Role of the gene regulators TFEB and TFE3 in Macrophages and B cells

Abstract

In multicellular organisms, differential regulation of gene expression is necessary to generate cells with specialized functions and to respond to environmental challenges such as metabolic stress and microbial infection. The experiments in this thesis focus on the study of the gene regulators of the MIT family, TFEB TFE3 and Mitf, in the mammalian immune system. These molecules are transcription factors that share sequence homology, virtually identical DNA binding properties and often functionally overlap. Recently, TFEB has been implicated by others to be the “master regulator” of genes involved in autophagy and lysosomal biogenesis [1]. Autophagy is important for the development and function of the immune system: monocytes must up-regulate autophagy in order to differentiate into macrophages, which then need autophagy for efficient phagocytosis, antigen presentation via MHC II, host antiviral defense, antifungal responses and for defense against gram-negative bacteria[2]. Moreover, autophagy is also required for the survival of B1 cells and plasma cells [3, 4]. Given the importance of autophagy to macrophage function, in Aim1 I studied the role of TFEB and TFE3 in autophagy in macrophage development and function. I studied autophagy activated by metabolic stress and immunological stimuli in vitro using transformed mouse RAW 264.7 macrophages and primary macrophages (derived from the bone marrow (BM) and peritoneal cavity) from mice conditionally deficient in TFEB alone or both TFEB and TFE3 (dko). Using these mice, in a complementary in vivo approach, I evaluated macrophage differentiation using established mouse models for phagocytosis, sterile peritonitis and for endotoxin-induced sepsis. I found that TFEB and TFE3 are both dispensable for monocyte differentiation into macrophages and for stereotypical phagocytotic responses to particulates. However, TFEB- and TFE3-deficient macrophages had impaired proinflammatory cytokine responses such as IL-6 and TNF-α to bacterial endotoxin, indicating a critical role for these molecules in host defense. In Aim 2, I studied antibody responses and phagocytic properties of B cells in which the MiT proteins have been inactivated. Preliminary studies by a previous student in the laboratory showed thatMarginal Zone (MZ) B cells and B1a B cells are reduced in mice in which TFEB and TFE3 are inhibited in the B lineage. MZ B cells are critical for protective antibody responses against encapsulated bacteria, whereas B1 cells provide important antibody protection to barrier sites such as the peritoneal cavity. In both cases, these B cell populations can respond to non-protein containing antigens without relying on help from CD4 T cells, a type of antibody response known as “thymus-independent” (TI) because CD4 T cells require the thymus to develop. B1 cells are known to depend on autophagy for their development and homeostasis, and B1 cells are known to be phagocytic. In contrast, follicular (FO) B cells, the predominant B cell subset, primarily respond to protein antigens in a CD4 T cell dependent manner (“thymus-dependent,” antigens, TD). We observed B cell hyper responsiveness and splenomegaly, which we hypothesize, were due to a defect in FO B cells caused by Mitf inactivation. However, the underlying mechanisms to explain the selective loss of MZ and B1 B cells in TFE3/TFEB inhibited mice as well as the FO B cell defect, and the consequences of these losses on antibody responses are unclear. Therefore, in Aim 2, I analyzed antibody responses to TI and TD antigens in mice in which MiT family is inhibited in B cells or in both B and T cells. I also analyzed the phagocytic properties of TFE3/TFEB-deficient B cells in vivo and in vitro using fluorescent latex beads and E. coli particles. I found that despite the decrease in MZ B cells caused by MiT inhibition in B cells, unexpectedly, these mice had elevated IgG responses to TI antigens compared to WT mice. However, the IgG isotype produced in response was more reflective of FO rather than MZ B cells. In contrast, all IgG responses were impaired in mice that had MiT family inhibited in both B and T cells, attributed due to a defect in T cell help caused by impaired CD40L expression. Phagocytosis by peritoneal B cells was also impaired, which may explain the reduction in their homeostatic numbers over time. My studies provide important new information about regulation of proinflammatory responses in macrophages and B cells, which will inform the development of novel therapeutic strategies to combat microbial infections and autoimmune diseases.