Publications by authors named "Anne B Satterthwaite"

28 Publications

  • Page 1 of 1

Recent Advances in Lupus B Cell Biology: PI3K, IFNγ, and Chromatin.

Front Immunol 2020 14;11:615673. Epub 2021 Jan 14.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States.

In the autoimmune disease Systemic Lupus Erythematosus (SLE), autoantibodies are formed that promote inflammation and tissue damage. There has been significant interest in understanding the B cell derangements involved in SLE pathogenesis. The past few years have been particularly fruitful in three domains: the role of PI3K signaling in loss of B cell tolerance, the role of IFNγ signaling in the development of autoimmunity, and the characterization of changes in chromatin accessibility in SLE B cells. The PI3K pathway coordinates various downstream signaling molecules involved in B cell development and activation. It is governed by the phosphatases PTEN and SHIP-1. Murine models lacking either of these phosphatases in B cells develop autoimmune disease and exhibit defects in B cell tolerance. Limited studies of human SLE B cells demonstrate reduced expression of PTEN or increased signaling events downstream of PI3K in some patients. IFNγ has long been known to be elevated in both SLE patients and mouse models of lupus. New data suggests that IFNγR expression on B cells is required to develop autoreactive germinal centers (GC) and autoantibodies in murine lupus. Furthermore, IFNγ promotes increased transcription of BCL6, IL-6 and T-bet in B cells, which also promote GC and autoantibody formation. IFNγ also induces epigenetic changes in human B cells. SLE B cells demonstrate significant epigenetic reprogramming, including enhanced chromatin accessibility at transcription factor motifs involved in B cell activation and plasma cell (PC) differentiation as well as alterations in DNA methylation and histone modifications. Histone deacetylase inhibitors limit disease development in murine lupus models, at least in part their ability to prevent B cell class switching and differentiation into plasma cells. This review will discuss relevant discoveries of the past several years pertaining to these areas of SLE B cell biology.
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http://dx.doi.org/10.3389/fimmu.2020.615673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841329PMC
January 2021

PIK3IP1 Promotes Extrafollicular Class Switching in T-Dependent Immune Responses.

J Immunol 2020 10 4;205(8):2100-2108. Epub 2020 Sep 4.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390;

PI3K plays multiple roles throughout the life of a B cell. As such, its signaling is tightly regulated. The importance of this is illustrated by the fact that both loss- and gain-of-function mutations in PI3K can cause immunodeficiency in humans. PIK3IP1, also known as TrIP, is a transmembrane protein that has been shown to inhibit PI3K in T cells. Results from the ImmGen Consortium indicate that PIK3IP1 expression fluctuates throughout B cell development in a manner inversely correlated with PI3K activity; however, its role in B cells is poorly understood. In this study, we define the consequences of B cell-specific deletion of PIK3IP1. B cell development, basal Ig levels, and T-independent responses were unaffected by loss of PIK3IP1. However, there was a significant delay in the production of IgG during T-dependent responses, and secondary responses were impaired. This is likely due to a role for PIK3IP1 in the extrafollicular response because germinal center formation and affinity maturation were normal, and PIK3IP1 is not appreciably expressed in germinal center B cells. Consistent with a role early in the response, PIK3IP1 was downregulated at late time points after B cell activation, in a manner dependent on PI3K. Increased activation of the PI3K pathway was observed in PIK3IP1-deficient B cells in response to engagement of both the BCR and CD40 or strong cross-linking of CD40 alone. Taken together, these observations suggest that PIK3IP1 promotes extrafollicular responses by limiting PI3K signaling during initial interactions between B and T cells.
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http://dx.doi.org/10.4049/jimmunol.2000584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541772PMC
October 2020

Foxo3 Promotes Apoptosis of B Cell Receptor-Stimulated Immature B Cells, Thus Limiting the Window for Receptor Editing.

J Immunol 2018 08 27;201(3):940-949. Epub 2018 Jun 27.

Rheumatic Diseases Division, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX 75390;

Central tolerance checkpoints are critical for the elimination of autoreactive B cells and the prevention of autoimmunity. When autoreactive B cells encounter their Ag at the immature B cell stage, BCR cross-linking induces receptor editing, followed by apoptosis if edited cells remain autoreactive. Although the transcription factor Foxo1 is known to promote receptor editing, the role of the related factor Foxo3 in central B cell tolerance is poorly understood. We find that BCR-stimulated immature B cells from Foxo3-deficient mice demonstrate reduced apoptosis compared with wild type cells. Despite this, Foxo3 mice do not develop increased autoantibodies. This suggests that the increased survival of Foxo3 immature B cells allows additional rounds of receptor editing, resulting in more cells "redeeming" themselves by becoming nonautoreactive. Indeed, increased Igλ usage and increased recombining sequence recombination among Igλ-expressing cells were observed in Foxo3 mice, indicative of increased receptor editing. We also observed that deletion of high-affinity autoreactive cells was intact in the absence of Foxo3 in the anti-hen egg lysozyme (HEL)/membrane-bound HEL model. However, Foxo3 levels in B cells from systemic lupus erythematosus (SLE) patients were inversely correlated with disease activity and reduced in patients with elevated anti-dsDNA Abs. Although this is likely due in part to increased B cell activation in these SLE patients, it is also possible that low-affinity B cells that remain autoreactive after editing may survive inappropriately in the absence of Foxo3 and become activated to secrete autoantibodies in the context of other SLE-associated defects.
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http://dx.doi.org/10.4049/jimmunol.1701070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6057821PMC
August 2018

