Abstract
Purpose :
There is a dire need for novel treatments that restore vision and retinal ganglion cell (RGC) function to individuals suffering from axonopathy, a pathological feature in many diseases and disorders such as glaucoma and traumatic optic nerve injury. Recently, our lab identified a new subset of immature neutrophils that rescue damaged neurons from death, and stimulate the regrowth of transected axons. In this study, we sought to generate a homogenous population of neuro-protective/regenerative neutrophils from bone marrow (BM) precursors in vitro, that could be used as a cell-based therapy to reverse neurological damage in an optic nerve crush (ONC) injury model.
Methods :
Ly6G+ neutrophils isolated from naive mouse BM were polarized for 24 hours in media containing a combination of IL-4 and granulocyte-colony stimulating factor (G-CSF). Polarized neutrophils were then co-cultured with explanted RGCs ex vivo or adoptively transferred into the eyes of mice following ONC injury.
Results :
We found that Ly6G+ BM cells, stimulated with both IL-4 and G-CSF, upregulate markers of alternative activation and acquire pro-regenerative properties. IL-4/G-CSF polarized, but not unpolarized, BM neutrophils directly stimulate neurite outgrowth of explanted RGCs and induce regeneration of severed optic nerve axons in vivo upon intraocular injection into mice with ONC injury. This neuroregenerative effect is mediated via the release of soluble factors, as polarized neutrophils stimulate neurite outgrowth when separated from explanted neurons by a transwell. IL-4/G-CSF polarized neutrophils have a distinct transcriptome that encompasses genes encoding known neuroprotective and axonal growth factors, including insulin-like growth factor-1 (IGF-1) and heparin binding EGF-like growth factor (HB-EGF). Neutralization of IGF-1 and HB-EGF in combination, suppressed neutrophil driven optic nerve regeneration. Additionally, IL-4/G-CSF polarized CD34+ human BM cells express markers of alternative activation and stimulate neurite outgrowth in human cortical neuron co-cultures.
Conclusions :
Our results demonstrate that polarized BM neutrophils have the capacity to induce RGC regeneration and outgrowth. Ultimately, our findings demonstrate a proof of principle that customized immune cell-based therapies have the potential to ameliorate RGC loss and visual disability.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.