PtdIns3P phosphatases MTMR3 and MTMR4 negatively regulate innate immune responses to DNA through modulating STING trafficking.

The innate immune system plays an essential role in initial recognition of pathogen infection by producing inflammatory cytokines and type I interferons. cGAS is a cytoplasmic sensor for DNA derived from DNA viruses. cGAS binding with DNA induces the production of cGAMP, a second messenger that associates with STING in endoplasmic reticulum (ER). STING changes its cellular distribution from ER to perinuclear Golgi, where it activates the protein kinase TBK1 that catalyzes the phosphorylation of IRF3. Here we found that STING trafficking is regulated by myotubularin-related protein (MTMR) 3 and MTMR4, members of protein tyrosine phosphatases that dephosphorylate 3' position in phosphatidylinositol (PtdIns) and generate PtdIns5P from PtdIns3,5P2 and PtdIns from PtdIns3P. We established MTMR3 and MTMR4 double knockout (DKO) RAW264.7 macrophage cells and found that they exhibited increased type I interferon production after interferon-stimulatory DNA (ISD) stimulation and herpes simplex virus 1 infection concomitant with enhanced IRF3 phosphorylation. In DKO cells, STING rapidly trafficked from ER to Golgi after ISD stimulation. Notably, DKO cells exhibited enlarged cytosolic puncta positive for PtdIns3P and STING was aberrantly accumulated in this puncta. Taken together, these results suggest that MTMR3 and MTMR4 regulate the production of PtdIns3P, which plays a critical role in suppressing DNA-mediated innate immune responses via modulating STING trafficking.

Liposomes loaded with bioactive lipids enhance antibacterial innate immunity irrespective of drug resistance.

Phagocytosis is a key mechanism of innate immunity, and promotion of phagosome maturation may represent a therapeutic target to enhance antibacterial host response. Phagosome maturation is favored by the timely and coordinated intervention of lipids and may be altered in infections. Here we used apoptotic body-like liposomes (ABL) to selectively deliver bioactive lipids to innate cells, and then tested their function in models of pathogen-inhibited and host-impaired phagosome maturation. Stimulation of macrophages with ABLs carrying phosphatidic acid (PA), phosphatidylinositol 3-phosphate (PI3P) or PI5P increased intracellular killing of BCG, by inducing phagosome acidification and ROS generation. Moreover, ABLs carrying PA or PI5P enhanced ROS-mediated intracellular killing of Pseudomonas aeruginosa, in macrophages expressing a pharmacologically-inhibited or a naturally-mutated cystic fibrosis transmembrane conductance regulator. Finally, we show that bronchoalveolar lavage cells from patients with drug-resistant pulmonary infections increased significantly their capacity to kill in vivo acquired bacterial pathogens when ex vivo stimulated with PA- or PI5P-loaded ABLs. Altogether, these results provide the proof of concept of the efficacy of bioactive lipids delivered by ABL to enhance phagosome maturation dependent antimicrobial response, as an additional host-directed strategy aimed at the control of chronic, recurrent or drug-resistant infections.

Small GTPase Rab8a-recruited Phosphatidylinositol 3-Kinase γ Regulates Signaling and Cytokine Outputs from Endosomal Toll-like Receptors.

