Medium-throughput image-based phenotypic siRNA screen to unveil the molecular basis of B cell polarization.

Cell polarity is an essential and highly conserved process governing cell function. Cell polarization is generally triggered by an external signal that induces the relocation of the centrosome, thus defining the polarity axis of the cell. Here, we took advantage of B cells as a model to study cell polarity and perform a medium-throughput siRNA-based imaging screen to identify new molecular regulators of polarization. We first identified candidates based on a quantitative proteomic analysis of proteins differentially associated with the centrosome of resting non-polarized and stimulated polarized B cells. We then targeted 233 candidates in a siRNA screen and identified hits regulating the polarization of the centrosome and/or lysosomes in B cells upon stimulation. Our dataset of proteomics, images, and polarity indexes provides a valuable source of information for a broad community of scientists interested in the molecular mechanisms regulating cell polarity.

Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration.

The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function.

Galectin-8 Favors the Presentation of Surface-Tethered Antigens by Stabilizing the B Cell Immune Synapse.

Complete activation of B cells relies on their capacity to extract tethered antigens from immune synapses by either exerting mechanical forces or promoting their proteolytic degradation through lysosome secretion. Whether antigen extraction can also be tuned by local cues originating from the lymphoid microenvironment has not been investigated. We here show that the expression of Galectin-8-a glycan-binding protein found in the extracellular milieu, which regulates interactions between cells and matrix proteins-is increased within lymph nodes under inflammatory conditions where it enhances B cell arrest phases upon antigen recognition in vivo and promotes synapse formation during BCR recognition of immobilized antigens. Galectin-8 triggers a faster recruitment and secretion of lysosomes toward the B cell-antigen contact site, resulting in efficient extraction of immobilized antigens through a proteolytic mechanism. Thus, extracellular cues can determine how B cells sense and extract tethered antigens and thereby tune B cell responses in vivo.

Lysosome signaling controls the migration of dendritic cells.

Dendritic cells (DCs) patrol their environment by linking antigen acquisition by macropinocytosis to cell locomotion. DC activation upon bacterial sensing inhibits macropinocytosis and increases DC migration, thus promoting the arrival of DCs to lymph nodes for antigen presentation to T cells. The signaling events that trigger such changes are not fully understood. We show that lysosome signaling plays a critical role in this process. Upon bacterial sensing, lysosomal calcium is released by the ionic channel TRPML1 (transient receptor potential cation channel, mucolipin subfamily, member 1), which activates the actin-based motor protein myosin II at the cell rear, promoting fast and directional migration. Lysosomal calcium further induces the activation of the transcription factor EB (TFEB), which translocates to the nucleus to maintain TRPML1 expression. We found that the TRPML1-TFEB axis results from the down-regulation of macropinocytosis after bacterial sensing by DCs. Lysosomal signaling therefore emerges as a hitherto unexpected link between macropinocytosis, actomyosin cytoskeleton organization, and DC migration.

Trans-inhibition of activation and proliferation signals by Fc receptors in mast cells and basophils.

Allergic and autoimmune inflammation are associated with the activation of mast cells and basophils by antibodies against allergens or auto-antigens, respectively. Both cell types express several receptors for the Fc portion of antibodies, the engagement of which by antigen-antibody complexes controls their responses. When aggregated on the plasma membrane, high-affinity immunoglobulin E (IgE) receptors (FcεRI) and low-affinity IgG receptors (FcγRIIIA in mice, FcγRIIA in humans) induce these cells to release and secrete proinflammatory mediators, chemokines, and cytokines that account for clinical symptoms. When coaggregated with activating receptors on the same cells, other low-affinity IgG receptors (FcγRIIB in both species) inhibit mast cell and basophil activation. We found that FcγRIIB inhibited not only signals triggered by activating receptors with which they were coengaged (cis-inhibition), but also signals triggered by receptors engaged independently (trans-inhibition). Trans-inhibition acted upon the FcεRI-dependent activation of mouse mast cells, mouse basophils, and human basophils, and upon growth factor receptor (Kit)-dependent normal mouse mast cell proliferation, as well as the constitutive in vitro proliferation and the in vivo growth of oncogene (v-Abl)-transformed mastocytoma cells. Trans-inhibition was induced by receptors, whether inhibitory (FcγRIIB) or activating (FcεRI), which recruited the lipid phosphatase SHIP1. By hydrolyzing PI(3,4,5)P3, SHIP1 induced a global unresponsiveness that affected biological responses triggered by receptors that use phosphoinositide 3-kinase to signal. These data suggest that trans-inhibition controls numerous physiological and pathological processes, and that it may be used as a therapeutic tool in inflammation, especially but not exclusively, in allergy and autoimmunity.

