Inhibition of lipid phosphatase SHIP1 expands myeloid-derived suppressor cells and attenuates rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a debilitating autoimmune disease of unknown cause, characterized by infiltration and accumulation of activated immune cells in the synovial joints where cartilage and bone destructions occur. Myeloid-derived suppressor cells (MDSCs) are of myeloid origin and are able to suppress T cell responses. Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1) was shown to be involved in the regulation of MDSC differentiation. The purpose of the present study was to investigate the effect of inhibition of SHIP1 on the expansion of MDSCs in RA using a collagen-induced inflammatory arthritis (CIA) mouse model. In DBA/1 mice, treatment with a small molecule-specific SHIP1 inhibitor 3α-aminocholestane (3AC) induced a marked expansion of MDSCs in vivo. Both pretreatment with 3AC of DBA/1 mice prior to CIA induction and intervention with 3AC during CIA progression significantly reduced disease incidence and severity. Adoptive transfer of MDSCs isolated from 3AC-treated mice, but not naïve MDSCs from normal mice, into CIA mice significantly reduced disease incidence and severity, indicating that the 3AC-induced MDSCs were the cellular mediators of the observed amelioration of the disease. In conclusion, inhibition of SHIP1 expands MDSCs in vivo and attenuates development of CIA in mice. Small molecule-specific inhibition of SHIP1 may therefore offer therapeutic benefit to patients with RA and other autoimmune diseases.

The 5-Phosphatase SHIP2 Promotes Neutrophil Chemotaxis and Recruitment.

Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality in vitro and in neutrophil recruitment to sites of sterile inflammation in vivo, in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.

Suppression of IgE-mediated anaphylaxis and food allergy with monovalent anti-FcεRIα mAbs.

BACKGROUND: Mast cell and basophil activation by antigen cross-linking of FcεRI-bound IgE is central to allergy pathogenesis. We previously demonstrated global suppression of this process by rapid desensitization with anti-FcεRIα mAbs. OBJECTIVES: We sought to determine whether use of monovalent (mv) anti-FcεRIα mAbs increases desensitization safety without loss of efficacy. METHODS: mv anti-human (hu) FcεRIα mAbs were produced with mouse-derived immunoglobulin variable regions and huIgG1 or huIgG4 C regions and were used to suppress murine IgE-mediated anaphylaxis and food allergy. mAbs were administered as a single dose or as serially increasing doses to mice that express hu instead of mouse FcεRIα; mice that additionally have an allergy-promoting IL-4Rα mutation; and hu cord blood-reconstituted immunodeficient, hu cytokine-secreting, mice that have large numbers of activated hu mast cells. Anaphylaxis susceptibility was sometimes increased by treatment with IL-4 or a ß-adrenergic receptor antagonist. RESULTS: mv anti-hu FcεRIα mAbs are considerably less able than divalent mAbs are to induce anaphylaxis and deplete mast cell and basophil IgE, but mv mAbs still strongly suppress IgE-mediated disease. The mv mAbs can be safely administered as a single large dose to mice with typical susceptibility to anaphylaxis, while a rapid desensitization approach safely suppresses disease in mice with increased susceptibility. Our huIgG4 variant of mv anti-huFcεRIα mAb is safer than our huIgG1 variant is, apparently because reduced interactions with FcεRs decrease ability to indirectly cross-link FcεRI. CONCLUSIONS: mv anti-FcεRIα mAbs more safely suppress IgE-mediated anaphylaxis and food allergy than divalent variants of the same mAbs do. These mv mAbs may be useful for suppression of huIgE-mediated disease.

Monomer hapten and hapten-specific IgG inhibit mast cell activation evoked by multivalent hapten with different mechanisms.

Aggregation of IgE bound to high affinity IgE receptor (FcεRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FcεRI immediately terminated degranulation, and formation of co-ligated FcεRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FcεRI and FcγRIIB after FcεRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

IgG-Independent Co-aggregation of FcεRI and FcγRIIB Results in LYN- and SHIP1-Dependent Tyrosine Phosphorylation of FcγRIIB in Murine Bone Marrow-Derived Mast Cells.

