Martina Kovarova

Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation

FcεRI activation of mast cells is thought to involve Lyn and Syk kinases proximal to the receptor and the signaling complex organized by the linker for activation of T cells (LAT). We report here that FcεRI also uses a Fyn kinase–dependent pathway that does not require Lyn kinase or the adapter LAT for its initiation, but is necessary for mast cell degranulation. Lyn-deficiency enhanced Fyn-dependent signals and degranulation, but inhibited the calcium response. Fyn-deficiency impaired degranulation, whereas Lyn-mediated signaling and calcium was normal. Thus, FcεRI-dependent mast cell degranulation involves cross-talk between Fyn and Lyn kinases.

Negative Regulation of Immunoglobulin E–dependent Allergic Responses by Lyn Kinase

A role for Lyn kinase as a positive regulator of immunoglobulin (Ig)E-dependent allergy has long been accepted. Contrary to this belief, Lyn kinase was found to have an important role as a negative regulator of the allergic response. This became apparent from the hyperresponsive degranulation of lyn − / − bone marrow–derived mast cells, which is driven by hyperactivation of Fyn kinase that occurs, in part, through the loss of negative regulation by COOH-terminal Src kinase (Csk) and the adaptor, Csk-binding protein. This phenotype is recapitulated in vivo as young lyn − / − mice showed an enhanced anaphylactic response. In vivo studies also demonstrated that as lyn − / − mice aged, their serum IgE increased as well as occupancy of the high affinity IgE receptor (FcεRI). This was mirrored by increased circulating histamine, increased mast cell numbers, increased cell surface expression of the high affinity IgE receptor (FcεRI), and eosinophilia. The increased IgE production was not a consequence of increased Fyn kinase activity in lyn − / − mice because both lyn − / − and lyn − / − fyn − / − mice showed high IgE levels. Thus, lyn − / − mice and mast cells thereof show multiple allergy-associated traits, causing reconsideration of the possible efficacy in therapeutic targeting of Lyn in allergic disease.

Structure-Function Analysis of Lyn Kinase Association with Lipid Rafts and Initiation of Early Signaling Events after Fcɛ Receptor I Aggregation

ABSTRACT The first step in immunoreceptor signaling is represented by ligand-dependent receptor aggregation, followed by receptor phosphorylation mediated by tyrosine kinases of the Src family. Recently, sphingolipid- and cholesterol-rich plasma membrane microdomains, called lipid rafts, have been identified and proposed to function as platforms where signal transduction molecules may interact with the aggregated immunoreceptors. Here we show that aggregation of the receptors with high affinity for immunoglobulin E (FcɛRI) in mast cells is accompanied by a co-redistribution of the Src family kinase Lyn. The co-redistribution requires Lyn dual fatty acylation, Src homology 2 (SH2) and/or SH3 domains, and Lyn kinase activity, incis or in trans. Palmitoylation site-mutated Lyn, which is anchored to the plasma membrane but exhibits reduced sublocalization into lipid rafts, initiates the tyrosine phosphorylation of FcɛRI subunits, Syk protein tyrosine kinase, and the linker for activation of T cells, along with an increase in the concentration of intracellular Ca2+. However, Lyn mutated in both the palmitoylation and myristoylation sites does not anchor to the plasma membrane and is incapable of initiating FcɛRI phosphorylation and early signaling events. These data, together with our finding that a constitutively tyrosine-phosphorylated FcɛRI does not exhibit an increased association with lipid rafts, suggest that FcɛRI phosphorylation and early activation events can be initiated outside of lipid rafts.

NLRP1 dependent pyroptosis leads to acute lung injury and morbidity in mice

Acute inflammation in response to both exogenous and endogenous danger signals can lead to the assembly of cytoplasmic inflammasomes that stimulate the activation of caspase-1. Subsequently, caspase-1 facilitates the maturation and release of cytokines and also, under some circumstances, the induction of cell death by pyroptosis. Using a mouse line lacking expression of NLRP1, we show that assembly of this inflammasome in cells is triggered by a toxin from anthrax and that it initiates caspase-1 activation and release of IL-1β. Furthermore, NLRP1 inflammasome activation also leads to cell death, which escalates over 3 d following exposure to the toxin and culminates in acute lung injury and death of the mice. We show that these events are not dependent on production of IL-1β by the inflammasome but are dependent on caspase-1 expression. In contrast, muramyl dipeptide-mediated inflammasome formation is not dependent on NLRP1 but NLRP3. Taken together, our findings show that assembly of the NLRP1 inflammasome is sufficient to initiate pyroptosis, which subsequently leads to a self-amplifying cascade of cell injury within the lung from which the lung cannot recover, eventually resulting in catastrophic consequences for the organism.

cPGES/p23 is required for glucocorticoid receptor function and embryonic growth but not prostaglandin E2 synthesis.