Bruton's Tyrosine Kinase, a Component of B Cell Signaling Pathways, Has Multiple Roles in the Pathogenesis of Lupus.

Front Immunol 2017 22;8:1986. Epub 2018 Jan 22.

Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, United States.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the loss of adaptive immune tolerance to nucleic acid-containing antigens. The resulting autoantibodies form immune complexes that promote inflammation and tissue damage. Defining the signals that drive pathogenic autoantibody production is an important step in the development of more targeted therapeutic approaches for lupus, which is currently treated primarily with non-specific immunosuppression. Here, we review the contribution of Bruton's tyrosine kinase (Btk), a component of B and myeloid cell signaling pathways, to disease in murine lupus models. Both gain- and loss-of-function genetic studies have revealed that Btk plays multiple roles in the production of autoantibodies. These include promoting the activation, plasma cell differentiation, and class switching of autoreactive B cells. Small molecule inhibitors of Btk are effective at reducing autoantibody levels, B cell activation, and kidney damage in several lupus models. These studies suggest that Btk may promote end-organ damage both by facilitating the production of autoantibodies and by mediating the inflammatory response of myeloid cells to these immune complexes. While Btk has not been associated with SLE in GWAS studies, SLE B cells display signaling defects in components both upstream and downstream of Btk consistent with enhanced activation of Btk signaling pathways. Taken together, these observations indicate that limiting Btk activity is critical for maintaining B cell tolerance and preventing the development of autoimmune disease. Btk inhibitors, generally well-tolerated and approved to treat B cell malignancy, may thus be a useful therapeutic approach for SLE.
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http://dx.doi.org/10.3389/fimmu.2017.01986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5786522PMC
January 2018

Pathways leading to an immunological disease: systemic lupus erythematosus.

Rheumatology (Oxford) 2017 04;56(suppl_1):i55-i66

The Rheumatic Diseases Division, Department of Internal Medicine, UT Southwestern Medical Center at Dallas, TX, USA.

SLE is a chronic autoimmune disease caused by perturbations of the immune system. The clinical presentation is heterogeneous, largely because of the multiple genetic and environmental factors that contribute to disease initiation and progression. Over the last 60 years, there have been a number of significant leaps in our understanding of the immunological mechanisms driving disease processes. We now know that multiple leucocyte subsets, together with inflammatory cytokines, chemokines and regulatory mediators that are normally involved in host protection from invading pathogens, contribute to the inflammatory events leading to tissue destruction and organ failure. In this broad overview, we discuss the main pathways involved in SLE and highlight new findings. We describe the immunological changes that characterize this form of autoimmunity. The major leucocytes that are essential for disease progression are discussed, together with key mediators that propagate the immune response and drive the inflammatory response in SLE.
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http://dx.doi.org/10.1093/rheumatology/kew427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410978PMC
April 2017

Leukocyte Beta-Catenin Expression Is Disturbed in Systemic Lupus Erythematosus.

PLoS One 2016 22;11(8):e0161682. Epub 2016 Aug 22.

The Department of Internal Medicine Rheumatic Diseases Division, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Wnt/β-catenin signaling is relatively understudied in immunity and autoimmunity. β-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished β-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/β-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of β-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of β-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161682PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4993388PMC
August 2017

Heightened cleavage of Axl receptor tyrosine kinase by ADAM metalloproteases may contribute to disease pathogenesis in SLE.

Clin Immunol 2016 08 27;169:58-68. Epub 2016 May 27.

The Department of Internal Medicine, Rheumatic Diseases Division, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States; The Department of Biomedical Engineering, University of Houston, Houston, TX 77204-5060, United States. Electronic address:

