Class I PI3K enzymes play critical roles in B cell activation by phosphorylating plasma membrane lipids to generate two distinct phosphoinositide (PI) products, PI(3,4,5)P3 and PI(3,4)P2. These PIs each bind distinct but overlapping sets of intracellular proteins that control cell survival, cytoskeletal reorganization, and metabolic activity. The tandem PH domain containing proteins (TAPPs) bind with high specificity to PI(3,4)P2, and their genetic uncoupling from PI(3,4)P2 in TAPP knock in (KI) mice was previously found to cause chronic B cell activation, abnormal germinal centers (GCs), and autoimmunity. In this article, we find that TAPPs provide feedback regulation affecting PI3K signaling and metabolic activation of B cells. Upon activation, TAPP KI B cells show enhanced metabolic activity associated with increased extracellular acidification rate, increased expression of glucose transporter GLUT1, and increased glucose uptake. TAPP KI B cells show markedly increased activation of the PI3K-regulated kinases Akt, GSK3β, and p70-S6K. Conversely, overexpression of the C-terminal TAPP PH domains in B cells can inhibit Akt phosphorylation by a mechanism requiring the TAPP PI(3,4)P2-binding pocket. Inhibition of the PI3K pathway in TAPP KI B cells reduced GLUT1 expression and glucose uptake, whereas inhibition of Akt alone was not sufficient to normalize these responses. TAPP KI GC B cells also show increased GLUT1 and glucose uptake, and treatment with the inhibitor of glycolysis 2-deoxy-D-glucose reduced chronic GC responses and autoantibody production within these mice. Our findings show that TAPP-PI(3,4)P2 interaction controls activation of glycolysis and highlights the significance of this pathway for B cell activation, GC responses, and autoimmunity.
TAPP1 and TAPP2 (where TAPP is tandem PH domain containing protein) are dual PH domain adaptors that selectively bind PI(3,4)P2 (phosphatidylinositol (3,4)-bisphosphate). PI(3,4)P2 is a lipid messenger generated by phosphoinositide 3-kinase (PI3K)and IntroductionThe phosphoinositide 3-kinase (PI3K) signaling pathway is linked to a number of key cellular processes including lymphocyte cell survival, proliferation, differentiation, and motility [1][2][3].Correspondence: Dr. Aaron J. Marshall e-mail: marshall@ms.umanitoba.ca PI3K functions by phosphorylating the D3 position of membrane phosphoinositides (PIs) generating PI(3)P, PI(3,4)P2, and PI(3,4,5)P3. These D3 PIs regulate various cellular processes through the recruitment of effector proteins containing PI-binding domains [4][5][6]. Regulation of this pathway in immune cells is crucial for prevention of leukocyte-derived cancers as well as development of autoimmune diseases [7][8][9][10].The B-cell antigen receptor (BCR) activates PI3K [11] and this pathway plays critical roles in B lymphocyte development and function [12,13]. The PI3K pathway is critical for C 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu Eur. J. Immunol. 2012. 42: 2760-2770 Leukocyte signaling 2761"tonic" signaling through the BCR, which is required to maintain the survival of mature B cells [14]. PI phosphatases such as PTEN and SHIP function to restrain PI3K signaling by limiting the amount of PI(3,4,5)P3 available for binding [15,16]. In B cells, inhibitory signaling via the receptor FcγRIIB requires binding of SHIP to an ITIM motif in its cytoplasmic tail [17]. SHIP-deficient mice show elevated immunoglobulin levels and increased B-cell proliferation upon BCR-FcγRIIB cross-linking, indicating that SHIP functions as a negative regulator in B cells [18]. PI second messengers regulate various cellular activities by functioning as membrane docking sites for proteins containing PIbinding modules such as PH, PX, or FYVE domains [19]. While PI(3,4,5)P3 is clearly implicated in activation of signaling pathways, the signaling events downstream of PI(3,4)P2 are not well understood. A key PI-dependant protein kinase Akt/PKB can bind both PI(3,4,5)P3 and PI(3,4)P2, and the latter lipid has been suggested to contribute to Akt activation [20,21]; however the direct versus indirect effects of PI(3,4)P2 on Akt activity remains controversial. SHIP, which hydrolyzes PIP3 to generate PI(3,4)P2, has been found in several systems to inhibit Akt activation [18,22], suggesting that PIP3 levels may often be the limiting factor regulating Akt activity.A few proteins are known to selectively bind to PI(3,4)P2, but not PIP3, including PX domain proteins involved in NADPH oxidase regulation [23] and TAPP1 and TAPP2 (where TAPP is tandem PH domain containing proteins) [24]. We previously found that TAPP adapters are expressed in B cells and are recruited to the plasma membrane in response to BCR cross-linking, in a delayed and sustained manner correlating with PI(3,4)P2 levels [25]. TAPP2 membra...