Dynamics of the membrane-cytoskeleton interface in MHC class II-restricted antigen presentation.

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC-T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.

Actin nucleation at the centrosome controls lymphocyte polarity.

Cell polarity is required for the functional specialization of many cell types including lymphocytes. A hallmark of cell polarity is the reorientation of the centrosome that allows repositioning of organelles and vesicles in an asymmetric fashion. The mechanisms underlying centrosome polarization are not fully understood. Here we found that in resting lymphocytes, centrosome-associated Arp2/3 locally nucleates F-actin, which is needed for centrosome tethering to the nucleus via the LINC complex. Upon lymphocyte activation, Arp2/3 is partially depleted from the centrosome as a result of its recruitment to the immune synapse. This leads to a reduction in F-actin nucleation at the centrosome and thereby allows its detachment from the nucleus and polarization to the synapse. Therefore, F-actin nucleation at the centrosome--regulated by the availability of the Arp2/3 complex--determines its capacity to polarize in response to external stimuli.

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse.

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR-antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR-antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.

Antibodies against growth factor receptors can inhibit the proliferation of transformed cells via a cis-interaction with inhibitory FcR.

When dimerized by Stem Cell Factor (SCF), the Receptor Tyrosine Kinase Kit triggers the proliferation of hematopoietic progenitors, including pro-B cells, and of some differentiated cells, including mast cells. We found previously that anti-Kit antibodies can mimic SCF and that anti-Kit-induced mast cell proliferation can be inhibited by the low-affinity IgG receptors FcγRIIB, when the two receptors are co-aggregated by IgG immune complexes. We show here that the same immune complexes inhibited anti-Kit-induced proliferation of Ba/F3 pro-B cells expressing wt Kit and FcγRIIB and that inhibition required the intracytoplasmic domain of FcγRIIB. Constitutively active Kit mutants are oncogenic. We show that Kit-dependent, ligand-independent proliferation of Ba/F3 cells expressing a constitutively dimerized Kit mutant was also inhibited by IgG immune complexes via FcγRIIB. FcγRIIB-dependent negative regulation therefore also affects Kit-dependent proliferation of transformed cells. Interestingly, the co-aggregation of Kit with FcγRIIB by immune complexes containing SCF also inhibited both growth factor-dependent and growth factor-independent proliferation of Ba/F3 cells expressing wt or mutated Kit, respectively. These results provide the basis for novel immunotherapeutical approaches of FcγRIIB-expressing tumors.

A strain of Lactobacillus casei inhibits the effector phase of immune inflammation.

Some nonpathogenic bacteria were found to have protective effects in mouse models of allergic and autoimmune diseases. These "probiotics" are thought to interact with dendritic cells during Ag presentation, at the initiation of adaptive immune responses. Many other myeloid cells are the effector cells of immune responses. They are responsible for inflammation that accounts for symptoms in allergic and autoimmune diseases. We investigated in this study whether probiotics might affect allergic and autoimmune inflammation by acting at the effector phase of adaptive immune responses. The effects of one strain of Lactobacillus casei were investigated in vivo on IgE-induced passive systemic anaphylaxis and IgG-induced passive arthritis, two murine models of acute allergic and autoimmune inflammation, respectively, which bypass the induction phase of immune responses, in vitro on IgE- and IgG-induced mouse mast cell activation and ex vivo on IgE-dependent human basophil activation. L. casei protected from anaphylaxis and arthritis, and inhibited mouse mast cell and human basophil activation. Inhibition required contact between mast cells and bacteria, was reversible, and selectively affected the Lyn/Syk/linker for activation of T cells pathway induced on engagement of IgE receptors, leading to decreased MAPK activation, Ca(2+) mobilization, degranulation, and cytokine secretion. Also, adoptive anaphylaxis induced on Ag challenge in mice injected with IgE-sensitized mast cells was abrogated in mice injected with IgE-sensitized mast cells exposed to bacteria. These results demonstrate that probiotics can influence the effector phase of adaptive immunity in allergic and autoimmune diseases. They might, therefore, prevent inflammation in patients who have already synthesized specific IgE or autoantibodies.