Systemic lupus erythematosus (SLE) is characterized by antibody-mediated chronic inflammation in the kidney, lung, skin, and other organs to cause inflammation and damage. Several inflammatory pathways are dysregulated in SLE, and understanding these pathways may improve diagnosis and treatment. In one such pathway, Axl tyrosine kinase receptor responds to Gas6 ligand to block inflammation in leukocytes. A soluble form of the Axl receptor ectodomain (sAxl) is elevated in serum from patients with SLE and lupus-prone mice. We hypothesized that sAxl in SLE serum originates from the surface of leukocytes and that the loss of leukocyte Axl contributes to the disease. We determined that macrophages and B cells are a source of sAxl in SLE and in lupus-prone mice. Shedding of the Axl ectodomain from the leukocytes of lupus-prone mice is mediated by the matrix metalloproteases ADAM10 and TACE (ADAM17). Loss of Axl from lupus-prone macrophages renders them unresponsive to Gas6-induced anti-inflammatory signaling in vitro. This phenotype is rescued by combined ADAM10/TACE inhibition. Mice with Axl-deficient macrophages develop worse disease than controls when challenged with anti-glomerular basement membrane (anti-GBM) sera in an induced model of nephritis. ADAM10 and TACE also mediate human SLE PBMC Axl cleavage. Collectively, these studies indicate that increased metalloprotease-mediated cleavage of leukocyte Axl may contribute to end organ disease in lupus. They further suggest dual ADAM10/TACE inhibition as a potential therapeutic modality in SLE.
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http://dx.doi.org/10.1016/j.clim.2016.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193537PMC
August 2016

The Role of the Transcription Factor Ets1 in Lupus and Other Autoimmune Diseases.

Crit Rev Immunol 2016 ;36(6):485-510

Department of Internal Medicine, Rheumatic Diseases Division; Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by excess B- and T-cell activation, the development of autoantibodies against self-antigens including nuclear antigens, and immune complex deposition in target organs, which triggers an inflammatory response and tissue damage. The genetic and environmental factors that contribute to the development of SLE have been studied extensively in both humans and mouse models of the disease. One of the important genetic contributions to SLE development is an alteration in the expression of the transcription factor Ets1, which regulates the functional differentiation of lymphocytes. Here, we review the genetic, biochemical, and immunological studies that have linked low levels of Ets1 to aberrant lymphocyte differentiation and to the pathogenesis of SLE.
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http://dx.doi.org/10.1615/CritRevImmunol.2017020284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695541PMC
January 2016

Genetic Interaction between Lyn, Ets1, and Btk in the Control of Antibody Levels.

J Immunol 2015 Sep 24;195(5):1955-63. Epub 2015 Jul 24.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390; Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390

Tight control of B cell differentiation into plasma cells (PCs) is critical for proper immune responses and the prevention of autoimmunity. The Ets1 transcription factor acts in B cells to prevent PC differentiation. Ets1(-/-) mice accumulate PCs and produce autoantibodies. Ets1 expression is downregulated upon B cell activation through the BCR and TLRs and is maintained by the inhibitory signaling pathway mediated by Lyn, CD22 and SiglecG, and SHP-1. In the absence of these inhibitory components, Ets1 levels are reduced in B cells in a Btk-dependent manner. This leads to increased PCs, autoantibodies, and an autoimmune phenotype similar to that of Ets1(-/-) mice. Defects in inhibitory signaling molecules, including Lyn and Ets1, are associated with human lupus, although the effects are more subtle than the complete deficiency that occurs in knockout mice. In this study, we explore the effect of partial disruption of the Lyn/Ets1 pathway on B cell tolerance and find that Lyn(+/-)Ets1(+/-) mice demonstrate greater and earlier production of IgM, but not IgG, autoantibodies compared with Lyn(+/-) or Ets1(+/-) mice. We also show that Btk-dependent downregulation of Ets1 is important for normal PC homeostasis when inhibitory signaling is intact. Ets1 deficiency restores the decrease in steady state PCs and Ab levels observed in Btk(-/-) mice. Thus, depending on the balance of activating and inhibitory signals to Ets1, there is a continuum of effects on autoantibody production and PC maintenance. This ranges from full-blown autoimmunity with complete loss of Ets1-maintaining signals to reduced PC and Ab levels with impaired Ets1 downregulation.
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http://dx.doi.org/10.4049/jimmunol.1500165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4546901PMC
September 2015

A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels.

J Immunol 2014 Jul 13;193(2):909-920. Epub 2014 Jun 13.

Department of Biochemistry, Center of Excellence in Bioinformatics and Life Sciences, State University of New York at Buffalo, Buffalo, NY 14203.

Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.
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http://dx.doi.org/10.4049/jimmunol.1400666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4082765PMC
July 2014

SLE peripheral blood B cell, T cell and myeloid cell transcriptomes display unique profiles and each subset contributes to the interferon signature.

PLoS One 2013 24;8(6):e67003. Epub 2013 Jun 24.

Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that is characterized by defective immune tolerance combined with immune cell hyperactivity resulting in the production of pathogenic autoantibodies. Previous gene expression studies employing whole blood or peripheral blood mononuclear cells (PBMC) have demonstrated that a majority of patients with active disease have increased expression of type I interferon (IFN) inducible transcripts known as the IFN signature. The goal of the current study was to assess the gene expression profiles of isolated leukocyte subsets obtained from SLE patients. Subsets including CD19(+) B lymphocytes, CD3(+)CD4(+) T lymphocytes and CD33(+) myeloid cells were simultaneously sorted from PBMC. The SLE transcriptomes were assessed for differentially expressed genes as compared to healthy controls. SLE CD33(+) myeloid cells exhibited the greatest number of differentially expressed genes at 208 transcripts, SLE B cells expressed 174 transcripts and SLE CD3(+)CD4(+) T cells expressed 92 transcripts. Only 4.4% (21) of the 474 total transcripts, many associated with the IFN signature, were shared by all three subsets. Transcriptional profiles translated into increased protein expression for CD38, CD63, CD107a and CD169. Moreover, these studies demonstrated that both SLE lymphoid and myeloid subsets expressed elevated transcripts for cytosolic RNA and DNA sensors and downstream effectors mediating IFN and cytokine production. Prolonged upregulation of nucleic acid sensing pathways could modulate immune effector functions and initiate or contribute to the systemic inflammation observed in SLE.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0067003PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3691135PMC
February 2014

IL-21 promotes the production of anti-DNA IgG but is dispensable for kidney damage in lyn-/- mice.

Eur J Immunol 2013 Feb 18;43(2):382-93. Epub 2012 Dec 18.

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-8884, USA.

The autoimmune disease systemic lupus erythematosus is characterized by loss of tolerance to nuclear Ags and a heightened inflammatory environment, which together result in end organ damage. Lyn-deficient mice, a model of systemic lupus erythematosus, lack an inhibitor of B-cell and myeloid cell activation. This results in B-cell hyper-responsiveness, plasma cell accumulation, autoantibodies, and glomerulonephritis (GN). IL-21 is associated with autoimmunity in mice and humans and promotes B-cell differentiation and class switching. Here, we explore the role of IL-21 in the autoimmune phenotypes of lyn(-/-) mice. We find that IL-21 mRNA is reduced in the spleens of lyn(-/-) IL-6(-/-) and lyn(-/-) Btk(lo) mice, neither of which produce pathogenic autoantibodies or develop significant GN. While IL-21 is dispensable for plasma cell accumulation and IgM autoantibodies in lyn(-/-) mice, it is required for anti-DNA IgG antibodies and some aspects of T-cell activation. Surprisingly, GN still develops in lyn(-/-) IL-21(-/-) mice. This likely results from the presence of IgG autoantibodies against a limited set of non-DNA Ags. These studies identify a specific role for IL-21 in the class switching of anti-DNA B cells and demonstrate that neither IL-21 nor anti-DNA IgG is required for kidney damage in lyn(-/-) mice.
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http://dx.doi.org/10.1002/eji.201142095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768150PMC
February 2013

Modulating proximal cell signaling by targeting Btk ameliorates humoral autoimmunity and end-organ disease in murine lupus.

Arthritis Res Ther 2012 Nov 8;14(6):R243. Epub 2012 Nov 8.

Introduction: Systemic lupus erythematosus is a chronic autoimmune disease characterized by an abundance of autoantibodies against nuclear antigens. Bruton's tyrosine kinase (Btk) is a proximal transducer of the BCR signal that allows for B-cell activation and differentiation. Recently, selective inhibition of Btk by PCI-32765 has shown promise in limiting activity of multiple cells types in various models of cancer and autoimmunity. The aim of this study was to determine the effect of Btk inhibition by PCI-32765 on the development of lupus in lupus-prone B6.Sle1 and B6.Sle1.Sle3 mice.

Methods: B6.Sle1 or B6.Sle1.Sle3 mice received drinking water containing either the Btk inhibitor PCI-32765 or vehicle for 56 days. Following treatment, mice were examined for clinical and pathological characteristics of lupus. The effect of PCI-32765 on specific cell types was also investigated.

Results: In this study, we report that Btk inhibition dampens humoral autoimmunity in B6.Sle1 monocongenic mice. Moreover, in B6.Sle1.Sle3 bicongenic mice that are prone to severe lupus, Btk inhibition also dampens humoral and cellular autoimmunity, as well as lupus nephritis.

Conclusions: These findings suggest that partial crippling of cell signaling in B cells and antigen presenting cells (APCs) may be a viable alternative to total depletion of these cells as a therapeutic modality for lupus.
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http://dx.doi.org/10.1186/ar4086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3674619PMC
November 2012

Separate checkpoints regulate splenic plasma cell accumulation and IgG autoantibody production in Lyn-deficient mice.

Eur J Immunol 2010 Jul;40(7):1897-905

Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA.

