Genetically engineered pro-regenerative macrophages as a treatment for spinal cord injury

Abstract

Macrophages play a critical role as essential effectors of the innate immune system. In response to different stimuli, they adopt a pro-inflammatory M1 or an anti-inflammatory M2 phenotype. After a spinal cord injury (SCI), the neuroprotective M2 macrophages are outbalanced by the neurotoxic M1 macrophages, which limit functional recovery. To increase the amount of M2 macrophages after SCI, this study aims to create super macrophages by overexpressing M2 genes or migration-mediating receptors in naive macrophages to induce polarisation towards an M2 phenotype. Monocyte/macrophage-like RAW264.7 cells were transfected with M2 markers FIZZ1 or YM1. Bone marrow-derived macrophages (BMDMs) were transduced with CCR2, a chemokine receptor. RT-qPCR was used to determine the gene expression of macrophage markers. Protein expression was evaluated through western blot. Although overexpression of FIZZ1 or YM1 was achieved in RAW264.7 cells, no significantly higher expression of M2 markers (CD206, Arg-1, TGF-β, CD163) was found in transfected cells compared to untreated controls. M1 markers IL-6, CD38, and CD86 were significantly higher expressed in M1 stimulated cells compared to FIZZ1 or YM1 transfected cells, while expression of M1 markers TNF-α and iNOS did not differ between these groups. In addition, CCR2 overexpression was not achieved in BMDMs. To conclude, overexpression of FIZZ1 or YM1 in RAW264.7 cells cannot induce a phenotypical switch into M2 but, unexpectedly, might trigger pro-inflammatory characteristics.