Final Report Summary - TAM IMMUNOLOGY (Role of TAM receptors in the differentiation and function of tissue macrophage subpopulations: Implications for the development of inflammatory disease)
Chronic inflammation and dysregulation of the immune response underlie multiple human pathologies, including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Macrophages have been implicated in the pathogenesis of these autoimmune diseases, with different subsets of these cells playing diverse roles. Macrophages are professional phagocytic cells involved in the elimination of pathogens and apoptotic cells. They can display specialized forms of activation and plasticity in response to host homeostatic signals or exogenous challenges, and they play essential roles in the innate immune response and the maintenance of tissue homeostasis. The TAM family of receptor tyrosine kinases - Tyro3, Axl and Mer - together with their activating ligands Gas6 and Protein S - are expressed in antigen-presenting cells (APCs) in multiple tissues, where they play pivotal roles in immunomodulation and regulation of tissue homeostasis. Previous studies demonstrate that TAMs act as pleiotropic inhibitors of the innate immune response. Macrophages and dendritic cells are constitutively active in TAM-deficient mice, which present with systemic inflammation and autoimmunity. In addition, recent studies suggest a dysregulation of TAM pathways in diverse cohorts of patients with autoimmune diseases. However, the molecular mechanisms underlying these phenomena remain poorly understood. Using different approaches we have explored the role of TAMs in tissue-specific macrophage differentiation and function, and ultimately, their contribution to human immune disorders.
The splenic marginal zone (MZ) is a unique microenvironment where resident immune cells are exposed to the blood. Marginal zone macrophages (MZMs) play a crucial role in clearing blood-borne antigens and apoptotic material from the circulation. We have recently discovered that TAM signaling is essential for the development of these macrophages in the Marginal Zone of the spleen. Preliminary experiments conducted in our laboratory have shown that TAM-deficient mice specifically lack the expression of splenic Marginal Zone macrophages. These dramatic and unexpected results support the hypothesis that TAM signaling participates in MZ macrophage differentiation and function. In this project we tested this hypothesis and characterized the molecular mechanism/s involved by pursuing the following four main objectives: (1) Perform a detailed characterization of splenic architecture in TAM (receptor and ligand)-deficient mice in embryonic, neonatal and adult mice. (2) Investigate the origin of splenic MZ macrophage populations and TAM signaling contribution to their development. (3) Generate a mouse model of TAM receptor deficiency in hematopoietic stem cells progenitors using the Cre/loxP System. (4) Analyze, in vivo, the function of MZ macrophages in animal models of inflammation and autoimmunity and the consequences of TAM deficiency.
We have analyzed splenic structure and organization in both WT and TAM-deficient genotypes and characterized their spleen development from birth to adulthood. We studied the expression of TAM receptors and their ligands in different splenic cell populations, as well as the expression of MZ markers in newborn and adult mice. Apart from splenic architecture, we also evaluated other lymphoid (lymph nodes, Peyer’s parches) and non-lymphoid tissues (liver, lung, and intestine) to explore the contribution of TAM signaling to tissue-specific macrophage differentiation. We have investigated the influence of TAM signaling in interactions between the different subpopulations of cells in the marginal zone by evaluating the effect of TAM deficiency in the development and/or organization of MZ-B cells and metalophillic macrophages. In this regard, we have also evaluated the effect of TAM deficiency in the expression levels of a battery of cytokines, chemokines and other molecules known to play an essential role during splenic ontogeny. Additionally, we have analyzed the profile of hematopoietic stem cells progenitors in WT and TAM deficient mice and generated bone marrow chimeric mice in order to evaluate the splenic configuration of those animals. In this same context, we have examined the profile and frequency of different subsets of monocytes in bone marrow and blood of WT and TAM deficient mice as these cells have been described to be potential precursors of MZ macrophages. During this period we have also generated two colonies of myeloid cell-specific knock-out mice for two of the TAM receptors (Axl and Mer) using two different Cre drivers controlled by the Cx3cr1 promoter - one of them inducible by tamoxifen treatment and the second one with a constitutively active Cre-recombinase enzyme. These new animal models have been used to analyze the spleen development and the in vivo consequences of TAM deficiency in lymphoid tissues. Finally, we have conducted studies to analyze the splenic retention/capture of blood-borne antigens in TAM-deficient mice and performed experiments to evaluate the sensitivity of WT and TAM deficient mice to infections induced by live pathogens.
We draw six conclusions from our results. First, TAM deficient mice show defective organization of the splenic marginal zone with a significant decrease in the MARCO+SIGNR1+ Marginal Zone Macrophage population. Second, the loss of MZMs in TAM deficient mice results in an inadequate clearance of circulating particles and pathogens. Third, there is a clear gene dosage effect and mice deficient in all three receptors display the most dramatic phenotype. Fourth, the genetic ablation of TAMs leads to early postnatal alterations in the development of the splenic architecture. Fifth, specific TAM expression in myeloid cells is important for the generation and correct localization of MZ macrophages. Finally, bone marrow chimeras reveal that hematopoietic-specific expression of TAMs restores the MZ microenvironment in TAM-deficient mice.