Accumulation of plasma cells and autoantibodies against nuclear antigens characterize both human and murine lupus. Understanding how these processes are controlled may reveal novel therapeutic targets for this disease. Mice deficient in Lyn, a negative regulator of B and myeloid cell activity, develop lupus-like autoimmune disease. Here, we show that lyn(-) (/) (-) mice exhibit increased splenic plasmablasts and plasma cells and produce IgM against a wide range of self-antigens. Both events require Btk, a target of Lyn-dependent inhibitory pathways. A Btk-dependent increase in the expression of the plasma cell survival factor IL-6 by lyn(-) (/) (-) splenic myeloid cells was also observed. Surprisingly, IL-6 was not required for plasma cell accumulation or polyclonal IgM autoreactivity in lyn(-/-) mice. IL-6 was, however, necessary for the production of IgG autoantibodies, which we show are focused towards a limited set of nucleic acid-containing and glomerular autoantigens in lyn(-) (/) (-) mice. A similar uncoupling of plasma cell accumulation from IgG autoantibodies was seen in lyn(+/-) mice. Plasma cell accumulation and polyclonal IgM autoreactivity are therefore controlled separately from, and are insufficient for, the production of IgG against lupus-associated autoantigens. Regulators of either of these two checkpoints may be attractive therapeutic targets for lupus.
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http://dx.doi.org/10.1002/eji.200940043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3057185PMC
July 2010

Foxo3-/- mice demonstrate reduced numbers of pre-B and recirculating B cells but normal splenic B cell sub-population distribution.

Int Immunol 2009 Jul 5;21(7):831-42. Epub 2009 Jun 5.

Department of Internal Medicine, Division of Rheumatology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

B cell antigen receptor (BCR) cross-linking promotes proliferation and survival of mature B cells. Phosphoinositide-3-kinase-mediated down-regulation of pro-apoptotic and anti-mitogenic genes such as the Foxo family of transcription factors is an important component of this process. Previously, we demonstrated that BCR signaling decreases expression of transcripts for Foxo1, Foxo3 and Foxo4. We now show that BCR-induced down-regulation of Foxo3 and Foxo4 mRNA expression occurs via distinct mechanisms from those established for Foxo1. While Foxo1, Foxo3 and Foxo4 bind the same DNA sequence, the differential control of their expression upon B cell activation suggests that they may have unique functions in the B lineage. To begin to address this issue, we evaluated B cell development and function in Foxo3-/- mice. No effect of Foxo3 deficiency was observed with respect to the following parameters in the splenic B cell compartment: sub-population distribution, proliferation, in vitro differentiation and expression of the Foxo target genes cyclin G2 and B cell translocation gene 1. However, Foxo3-/- mice demonstrated increased basal levels of IgG2a, IgG3 and IgA. A significant reduction in pre-B cell numbers was also observed in Foxo3-/- bone marrow. Finally, recirculating B cells in the bone marrow and peripheral blood were decreased in Foxo3-/- mice, perhaps due to lower than normal expression of receptor for sphingosine-1 phosphate, which mediates egress from lymphoid organs. Thus, Foxo3 makes a unique contribution to B cell development, B cell localization and control of Ig levels.
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http://dx.doi.org/10.1093/intimm/dxp049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699488PMC
July 2009

Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells.

Mol Immunol 2008 Dec 11;46(2):233-41. Epub 2008 Oct 11.

Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, United States.

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk-/- mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk-/- mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk-/- mice. A transgene expressing a low level of Btk in B cells (Btk(lo)) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk(lo) B cells. anti-DNA IgG was not observed in 56R.Btk(lo) mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.
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http://dx.doi.org/10.1016/j.molimm.2008.08.278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893544PMC
December 2008

Shared signaling networks active in B cells isolated from genetically distinct mouse models of lupus.

J Clin Invest 2007 Aug;117(8):2186-96

Division of Rheumatology, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8884, USA.

Though B cells play key roles in lupus pathogenesis, the molecular circuitry and its dysregulation in these cells as disease evolves remain poorly understood. To address this, a comprehensive scan of multiple signaling axes using multiplexed Western blotting was undertaken in several different murine lupus strains. PI3K/AKT/mTOR (mTOR, mammalian target of rapamycin), MEK1/Erk1/2, p38, NF-kappaB, multiple Bcl-2 family members, and cell-cycle molecules were observed to be hyperexpressed in lupus B cells in an age-dependent and lupus susceptibility gene-dose-dependent manner. Therapeutic targeting of the AKT/mTOR axis using a rapamycin (sirolimus) derivative ameliorated the serological, cellular, and pathological phenotypes associated with lupus. Surprisingly, the targeting of this axis was associated with the crippling of several other signaling axes. These studies reveal that lupus pathogenesis is contingent upon the activation of an elaborate network of signaling cascades that is shared among genetically distinct mouse models and raise hope that targeting pivotal nodes in these networks may offer therapeutic benefit.
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http://dx.doi.org/10.1172/JCI30398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1913486PMC
August 2007

Btk and phospholipase C gamma 2 can function independently during B cell development.

Eur J Immunol 2007 Apr;37(4):1033-42

Department of Internal Medicine, UT Southwestern Medical Center, Dallas, TX 75390-8884, USA.