C5a receptor enables participation of mast cells in immune complex arthritis independently of Fcγ receptor modulation.

OBJECTIVE: Mast cells are tissue-resident immune sentinels that are implicated in the pathogenesis of inflammatory joint disease. The aim of this study was to test our hypothesis that complement fragments could be key activators of synovial mast cells in autoimmune arthritis. METHODS: In vivo studies used the murine K/BxN arthritis model, a distal symmetric polyarthritis mediated by IgG immune complexes. Expression of C5aR on synovial mast cells was determined by immunohistochemical and functional studies. C5aR(-/-) and control mast cells were engrafted into mast cell-deficient WBB6 F1-Kit(w) /Kit(W-v) (W/Wv) mice to examine the requirement for this receptor in arthritis. C5aR-dependent activation of mast cells was investigated in C5aR(-/-) animals and in murine and human mast cell cultures. RESULTS: Murine synovial mast cells express functional C5aR. Unlike their wild-type counterparts, C5aR(-/-) mast cells adoptively transferred into W/Wv mice were not competent to restore arthritis, despite equivalent synovial engraftment. Activation of C5aR(-/-) mast cells by K/BxN serum in vivo remained intact, indicating that C5aR is dispensable for normal IgG-mediated triggering. Consistent with this result, cultured mast cells treated with C5a failed to modulate the expression of Fcγ receptors (FcγR) or to otherwise alter the activation threshold. In human mast cells, C5a promoted the production of the neutrophil chemotaxin interleukin-8, and recruitment of neutrophils at 24 hours after serum administration was impaired in C5aR(-/-) mice, suggesting that enhanced neutrophil chemoattractant production underlies the requirement for C5aR on mast cells in arthritis. CONCLUSION: Stimulation via C5aR is required to unleash the proinflammatory activity of synovial mast cells in immune complex arthritis, albeit via a mechanism that is distinct from C5a-modulated expression of FcγR.

A novel druglike spleen tyrosine kinase binder prevents anaphylactic shock when administered orally.

BACKGROUND: The spleen tyrosine kinase (Syk) is recognized as a potential pharmaceutical target for the treatment of type I hypersensitivity reactions including allergic rhinitis, urticaria, asthma, and anaphylaxis because of its critical position upstream of immunoreceptor signaling complexes that regulate inflammatory responses in leukocytes. OBJECTIVE: Our aim was to improve the selectivity of anti-Syk therapies by impeding the interaction of Syk with its cellular partners, instead of targeting its catalytic site. METHODS: We have previously studied the inhibitory effects of the anti-Syk intracellular antibody G4G11 on Fc epsilonRI-induced release of allergic mediators. A compound collection was screened by using an antibody displacement assay to identify functional mimics of G4G11 that act as potential inhibitors of the allergic response. The effects of the selected druglike compounds on mast cell activation were evaluated in vitro and in vivo. RESULTS: We discovered compound 13, a small molecule that inhibits Fc epsilonRI-induced mast cell degranulation in vitro and anaphylactic shock in vivo. Importantly, compound 13 was efficient when administered orally to mice. Structural analysis, docking, and site-directed mutagenesis allowed us to identify the binding cavity of this compound, located at the interface between the 2 Src homology 2 domains and the interdomain A of Syk. CONCLUSION: We have isolated a new class of druglike compounds that modulate the interaction of Syk with some of its macromolecular substrates implicated in the degranulation pathway in mast cells.

Ex vivo and in vitro primary mast cells.