ABSTRACT A number of studies have identified cytosolic prostaglandin E2 synthase (cPGES)/p23 as a cytoplasmic protein capable of metabolism of prostaglandin E2 (PGE2) from the cyclooxygenase metabolite prostaglandin endoperoxide (PGH2). However, this protein has also been implicated in a number of other pathways, including stabilization of the glucocorticoid receptor (GR) complex. To define the importance of the functions assigned to this protein, mice lacking detectible cPGES/p23 expression were generated. cPGES/p23−/− pups die during the perinatal period and display retarded lung development reminiscent of the phenotype of GR-deficient neonates. Furthermore, GR-sensitive gluconeogenic enzymes are not induced in the prenatal period. However, unlike GR-deficient embryos, cPGES/p23−/− embryos are small and a proliferation defect is observed in cPGES/p23−/− fibroblasts. Analysis of arachidonic acid metabolites in embryonic tissues and primary fibroblasts failed to support a function for this protein in PGE2 biosynthesis. Thus, while the growth retardation of the cPGES/p23−/− pups and decreased proliferation of primary fibroblasts identify functions for this protein in addition to GR stabilization, it is unlikely that these functions include metabolism of PGH2 to PGE2.

Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness

Mutation of the 3β-hydroxysterol Δ7-reductase gene (Dhcr7 −/−) results in Smith-Lemli-Opitz syndrome (SLOS). Patients, and genetically altered mice, are unable to produce cholesterol and accumulate 7-dehydrocholesterol (DHC) in serum and tissue. This causes multiple growth and developmental abnormalities as well as immune system anomalies including allergy. Because cholesterol is a key component of liquid-ordered membranes (lipid rafts) and these domains have been implicated in regulating mast cell activation, we examined whether mast cell responsiveness is altered in this model. Mast cells derived from Dhcr7 −/− mice (DHCR KO) showed constitutive cytokine production and hyper-degranulation after stimulation of the high affinity IgE receptor (FcɛRI). DHCR KO mast cells, but not wild-type mast cells, accumulated DHC in lipid rafts. DHC partially disrupted lipid raft stability and displaced Lyn kinase protein and activity from lipid rafts. This led to down-regulation of some Lyn-dependent signaling events but increased Fyn kinase activity and Akt phosphorylation. The Lyn-dependent phosphorylation of Csk-binding protein, which negatively regulates Fyn activity, was decreased. This phenotype reproduces some of the characteristics of Lyn-null mast cells, which also demonstrate hyper-degranulation. These findings provide the first evidence of lipid raft dysfunction in SLOS and may explain the observed association of allergy with SLOS.

Impaired Host Defense in Mice Lacking ONZIN

ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin−/− mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin−/− mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin−/− neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.

Cooperation between mast cells and neurons is essential for antigen-mediated bronchoconstriction

Mast cells are important sentinels guarding the interface between the environment and the body: a breach in the integrity of this interface can lead to the release of a plethora of mediators that engage the foreign agent, recruit leukocytes, and initiate adaptive physiological changes in the organism. While these capabilities make mast cells critical players in immune defense, it also makes them important contributors to the pathogenesis of diseases such as asthma. Mast cell mediators induce dramatic changes in smooth muscle physiology, and the expression of receptors for these factors by smooth muscle suggests that they act directly to initiate constriction. Contrary to this view, we show herein that mast cell-mediated bronchoconstriction is observed only in animals with intact innervation of the lung and that serotonin release alone is required for this action. While ablation of sensory neurons does not limit bronchoconstriction, constriction after Ag challenge is absent in mice in which the cholinergic pathways are compromised. Linking mast cell function to the cholinergic system likely provides an important means of modulating the function of these resident immune cells to physiology of the lung, but may also provide a safeguard against life-threatening anaphylaxis during mast cell degranulation.

A molecular understanding of mast cell activation and the promise of anti-allergic therapeutics.

Mast cells are central to allergic disease. Their immediate (exocytosis of granule-stored allergic-mediators) and delayed (de novo synthesis of inflammatory mediators) response to an allergen underlies the symptoms seen in acute; and chronic allergic disease. Thus, intervention in the allergen-mediated activation of mast cells is a long sought after goal in the treatment and management of allergic disease. The recent gain in deciphering the molecular mechanisms underlying immunoglobulin E (IgE)-mediated mast cell activation has provided optimism for the development of new therapeutic strategies. Among the most promising is the use of humanized anti-IgE antibodies that inhibit binding of IgE to its high affinity receptor (FcepsilonRI) on the mast cell. Other strategies target molecules proximal to FcepsilonRI, whose activities are central in mast cell activation. One such molecule, Syk kinase, has been targeted by various approaches including a small molecule inhibitor that specifically abrogates mast cell degranulation. More recently, various molecules that function to promote protein-protein interactions (adapters) were demonstrated as essential to mast cell degranulation and cytokine production. It remains to be seen if these molecules hold therapeutic promise for disease intervention. Additional studies identifying molecules required for mast cell granule fusion and content exocytosis also bodes well for discovery of new therapeutic targets. While our understanding of IgE-mediated mast cell activation is still at its inception, the modest success in identifying molecules essential to this process affords some confidence for better treatment of allergic disease.

Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection.