RESUMEN
Multiphoton intravital microscopy (MP-IVM) is an imaging technique used for the observation of living organisms at a microscopic resolution. The tissue of interest is exposed through a window allowing imaging of cells in real time. Using MP-IVM, the temporospatial kinetics of leukocyte transendothelial migration can be visualized and quantitated using reporter mice and cell-specific fluorophore-conjugated monoclonal antibodies to track the leukocytes within and outside of vascular beds. Here we describe a method used to study neutrophil transendothelial migration and blood-brain barrier permeability in a mouse model of herpes simplex virus I (HSV) encephalitis.
Asunto(s)
Barrera Hematoencefálica , Modelos Animales de Enfermedad , Encefalitis por Herpes Simple , Microscopía Intravital , Microscopía de Fluorescencia por Excitación Multifotónica , Neutrófilos , Migración Transendotelial y Transepitelial , Animales , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/virología , Barrera Hematoencefálica/patología , Ratones , Microscopía Intravital/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neutrófilos/metabolismo , Encefalitis por Herpes Simple/patología , Encefalitis por Herpes Simple/virología , Encefalitis por Herpes Simple/metabolismo , Herpesvirus Humano 1/fisiología , PermeabilidadRESUMEN
Viral myocarditis, an inflammatory disease of the heart, causes significant morbidity and mortality. Type I interferon (IFN)-mediated antiviral responses protect against myocarditis, but the mechanisms are poorly understood. We previously identified A Disintegrin And Metalloproteinase domain 9 (ADAM9) as an important factor in viral pathogenesis. ADAM9 is implicated in a range of human diseases, including inflammatory diseases; however, its role in viral infection is unknown. Here, we demonstrate that mice lacking ADAM9 are more susceptible to encephalomyocarditis virus (EMCV)-induced death and fail to mount a characteristic type I IFN response. This defect in type I IFN induction is specific to positive-sense, single-stranded RNA (+ ssRNA) viruses and involves melanoma differentiation-associated protein 5 (MDA5)-a key receptor for +ssRNA viruses. Mechanistically, ADAM9 binds to MDA5 and promotes its oligomerization and thereby downstream mitochondrial antiviral-signaling protein (MAVS) activation in response to EMCV RNA stimulation. Our findings identify a role for ADAM9 in the innate antiviral response, specifically MDA5-mediated IFN production, which protects against virus-induced cardiac damage, and provide a potential therapeutic target for treatment of viral myocarditis.