LPS-mediated activation of Toll-like receptor 4 (TLR4) in macrophages results in the coordinated release of proinflammatory cytokines, followed by regulatory mediators, to ensure that this potentially destructive pathway is tightly regulated. We showed previously that Rab8a recruits PI3Kγ for Akt-dependent signaling during TLR4 activation to limit the production of the proinflammatory cytokines IL-6 and IL-12p40 while enhancing the release of the regulatory/anti-inflammatory cytokine IL-10. Here we broaden the array of immune receptors controlled by Rab8a-PI3Kγ and further define the Rab-mediated membrane domains required for signaling. With CRISPR/Cas9-mediated gene editing to stably knock out and recover Rab8a in macrophage cell lines, we match Akt signaling profiles with cytokine outputs, confirming that Rab8a is a novel regulator of the Akt/mammalian target of rapamycin (mTOR) pathway downstream of multiple TLRs. Upon developing a Rab8a activation assay, we show that TLR3 and 9 agonists also activate Rab8a. Live-cell imaging reveals that Rab8a is first recruited to the plasma membrane and dorsal ruffles, but it is retained during collapse of ruffles to form macropinosomes enriched for phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), suggesting that the macropinosome is the location where Rab8a is active. We pinpoint macropinosomes as the sites for Rab8-mediated biasing of inflammatory signaling responses via inducible production of anti-inflammatory cytokines. Thus, Rab8a and PI3Kγ are positioned in multiple TLR pathways, and this signaling axis may serve as a pharmacologically tractable target during infection and inflammation.

Phosphoinositol 3-phosphate acts as a timer for reactive oxygen species production in the phagosome.

Production of reactive oxygen species (ROS) in the phagosome by the NADPH oxidase is critical for mammalian immune defense against microbial infections and phosphoinositides are important regulators in this process. Phosphoinositol 3-phosphate (PI(3)P) regulates ROS production at the phagosome via p40phox by an unknown mechanism. This study tested the hypothesis that PI(3)P controls ROS production by regulating the presence of p40phox and p67phox at the phagosomal membrane. Pharmacologic inhibition of PI(3)P synthesis at the phagosome decreased the ROS production both in differentiated PLB-985 cells and human neutrophils. It also releases p67phox, the key cytosolic subunit of the oxidase, and p40phox from the phagosome. The knockdown of the PI(3)P phosphatase MTM1 or Rubicon or both increases the level of PI(3)P at the phagosome. That increase enhances ROS production inside the phagosome and triggers an extended accumulation of p67phox at the phagosome. Furthermore, the overexpression of MTM1 at the phagosomal membrane induces the disappearance of PI(3)P from the phagosome and prevents sustained ROS production. In conclusion, PI(3)P, indeed, regulates ROS production by maintaining p40phox and p67phox at the phagosomal membrane.

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.

The Multifaceted Roles of PI3Kγ in Hypertension, Vascular Biology, and Inflammation.

PI3Kγ is a multifaceted protein, crucially involved in cardiovascular and immune systems. Several studies described the biological and physiological functions of this enzyme in the regulation of cardiovascular system, while others stressed its role in the modulation of immunity. Although PI3Kγ has been historically investigated for its role in leukocytes, the last decade of research also dedicated efforts to explore its functions in the cardiovascular system. In this review, we report an overview recapitulating how PI3Kγ signaling participates in the regulation of vascular functions involved in blood pressure regulation. Moreover, we also summarize the main functions of PI3Kγ in immune responses that could be potentially important in the interaction with the cardiovascular system. Considering that vascular and immune mechanisms are increasingly emerging as intertwining players in hypertension, PI3Kγ could be an intriguing pathway acting on both sides. The availability of specific inhibitors introduces a perspective of further translational research and clinical approaches that could be exploited in hypertension.

Lymphoid susceptibility to the Aggregatibacter actinomycetemcomitans cytolethal distending toxin is dependent upon baseline levels of the signaling lipid, phosphatidylinositol-3,4,5-triphosphate.