Finally, our findings about the role of TAM receptors in the development of marginal zone macrophages in the spleen are relevant to human therapy. TAM inhibitors are in development for cancer therapies and treatment of enveloped virus infections. Conversely, TAM activators have been proposed as treatments for autoimmune indications. The results derived from our project have important implications for the therapeutic application of inhibiting and activating TAM modulators, particularly with respect to their target specificity and the avoidance of possible treatment side effects in the immune system. Long-term inhibition of Axl and Mer in the course of a cancer therapy should be evaluated for the development of defective splenic clearance of blood-borne pathogens, impaired vision, reduced male fertility, and autoimmune disease as side effects. Long-term inhibition of only Axl may result in fewer adverse reactions. These and related considerations suggest that TAM modulation is an especially promising approach to the treatment of human disease.
The splenic marginal zone (MZ) is a unique microenvironment where resident immune cells are exposed to the blood. Marginal zone macrophages (MZMs) play a crucial role in clearing blood-borne antigens and apoptotic material from the circulation. We have recently discovered that TAM signaling is essential for the development of these macrophages in the Marginal Zone of the spleen. Preliminary experiments conducted in our laboratory have shown that TAM-deficient mice specifically lack the expression of splenic Marginal Zone macrophages. These dramatic and unexpected results support the hypothesis that TAM signaling participates in MZ macrophage differentiation and function. In this project we tested this hypothesis and characterized the molecular mechanism/s involved by pursuing the following four main objectives: (1) Perform a detailed characterization of splenic architecture in TAM (receptor and ligand)-deficient mice in embryonic, neonatal and adult mice. (2) Investigate the origin of splenic MZ macrophage populations and TAM signaling contribution to their development. (3) Generate a mouse model of TAM receptor deficiency in hematopoietic stem cells progenitors using the Cre/loxP System. (4) Analyze, in vivo, the function of MZ macrophages in animal models of inflammation and autoimmunity and the consequences of TAM deficiency.
We have analyzed splenic structure and organization in both WT and TAM-deficient genotypes and characterized their spleen development from birth to adulthood. We studied the expression of TAM receptors and their ligands in different splenic cell populations, as well as the expression of MZ markers in newborn and adult mice. Apart from splenic architecture, we also evaluated other lymphoid (lymph nodes, Peyer’s parches) and non-lymphoid tissues (liver, lung, and intestine) to explore the contribution of TAM signaling to tissue-specific macrophage differentiation. We have investigated the influence of TAM signaling in interactions between the different subpopulations of cells in the marginal zone by evaluating the effect of TAM deficiency in the development and/or organization of MZ-B cells and metalophillic macrophages. In this regard, we have also evaluated the effect of TAM deficiency in the expression levels of a battery of cytokines, chemokines and other molecules known to play an essential role during splenic ontogeny. Additionally, we have analyzed the profile of hematopoietic stem cells progenitors in WT and TAM deficient mice and generated bone marrow chimeric mice in order to evaluate the splenic configuration of those animals. In this same context, we have examined the profile and frequency of different subsets of monocytes in bone marrow and blood of WT and TAM deficient mice as these cells have been described to be potential precursors of MZ macrophages. During this period we have also generated two colonies of myeloid cell-specific knock-out mice for two of the TAM receptors (Axl and Mer) using two different Cre drivers controlled by the Cx3cr1 promoter - one of them inducible by tamoxifen treatment and the second one with a constitutively active Cre-recombinase enzyme. These new animal models have been used to analyze the spleen development and the in vivo consequences of TAM deficiency in lymphoid tissues. Finally, we have conducted studies to analyze the splenic retention/capture of blood-borne antigens in TAM-deficient mice and performed experiments to evaluate the sensitivity of WT and TAM deficient mice to infections induced by live pathogens.
We draw six conclusions from our results. First, TAM deficient mice show defective organization of the splenic marginal zone with a significant decrease in the MARCO+SIGNR1+ Marginal Zone Macrophage population. Second, the loss of MZMs in TAM deficient mice results in an inadequate clearance of circulating particles and pathogens. Third, there is a clear gene dosage effect and mice deficient in all three receptors display the most dramatic phenotype. Fourth, the genetic ablation of TAMs leads to early postnatal alterations in the development of the splenic architecture. Fifth, specific TAM expression in myeloid cells is important for the generation and correct localization of MZ macrophages. Finally, bone marrow chimeras reveal that hematopoietic-specific expression of TAMs restores the MZ microenvironment in TAM-deficient mice.
Finally, our findings about the role of TAM receptors in the development of marginal zone macrophages in the spleen are relevant to human therapy. TAM inhibitors are in development for cancer therapies and treatment of enveloped virus infections. Conversely, TAM activators have been proposed as treatments for autoimmune indications. The results derived from our project have important implications for the therapeutic application of inhibiting and activating TAM modulators, particularly with respect to their target specificity and the avoidance of possible treatment side effects in the immune system. Long-term inhibition of Axl and Mer in the course of a cancer therapy should be evaluated for the development of defective splenic clearance of blood-borne pathogens, impaired vision, reduced male fertility, and autoimmune disease as side effects. Long-term inhibition of only Axl may result in fewer adverse reactions. These and related considerations suggest that TAM modulation is an especially promising approach to the treatment of human disease.