BackgroundBam32, a 32 kDa adaptor molecule, plays important role in B cell receptor signalling, T cell receptor signalling and antibody affinity maturation in germinal centres. Since antibodies against trypanosome variant surface glycoproteins (VSG) are critically important for control of parasitemia, we hypothesized that Bam32 deficient (Bam32-/-) mice would be susceptible to T. congolense infection.Methodology/Principal FindingsWe found that T. congolense-infected Bam32-/- mice successfully control the first wave of parasitemia but then fail to control subsequent waves and ultimately succumb to their infection unlike wild type (WT) C57BL6 mice which are relatively resistant. Although infected Bam32-/- mice had significantly higher hepatomegaly and splenomegaly, their serum AST and ALT levels were not different, suggesting that increased liver pathology may not be responsible for the increased susceptibility of Bam32-/- mice to T. congolense. Using direct ex vivo flow cytometry and ELISA, we show that CD4+ T cells from infected Bam32-/- mice produced significantly increased amounts of disease-exacerbating proinflammatory cytokines (including IFN-γ, TNF-α and IL-6). However, the percentages of regulatory T cells and IL-10-producing CD4+ cells were similar in infected WT and Bam32-/- mice. While serum levels of parasite-specific IgM antibodies were normal, the levels of parasite-specific IgG, (particularly IgG1 and IgG2a) were significantly lower in Bam32-/- mice throughout infection. This was associated with impaired germinal centre response in Bam32-/- mice despite increased numbers of T follicular helper (Tfh) cells. Adoptive transfer studies indicate that intrinsic B cell defect was responsible for the enhanced susceptibility of Bam32-/- mice to T. congolense infection.Conclusions/SignificanceCollectively, our data show that Bam32 is important for optimal anti-trypanosome IgG antibody response and suppression of disease-promoting proinflammatory cytokines and its deficiency leads to inability to control T. congolense infection in mice.
Follicular dendritic cell secreted protein (FDC-SP) is a secreted peptide predominantly expressed in mucosal tissues. We previously reported that FDC-SP transgenic mice have altered B-cell responses to systemic immunization; however, the role of FDC-SP in mucosal immunity is unknown. Here, we report that FDC-SP functions in regulating immunoglobulin A production. FDC-SP transgenic mice show decreased IgA levels in serum, saliva, and bronchoalveolar lavage fluid. Reciprocally, FDC-SP-deficient mice show significantly increased IgA levels in serum and intestinal lavage, associated with accumulation of IgA+ cells in blood, bone marrow, Peyer's patches, and lymph nodes. FDC-SP-deficient mice generated higher titers of antigen-specific IgA but normal IgG1 responses upon immunization. Purified FDC-SP transgenic B cells generated decreased IgA responses to transforming growth factor β (TGFβ)+interleukin 5 (IL5) stimulation. Consistent with a direct effect of FDC-SP on B cells, recombinant FDC-SP suppressed B-cell IgA production in vitro. Six- to 14-month-old FDC-SP-deficient mice show IgA deposition in kidney glomeruli, which was associated with proteinuria and pathology consistent with mild IgA nephropathy (IgAN). Our results demonstrate a novel biological activity of FDC-SP in controlling B-cell IgA production and identify FDC-SP-deficient mice as a novel mouse model of IgAN.
Control of B-cell signal transduction is critical to prevent production of pathological autoantibodies. Tandem PH domain containing proteins (TAPPs) specifically bind PI(3,4)P2, a phosphoinositide product generated by PI 3-kinases and the phosphatase SHIP. TAPP KI mice bearing PH domain-inactivating mutations in both TAPP1 and TAPP2 genes, Our findings demonstrate a B cell-intrinsic role of TAPP-PI(3,4)P2 interaction in regulating GC responses and autoantibody production and suggest that uncontrolled Akt activity in B cells can drive autoimmunity.
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