Nanoparticle-Delivered IRF5 siRNA Facilitates M1 to M2 Transition, Reduces Demyelination and Neurofilament Loss, and Promotes Functional Recovery After Spinal Cord Injury in Mice.

Abstract

Macrophage activation and persistent inflammation contribute to the pathogenesis of spinal cord injury (SCI), and different phenotypes of macrophages play diverse roles in the pathological process of SCI. After SCI, there is an acute phase of alternatively activated (M2) macrophage infiltration, followed by a long-lasting phase of classically activated (M1) macrophage accumulation in the wound. The long-lasting predominance of M1 macrophages may derail healing and compromise organ functions. Based on the previous findings that the transcription factor interferon regulatory factor 5 (IRF5) up-regulates genes associated with M1 macrophages, we attempted to examine the effect of silencing IRF5 on SCI progression. IRF5 expression was assessed with Western blotting or immunohistochemistry. Macrophage phenotypes were measured with flow cytometry or immunohistochemistry. M1- or M2-related cytokines were measured with a Luminex assay kit. IRF5 siRNA was delivered into the macrophages infiltrated into the wound of SCI mice through lipidoid nanoparticle. Locomotor functions were measured with Basso Mouse Scale (BMS) scoring. Myelination was assessed with luxol fast blue staining. Myelin binding protein, neurofilaments, synaptic markers, and cytokines in the wound area were measured with Western blotting. The Mann-Whitney U test was used for statistical analyses. After SCI, significant elevation of IRF5 was evident on day 1, peaked on day 7, and gradually decreased thereafter. Similar dynamic change in the expression of CD86, a typical M1 marker, was observed. In contrast, there was a transient increase in the expression of CD206, a typical M2 marker, which peaked 6h after SCI, and returned to baseline within 1day. Macrophages isolated from the epicenter at day 3 after SCI were predominantly M1 phenotype, and a siRNA-mediated knockdown of IRF5 resulted in a reduced expression of M1 macrophage markers and increased expression of M2 macrophage markers. Nanoparticle-mediated delivery of IRF5 siRNA to SCI mouse model resulted in a dramatic decrease in the number of M1 macrophages and a significant increase in the number of M2 macrophages in the wound. This was associated with a robust inflammation resolution, attenuation of demyelination and neurofilament loss, and significant improvement of locomotor function (p < 0.05). IRF5 may serve as a therapeutic target to promote post-SCI recovery.