Abstract
Purpose :
Evidence has emerged in favor of microglia, the resident immune cells in the retina, involvement in neurogenesis in general and retinal ganglion cell (RGC) differentiation in particular through complement-mediated phagocytosis. However, there is a knowledge gap about the role of proinflammatory cytokines, key components of microglia secretome, in RGC development. Here, we demonstrate that pro-inflammatory cytokines, TNFα and IL-1β, elaborated by human microglia (hμG), facilitate neuritogenesis in developing human retinal ganglion cells (RGCs) and axon regeneration by recruiting the AKT/mTOR pathway.
Methods :
hRGCs and hμG were directly differentiated from hESBrn3b-tdT cells and hiPS cells, respectively, by recapitulating developmental mechanism. hRGCs were generated in the presence of recombinant TNFα (50ng/ml) and IL-1β (50ng/ml) and their influence on hRGC number, neuritogenesis, and functional maturation was examined by immunofluorescence, Sholl, and multi-electrode array (MEA) analyses, respectively. Experiments were repeated with unchallenged hμG-derived condition medium (CM) with or without antibody-mediated neutralization of cytokines. Third, the influence of hμG-derived CM on axon regeneration was examined in microfluidic model of RGC axon regeneration (Subramani et al., 2023 Front. Cell. Dev. Biol. 11:1214104).
Results :
We observed, as compared to control, that recombinant or hμG-derived CM pro-inflammatory cytokines (1) had no influence on the efficiency of generation of hRGCs, (2) significantly increased the complexity and length of the neurites in general and axons in particular, (3) significantly increased the mean firing rate and spontaneous spikes generation after six days of MEA recording (4) significantly increased the indices of regeneration (number and length of regenerated axons) after axotomy and (5) activated AKT/mTOR pathway for the cellular effects.
Conclusions :
To our knowledge, this is the first demonstration of the influence of microglia-derived pro-inflammatory cytokines on hRGC development, which can be recapitulated to promote axon regeneration. The cytokines recruit the AKT/mTOR pathway, known to regulate RGC development and regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.