FcεRI β‐chain ITAM amplifies PI3K‐signaling to ensure synergistic degranulation response via FcεRI and adenosine receptors

Simultaneous stimulation with antigen and adenosine in mast cells induces a synergistic degranulation response at a low antigen dose that is insufficient to cause secretion by itself. This kind of stimulation is thought to be relevant to the immediate asthmatic response upon bronchial challenge with low‐dose allergen. In this context, FcεRI‐ and adenosine receptor‐mediated signalings cooperate to increase degranulation in mast cells. In the present study, we prepared mast cells that have mutations (Y219F/Y225F/Y229F) in three tyrosine residues of the FcεRI β‐chain (FcRβ)‐ITAM in order to elucidate the molecular mechanisms of degranulation response synergistically elicited by costimulation with low‐dose antigen and adenosine. Introduction of mutations in the FcRβ‐ITAM abolished the synergistic degranulation response. Upon costimulation with low‐dose antigen and adenosine, tyrosine phosphorylation of Grb2‐associated binder 2, which is located upstream of PI3K‐signaling, was significantly increased, but severely diminished in FcRβ‐ITAM mutant cells. These findings indicate that FcRβ acts as a critical element in mast cell synergistic degranulation response through FcεRI and adenosine receptors, and that PI3K‐signaling through FcRβ‐ITAM is a crucial participant in augmentation of FcεRI‐mediated degranulation by adenosine.     

PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation

Phosphatidylinositol‐3‐kinase gamma (PI3Kγ) is a leukocyte‐specific lipid kinase with signaling function downstream of G protein‐coupled receptors to regulate cell trafficking, but its role in T cells remains unclear. To investigate the requirement of PI3Kγ kinase activity in T‐cell function, we studied T cells from PI3Kγ kinase‐dead knock‐in (PI3Kγ^KD/KD) mice expressing the kinase‐inactive PI3Kγ protein. We show that CD4⁺ and CD8⁺ T cells from PI3Kγ^KD/KD mice exhibit impaired TCR/CD28‐mediated activation that could not be rescued by exogenous IL‐2. The defects in proliferation and cytokine production were also evident in naïve and memory T cells. Analysis of signaling events in activated PI3Kγ^KD/KD T cells revealed a reduction in phosphorylation of protein kinase B (AKT) and ERK1/2, a decrease in lipid raft formation, and a delay in cell cycle progression. Furthermore, PI3Kγ^KD/KD CD4⁺ T cells displayed compromised differentiation toward Th1, Th2, Th17, and induced Treg cells. PI3Kγ^KD/KD mice also exhibited an impaired response to immunization and a reduced delayed‐type hypersensitivity to Ag challenge. These findings indicate that PI3Kγ kinase activity is required for optimal T‐cell activation and differentiation, as well as for mounting an efficient T cell‐mediated immune response. The results suggest that PI3Kγ kinase inhibitors could be beneficial in reducing the undesirable immune response in autoimmune diseases.     

Leukocyte transmigration is modulated by chemokine‐mediated PI3Kγ‐dependent phosphorylation of vimentin

Phosphoinositide 3‐kinase γ (PI3Kγ) plays a fundamental role in mediating leukocyte migration to inflammation sites. However, the downstream cytoplasmic events triggered by its signaling activity are still largely obscure. To address this issue, tyrosine and serine/threonine phosphorylated proteins of chemokine‐stimulated WT or PI3Kγ‐null macrophages were investigated. Among the proteins analyzed, the intermediate filament vimentin was found as a downstream effector of the PI3Kγ signaling pathway. Specific analysis of the phosphorylation state of vimentin in macrophages showed that this protein becomes rapidly phosphorylated in both tyrosine and serine residues upon chemokine stimulation. In the absence of PI3Kγ or the kinase activity of PI3Kγ (PI3Kγ^KD/KD), phosphorylation of vimentin was reduced. PI3Kγ‐null macrophages displayed impaired chemokine‐driven vimentin fiber disassembly as well as reduced ability to transmigrate across endothelial cells. While WT macrophages infected with a vimentin mutant resistant to N‐terminal serine phosphorylation showed a reduction in transendothelial migration, infection of PI3Kγ‐null macrophages with a vimentin mutant mimicking serine phosphorylation of N‐terminal residues rescued the transendothelial migration defect. These results define vimentin N‐terminal phosphorylation and fiber reorganization as a target of chemokine‐dependent PI3Kγ signaling in leukocytes.     