The pre-BCR and the BCR regulate B cell development via a signalosome nucleated by the adaptor protein B cell linker protein (BLNK). Formation of this complex facilitates activation of phospholipase C (PLC) gamma2 by Bruton's tyrosine kinase (Btk). To determine whether Btk and PLCgamma2 also have separate functions, we generated Btk(-/-)PLCgamma2(-/-) mice. They demonstrated a block in development at the pre-B stage and increased pre-BCR surface expression. This phenotype was more severe than that of Btk(-/-) or PLCgamma2(-/-) mice. Although both Btk and PLCgamma2 were required for proliferation of splenic B cells in response to BCR cross-linking, they contributed differently to anti-IgM-induced phosphorylation of ERK. Btk(-/-) and PLCgamma2(-/-) mice each had a reduced frequency of Iglambda-expressing B cells and impaired migration of pre-B cells towards stromal cell-derived factor 1. However, the increase in pre-B cell malignancy that occurs in BLNK(-/-) mice in the absence of Btk was not observed in the absence of PLCgamma2. Thus, Btk and PLCgamma2 act both in concert and independently throughout B cell development.
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http://dx.doi.org/10.1002/eji.200636451DOI Listing
April 2007

Btk regulates multiple stages in the development and survival of B-1 cells.

Mol Immunol 2007 Apr 4;44(10):2719-28. Epub 2007 Jan 4.

Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

B-1 cells are important players in the first line of defense against pathogens. According to current models for the origin of B-1 cells, they either represent a separate lineage from conventional B-2 cells or differentiate from conventional B-2 cells via an intermediate, B-1(int), in response to positive selection by antigen. Here we show that Btk, a Tec family kinase that mediates B cell antigen receptor (BCR) signaling, is required at multiple stages of B-1 cell development. VH12 anti-phosphatidylcholine (PtC) IgH transgenic mice provide a model for the induced differentiation of B-1 cells. This transgene selects for PtC-reactive cells and induces them to adopt a B-1 phenotype. Both processes have been shown to depend on Btk. To determine whether this is secondary to a requirement for Btk in the development of mature B-2 cells, we crossed VH12 transgenic mice to mice expressing low levels of Btk. B-2 cell development occurs normally in Btk(lo) mice despite reduced responsiveness to BCR crosslinking. Analysis of VH12.Btk(lo) mice reveals that Btk regulates the B-1(int) to B-1 transition and/or the survival of splenic B-1 cells, in part via a mechanism independent of its role in BCR signaling. We also show that Btk mediates the survival of, and expression of IL-10 by, those B-1 cells that do develop and migrate to the peritoneum. Multiple roles for Btk in B-1 cell development and maintenance may explain the particular sensitivity of this population to mutations in components of Btk signaling pathways.
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http://dx.doi.org/10.1016/j.molimm.2006.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2567238PMC
April 2007

B cell receptor signaling down-regulates forkhead box transcription factor class O 1 mRNA expression via phosphatidylinositol 3-kinase and Bruton's tyrosine kinase.

J Immunol 2007 Jan;178(2):740-7

Department of Internal Medicine, Division of Rheumatology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

BCR cross-linking promotes mature B cell proliferation and survival. PI3K-mediated down-regulation of proapoptotic and antimitogenic genes such as forkhead box transcription factor class O 1 (FOXO1) is an important component of this process. Previously, BCR-induced phosphorylation of FOXO1 was shown to lead to a block in nuclear localization and subsequent protein degradation. We demonstrate that the BCR also signals through PI3K to down-regulate FOXO1 mRNA expression. Bruton's tyrosine kinase (Btk), a downstream effector of PI3K, signals through B cell linker protein (BLNK) and phospholipase C (PLC)gamma2 to mediate B cell proliferation and survival in response to BCR cross-linking. BCR-induced down-regulation of FOXO1 mRNA was impaired in murine knockouts of Btk, BLNK, and PLCgamma2. Because B cells in these models are predominantly immature, experiments were also performed using mature B cells expressing low levels of Btk and BLNK. Similar results were obtained. Inhibitors of downstream components of the Btk/BLNK/PLCgamma2 pathway were used to define the mechanism by which Btk signaling inhibits FOXO1 expression. The protein kinase Cbeta inhibitor Gö6850 had minimal effects on BCR-mediated FOXO1 mRNA down-regulation. However, cyclosporin A, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, had similar effects on FOXO1 mRNA expression as the PI3K inhibitor LY294002. Neither Btk deficiency nor cyclosporin A prevented FOXO1 protein phosphorylation, indicating that PI3K down-regulates FOXO1 via two independent pathways. We show that the Btk/BLNK/PLCgamma2 pathway mediates BCR-induced changes in expression of the FOXO1 target gene cyclin G2. These observations support the hypothesis that Btk mediates BCR-induced proliferation and survival in part via inhibition of FOXO expression.
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http://dx.doi.org/10.4049/jimmunol.178.2.740DOI Listing
January 2007

Autoreactive B cell responses to RNA-related antigens due to TLR7 gene duplication.

Science 2006 Jun 18;312(5780):1669-72. Epub 2006 May 18.

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases (NIAID), NIH, Rockville, MD 20852, USA.

