostasis and Disease Contexts.

The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.

The dynamics of Rho GTPase signaling and implications for targeting cancer and the tumor microenvironment.

Numerous large scale genomics studies have demonstrated that cancer is a molecularly heterogeneous disease, characterized by acquired changes in the structure and DNA sequence of tumor genomes. More recently, the role of the equally complex tumor microenvironment in driving the aggressiveness of this disease is increasingly being realized. Tumor cells are surrounded by activated stroma, creating a dynamic environment that promotes cancer development, metastasis and chemoresistance. The Rho family of small GTPases plays an essential role in the regulation of cell shape, cytokinesis, cell adhesion, and cell motility. Importantly, these processes need to be considered in the context of a complex 3-dimensional (3D) environment, with reciprocal feedback and cross-talk taking place between the tumor cells and host environment. Here we discuss the role of molecular networks involving Rho GTPases in cancer, and the therapeutic implications of inhibiting Rho signaling in both cancer cells and the emerging concept of targeting the surrounding stroma.

The Rac-FRET mouse reveals tight spatiotemporal control of Rac activity in primary cells and tissues.

The small G protein family Rac has numerous regulators that integrate extracellular signals into tight spatiotemporal maps of its activity to promote specific cell morphologies and responses. Here, we have generated a mouse strain, Rac-FRET, which ubiquitously expresses the Raichu-Rac biosensor. It enables FRET imaging and quantification of Rac activity in live tissues and primary cells without affecting cell properties and responses. We assessed Rac activity in chemotaxing Rac-FRET neutrophils and found enrichment in leading-edge protrusions and unexpected longitudinal shifts and oscillations during protruding and stalling phases of migration. We monitored Rac activity in normal or disease states of intestinal, liver, mammary, pancreatic, and skin tissue, in response to stimulation or inhibition and upon genetic manipulation of upstream regulators, revealing unexpected insights into Rac signaling during disease development. The Rac-FRET strain is a resource that promises to fundamentally advance our understanding of Rac-dependent responses in primary cells and native environments.

Synaptotagmin 1 causes phosphatidyl inositol lipid-dependent actin remodeling in cultured non-neuronal and neuronal cells.

Here we demonstrate that a dramatic actin polymerizing activity caused by ectopic expression of the synaptic vesicle protein synaptotagmin 1 that results in extensive filopodia formation is due to the presence of a lysine rich sequence motif immediately at the cytoplasmic side of the transmembrane domain of the protein. This polybasic sequence interacts with anionic phospholipids in vitro, and, consequently, the actin remodeling caused by this sequence is interfered with by expression of a phosphatidyl inositol (4,5)-bisphosphate (PIP2)-targeted phosphatase, suggesting that it intervenes with the function of PIP2-binding actin control proteins. The activity drastically alters the behavior of a range of cultured cells including the neuroblastoma cell line SH-SY5Y and primary cortical mouse neurons, and, since the sequence is conserved also in synaptotagmin 2, it may reflect an important fine-tuning role for these two proteins during synaptic vesicle fusion and neurotransmitter release.

Internalization studies of chimeric neuropeptide Y receptors Y1 and Y2 suggest complex interactions between cytoplasmic domains.

Agonist stimulation readily internalizes neuropeptide Y receptor Y1 while there are contradictory results for the Y2 receptor. In order to explore putative functional differences between the Y1 and Y2 receptors we generated reciprocal chimeras by swapping the third intracellular loop, the carboxy terminus or both between human Y1 and Y2. Internalization was studied in a quantitative radioligand binding assay with removal of surface-bound ligand in an acidic-wash procedure. The internalization assay revealed a lower degree of internalization as well as slower kinetics for the Y2 receptor. Generally, reciprocal exchange of receptor segments did not convey properties of the donor receptor but tended to enhance internalization. Surprisingly, insertion of the Y2 carboxy terminus into Y1 gave almost complete internalization (92%), rather than reduced internalization, while the insertion of both segments resulted in internalization equal to the native Y1 receptor. These findings were confirmed by fluorescence microscopy of immuno-stained receptors tagged with a C-terminal FLAG epitope. However, after exposure to high agonist concentrations (100 nM) Y2 was internalized. Studies of Y2 and the closely related Y7 receptor confirmed low internalization for Y2 from chicken and teleost fishes as well as Y7 from two teleosts. The conservation across species of low internalization at physiological concentrations suggests that this is an ancient feature and of vital importance for Y2 function. We propose that amino acid motifs in the third intracellular loop as well as the C terminus of both Y1 and Y2 are able to drive agonist-promoted internalization and that there may be constraining motifs in the Y2 receptor.

Microtubule-dependent localization of profilin I mRNA to actin polymerization sites in serum-stimulated cells.

Specific localization of messenger RNA (mRNA) appears to be a general mechanism to accumulate certain proteins to subcellular compartments for participation in local processes, thereby maintaining cell polarity under strict spatiotemporal control. Transportation of mRNA with associated protein components (RNP granules) by the actin microfilament or the microtubule systems is one important mechanism to achieve this locally distributed protein production. Here we provide evidence for a microtubule-dependent localization of mRNA encoding the actin regulatory protein profilin to sites in mouse embryonic fibroblasts, which express enhanced actin polymerization.