Genetic ablation of PI3Kγ results in defective IL‐17RA signalling in T lymphocytes and increased IL‐17 levels

The signalling molecule PI3Kγ has been reported to play a key role in the immune system and the inflammatory response. In particular, it facilitates the migration of haemato‐poietic cells to the site of inflammation. In this study, we reveal a novel role for PI3Kγ in the regulation of the pro‐inflammatory cytokine IL‐17. Loss of PI3Kγ or expression of a catalytically inactive mutant of PI3Kγ in mice led to increased IL‐17 production both in vitro and in vivo in response to various stimuli. The kinetic profile was unaltered from WT cells, with no effect on proliferation or other cytokines. Elevated levels of IL‐17 were not due to an aberrant expansion of IL‐17‐producing cells. Furthermore, we also identified an increase in IL‐17RA expression on PI3Kγ^?/? CD4⁺ T cells, yet these cells exhibited impaired PI3K‐dependent signalling in response to IL‐17A, and subsequent NF‐κB phosphorylation. In vivo, instillation of recombinant IL‐17 into the airways of mice lacking PI3Kγ signalling also resulted in reduced phosphorylation of Akt. Cell influx in response to IL‐17 was also reduced in PI3Kγ^?/? lungs. These data demonstrate PI3Kγ‐dependent signalling downstream of IL‐17RA, which plays a pivotal role in regulating IL‐17 production in T cells.     

Acceleration of Autoimmune Diabetes by Cyclophosphamide is Associated with an Enhanced IFN-γ Secretion Pathway

Cyclophosphamide (CY), an alkylating cytostatic drug, is known for its ability to accelerate a number of experimental autoimmune diseases including spontaneous diabetes in NOD mice. The mechanism(s) by which CY renders autoreactive lymphocytes more pathogenic is largely unknown, but it has been postulated that the drug preferentially depletes regulatory (suppressor) T cells. It has been suggested that in cell-mediated autoimmune diseases, Th2-like lymphocytes secreting IL-4 and/or IL-10 provide protection, while Th1-like cells secreting IFN-γ are pathogenic. In this study, we analysed the effects of CY on autoimmune diabetes and cytokines in two mouse models: the spontaneous diabetes of NOD mice and the diabetes induced in C57BL/KsJ mice by multiple injections of low dose streptozotocin (LD-STZ). In both models, CY induced severe lymphopenia and accelerated the progression to hyperglycemia. This was associated with changes in splenic cytokine patterns indicating a shift towards the IFN-γ-secreting phenotype. We provide here evidence that IFN-γ producers are relatively resistant to depletion by CY and that Th0 clones can be shifted towards Th1. However, direct exposure of T lymphocytes to CY may not be a necessary condition for exacerbation of diabetes; NOD.scid mice treated with CY before adoptive transfer of NOD splenocytes developed diabetes at a higher rate than did controls. Thus, the acceleration of diabetes by CY seems to be a complex event, which includes the relatively high resistance of IFN-γ producers to the drug, their rapid reconstitution, and a Th1 shift of surviving T cell clones.     

Activating PI3Kδ mutations in a cohort of 669 patients with primary immunodeficiency

The gene PIK3CD codes for the catalytic subunit of phosphoinositide 3‐kinase δ (PI3K δ ), and is expressed solely in leucocytes. Activating mutations of PIK3CD have been described to cause an autosomal dominant immunodeficiency that shares clinical features with common variable immunodeficiency (CVID). We screened a cohort of 669 molecularly undefined primary immunodeficiency patients for five reported mutations (four gain‐of‐function mutations in PIK3CD and a loss of function mutation in PIK3R1) using pyrosequencing. PIK3CD mutations were identified in three siblings diagnosed with CVID and two sporadic cases with a combined immunodeficiency (CID). The PIK3R1 mutation was not identified in the cohort. Our patients with activated PI3Kδ syndrome (APDS) showed a range of clinical and immunological findings, even within a single family, but shared a reduction in naive T cells. PIK3CD gain of function mutations are more likely to occur in patients with defective B and T cell responses and should be screened for in CVID and CID, but are less likely in patients with a pure B cell/hypogammaglobulinaemia phenotype.     

