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Phd Thesis the Role of Wiskott-Aldrich Syndrome Protein

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Phd Thesis the Role of Wiskott-Aldrich Syndrome Protein Università degli Studi di Trieste Graduate School in MOLECULAR BIOMEDICINE PhD Thesis The role of Wiskott-Aldrich Syndrome protein- mediated actin dynamics in controlling type-I IFN production in plasmacytoid dendritic cells Candidate: Francesca Prete Thesis supervisor: Dr. Federica Benvenuti XXIV cycle – Academic Year 2011 Alla famiglia, ad Eu, Fabio, Giulia, Michela, Roberta, Gabriele. A chi, anche se lontano, resta. In everything I seek to grasp The fundamental: The daily choice, the daily task, The sentimental. To plumb the essence of the past, The first foundations, The crux, the roots, the inmost hearts, The explanations. And, puzzling out the weave of fate, Events observer, To live, feel, love and meditate And to discover. Boris Pasternak CONTENTS INTRODUCTION .................................................................................................................... 1 1.1 Innate immunity vs adaptive immunity ............................................................................ 2 1.1.1 Toll-like receptors.................................................................................................... 2 1.2 Dendritic cells general features .................................................................................. 4 1.2.1 The “Langerhans cells paradigm” ............................................................................ 5 1.2.2 DCs classification ..................................................................................................... 6 1.3 Plasmacytoid dendritic cells (pDCs) ............................................................................. 11 1.3.1 PDCs in the immune response: general features .................................................... 11 1.3.2 Phenotypic characterization of pDCs ..................................................................... 14 1.3.3 PDCs development ................................................................................................. 14 1.3.4 PDCs localization and migration ............................................................................ 15 1.3.5 PDCs are professional type-I interferon-producing cells ....................................... 17 1.3.6 Regulation of IFN synthesis in pDCs ..................................................................... 19 1.3.7 TLR-mediated activation of pDCs.......................................................................... 19 1.4 PDCs-derived type-I IFNs in linking innate and adaptive immunity ........................ 23 1.4.1 pDCs regulate the function of cDCs by type-I IFN ................................................ 23 1.4.2 Regulation of B cell function by pDCs .................................................................. 24 1.4.3 Regulation of T and NK cell function by pDCs ..................................................... 25 1.4.4 pDCs/type-I IFN in autoimmunity ......................................................................... 25 1.5 Wiskott-Aldrich Syndrome (WAS) .............................................................................. 30 1.5.1 The cytoskeleton ..................................................................................................... 31 1.5.2 Actin filament assembly and disassembly .............................................................. 31 1.5.3 Immune cellular functions dependent on actin cytoskeletal dynamics ................... 33 1.5.4 Nucleation-promoting factors: the WASp family ................................................... 35 1.5.5 Wiskott-Aldrich syndrome, general traits ............................................................... 37 1.5.6 Cellular defects in WAS ......................................................................................... 38 1.5.7 Autoimmune manifestations in WAS ..................................................................... 41 AIMS ................................................................................................................................... 44 2 MATERIALS AND METHODS ......................................................................................... 45 3 RESULTS............................................................................................................................. 56 3.1 Analysis of the pDCs compartment in WKO mice ......................................................... 57 3.1.1 PDCs frequency, absolute numbers and turnover rate ............................................ 58 3.1.2 Basal activation state of pDCs ................................................................................ 60 3.1.3 WKO pDCs constitutively secrete type-I IFN ........................................................ 60 3.2 Analysis of the response to TLR9 stimulation by WASp-null pDCs .............................. 61 3.2.1 Progressive exhaustion of the TLR9/IFN-α pathway in pDCs from WKO mice ... 62 3.2.2 Analysis of the constitutive pDCs activation in WKO mice .................................. 66 3.2.3 WKO pDCs are intrinsically more responsive to TLR9 ......................................... 67 3.2.4 WASp plays a cell-autonomous role in pDCs ........................................................ 69 3.2.4 WASp-mediated actin dynamics selectively control TLR9/IFN-α axis in pDCs ... 71 3.3 Analysis of the endocytic pathway in WASp-null pDCs ................................................ 72 3.3.1 WASp-mediated actin dynamics regulate accumulation of CpG-A in early endosomes ....................................................................................................................... 73 3.3.2 WASp regulates the correct organization of the endocytic pathway ...................... 75 3.4 PDCs/type-I IFN alterations in WAS patients ............................................................... 78 3.4.1 PDCs in spleen and peripheral blood of WAS patients .......................................... 79 3.4.2 Type-I IFN signature in WAS patients ................................................................... 81 3.5 Evidences of a link between dysregulated PDCs/type-I IFN axis and altered innate and adaptive immunity in WKO mice .................................................................................. 83 3.5.1 Conventional DC (cDCs) phenotype in WKO mice ............................................... 84 3.5.2 cDCs functionality in WKO mice........................................................................... 86 3.5.3 B cells phenotype in WKO mice ............................................................................ 87 3.5.4 CD4+ T cell responses in WKO mice .................................................................... 87 3.5.5 CD8+ T cell responses in WKO mice ..................................................................... 90 4 DISCUSSION ........................................................................................................... 93 5 REFERENCES ....................................................................................................... 103 ABSTRACT Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by recurrent infections, and a marked predisposition to develop autoimmune phenomena. The disease is caused by mutations in WASp, a key regulator of actin polymerization expressed only in hematopoietic cells. A general impairment of hematopoietic cell functions contributes to the pathogenesis of the disease. Neutrophils, B cells, T cells and DCs deficient for WASp were all shown to have impaired homing ability, a cellular function that strictly depends on spatio-temporal regulation of actin polymerization. WASp null T cells present severe impairment in coupling TCR stimulation to proliferation and fail to organize signaling molecules within the immunological synapse (Badour et al., 2004; Dupre et al., 2002; Sims et al., 2007). B cells intrinsic defects include altered B cell receptor clustering, defective homeostasis of mature B cells and a specific reduction in the expression of the complement receptor (Park et al., 2005; Simon et al., 1992; Westerberg et al., 2001). WASp null DCs fail to assemble podosomes and display late migration from the periphery to lymph nodes (de Noronha et al., 2005). Moreover, WASp expression in DCs is critical to organize the dynamic cytoskeletal changes that facilitate DC-T cell interaction during antigen presentation (Pulecio et al., 2008; Bouma et al., 2011). Together, these cellular alterations provided clues to understand the reduced response to pathogens and the immunodeficiency of WAS patients. However, the mechanisms by which perturbation of actin dynamics promote autoimmune phenomena are less clear. Autoimmune complications occur in 40-72% of children with severe WAS phenotype. The most common autoimmune features that develop in WAS patients include hemolytic anemia, vasculitis, renal disease, and arthritis (Dupuis-Girod et al., 2003; Sullivan et al., 1994; Humblet- baron et al., 2007). Impairment of T and B cell tolerance have been reported in WAS patients and in Was-/- mice, but the exact cellular mechanisms that link loss of WASp function to autoimmunity have not been fully elucidated yet (Becker-Herman et al., 2011; Recher et al., 2012; Marangoni et al., 2007; Maillard et al., 2007; Humblet-Baron et al., 2007). It is increasingly recognized that excessive activation of pDCs and elevated type-I interferon (IFN) levels are pathogenic in several human autoimmune diseases such as SLE, psoriasis, Sjogren’s syndrome. Since WAS autoimmune manifestations partially overlap
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