Antibodies against nuclear self-antigens are characteristic of systemic autoimmunity, although mechanisms promoting their generation and selection are unclear. Here, we report that B cells containing the Y-linked autoimmune accelerator (Yaa) locus are intrinsically biased toward nucleolar antigens because of increased expression of TLR7, a single-stranded RNA-binding innate immune receptor. The TLR7 gene is duplicated in Yaa mice because of a 4-Megabase expansion of the pseudoautosomal region. These results reveal high divergence in mouse Y chromosomes and represent a good example of gene copy number qualitatively altering a polygenic disease manifestation.
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http://dx.doi.org/10.1126/science.1124978DOI Listing
June 2006

Btk plays a crucial role in the amplification of Fc epsilonRI-mediated mast cell activation by kit.

J Biol Chem 2005 Dec 21;280(48):40261-70. Epub 2005 Sep 21.

Laboratory of Allergic Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-1881, USA.

Stem cell factor (SCF) acts in synergy with antigen to enhance the calcium signal, degranulation, activation of transcription factors, and cytokine production in human mast cells. However, the underlying mechanisms for this synergy remain unclear. Here we show, utilizing bone marrow-derived mast cells (BMMCs) from Btk and Lyn knock-out mice, that activation of Btk via Lyn plays a key role in promoting synergy. As in human mast cells, SCF enhanced degranulation and cytokine production in BMMCs. In Btk-/- BMMCs, in which there was a partial reduction in the capacity to degranulate in response to antigen, SCF was unable to enhance the residual antigen-mediated degranulation. Furthermore, as with antigen, the ability of SCF to promote cytokine production was abrogated in the Btk-/- BMMCs. The impairment of responses in Btk-/- cells correlated with an inability of SCF to augment phospholipase Cgamma1 activation and calcium mobilization, and to phosphorylate NFkappaB and NFAT for cytokine gene transcription in these cells. Similar studies with Lyn-/- and Btk-/-/Lyn-/- BMMCs indicated that Lyn was a regulator of Btk for these responses. These data demonstrate, for the first time, that Btk is a key regulator of a Kit-mediated amplification pathway that augments Fc epsilonRI-mediated mast cell activation.
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http://dx.doi.org/10.1074/jbc.M506063200DOI Listing
December 2005

NTAL phosphorylation is a pivotal link between the signaling cascades leading to human mast cell degranulation following Kit activation and Fc epsilon RI aggregation.

Blood 2004 Jul 9;104(1):207-14. Epub 2004 Mar 9.

Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Aggregation of high-affinity receptors for immunoglobulin E (Fc epsilon RI) on the surface of mast cells results in degranulation, a response that is potentiated by binding of stem cell factor (SCF) to its receptor Kit. We observed that one of the major initial signaling events associated with Fc epsilon RI-mediated activation of human mast cells (HuMCs) is the rapid tyrosine phosphorylation of a protein of 25 to 30 kDa. The phosphorylation of this protein was also observed in response to SCF. This protein was identified as non-T-cell activation linker (NTAL), an adaptor molecule similar to linker for activated T cells (LAT). Unlike the Fc epsilon RI response, SCF induced NTAL phosphorylation in the absence of detectable LAT phosphorylation. When SCF and antigen were added concurrently, there was a marked synergistic effect on NTAL phosphorylation, however, SCF did not enhance the phosphorylation of LAT induced by Fc epsilon RI aggregation. Fc epsilon RI- and SCF-mediated NTAL phosphorylation appear to be differentially regulated by Src kinases and/or Kit kinase, respectively. Diminution of NTAL expression by silencing RNA oligonucleotides in HuMCs resulted in a reduction of both Kit- and Fc epsilon RI-mediated degranulation. NTAL, thus, appears to be an important link between the signaling pathways that are initiated by these receptors, culminating in mast cell degranulation.
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http://dx.doi.org/10.1182/blood-2003-08-2769DOI Listing
July 2004

Reduced dosage of Bruton's tyrosine kinase uncouples B cell hyperresponsiveness from autoimmunity in lyn-/- mice.

J Immunol 2003 Aug;171(4):1850-8

Harold Simmons Arthritis Research Center, Department of Internal Medicine and Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

The development of autoimmunity is correlated with heightened sensitivity of B cells to B cell Ag receptor (BCR) cross-linking. BCR signals are down-regulated by Lyn, which phosphorylates inhibitory receptors. lyn(-/-) mice have reduced BCR signaling thresholds and develop autoantibodies, glomerulonephritis, splenomegaly due to myeloid hyperplasia, and increased B-1 cell numbers. Bruton's tyrosine kinase (Btk), a critical component of BCR signaling pathways, is required for autoantibody production in lyn(-/-) mice. It is unclear whether Btk mediates autoimmunity at the level of BCR signal transduction or B cell development, given that lyn(-/-)Btk(-/-) mice have a severe reduction in conventional B and B-1 cell numbers. To address this issue, we crossed a transgene expressing a low dosage of Btk (Btk(low)) in B cells to lyn(-/-)Btk(-/-) mice. Conventional B cell populations were restored to levels similar to those in lyn(-/-) mice. These cells were as hypersensitive to BCR cross-linking as lyn(-/-) B cells as measured by proliferation, Ca(2+) flux, and activation of extracellular signal-regulated kinase and Akt. However, lyn(-/-)Btk(low) mice did not produce anti-ssDNA, anti-dsDNA, anti-histone, or anti-histone/DNA IgM or IgG. They also lacked B-1 cells and did not exhibit splenomegaly. Thus, B cell hyperresponsiveness is insufficient for autoimmunity in lyn(-/-) mice. These studies implicate B-1 and/or myeloid cells as key contributors to the lyn(-/-) autoimmune phenotype.
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http://dx.doi.org/10.4049/jimmunol.171.4.1850DOI Listing
August 2003

