Evaluation of M2-like macrophage enrichment after diffuse traumatic brain injury through transient interleukin-4 expression from engineered mesenchymal stromal cells

Abstract

Background Appropriately modulating inflammation after traumatic brain injury (TBI) may prevent disabilities for the millions of those inflicted annually. In TBI, cellular mediators of inflammation, including macrophages and microglia, possess a range of phenotypes relevant for an immunomodulatory therapeutic approach. It is thought that early phenotypic modulation of these cells will have a cascading healing effect. In fact, an anti-inflammatory, “M2-like” macrophage phenotype after TBI has been associated with neurogenesis, axonal regeneration, and improved white matter integrity (WMI). There already exist clinical trials seeking an M2-like bias through mesenchymal stem/stromal cells (MSCs). However, MSCs do not endogenously synthesize key signals that induce robust M2-like phenotypes such as interleukin-4 (IL-4). Methods To enrich M2-like macrophages in a clinically relevant manner, we augmented MSCs with synthetic IL-4 mRNA to transiently express IL-4. These IL-4 expressing MSCs (IL-4 MSCs) were characterized for expression and functionality and then delivered in a modified mouse TBI model of closed head injury. Groups were assessed for functional deficits and MR imaging. Brain tissue was analyzed through flow cytometry, multi-plex ELISA, qPCR, histology, and RNA sequencing. Results We observed that IL-4 MSCs indeed induce a robust M2-like macrophage phenotype and promote anti-inflammatory gene expression after TBI. However, here we demonstrate that acute enrichment of M2-like macrophages did not translate to improved functional or histological outcomes, or improvements in WMI on MR imaging. To further understand whether dysfunctional pathways underlie the lack of therapeutic effect, we report transcriptomic analysis of injured and treated brains. Through this, we discovered that inflammation persists despite acute enrichment of M2-like macrophages in the brain. Conclusion The results demonstrate that MSCs can be engineered to induce a stronger M2-like macrophage response in vivo. However, they also suggest that acute enrichment of only M2-like macrophages after diffuse TBI cannot orchestrate neurogenesis, axonal regeneration, or improve WMI. Here, we also discuss our modified TBI model and methods to assess severity, behavioral studies, and propose that IL-4 expressing MSCs may also have relevance in other cavitary diseases or in improving biomaterial integration into tissues.

Challenges and Improvements of Novel Therapies for Ischemic Stroke

“…Consequently, researchers focus on structure transformation to reduce inflammatory damage. Overexpression of IL-4 in MSCs before intrahippocampal injections obviously induced the M2 polarization of microglia and an anti-inflammation status as well as the activation of macroglia, leading to better motor function recovery and less MSCs death ( Enam et al, 2020 ). In the same way, delivering recombinant IL-10 alongside MSCs increased anti-inflammation effects as indicated by fewer astrocytes and glial scar formation ( Peruzzaro et al, 2019 ).…”

Mesenchymal Stem Cells for Neurological Disorders

“…They also promoted microglia to express M2 phenotypic markers and reduce the production of TNF‐ α in the injured brain. [ 58,59 ] It is known that hypoxia complicates TBI contributing to secondary brain injury. Genetically modified MSCs to overexpress hypoxia‐inducible factor 1 α (HIF‐1 α ) revealed more remarkable improvement in TBI mice's neurological recovery compared with native MSC transplantation.…”

Recent Advances in Stem Cell Therapies to Address Neuroinflammation, Stem Cell Survival, and the Need for Rehabilitative Therapies to Treat Traumatic Brain Injuries

“…However, as Enam concluded with their in vivo experiments, while this method did induce cytokine and gene level changes, it was not sufficient to change any functional or histological outcomes following a TBI. Additional evidence from transcriptomic studies revealed persistent inflammatory pathway conditions and a lack of neuroregeneration [ 33 ].…”

“…Additional recent studies involving the direct transplant of genetically modified MSCs to the area of a TBI, were performed by Peruzzaro et al, (2019) and Maiti et al, (2019) [ 32 , 33 ]. Each of these studies used bone marrow-derived MSCs that were virally transfected to overexpress human IL-10, an anti-inflammatory cytokine.…”