Medical Biology - Research Publications
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ItemCARD11 is dispensable for homeostatic responses and suppressive activity of peripherally induced FOXP3+ regulatory T cells(WILEY, 2019-09)FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.
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ItemNo Preview AvailableA type III effector antagonizes death receptor signalling during bacterial gut infection(NATURE PUBLISHING GROUP, 2013-09-12)Successful infection by enteric bacterial pathogens depends on the ability of the bacteria to colonize the gut, replicate in host tissues and disseminate to other hosts. Pathogens such as Salmonella, Shigella and enteropathogenic and enterohaemorrhagic (EPEC and EHEC, respectively) Escherichia coli use a type III secretion system (T3SS) to deliver virulence effector proteins into host cells during infection that promote colonization and interfere with antimicrobial host responses. Here we report that the T3SS effector NleB1 from EPEC binds to host cell death-domain-containing proteins and thereby inhibits death receptor signalling. Protein interaction studies identified FADD, TRADD and RIPK1 as binding partners of NleB1. NleB1 expressed ectopically or injected by the bacterial T3SS prevented Fas ligand or TNF-induced formation of the canonical death-inducing signalling complex (DISC) and proteolytic activation of caspase-8, an essential step in death-receptor-induced apoptosis. This inhibition depended on the N-acetylglucosamine transferase activity of NleB1, which specifically modified Arg 117 in the death domain of FADD. The importance of the death receptor apoptotic pathway to host defence was demonstrated using mice deficient in the FAS signalling pathway, which showed delayed clearance of the EPEC-like mouse pathogen Citrobacter rodentium and reversion to virulence of an nleB mutant. The activity of NleB suggests that EPEC and other attaching and effacing pathogens antagonize death-receptor-induced apoptosis of infected cells, thereby blocking a major antimicrobial host response.
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ItemDestruction of tumor vasculature and abated tumor growth upon VEGF blockade is driven by proapoptotic protein Bim in endothelial cells(ROCKEFELLER UNIV PRESS, 2011-07-04)For malignant growth, solid cancers must stimulate the formation of new blood vessels by producing vascular endothelial growth factor (VEGF-A), which is required for the survival of tumor-associated vessels. Novel anticancer agents that block VEGF-A signaling trigger endothelial cell (EC) apoptosis and vascular regression preferentially within tumors, but how the ECs die is not understood. In this study, we demonstrate that VEGF-A deprivation, provoked either by drug-induced tumor shrinkage or direct VEGF-A blockade, up-regulates the proapoptotic BH3 (Bcl-2 homology 3)-only Bcl-2 family member Bim in ECs. Importantly, the tumor growth inhibitory activity of a VEGF-A antagonist required Bim-induced apoptosis of ECs. These findings thus reveal the mechanism by which VEGF-A blockade induces EC apoptosis and impairs tumor growth. They also indicate that drugs mimicking BH3-only proteins may be exploited to kill tumor cells not only directly but also indirectly by ablating the tumor vasculature.
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ItemType I Interferon Drives Dendritic Cell Apoptosis via Multiple BH3-Only Proteins following Activation by PolyIC In Vivo(PUBLIC LIBRARY SCIENCE, 2011-06-02)BACKGROUND: DC are activated by pathogen-associated molecular patterns (PAMPs), and this is pivotal for the induction of adaptive immune responses. Thereafter, the clearance of activated DC is crucial to prevent immune pathology. While PAMPs are of major interest for vaccine science due to their adjuvant potential, it is unclear whether and how PAMPs may affect DC viability. We aimed to elucidate the possible apoptotic mechanisms that control activated DC lifespan in response to PAMPs, particularly in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We report that polyinosinic:polycytidylic acid (PolyIC, synthetic analogue of dsRNA) induces dramatic apoptosis of mouse splenic conventional DC (cDC) in vivo, predominantly affecting the CD8α subset, as shown by flow cytometry-based analysis of splenic DC subsets. Importantly, while Bim deficiency conferred only minor protection, cDC depletion was prevented in mice lacking Bim plus one of three other BH3-only proteins, either Puma, Noxa or Bid. Furthermore, we show that Type I Interferon (IFN) is necessary and sufficient for DC death both in vitro and in vivo, and that TLR3 and MAVS co-operate in IFNß production in vivo to induce DC death in response to PolyIC. CONCLUSIONS/SIGNIFICANCE: These results demonstrate for the first time in vivo that apoptosis restricts DC lifespan following activation by PolyIC, particularly affecting the CD8α cDC subset. Such DC apoptosis is mediated by the overlapping action of pro-apoptotic BH3-only proteins, including but not solely involving Bim, and is driven by Type I IFN. While Type I IFNs are important anti-viral factors, CD8α cDC are major cross-presenting cells and critical inducers of CTL. We discuss such paradoxical finding on DC death with PolyIC/Type I IFN. These results could contribute to understand immunosuppression associated with chronic infection, and to the optimization of DC-based therapies and the clinical use of PAMPs and Type I IFNs.