C‐Reactive Protein Triggers Calcium Signalling in Human Neutrophilic Granulocytes via FcγRIIa in an Allele‐Specific Way

C‐reactive protein (CRP) binds to Fcγ‐receptors, FcγRIIa (CD32) with high affinity and to FcγRIa (CD64) with low affinity. The binding to CD32 has been shown to be allele specific, that is, it binds to R/R131 but not to H/H131. Little is known about the cooperation of CRP and neutrophilic granulocytes (PMNs) in inflammatory reactions. The purpose of the present study was to examine CRP signalling in human PMNs, and whether this signalling is also allele specific. Cytosolic calcium of PMN was measured in a single‐cell digital imaging system. Receptor expression and polymorphism were studied by real‐time RT‐PCR, flow cytometry and standard PCR. C‐reactive protein induced cytosolic calcium signals in PMNs from homozygote R/R131donors, but not in PMNs from heterozygote R/H131 donors. However, after the heterozygote PMNs had been incubated with IFN‐γ (100 U/ml) for 2 h, both the proportion of cells responding and the size of the CRP‐induced calcium signals increased. IFN‐γ increased mRNA expression of CD64 about fivefold and surface protein expression of CD64 about fourfold. The calcium signal elicited by CRP was augmented by PMN adhesion to fibronectin, but almost totally abrogated by sphingosine kinase inhibitors. The signals were partly dependent on calcium influx. In conclusion, calcium signalling instigated by CRP in human PMN is FcγRIIa allele specific, as R/R131 responded to CRP, whereas R/H131 did not. However, increased expression of FcγRIa (CD64), stimulated by IFN‐γ, can augment calcium signalling by CRP in low‐responders. This suggests that the state of the PMNs, as well as the genetic origin, affect sensitivity for CRP.     

Mincle,the receptor for mycobacterial cord factor,forms a functional receptor complex with MCL and FcεRI‐γ

Upon receptor activation, the myeloid C‐type lectin receptor Mincle signals via the Syk‐CARD9‐Bcl10‐MALT1 pathway. It does so by recruiting the ITAM‐bearing FcεRI‐γ. The related receptor macrophage C‐type Lectin (MCL) has also been shown to be associated with Syk and to be dependent upon this signaling axis. We have previously shown that MCL co‐precipitates with FcεRI‐γ, but were unable to show a direct association, suggesting that MCL associates with FcεRI‐γ via another molecule. Here, we have used rat primary cells and cell lines to investigate this missing link. A combination of flow cytometric and biochemical analysis showed that Mincle and MCL form heteromers on the cell surface. Furthermore, association with MCL and FcεRI‐γ increased Mincle expression and enhanced phagocytosis of Ab‐coated beads. The results presented in this paper suggest that the Mincle/MCL/FcεRI‐γ complex is the functionally optimal form for these C‐type lectin receptors on the surface of myeloid cells.     

Food Allergen–Induced Mast Cell Degranulation is Dependent on PI3K‐Mediated Reactive Oxygen Species Production and Upregulation of Store‐Operated Calcium Channel Subunits

