Multiple sclerosis (MS) is a multifactorial disease of unknown etiology that affects the central nervous system (CNS), causing neuroinflammation and demyelination. MS is characterized by a strong inflammatory component mediated by the immune system. The immune system is composed of various cell classes, including antigen- presenting cells (APCs), which are responsible for recognizing exogenous and endogenous molecules, processing them, and presenting them via the major histocompatibility complex class II (MHC class II) to activate the immune response. The professional APCs include dendritic cells, macrophages, microglia (in the CNS), and B lymphocytes. Once the antigen is processed, these cells can activate naïve CD4+ T lymphocytes. In MS, it is believed that microglia and macrophages can process myelin, initiating an inflammatory process against it, leading to the formation of autoreactive T lymphocytes that sustain the inflammatory process, further damaging the myelin sheath. This process also recruits additional monocytes that, by phagocytizing myelin fragments, fuel the processing cycle that subsequently activates more T lymphocytes. Monocytes, macrophages, and microglia express numerous receptors involved in phagocytosis, including the MS4A family cluster. This cluster comprises several genes, such as MS4A4A and MS4A6A, which are highly expressed by APCs. Their murine homologs, Ms4a4a and Ms4a6d, are described as highly expressed in models of neuroinflammation and demyelination and are crucial in macrophage polarization. Both appear to be involved in anti-inflammatory processes and the phagocytosis of lipid substrates, but their role in the phagocytosis, degradation, and processing of myelin or its components is unknown.
In the first objective, the phagocytic capacity of murine macrophages differentiated from bone marrow progenitors will be tested when stimulated with myelin. This experiment will be conducted on macrophages derived from Ms4a4a-KO (Knock-Out) and Ms4a6d-KO mice. Another experimental set will evaluate the ability of these macrophages to promote the proliferation of autoreactive T lymphocytes when stimulated with myelin components.
In the second objective, the phagocytic capacity of human macrophages derived from anonymous donors will be tested when stimulated with myelin. These macrophages will undergo gene silencing for MS4A4A and/or MS4A6A sing siRNA (silencing-interfering RNA). The effectiveness will be assessed via flow cytometry, western blot, and real-time PCR. Subsequently, these macrophages will be stimulated with myelin to study the action of these genes in the processing, phagocytosis, and degradation of myelin.
In the first objective, the phagocytic capacity of murine macrophages differentiated from bone marrow progenitors will be tested when stimulated with myelin. This experiment will be conducted on macrophages derived from Ms4a4a-KO (Knock-Out) and Ms4a6d-KO mice. Another experimental set will evaluate the ability of these macrophages to promote the proliferation of autoreactive T lymphocytes when stimulated with myelin components.
In the second objective, the phagocytic capacity of human macrophages derived from anonymous donors will be tested when stimulated with myelin. These macrophages will undergo gene silencing for MS4A4A and/or MS4A6A sing siRNA (silencing-interfering RNA). The effectiveness will be assessed via flow cytometry, western blot, and real-time PCR. Subsequently, these macrophages will be stimulated with myelin to study the action of these genes in the processing, phagocytosis, and degradation of myelin.