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ItemEvidence against upstream regulation of the unfolded protein response (UPR) by pro-apoptotic BIM and PUMA(NATURE PUBLISHING GROUP, 2014-07)
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ItemCharacterisation of a novel A1-specific monoclonal antibody(NATURE PUBLISHING GROUP, 2014-12)
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ItemNFκB1 is essential to prevent the development of multiorgan autoimmunity by limiting IL-6 production in follicular B cells(ROCKEFELLER UNIV PRESS, 2016-04-04)We examined the role of NFκB1 in the homeostasis and function of peripheral follicular (Fo) B cells. Aging mice lacking NFκB1 (Nfκb1(-/-)) develop lymphoproliferative and multiorgan autoimmune disease attributed in large part to the deregulated activity of Nfκb1(-/-)Fo B cells that produce excessive levels of the proinflammatory cytokine interleukin 6 (IL-6). Despite enhanced germinal center (GC) B cell differentiation, the formation of GC structures was severely disrupted in the Nfκb1(-/-)mice. Bone marrow chimeric mice revealed that the Fo B cell-intrinsic loss of NFκB1 led to the spontaneous generation of GC B cells. This was primarily the result of an increase in IL-6 levels, which promotes the differentiation of Fo helper CD4(+)T cells and acts in an autocrine manner to reduce antigen receptor and toll-like receptor activation thresholds in a population of proliferating IgM(+)Nfκb1(-/-)Fo B cells. We demonstrate that p50-NFκB1 represses Il-6 transcription in Fo B cells, with the loss of NFκB1 also resulting in the uncontrolled RELA-driven transcription of Il-6.Collectively, our findings identify a previously unrecognized role for NFκB1 in preventing multiorgan autoimmunity through its negative regulation of Il-6 gene expression in Fo B cells.
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ItemThe linear ubiquitin chain assembly complex: a new function in thymic T cell differentiation and regulatory T cell homeostasis(WILEY-BLACKWELL, 2016-08)The linear ubiquitin chain assembly complex (LUBAC) is essential for innate immunity in mice and humans, yet its role in adaptive immunity is unclear. Here we show that the LUBAC components HOIP, HOIL-1 and SHARPIN have essential roles in late thymocyte differentiation, FOXP3+ regulatory T (Treg)-cell development and Treg cell homeostasis. LUBAC activity is not required to prevent TNF-induced apoptosis or necroptosis but is necessary for the transcriptional programme of the penultimate stage of thymocyte differentiation. Treg cell-specific ablation of HOIP causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis. These data reveal stage-specific requirements for LUBAC in coordinating the signals required for T-cell differentiation.
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ItemImpact of the combined loss of BOK, BAX and BAK on the hematopoietic system is slightly more severe than compound loss of BAX and BAK(NATURE PUBLISHING GROUP, 2015-10)It is well established that BAX and BAK play crucial, overlapping roles in the intrinsic pathway of apoptosis. Gene targeted mice lacking both BAX and BAK have previously been generated, but the majority of these animals died perinatally. BOK is a poorly studied relative of BAX and BAK that shares extensive amino acid sequence homology to both proteins, but its function remains largely unclear to date. To determine whether BOK plays an overlapping role with BAX and BAK, we utilized a hematopoietic reconstitution model where lethally irradiated wild type mice were transplanted with Bok(-/-)Bax(-/-)Bak(-/-) triple knockout (TKO) fetal liver cells, and compared alongside mice reconstituted with a Bax(-/-)Bak(-/-) double knockout (DKO) hematopoietic compartment. We report here that mice with a TKO and DKO hematopoietic system died at a similar rate and much earlier than control animals, mostly due to severe autoimmune pathology. Both TKO and DKO reconstituted mice also had altered frequencies of various leukocyte subsets in the thymus, bone marrow and spleen, displayed leukocyte infiltrates and autoimmune pathology in multiple tissues, as well as elevated levels of anti-nuclear autoantibodies. Interestingly, the additional deletion of BOK (on top of BAX and BAK loss) led to a further increase in peripheral blood lymphocytes, as well as enhanced lymphoid infiltration in some organs. These findings suggest that BOK may have some functions that are redundant with BAX and BAK in the hematopoietic system.
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ItemSoluble FAS ligand is not required for pancreatic islet inflammation or beta-cell destruction in non-obese diabetic mice(NATURE PUBLISHING GROUP, 2019-09-23)CD8+ T cells play a central role in beta-cell destruction in type 1 diabetes. CD8+ T cells use two main effector pathways to kill target cells, perforin plus granzymes and FAS ligand (FASL). We and others have established that in non-obese diabetic (NOD) mice, perforin is the dominant effector molecule by which autoreactive CD8+ T cells kill beta cells. However, blocking FASL pharmacologically was shown to protect NOD mice from diabetes, indicating that FASL may have some role. FASL can engage with its receptor FAS on target cells either as membrane bound or soluble FASL. It has been shown that membrane-bound FASL is required to stimulate FAS-induced apoptosis in target cells, whereas excessive soluble FASL can induce NF-κB-dependent gene expression and inflammation. Because islet inflammation is a feature of autoimmune diabetes, we tested whether soluble FASL could be important in disease pathogenesis independent of its cell death function. We generated NOD mice deficient in soluble FASL, while maintaining expression of membrane-bound FASL due to a mutation in the FASL sequence required for cleavage by metalloproteinase. NOD mice lacking soluble FASL had normal numbers of lymphocytes in their spleen and thymus. Soluble FASL deficient NOD mice had similar islet inflammation as wild-type NOD mice and were not protected from diabetes. Our data indicate that soluble FASL is not required in development of autoimmune diabetes.