The importance of Ca²⁺ influx via store‐operated calcium channels (SOCs) leading to mast cell degranulation is well known in allergic disease. However, the underlying mechanisms are not fully understood. With food‐allergic rat model, the morphology of degranulated mast cell was analysed by toluidine blue stain and electron microscope. Ca²⁺ influx via SOCs was checked by Ca²⁺ imaging confocal microscope. Furthermore, the mRNA and protein expression of SOCs subunits were investigated using qPCR and Western blot. We found that ovalbumin (OVA) challenge significantly increased the levels of Th2 cytokines and OVA‐specific IgE in allergic animals. Parallel to mast cell activation, the levels of histamine in serum and supernatant of rat peritoneal lavage solution were remarkably increased after OVA treatment. Moreover, the Ca²⁺ entry through SOCs evoked by thapsigargin was increased in OVA‐challenged group. The mRNA and protein expressions of SOC subunits, stromal interaction molecule 1 (STIM1) and Orail (calcium‐release‐activated calcium channel protein 1), were dramatically elevated under food‐allergic condition. Administration of Ebselen, a scavenger of reactive oxygen species (ROS), significantly attenuated OVA sensitization‐induced intracellular Ca²⁺ rise and upregulation of SOCs subunit expressions. Intriguingly, pretreatment with PI3K‐specific inhibitor (Wortmannin) partially abolished the production of ROS and subsequent elevation of SOCs activity and their subunit expressions. Taken together, these results imply that enhancement of SOC‐mediated Ca²⁺ influx induces mast cell activation, contributing to the pathogenesis of OVA‐stimulated food allergy. PI3K‐dependent ROS generation involves in modulating the activity of SOCs by increasing the expressions of their subunit.     

Constitutive activation of the PI3K‐AKT pathway and cardiovascular abnormalities in an individual with Kosaki overgrowth syndrome

Kosaki overgrowth syndrome is a recently described syndrome characterized by distinctive facial features, brain white matter lesions, and developmental delay. Germline activating heterozygous PDGFRB mutations have been reported in this condition. Systemic connective tissue‐type findings have been described in some individuals. We describe a 19‐year‐old Caucasian female with a history of hydrocephalus, Dandy–Walker malformation, cervical spine arachnoid cyst, progressive scoliosis, and overgrowth. Her physical exam included distinctive craniofacial dysmorphism, as well as soft and hyperextensible skin. Cardiovascular imaging during adolescence revealed saccular aneurysms in both coronary artery systems and subtle tortuosity of the cervical vertebral arteries. Exome sequencing trio analysis identified a de novo previously reported pathogenic variant in PDGFRB, c.1696T>C (p.[Trp566Arg]). Further functional studies included platelet‐derived growth factor cellular metabolic pathway activity that confirmed the variant causes a constitutive activation of the PI3K‐AKT pathway. This is the first report to characterize the activating nature of this PDGFRB variant. We also highlight the connective tissue findings seen in Kosaki overgrowth syndrome and recommend baseline echocardiographic evaluation in all individuals with this condition with particular emphasis on coronary arteries.     

Quantification of α‐subunit isoforms of Na,K‐ATPase in rat resistance vessels

Aim: Rat mesenteric resistance vessels (RV) were characterized with respect to concentration of individual α‐subunit isoforms of Na,K‐ATPase. Methods: Total vessel homogenates were used to avoid any loss or subfractionation of membranes. They were applied to sodium dodecyl sulphate gels and, for calibration, in parallel lanes were run purified rat Na,K‐ATPase preparations with known isoform distribution and content. The capacity per mg protein for Na⁺‐dependent ³²P‐phosphorylation of Na,K‐ATPase isolated from rat kidney was used for α₁ calibration and that for high‐affinity (³H)ouabain binding of Na,K‐ATPase isolated from rat brain was used for (α₂ + α₃) calibration. Western blots containing homogenate proteins and reference enzyme were incubated with isoform‐specific antibodies and radiolabelled secondary antibodies. The signals from adjacent α spots were used for qualitative and quantitative characterization of rat vessels. Results: A concentration of 100.7 ± 14.4 pmol (n = 11) per g wet weight of the α₁‐isoform containing Na,K‐ATPase was found in RV from 12–14‐week rats. A much lower and more unreliable content of α₂‐ and α₃‐isoforms was found. These ouabain‐sensitive isoforms seem to represent a maximum of 5–10% each compared with the ouabain‐insensitive rat α₁‐isoform. Conclusions: The isoform pattern in RV, in which the isoform with high/intermediate Na⁺‐affinity is the absolutely dominating one representing nearly all sodium pumps in this tissue, is very different from that seen in rat skeletal muscles. Due to the high content of the ouabain‐insensitive α₁‐isoform in rat RV this species would seem a less relevant model in studies addressing a role of cardiac glycosides and putative endogenous ouabain‐like factors in hypertension.     