The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) induces G2 arrest and apoptosis in lymphocytes and other cell types. We have shown that the active subunit, CdtB, exhibits phosphatidylinositol-3,4,5-triphosphate (PIP3) phosphatase activity and depletes lymphoid cells of PIP3. Hence we propose that Cdt toxicity results from depletion of this signaling lipid and perturbation of phosphatidylinositol-3-kinase (PI-3K)/PIP3/Akt signaling. We have now focused on the relationship between cell susceptibility to CdtB and differences in the status of baseline PIP3 levels. Our studies demonstrate that the baseline level of PIP3, and likely the dependence of cells on steady-state activity of the PI-3K signaling pathway for growth and survival, influence cell susceptibility to the toxic effects of Cdt. Jurkat cells with known defects in both PIP3 degradative enzymes, PTEN and SHIP1, not only contain high baseline levels of PIP3, pAkt, and pGSK3ß, but also exhibit high sensitivity to Cdt. In contrast, HUT78 cells, with no known defects in this pathway, contain low levels of PIP3, pAkt, and pGSK3ß and likely minimal dependence on the PI-3K signaling pathway for growth and survival, and exhibit reduced susceptibility to Cdt. These differences in susceptibility to Cdt cannot be explained by differential toxin binding or internalization of the active subunit. Indeed, we now demonstrate that Jurkat and HUT78 cells bind toxin at comparable levels and internalize relatively equal amounts of CdtB. The relevance of these observations to the mode of action of Cdt and its potential role as a virulence factor is discussed.

Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs.

Bruton's Tyrosine Kinase (BTK) and Vav1 contribute to Dectin1-dependent phagocytosis of Candida albicans in macrophages.

Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.

The kindlin 3 pleckstrin homology domain has an essential role in lymphocyte function-associated antigen 1 (LFA-1) integrin-mediated B cell adhesion and migration.

The protein kindlin 3 is mutated in the leukocyte adhesion deficiency III (LAD-III) disorder, leading to widespread infection due to the failure of leukocytes to migrate into infected tissue sites. To gain understanding of how kindlin 3 controls leukocyte function, we have focused on its pleckstrin homology (PH) domain and find that deletion of this domain eliminates the ability of kindlin 3 to participate in adhesion and migration of B cells mediated by the leukocyte integrin lymphocyte function-associated antigen 1 (LFA-1). PH domains are often involved in membrane localization of proteins through binding to phosphoinositides. We show that the kindlin 3 PH domain has binding affinity for phosphoinositide PI(3,4,5)P3 over PI(4,5)P2. It has a major role in membrane association of kindlin 3 that is enhanced by the binding of LFA-1 to intercellular adhesion molecule 1 (ICAM-1). A splice variant, kindlin 3-IPRR, has a four-residue insert in the PH domain at a critical site that influences phosphoinositide binding by enhancing binding to PI(4,5)P2 as well as by binding to PI(3,4,5)P3. However kindlin 3-IPRR is unable to restore the ability of LAD-III B cells to adhere to and migrate on LFA-1 ligand ICAM-1, potentially by altering the dynamics or PI specificity of binding to the membrane. Thus, the correct functioning of the kindlin 3 PH domain is central to the role that kindlin 3 performs in guiding lymphocyte adhesion and motility behavior, which in turn is required for a successful immune response.

SNX3 recruits to phagosomes and negatively regulates phagocytosis in dendritic cells.

Phagocytes such as dendritic cells (DC) and macrophages employ phagocytosis to take up pathogenic bacteria into phagosomes, digest the bacteria and present the bacteria-derived peptide antigens to the adaptive immunity. Hence, efficient antigen presentation depends greatly on a well-regulated phagocytosis process. Lipids, particularly phosphoinositides, are critical components of the phagosomes. Phosphatidylinositol-3,4,5-triphosphate [PI(3,4,5)P3 ] is formed at the phagocytic cup, and as the phagosome seals off from the plasma membrane, rapid disappearance of PI(3,4,5)P3 is accompanied by high levels of phosphatidylinositol-3-phosphate (PI3P) formation. The sorting nexin (SNX) family consists of a diverse group of Phox-homology (PX) domain-containing cytoplasmic and membrane-associated proteins that are potential effectors of phosphoinositides. We hypothesized that SNX3, a small sorting nexin that contains a single PI3P lipid-binding PX domain as its only protein domain, localizes to phagosomes and regulates phagocytosis in DC. Our results show that SNX3 recruits to nascent phagosomes and silencing of SNX3 enhances phagocytic uptake of bacteria by DC. Furthermore, SNX3 competes with PI3P lipid-binding protein, early endosome antigen-1 (EEA1) recruiting to membranes. Our results indicate that SNX3 negatively regulates phagocytosis in DC possibly by modulating recruitment of essential PI3P lipid-binding proteins of the phagocytic pathways, such as EEA1, to phagosomal membranes.

An anti-phosphoinositide-specific monoclonal antibody that neutralizes HIV-1 infection of human monocyte-derived macrophages.

HIV-1 entry into cells requires the interaction of both HIV-1 envelope proteins and membrane lipids. We investigated the mechanism of neutralization of HIV-1 infection of primary monocyte-derived macrophages (MDM) by a murine monoclonal antibody (mAb) WR321. WR321 specifically binds phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate. These phosphoinositides are present not only on the inner surface of the plasma membranes of cells but also on the surface of virions. HIV-1 acquires these lipids during the budding process. Pre-incubation of WR321 with the virus but not with MDM neutralized HIV-1 infection of MDM. Our results demonstrate that WR321 was internalized only when it was bound to HIV-1. WR321 did not prevent the entry of HIV-1 into MDM. However, once WR321 was internalized along with HIV-1 the mAb acted intracellulary to prevent the release of virions from MDM and also triggered the release of ß-chemokines.

PIP3 regulation as promising targeted therapy of mast-cell-mediated diseases.

It is well established that mast cells play a key regulatory role in allergy and inflammation involving engagement of antigen with IgE bound to high-affinity IgE receptors (FcεRI). The most aggressive efforts in regulating mast cell function have focused on selectively inhibiting cell activation and subsequent mediator synthesis and release, or alternatively, blocking the action of proinflammatory mediators in order to prevent or reduce disease severity. More recently, the goal for rationally designed pharmacotherapy has shifted focus to targeting and disrupting signaling pathways leading to inhibition of specific cell function(s). In this context, the PI-3K/PIP3/Akt pathway represents a potent target for pharmacologic intervention in mast cell-mediated inflammatory disorders. A pivotal component of this cascade is the activation of phosphatidylinositol-3-kinase (PI-3K) leading to a rise in intracellular levels of phosphatidylinositol 3,4,5-trisphosphate (PIP3). PIP3 has broad effects on mast cell signaling and function as well as on proliferation and survival. We propose that PIP3 represents a potent target for developing therapeutic approaches to down regulate mast cell function and, in turn, reduce the severity of mast cell dependent disease. In this article we review approaches that have been taken to regulate the PI-3K pathway in mast cells. Moreover, we review a novel approach to target the signaling lipid, PIP3, and deplete intracellular levels of this phosphoinositol using a chimeric toxin composed of the FcεRI binding region of IgE and the active subunit of the cytolethal distending toxin, CdtB, which we have recently demonstrated to function as a PIP3 phosphatase.

Inositol hexakisphosphate kinase 1 regulates neutrophil function in innate immunity by inhibiting phosphatidylinositol-(3,4,5)-trisphosphate signaling.

Inositol phosphates are widely produced throughout animal and plant tissues. Diphosphoinositol pentakisphosphate (InsP7) contains an energetic pyrophosphate bond. Here we demonstrate that disruption of inositol hexakisphosphate kinase 1 (InsP6K1), one of the three mammalian inositol hexakisphosphate kinases (InsP6Ks) that convert inositol hexakisphosphate (InsP6) to InsP7, conferred enhanced phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3)-mediated membrane translocation of the pleckstrin homology domain of the kinase Akt and thus augmented downstream PtdIns(3,4,5)P3 signaling in mouse neutrophils. Consequently, these neutrophils had greater phagocytic and bactericidal ability and amplified NADPH oxidase-mediated production of superoxide. These phenotypes were replicated in human primary neutrophils with pharmacologically inhibited InsP6Ks. In contrast, an increase in intracellular InsP7 blocked chemoattractant-elicited translocation of the pleckstrin homology domain to the membrane and substantially suppressed PtdIns(3,4,5)P3-mediated cellular events in neutrophils. Our findings establish a role for InsP7 in signal transduction and provide a mechanism for modulating PtdIns(3,4,5)P3 signaling in neutrophils.