Mast cells are cells of the innate immune system whose biological responses are markedly modulated by effector molecules of adaptive immunity, i.e., antibodies. They thus contribute to anti-infectious defense but also to antibody-dependent inflammatory responses. They are especially well known as inducers of allergic reactions. They are widely distributed in most tissues, but in low numbers. They are not readily purified, and with a poor yield. For these reasons, means to generate large numbers of homogenous non-transformed mast cells have been developed. We describe here (1) fractionation methods suitable for purifying mouse or rat peritoneal mast cells and for purifying human mast cells of various origins, and (2) conditions for generating pure cultured mast cell populations from mouse, rat, and human tissues.

Non-T cell activation linker promotes mast cell survival by dampening the recruitment of SHIP1 by linker for activation of T cells.

The linker for activation of T cells (LAT) and the non-T cell activation linker (NTAL) are two transmembrane adapters which organize IgE receptor (FcepsilonRI) signaling complexes in mast cells. LAT positively regulates, whereas NTAL negatively regulates mast cell activation. We previously found that the four distal tyrosines of LAT can generate negative signals. We show here that two of these tyrosines provide two binding sites for SHIP1, that LAT recruits SHIP1 in vivo, and that SHIP1 recruitment is enhanced in NTAL-deficient cells. We show that NTAL negatively regulates mast cell activation by decreasing the recruitment, by LAT, of molecules involved in FcepsilonRI-dependent positive signaling. We show that NTAL also decreases the recruitment of SHIP1 by LAT, leading to an increased phosphorylation of the antiapoptotic molecule Akt, and positively regulates mast cell survival. We finally show that the positive effect of NTAL on Akt phosphorylation and mast cell survival requires LAT. Our data thus document the mechanisms by which LAT and NTAL can generate both positive and negative signals which differentially regulate mast cell activation and survival. They also provide molecular bases for the recruitment of SHIP1 in FcepsilonRI signaling complexes. SHIP1 is a major negative regulator of mast cell activation and, hence, of allergic reactions.

The mast cell IgG receptors and their roles in tissue inflammation.

Mast cells are effector cells of the innate immune system, but because they express Fc receptors (FcRs), they can be engaged in adaptive immunity by antibodies. Mast cell FcRs include immunoglobulin E (IgE) and IgG receptors and, among these, activating and inhibitory receptors. The engagement of mast cell IgG receptors by immune complexes may or may not trigger cell activation, depending on the type of mast cell. The coengagement of IgG and IgE receptors results in inhibition of mast cell activation. The Src homology-2 domain-containing inositol 5-phosphatase-1 is a major effector of negative regulation. Biological responses of mast cells depend on the balance between positive and negative signals that are generated in FcR complexes. The contribution of human mast cell IgG receptors in allergies remains to be clarified. Increasing evidence indicates that mast cells play critical roles in IgG-dependent tissue-specific autoimmune diseases. Convincing evidence was obtained in murine models of multiple sclerosis, rheumatoid arthritis, bullous pemphigoid, and glomerulonephritis. In these models, the intensity of lesions depended on the relative engagement of activating and inhibitory IgG receptors. In vitro models of mature tissue-specific murine mast cells are needed to investigate the roles of mast cells in these diseases. One such model unraveled unique differentiation/maturation-dependent biological responses of serosal-type mast cells.

Peritoneal cell-derived mast cells: an in vitro model of mature serosal-type mouse mast cells.

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcgammaRIIIA and were negatively regulated by FcgammaRIIB. We found that a moderate FcgammaRIIB-dependent negative regulation, due not to a higher FcgammaRIIIA/FcgammaRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.

The engagement of activating FcgammaRs inhibits primate lentivirus replication in human macrophages.

