Alternatively activated neutrophils drive recruitment and polarization of monocytes to stimulate axon regeneration.

Abstract

Purpose : Axonopathy is an early and prominent pathological feature of optic neuritis, multiple sclerosis (MS), and traumatic central nervous system (CNS) injury. Although neuroinflammation is believed to be the primary driver of damage in traumatic CNS injury, MS and other neurological conditions, animal model studies have shown that certain immune cell subsets can have neuroprotective effects. Normally RGC axons do not regrow following crush injury. However, robust axonal regeneration ensues in association with zymosan induced intravitreal inflammation.

Methods : Intraocular inflammatory cells were harvested from mice at serial time points following ONC/i.o. zymomsan for analysis. Primary retinal ganglion cells (RGC) were co-cultured with immune cell subsets purified from the intraocular infiltrates, or with immune cell subset conditioned media (CM). After 24 hours, RGC neurite outgrowth was measured. In separate experiments, purified immune cells were adoptively transferred into the eyes of mice with ONC injury. Optic nerves were collected 14 days later and axonal density was measured at serial distances from the crush site via fluorescently conjugated CTB tracer uptake.

Results : We identified a novel subset of Ly6G^low CD14+ neutrophils in the infiltrates that correlate with the extent of RGC axon regeneration. Ly6G^low neutrophils adoptively transferred into the eye after ONC, recruited arginase(arg)+ CD206+ monocytes to the vitreous fluid and stimulated axon regeneration in vivo. We are able to selectively deplete these Arg+ CD206+ monocytes in an Arg-DTR mouse which then prevents regeneration. Arg+ monocytes, or their CM, directly stimulated RGC neurite outgrowth in vitro. In vitro, these alternatively activated neutrophils are directly responsible for stimulating monocytes towards alternative polarization, showing that the polarization of monocytes is a direct result of the neutrophil polarization state.

Conclusions : A novel subset of Ly6G^lowCD14+ neutrophils is capable of driving the regeneration of transected CNS axons. These cells recruit and polarize monocytes and work together to promote axon regeneration. These myeloid cells secrete pro-regenerative neurotrophic factors, and can be selectively depleted with an Arg-DTR mouse. Our findings may ultimately lead to the development of novel immunomodulatory drugs, or the repurposing of immunomodulatory drugs in clinical use.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.