Antigen-specific enhancement of natural human IgG antibodies to phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol-4-phosphate, cholesterol, and lipid A by a liposomal vaccine containing lipid A.

Natural IgG antibodies (NA) to lipids are ubiquitously distributed in sera of healthy humans and are believed to serve beneficial functions. Although NA to lipids generally exhibit germ line or near germ line binding specificities, the antibodies commonly increase transiently in the acute phases of most, if not all, infectious diseases and may serve as a first line of defense. In order to determine whether similar anti-lipid antibodies can be induced by a vaccine in humans, we examined stored sera obtained from volunteers who had previously received a candidate vaccine to Plasmodium falciparum. The vaccine had consisted of liposomes that contained both the recombinant protein antigen and also contained monophosphoryl lipid A (MPLA) as an adjuvant. All of the pre-immune sera contained NA to one or more of the liposomal lipids in the vaccine: dimyristol phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. After initial immunization, followed by a boost, increased levels of IgG antibodies to all of the liposomal lipids, especially DMPG and MPLA, were observed by ELISA. Antibodies to phosphatidylinositol-4-phosphate (PIP) above the normal pre-immune NA to PIP were also observed. Although PIP was not present in the immunizing liposomes, based on the adsorption of anti-PIP antibodies by DMPG the anti-PIP antibodies were thought to represent cross-reacting anti-DMPG antibodies. The immune response was apparently antigen-specific in that NA to unrelated lipids, other than PIP, that were not present in the liposomes, galactosyl ceramide and ganglioside GM1, were not increased by the immunization. We conclude that antibodies to DMPC, DMPG, PIP, cholesterol, and MPLA can be induced in humans by immunization with liposomes containing MPLA.

Human regulatory T cells rapidly suppress T cell receptor-induced Ca(2+), NF-κB, and NFAT signaling in conventional T cells.

CD4(+)CD25(hi)Foxp3(+) regulatory T cells (T(regs)) are critical mediators of self-tolerance, which is crucial for the prevention of autoimmune disease, but T(regs) can also inhibit antitumor immunity. T(regs) inhibit the proliferation of CD4(+)CD25(-) conventional T cells (T(cons)), as well as the ability of these cells to produce effector cytokines; however, the molecular mechanism of suppression remains unclear. Here, we showed that human T(regs) rapidly suppressed the release of calcium ions (Ca(2+)) from intracellular stores in response to T cell receptor (TCR) activation in T(cons). The inhibition of Ca(2+) signaling resulted in decreased dephosphorylation, and thus decreased activation, of the transcription factor nuclear factor of activated T cells 1 (NFAT1) and reduced the activation of nuclear factor κB (NF-κB). In contrast, Ca(2+)-independent events in T(cons), such as TCR-proximal signaling and activation of the transcription factor activator protein 1 (AP-1), were not affected during coculture with T(regs). Despite suppressing intracellular Ca(2+) mobilization, coculture with T(regs) did not block the generation of inositol 1,4,5-trisphosphate in TCR-stimulated T(cons). The T(reg)-induced suppression of the activity of NFAT and NF-κB and of the expression of the gene encoding the cytokine interleukin-2 was reversed in T(cons) by increasing the concentration of intracellular Ca(2+). Our results elucidate a previously unrecognized and rapid mechanism of T(reg)-mediated suppression. This increased understanding of T(reg) function may be exploited to generate possible therapies for the treatment of autoimmune diseases and cancer.

Lipid signaling in T-cell development and function.