We previously reported that the stimulation of monocyte-derived macrophages (MDM) by plate-bound i.v. Igs inhibits HIV-1 replication. In this study, we show that IgG immune complexes also suppress HIV-1 replication in MDMs and that activating receptors for the Fc portion of IgG-FcgammaRI, FcgammaRIIA, and FcgammaRIII-are responsible for the inhibition. MDM stimulation through FcgammaRs induces activation signals and the secretion of HIV-1 modulatory cytokines, such as M-CSF, TNF-alpha, and macrophage-derived chemokine. However, none of these cytokines contribute to HIV-1 suppression. HIV-1 entry and postintegration steps of viral replication are not affected, whereas reduced levels of reverse transcription products and of integrated proviruses, as determined by real-time PCR analysis, account for the suppression of HIV-1 gene expression in FcgammaR-activated MDMs. We found that FcgammaR-dependent activation of MDMs also inhibits the replication of HIV-2, SIVmac, and SIVagm, suggesting a common control mechanism for primate immunodeficiency lentiviruses in activated macrophages.

Linker for activation of T cells integrates positive and negative signaling in mast cells.

The transmembrane adapter linker for activation of T cells (LAT) is thought to couple immunoreceptors to intracellular signaling pathways. In mice, its intracytoplasmic domain contains nine tyrosines which, when phosphorylated upon receptor aggregation, recruit Src-homology 2 domain-containing cytosolic enzymes and adapters. The four distal tyrosines are critical for both TCR and FcepsilonRI signaling. Unexpectedly, knock-in mice expressing LAT with a point mutation of the first or of the last three of these tyrosines exhibited an abnormal T cell development characterized by a massive expansion of TH2-like alphabeta or gammadelta T cells, respectively. This phenotype suggests that, besides positive signals, LAT might support negative signals that normally regulate terminal T cell differentiation and proliferation. We investigated here whether LAT might similarly regulate mast cell activation, by generating not only positive but also negative signals, following FcR engagement. To this end, we examined IgE- and/or IgG-induced secretory and intracellular responses of mast cells derived from knock-in mice expressing LAT with combinations of tyrosine mutations (Y136F, Y(175, 195, 235)F, or Y(136, 175, 195, 235)F). A systematic comparison of pairs of mutants enabled us to dissect the respective roles played by the five proximal and the four distal tyrosines. We found that LAT tyrosines differentially contribute to exocytosis and cytokine secretion and differentially regulate biological responses of mucosal- and serosal-type mast cells. We also found that, indeed, both positive and negative signals may emanate from distinct tyrosines in LAT, whose integration modulates mast cell secretory responses.

Negative regulation of mast cell proliferation by FcgammaRIIB.

FcgammaRIIB are single-chain low-affinity receptors for the Fc portion of IgG antibodies that are widely expressed by hematopoietic cells including mast cells. We previously demonstrated that FcgammaRIIB negatively regulate cell activation triggered by receptors that possess Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) including high-affinity IgE receptors (FcepsilonRI). FcgammaRIIB possess an Immunoreceptor Tyrosine-based Inhibition Motif (ITAM) whose deletion or mutation abolishes inhibition. When coaggregated with FcepsilonRI, the FcgammaRIIB ITIM is tyrosyl-phosphorylated by the src family protein tyrosine kinase lyn, and recruits the SH2 domain-containing inositol 5-phosphatase SHIP that accounts for inhibition of cell activation. We found recently that, when coaggregated with Kit, FcgammaRIIB can also inhibit mast cell proliferation: thymidine incorporation is inhibited, cells do not enter the G1 phase of the cell cycle, the induction of cyclins D2, D3 and A is inhibited, the activation of the MAP kinases Erk1/2, JNK and p38 is decreased, Akt phosphorylation is inhibited, and SHIP coprecipitates with FcgammaRIIB. Although inhibition of Akt phosphorylation and Erk activation was abrogated in SHIP(-/-) cells, inhibition of thymidine incorporation was only partially reduced. FcgammaRIIB-dependent inhibition of Kit-mediated mast cell proliferation was however mimicked by FcgammaRIIB whose intracytoplasmic domain was replaced by the catalytic domain of SHIP. We also found that FcgammaRIIB can inhibit the proliferation of cells whose proliferation was rendered growth factor-independent because they express a mutated form of Kit that renders this RTK constitutively activated. Based on these results we developed models aiming at using FcgammaRIIB as targets for new therapeutic approaches of disease associated with mast cell activation such as allergies and diseases associated with mast cell proliferation such as mastocytosis, mastocytomas or mast cell leukemias.