Human IL4 mRNA

Proper regulation of inflammation following traumatic brain injury (TBI) may help avoid disability in the millions of patients who suffer from TBI each year. In TBI, inflammatory cell mediators, including macrophages and microglia, have a range of phenotypes that are relevant to immunomodulatory therapeutic approaches. It is thought that early phenotypic modulation of these cells will produce a cascade of healing effects. Indeed, an anti-inflammatory “M2-like” macrophage phenotype after TBI is associated with improved neurogenesis, axonal regeneration, and white matter integrity (WMI). Clinical trials are currently attempting to achieve M2-like macrophage differentiation from mesenchymal stem/stromal cells (MSCs). To enrich for M2-like macrophages in a clinically relevant manner, researchers used synthetic IL-4 mRNA to enhance MSCs to transiently express IL-4. The expression and function of these IL-4-expressing MSCs (IL-4 MSCs) were characterized and then delivered into a modified mouse closed head injury TBI model. Here, the researchers observed that IL-4 MSCs indeed induced a robust M2-like macrophage phenotype after TBI and promoted the expression of anti-inflammatory genes. However, the acute enrichment of M2-like macrophages did not translate into improved functional or histological outcomes, nor into improved WMI on MRI imaging. To further understand whether dysfunctional pathways were the root cause of the poor treatment effect, the researchers reported transcriptome analysis of injured and treated brains. Through analysis, it was found that despite the acute enrichment of M2-like macrophages in the brain, inflammation still existed.

Here, the researchers determined whether IL-4 secreted by transfected MSCs was functional. IL-4 is a potent stimulator of the M2 cell surface marker CD206 on macrophages. They set up four groups: plain medium, medium conditioned with naïve MSCs (MSC CM), MSC CM supplemented with 100 ng of recombinant IL-4 (rIL-4 CM), and medium conditioned with MSCs transfected with IL-4 mRNA (eIL-4 CM) (Figure 1a). The medium was incubated with J774A.1 macrophage cultures for 24 hours before harvesting, staining, and analyzing in a flow cytometer. Macrophage gating strategy included fluorescence-minus-one (FMO) groups to distinguish positive staining. As hypothesized, CD206 expression was significantly increased in macrophages treated with rIL-4 CM or eIL-4 CM (Figure 1b, c). As expected, CD163, another M2-like marker (usually stimulated by IL-10), was not upregulated by IL-4 (Figure 1d). Control experiments showed that the morphology and division rate of macrophages were not affected by MSC-specific medium. These studies indicate that IL-4 produced by MSCs transfected with synthetic IL-4 mRNA can induce an M2-like phenotype in macrophages.

Figure 1. Functionality of IL-4 expressed by IL-4 mRNA-transfected MSCs. (Enam S F, et al., 2020)