Second messenger molecules relay, amplify, and diversify cell surface receptor signals. Two important examples are phosphorylated D-myo-inositol derivatives, such as phosphoinositide lipids within cellular membranes, and soluble inositol phosphates. Here, we review how phosphoinositide metabolism generates multiple second messengers with important roles in T-cell development and function. They include soluble inositol(1,4,5)trisphosphate, long known for its Ca(2+)-mobilizing function, and phosphatidylinositol(3,4,5)trisphosphate, whose generation by phosphoinositide 3-kinase and turnover by the phosphatases PTEN and SHIP control a key "hub" of TCR signaling. More recent studies unveiled important second messenger functions for diacylglycerol, phosphatidic acid, and soluble inositol(1,3,4,5)tetrakisphosphate (IP(4)) in immune cells. Inositol(1,3,4,5)tetrakisphosphate acts as a soluble phosphatidylinositol(3,4,5)trisphosphate analog to control protein membrane recruitment. We propose that phosphoinositide lipids and soluble inositol phosphates (IPs) can act as complementary partners whose interplay could have broadly important roles in cellular signaling.

PtdIns3P and Rac direct the assembly of the NADPH oxidase on a novel, pre-phagosomal compartment during FcR-mediated phagocytosis in primary mouse neutrophils.

The generation of reactive oxygen species (ROS) by the nicotinamide adenine dinucleotide phosphate oxidase is an important mechanism by which neutrophils kill pathogens. The oxidase is composed of a membrane-bound cytochrome and 4 soluble proteins (p67(phox), p40(phox), p47(phox), and GTP-Rac). These components form an active complex at the correct time and subcellular location through a series of incompletely understood mutual interactions, regulated, in part, by GTP/GDP exchange on Rac, protein phosphorylation, and binding to lipid messengers. We have used a variety of assays to follow the spatiotemporal assembly of the oxidase in genetically engineered primary mouse neutrophils, during phagocytosis of both serum- and immunoglobulin G-opsonized targets. The oxidase assembles directly on serum-Staphylococcus aureus-containing phagosomes within seconds of phagosome formation; this process is only partially dependent (∼ 30%) on PtdIns3P binding to p40(phox), but totally dependent on Rac1/2 binding to p67(phox). In contrast, in response to immunoglobulin G-targets, the oxidase first assembles on a tubulovesicular compartment that develops at sites of granule fusion to the base of the emerging phagosome; oxidase assembly and activation is highly dependent on both PtdIns3P-p40(phox) and Rac2-p67(phox) interactions and delivery to the phagosome is regulated by Rab27a. These results define a novel pathway for oxidase assembly downstream of FcR-activation.

Inhibition of mast cell degranulation by a chimeric toxin containing a novel phosphatidylinositol-3,4,5-triphosphate phosphatase.

It is well established that many cell functions are controlled by the PI-3K signaling pathway and the signaling lipid, phosphatidylinositol-3,4,5-triphosphate (PIP3). This is particularly true for mast cells which play a key regulatory role in allergy and inflammation through activation via high-affinity IgE receptors (FcɛRI) leading to activation of signaling cascades and subsequent release of histamine and other pro-inflammatory mediators. A pivotal component of this cascade is the activation of PI-3K and a rise in intracellular levels of PIP3. In this study, we developed a novel chimeric toxin that selectively binds to mast cells and which functions as a PIP3 phosphatase. Specifically, the chimeric toxin was composed of the FcɛRI binding region of IgE and the active subunit of the cytolethal distending toxin, CdtB, which we have recently demonstrated to function as a PIP3 phosphatase. We demonstrate that the chimeric toxin retains PIP3 phosphatase activity and selectively binds to mast cells. Moreover, the toxin is capable of altering intracellular levels of PIP3, block antigen-induced Akt phosphorylation and degranulation. These studies provide further evidence for the pivotal role of PIP3 in regulating mast cell activation and for this signaling lipid serving as a novel target for therapeutic intervention of mast cell-mediated disease. Moreover, these studies provide evidence for the utilization of CdtB as a novel therapeutic agent for targeting the PI-3K signaling pathway.