Genetic variation of human neutrophil Fcγ receptors and SIRPα in antibody‐dependent cellular cytotoxicity towards cancer cells

The efficacy of cancer therapeutic antibodies varies considerably among patients. Anti‐cancer antibodies act through different mechanisms, including antibody‐dependent cellular cytotoxicity (ADCC) triggered via Fcγ receptors (FcγR). This phagocyte ADCC can be promoted by interference with CD47‐SIRPα interactions, but the magnitude of this enhancement also varies among individuals. Both FcγR and SIRPα display considerable genetic variation, and we investigated whether this explains some of the variability in ADCC. Because of linkage disequilibrium between FcγR variants the interpretation of previous reports suggesting a potential link between FcγR polymorphisms and ADCC has been troublesome. We performed an integrated genetic analysis that enables stratification. ADCC by activated human neutrophils towards Trastuzumab‐coated breast cancer cells was predominantly dependent on FcγRIIa. Neutrophils from individuals with the FcγRIIa‐131H polymorphic variant displayed significantly higher killing capacity relative to those with FcγRIIa‐131R. Furthermore, ADCC was consistently enhanced by targeting CD47‐SIRPα interactions, and there were no significant functional differences between the two most prevalent SIRPα polymorphic variants. Thus, neutrophil ADCC capacity is directly related to the FcγRIIa polymorphism, and targeting CD47‐SIRPα interactions enhances ADCC independently of FcγR and SIRPα genotype, thereby further suggesting that CD47‐SIRPα interference might be a generic strategy for potentiating the efficacy of antibody therapy in cancer.     

Suppression of SPIN1‐mediated PI3K–Akt pathway by miR‐489 increases chemosensitivity in breast cancer

Drug resistance is one of the major obstacles for improving the prognosis of breast cancer patients. Increasing evidence has linked the association of aberrantly expressed microRNAs (miRNAs) with tumour development and progression as well as chemoresistance. Despite recent advances, there is still little known about the potential role and mechanism of miRNAs in breast cancer chemoresistance. Here we describe that 16 miRNAs were found to be significantly down‐regulated and 11 up‐regulated in drug‐resistant breast cancer tissues compared with drug‐sensitive tissues, using a miRNA microarray. The results also showed miR‐489 to be one of the most down‐regulated miRNAs in drug‐resistant tissues and cell lines, as confirmed by miRNA microarray screening and real‐time quantitative PCR. A decrease in miR‐489 expression was associated with chemoresistance as well as lymph node metastasis, increased tumour size, advanced pTNM stage and poor prognosis in breast cancer. Functional analysis revealed that miR‐489 increased breast cancer chemosensitivity and inhibited cell proliferation, migration and invasion, both in vitro and in vivo. Furthermore, SPIN1, VAV3, BCL2 and AKT3 were found to be direct targets of miR‐489. SPIN1 was significantly elevated in drug‐resistant and metastatic breast cancer tissues and inversely correlated with miR‐489 expression. High expression of SPIN1 was associated with higher histological grade, lymph node metastasis, advanced pTNM stage and positive progesterone receptor (PR) status. Increased SPIN1 expression enhanced cell migration and invasion, inhibited apoptosis and partially antagonized the effects of miR‐489 in breast cancer. PIK3CA, AKT, CREB1 and BCL2 in the PI3K–Akt signalling pathway, demonstrated to be elevated in drug‐resistant breast cancer tissues, were identified as downstream effectors of SPIN1. It was further found that either inhibition of SPIN1 or overexpression of miR‐489 suppressed the PI3K–Akt signalling pathway. These data indicate that miR‐489 could reverse the chemoresistance of breast cancer via the PI3K–Akt pathway by targeting SPIN1. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.