Haploinsufficiency of B cell linker protein enhances B cell signaling defects in mice expressing a limiting dosage of Bruton's tyrosine kinase.

Int Immunol 2003 Mar;15(3):383-92

Simmons Arthritis Research Center, Department of Internal Medicine and Center for Immunology, UT Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.

Current models of lymphocyte activation suggest that formation of a signaling complex, or "signalosome", composed of Syk, Bruton's tyrosine kinase (Btk), phospholipase gamma2 and the adaptor protein B cell linker protein (BLNK) is critical for transmission of signals from the BCR. However, impaired B cell development in mice lacking each individual signalosome component has made it difficult to study the functional consequences of the formation of this complex in mature B cells. Sensitized genetic systems, commonly used in Drosophila, define signaling pathways by combining partial loss of function mutations in the components of interest. This allows genetic interactions to be observed in the absence of pleiotropic or lethal effects of complete deficiency of either gene. We used this approach to demonstrate that Btk and BLNK are limiting components of a common signaling pathway that mediates the mitogenic response of mature B cells to antigen. B cells from transgenic mice expressing a limiting dosage of Btk (Btk(lo)) have normal numbers of mature B cells that have reduced, but measurable, responses to BCR cross-linking. Haploinsufficiency of BLNK did not affect the development of Btk(lo) B cells. However, it exacerbated their defects in BCR-induced Ca(2+) flux, IkappaB degradation, and up-regulation of cyclin D2, bcl-x(L) and A1 leading to dramatic impairment of B cell mitogenic responses. In contrast, no effect of reduced Btk and BLNK dosage was observed on extracellular signal-regulated kinase activation. These results suggest that the signals regulating the maintenance and activation of mature B cells are differentially sensitive to the strength of the signal emanating from the signalosome.
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http://dx.doi.org/10.1093/intimm/dxg034DOI Listing
March 2003

Complementary roles for CD19 and Bruton's tyrosine kinase in B lymphocyte signal transduction.

J Immunol 2002 Jun;168(11):5465-76

Department of Immunology, Duke University Medical Center, Durham, NC 27710, USA.

CD19 and Bruton's tyrosine kinase (Btk) may function along common signaling pathways in regulating intrinsic and B cell Ag receptor (BCR)-induced signals. To identify physical and functional interactions between CD19 and Btk, a CD19-negative variant of the A20 B cell line was isolated, and CD19-deficient (CD19(-/-)) and CD19-overexpressing mice with the X-linked immunodeficient (Xid; Btk) mutation were generated. In A20 cells, Btk physically associated with CD19 following BCR engagement. CD19 and Btk interactions were not required for initial Btk phosphorylation, but CD19 expression maintained Btk in an activated state following BCR engagement. In primary B cells, CD19 signaling also required downstream Btk function since CD19-induced intracellular Ca(2+) ([Ca(2+)](i)) responses were modest in Xid B cells. In addition, CD19 overexpression did not normalize the Xid phenotype and most phenotypic and functional hallmarks of CD19 overexpression were not evident in these mice. However, CD19 and Btk also regulate independent signaling pathways since their combined loss had additive inhibitory effects on BCR-induced [Ca(2+)](i) responses and CD19 deficiency induced a severe immunodeficiency in Xid mice. Thus, CD19 expression amplifies or prolongs Btk-mediated signaling, rather than serving as a required agent for Btk activation. Consistent with this, phosphatidylinositol 3-monophosphate kinase and Akt activation were normal in CD19(-/-) B cells following IgM engagement, although their kinetics of activation was altered. Thus, these biochemical and compound gene dosage studies indicate that Btk activation and [Ca(2+)](i) responses following BCR engagement are regulated through multiple pathways, including a CD19/Src family kinase-dependent pathway that promotes the longevity of Btk signaling.
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http://dx.doi.org/10.4049/jimmunol.168.11.5465DOI Listing
June 2002

Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes.

Proc Natl Acad Sci U S A 2002 Jan 26;99(1):359-64. Epub 2001 Dec 26.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.

Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle.
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http://dx.doi.org/10.1073/pnas.012605099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC117565PMC
January 2002