Defining the anti-inflammatory activity of a potent myxomaviral chemokine modulating protein, M-T7, through site directed mutagenesis

Viral chemokine modulating proteins provide new and extensive sources for therapeutics. Purified M-T7, a poxvirus-derived secreted immunomodulatory protein, reduces mononuclear cell invasion and atheroma in rodent models of angioplasty injury as well as aortic and renal transplant, improving renal allograft survival. M-T7 is a rabbit species-specific interferon gamma receptor (IFN gamma R) homolog, but also inhibits chemokine/glycosaminoglycan (GAG) interactions for C, CC and CXC chemokines, with cross-species specific inhibitory activity. M-T7 anti-atheroma activity is blunted in GAG deficient mouse aortic transplants, but not in CC chemokine receptor deficient transplants, supporting M-T7 interference in chemokine/GAG interactions as the basis of the atheroma-inhibitory activity. We have assessed point mutants of M-T7 both in vivo in a mouse angioplasty model and in vitro in tissue culture and binding assays, in order to better define the primary mechanism of anti-atheroma activity. Of these M-T7 mutants, the (RE)-E-171 and (EI)-I-209 M-T7 mutants lost inhibitory activity for plaque growth in hyperlipidemic ApoE(-/-) mice after angioplasty injury and (RE)-E-171, moreover, greatly exacerbated plaque growth and inflammation. (FD)-D-137 retained some inhibitory activity for plaque growth. In contrast, for cell migration assays, M-T7-His(6x), (FD)-D-137, (RE)-E-171, and (EI)-I-209 all inhibited CC chemokine (RANTES) mediated cell migration. For the ligand binding assays, (RE)-E-171 and (EI)-I-209 had significantly reduced binding to RANTES and IFN gamma, whereas (FD)-D-137 retained wild-type binding activity. Heparin treatment further reduced RANTES binding of all three M-T7 mutants. In summary, point mutations of M-T7, (RE)-E-171 and (EI)-I-209, exhibited reduced anti-inflammatory properties in vivo after mouse angioplasty with a loss of in vitro binding to RANTES and IFN gamma, indicating these point mutations partially disrupt M-T7 ligand-binding activities. Unexpectedly, the M-T7 mutants all retained inhibitory activity for human monocyte THP-1 cell migration ex vivo, suggesting additional inhibitory properties against human monocyte THP-1 cells that are independent of chemokine inhibition. (C) 2013 Elsevier Ltd. All rights reserved.