Acute or chronic inflammation is a cooperative immune response against infection, tissue injury or toxin, while it can lead tissue damage and human disease. Thus, regulation of immune cell functions likes macrophage or T cell, etc. is an important strategy to control overwhelming immune response. In...
Acute or chronic inflammation is a cooperative immune response against infection, tissue injury or toxin, while it can lead tissue damage and human disease. Thus, regulation of immune cell functions likes macrophage or T cell, etc. is an important strategy to control overwhelming immune response. In this study, NLR family member X1 (NLRX1) has been focused for investigation as a novel immune modulator of both macrophage and T cell. As a major methodology, recombinant protein synthesis utilizing cell-penetrating peptides like pIL-1αNLS, C10 or dNP2 was adopted for this study. In particular, Leucine rich repeat (LRR) domain of NLRX1 but not nucleotide binding domain (NBD) demonstrated regulatory function on IL-1β and IL-6 production by macrophages as well as Th1 differentiation leading successful inhibition of mouse model of sepsis and experimental autoimmune encephalomyelitis. Each of chapter with a specific topic was separately investigated as follows.
First, novel cell penetrating peptides from nuclear localization sequence (NLS) of human cytokine IL-1α (pIL-1αNLS) was identified by in silico filtering strategy. The pIL-1αNLS could efficiently deliver EGFP and dTomato proteins into the cytosol and the nucleus of Jurkat and HeLa cells, which mainly mediates endocytosis mechanism without significant toxicity. In addition, it efficiently delivered proteins in vivo including spleen, liver and intestines following systemic administration. Thus, unexpectedly NLS of cytokine has shown to have ability as a cell-penetrating peptide to deliver a cargo in vitro and in vivo.
Secondly, to extend former study and find out macrophage-preferable cell-penetrating peptide, C10 was identified in IL-10 cytokine by in silico screening, which contains leucine. It efficiently delivered EGFP and dTomato proteins into murine splenic macrophage in vitro and in vivo rather than other cell types. Next, C10 conjugated LRR or NBD of NLRX1 protein (C10-LRR) was generated and only LRR domain but not NBD improved survival rate of the mice with LPS-mediated lethal endotoxemia. C10-LRR significantly inhibited IL-6 and IL-1β production with regulation of NF-κB and inflammasome signaling in macrophages. Thus, macrophage-targeting C10 enables LRR to inhibit macrophage activation and sepsis in mice.
Thirdly, to prove its regulatory function in T cells and autoimmune disease model, a superior T cell penetrating and BBB-permeable peptide, dNP2 was conjugated with LRR domain of NLRX1 (dNP2-LRR), which showed significant delivery in the spinal cord and brain tissue. When dNP2-LRR was treated to EAE induced mice, it significantly ameliorated disease severity with reduced number of infiltrated T cells, macrophages and monocytes in central nervous system (CNS). In particular, dNP2-LRR specifically inhibited Th1 differentiation but not Th17 or iTreg in vitro implying its regulatory function in effector T cell differentiation. Thus, LRR domain of NLRX1 could be able to control T cell responses as well as macrophage functions.
Acute or chronic inflammation is a cooperative immune response against infection, tissue injury or toxin, while it can lead tissue damage and human disease. Thus, regulation of immune cell functions likes macrophage or T cell, etc. is an important strategy to control overwhelming immune response. In this study, NLR family member X1 (NLRX1) has been focused for investigation as a novel immune modulator of both macrophage and T cell. As a major methodology, recombinant protein synthesis utilizing cell-penetrating peptides like pIL-1αNLS, C10 or dNP2 was adopted for this study. In particular, Leucine rich repeat (LRR) domain of NLRX1 but not nucleotide binding domain (NBD) demonstrated regulatory function on IL-1β and IL-6 production by macrophages as well as Th1 differentiation leading successful inhibition of mouse model of sepsis and experimental autoimmune encephalomyelitis. Each of chapter with a specific topic was separately investigated as follows.
First, novel cell penetrating peptides from nuclear localization sequence (NLS) of human cytokine IL-1α (pIL-1αNLS) was identified by in silico filtering strategy. The pIL-1αNLS could efficiently deliver EGFP and dTomato proteins into the cytosol and the nucleus of Jurkat and HeLa cells, which mainly mediates endocytosis mechanism without significant toxicity. In addition, it efficiently delivered proteins in vivo including spleen, liver and intestines following systemic administration. Thus, unexpectedly NLS of cytokine has shown to have ability as a cell-penetrating peptide to deliver a cargo in vitro and in vivo.
Secondly, to extend former study and find out macrophage-preferable cell-penetrating peptide, C10 was identified in IL-10 cytokine by in silico screening, which contains leucine. It efficiently delivered EGFP and dTomato proteins into murine splenic macrophage in vitro and in vivo rather than other cell types. Next, C10 conjugated LRR or NBD of NLRX1 protein (C10-LRR) was generated and only LRR domain but not NBD improved survival rate of the mice with LPS-mediated lethal endotoxemia. C10-LRR significantly inhibited IL-6 and IL-1β production with regulation of NF-κB and inflammasome signaling in macrophages. Thus, macrophage-targeting C10 enables LRR to inhibit macrophage activation and sepsis in mice.
Thirdly, to prove its regulatory function in T cells and autoimmune disease model, a superior T cell penetrating and BBB-permeable peptide, dNP2 was conjugated with LRR domain of NLRX1 (dNP2-LRR), which showed significant delivery in the spinal cord and brain tissue. When dNP2-LRR was treated to EAE induced mice, it significantly ameliorated disease severity with reduced number of infiltrated T cells, macrophages and monocytes in central nervous system (CNS). In particular, dNP2-LRR specifically inhibited Th1 differentiation but not Th17 or iTreg in vitro implying its regulatory function in effector T cell differentiation. Thus, LRR domain of NLRX1 could be able to control T cell responses as well as macrophage functions.
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