Activation of the high-affinity receptor for IgE (FcεRI) follows a bell-shaped dose-response curve. Upon supra-optimal stimulation, mast cell effector responses are down-regulated by inhibitory molecules like the SH2-containing inositol-5'-phosphatase SHIP1 and the SRC-family-kinase LYN. To identify further molecules involved in a negative regulatory signalosome, we screened for proteins showing the same pattern of tyrosine phosphorylation as SHIP1, which is tyrosine-phosphorylated strongest upon supra-optimal antigen (Ag) stimulation. The low-affinity IgG receptor, FcγRIIB, was found to be most strongly phosphorylated under supra-optimal conditions. This phosphorylation is the consequence of passive, Ag/IgE-dependent and progressive co-localization of FcεRI and FcγRIIB, which is not dependent on IgG. Upon supra-optimal FcεRI cross-linking, FcγRIIB phosphorylation is executed by LYN and protected from dephosphorylation by SHIP1. Analysis of FcγRIIB-deficient bone marrow-derived mast cells revealed an ambiguous phenotype upon FcεRI cross-linking. Absence of FcγRIIB significantly diminished the level of SHIP1 phosphorylation and resulted in augmented Ca2+ mobilization. Though, degranulation and IL-6 production were only weakly altered. Altogether our data establish the LYN/FcγRIIB/SHIP1 signalosome in the context of FcεRI activation, particularly at supra-optimal Ag concentrations. The fact that SHIP1 tyrosine phosphorylation/activation not only depends on FcγRIIB, highlights the necessity for its tight backup control.

Structural characterization of two endopolysaccharides from Phellinus sp. and their immunologic effects by intragastric administration in a healthy mammalian model.

Two purified endopolysaccharides derived from cultured Phellinus sp., individually named SHIP-1 and 2, were structurally characterized, along with an evaluation of their in vivo influential immunomodulatory activity in a healthy mammalian model. The structure of SHIP-1 was mainly composed of →4)-α-d-Fucp-(1→, →3,6)-α-d-Araf-(1→ and →2,4)-ß-d-Galp-(→, with four residuals of α-d-Manp-(1→ and one α-d-Glcp-(1→ as sidegroups, while the planar structure and the heteronuclear multiple-bond correlation of SHIP-2 were not able to be analyzed. Biochemical analysis in the healthy mice model demonstrated that SHIP-1 increased the concentrations of the detected cytokines in a dosage-dependent manner but not in a time-dependent way. SHIP-2 exerted a positive effect in a dose-dependent manner over time for interferon gamma (IFN-γ) and interleukin (IL)-2 cytokine production at elevated dosages of 200, or 350 mg kg-1 d-1, while IFN-alpha(α) and IL-4 production was observed only in a dosage-dependent manner even at high dosages. Thus, SHIP-1 and 2 significantly improved the immune response through the intragastric administration of the tested high dosages by increasing the production of cytokines in the healthy mice, and these polysaccharides could possibly be used as an immunopotentiator in health foods or dietary supplements.

Detection of Experimental and Clinical Immune Complexes by Measuring SHIP-1 Recruitment to the Inhibitory FcγRIIB.

Fc γ receptors (FcγR) are involved in multiple aspects of immune cell regulation, are central to the success of mAb therapeutics, and underpin the pathology of several autoimmune diseases. However, reliable assays capable of accurately measuring FcγR interactions with their physiological ligands, IgG immune complexes (IC), are limited. A method to study and detect IC interactions with FcγRs was therefore developed. This method, designed to model the signaling pathway of the inhibitory FcγRIIB (CD32B), used NanoLuc Binary Interaction Technology to measure recruitment of the Src homology 2 domain-containing inositol phosphatase 1 to the ITIM of this receptor. Such recruitment required prior cross-linking of an ITAM-containing activatory receptor, and evoked luciferase activity in discrete clusters at the cell surface, recapitulating the known biology of CD32B signaling. The assay detected varying forms of experimental IC, including heat-aggregated IgG, rituximab-anti-idiotype complexes, and anti-trinitrophenol-trinitrophenol complexes in a sensitive manner (≤1 µg/ml), and discriminated between complexes of varying size and isotype. Proof-of-concept for the detection of circulating ICs in autoimmune disease was provided, as responses to sera from patients with systemic lupus erythematosus and rheumatoid arthritis were detected in small pilot studies. Finally, the method was translated to a stable cell line system. In conclusion, a rapid and robust method for the detection of IC was developed, which has numerous potential applications including the monitoring of IC in autoimmune diseases and the study of underlying FcγR biology.

Timescale Separation of Positive and Negative Signaling Creates History-Dependent Responses to IgE Receptor Stimulation.

The high-affinity receptor for IgE expressed on the surface of mast cells and basophils interacts with antigens, via bound IgE antibody, and triggers secretion of inflammatory mediators that contribute to allergic reactions. To understand how past inputs (memory) influence future inflammatory responses in mast cells, a microfluidic device was used to precisely control exposure of cells to alternating stimulatory and non-stimulatory inputs. We determined that the response to subsequent stimulation depends on the interval of signaling quiescence. For shorter intervals of signaling quiescence, the second response is blunted relative to the first response, whereas longer intervals of quiescence induce an enhanced second response. Through an iterative process of computational modeling and experimental tests, we found that these memory-like phenomena arise from a confluence of rapid, short-lived positive signals driven by the protein tyrosine kinase Syk; slow, long-lived negative signals driven by the lipid phosphatase Ship1; and slower degradation of Ship1 co-factors. This work advances our understanding of mast cell signaling and represents a generalizable approach for investigating the dynamics of signaling systems.

SHIP-1 Deficiency in AID+ B Cells Leads to the Impaired Function of B10 Cells with Spontaneous Autoimmunity.

Unlike conventional B cells, regulatory B cells exhibit immunosuppressive functions to downregulate inflammation via IL-10 production. However, the molecular mechanism regulating the production of IL-10 is not fully understood. In this study, we report the finding that activation-induced cytidine deaminase (AID) is highly upregulated in the IL-10-competent B cell (B10) cell from Innp5dfl/flAicdaCre/+ mice, whereas the 5' inositol phosphatase SHIP-1 is downregulated. Notably, SHIP-1 deficiency in AID+ B cells leads to a reduction in cell count and impaired IL-10 production by B10 cells. Furthermore, the Innp5dfl/flAicdaCre/+ mouse model shows B cell-dependent autoimmune lupus-like phenotypes, such as elevated IgG serum Abs, formation of spontaneous germinal centers, production of anti-dsDNA and anti-nuclear Abs, and the obvious deposition of IgG immune complexes in the kidney with age. We observe that these lupus-like phenotypes can be reversed by the adoptive transfer of B10 cells from control Innp5dfl/fl mice, but not from the Innp5dfl/flAicdaCre/+ mice. This finding highlights the importance of defective B10 cells in Innp5dfl/flAicdaCre/+ mice. Whereas p-Akt is significantly upregulated, MAPK and AP-1 activation is impaired in B10 cells from Innp5dfl/flAicdaCre/+ mice, resulting in the reduced production of IL-10. These results show that SHIP-1 is required for the maintenance of B10 cells and production of IL-10, and collectively suggests that SHIP-1 could be a new potential therapeutic target for the treatment of autoimmune diseases.

Early tyrosine phosphorylation events following adenosine A2A receptor in human neutrophils: identification of regulated pathways.

Activation of the adenosine 2A receptor (A2AR) elevates intracellular levels of cAMP and acts as a physiologic inhibitor of inflammatory neutrophil functions. In this study, we looked into the impact of A2AR engagement on early phosphorylation events. Neutrophils were stimulated with well-characterized proinflammatory agonists in the absence or presence of an A2AR agonist {3-[4-[2-[ [6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino] ethyl] phenyl] propanoic acid (CGS 21680)}, PGE2, or a mixture of the compounds RO 20-1724 and forskolin. As assessed by immunoblotting, several proteins were tyrosine phosphorylated; CGS 21680 markedly decreased tyrosine phosphorylation levels of 4 regions (37-45, 50-55, 60, and 70 kDa). Key signaling protein kinases-p38 MAPK, Erk-1/2, PI3K/Akt, Hck, and Syk-showed decreased phosphorylation, whereas Lyn, SHIP-1, or phosphatase and tensin homolog (PTEN) was spared. PGE2 or the intracellular cAMP-elevating combination of RO 20-1724 and forskolin mostly mimicked the effect of CGS 21680. Together, results unveil intracellular signaling pathways targeted by the A2AR, some of which might be key in modulating neutrophil functions.

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 Dok-3/Grb2 adaptor module promotes inducible association of the lipid phosphatase SHIP with the BCR in a coreceptor-independent manner.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) plays a key role in preventing autoimmune phenomena by limiting antigen-mediated B cell activation. SHIP function is thought to require the dual engagement of the BCR and negative regulatory coreceptors as only the latter appear capable of recruiting SHIP from the cytosol to the plasma membrane by the virtue of phosphorylated immunoreceptor tyrosine-based inhibitory motifs. Here, we demonstrate a coreceptor-independent membrane recruitment and function of SHIP in B cells. In the absence of coreceptor ligation, SHIP translocates to sites of BCR activation through a concerted action of the protein adaptor unit Dok-3/Grb2 and phosphorylated BCR signaling components. Our data reveal auto-inhibitory SHIP activation by the activated BCR and suggest an unexpected negative-regulatory capacity of immunoreceptor tyrosine-based activation motifs in Igα and Igß.

FcγRIIB-Independent Mechanisms Controlling Membrane Localization of the Inhibitory Phosphatase SHIP in Human B Cells.

SHIP is an important regulator of immune cell signaling that functions to dephosphorylate the phosphoinositide phosphatidylinositol 3,4,5-trisphosphate at the plasma membrane and mediate protein-protein interactions. One established paradigm for SHIP activation involves its recruitment to the phospho-ITIM motif of the inhibitory receptor FcγRIIB. Although SHIP is essential for the inhibitory function of FcγRIIB, it also has critical modulating functions in signaling initiated from activating immunoreceptors such as B cell Ag receptor. In this study, we found that SHIP is indistinguishably recruited to the plasma membrane after BCR stimulation with or without FcγRIIB coligation in human cell lines and primary cells. Interestingly, fluorescence recovery after photobleaching analysis reveals differential mobility of SHIP-enhanced GFP depending on the mode of stimulation, suggesting that although BCR and FcγRIIB can both recruit SHIP, this occurs via distinct molecular complexes. Mutagenesis of a SHIP-enhanced GFP fusion protein reveals that the SHIP-Src homology 2 domain is essential in both cases whereas the C terminus is required for recruitment via BCR stimulation, but is less important with FcγRIIB coligation. Experiments with pharmacological inhibitors reveal that Syk activity is required for optimal stimulation-induced membrane localization of SHIP, whereas neither PI3K or Src kinase activity is essential. BCR-induced association of SHIP with binding partner Shc1 is dependent on Syk, as is tyrosine phosphorylation of both partners. Our results indicate that FcγRIIB is not uniquely able to promote membrane recruitment of SHIP, but rather modulates its function via formation of distinct signaling complexes. Membrane recruitment of SHIP via Syk-dependent mechanisms may be an important factor modulating immunoreceptor signaling.

Continuous inhibitory signaling by both SHP-1 and SHIP-1 pathways is required to maintain unresponsiveness of anergic B cells.

Many autoreactive B cells persist in the periphery in a state of unresponsiveness called anergy. This unresponsiveness is rapidly reversible, requiring continuous BCR interaction with self-antigen and resultant regulatory signaling for its maintenance. Using adoptive transfer of anergic B cells with subsequent acute induction of gene deletion or expression, we demonstrate that the continuous activities of independent inhibitory signaling pathways involving the tyrosine phosphatase SHP-1 and the inositol phosphatase SHIP-1 are required to maintain anergy. Acute breach of anergy by compromise of either of these pathways leads to rapid cell activation, proliferation, and generation of short-lived plasma cells that reside in extrafollicular foci. Results are consistent with predicted/observed reduction in the Lyn-SHIP-1-PTEN-SHP-1 axis function in B cells from systemic lupus erythematosus patients.

CD11b immunophenotyping identifies inflammatory profiles in the mouse and human lungs.

The development of easily accessible tools for human immunophenotyping to classify patients into discrete disease endotypes is advancing personalized therapy. However, no systematic approach has been developed for the study of inflammatory lung diseases with often complex and highly heterogeneous disease etiologies. We have devised an internally standardized flow cytometry approach that can identify parallel inflammatory alveolar macrophage phenotypes in both the mouse and human lungs. In mice, lung innate immune cell alterations during endotoxin challenge, influenza virus infection, and in two genetic models of chronic obstructive lung disease could be segregated based on the presence or absence of CD11b alveolar macrophage upregulation and lung eosinophilia. Additionally, heightened alveolar macrophage CD11b expression was a novel feature of acute lung exacerbations in the SHIP-1(-/-) model of chronic obstructive lung disease, and anti-CD11b antibody administration selectively blocked inflammatory CD11b(pos) but not homeostatic CD11b(neg) alveolar macrophages in vivo. The identification of analogous profiles in respiratory disease patients highlights this approach as a translational avenue for lung disease endotyping and suggests that heterogeneous innate immune cell phenotypes are an underappreciated component of the human lung disease microenvironment.