Abstract
Purpose :
The retinal microenvironment changes during aging & disease states, through altered immune activation impede in retinal repair. An immunomodulator identified in our lab, MANF, promotes retinal repair by maintaining ocular immune homeostasis. However, the mechanism by which MANF modulates the retinal microenvironment is still unclear. MANF is evolutionally conserved in flies, mice & humans, and their levels decline with age. Mimicking this using MANF-het mice, having half the MANF levels lead to multisystem inflammatory phenotypes and other age-associated pathologies. Thus we sought to understand the molecular mechanism and pathways by which MANF modulates the retinal inflammation in aging to identify other therapeutic targets to improve success in retinal regeneration therapies.
Methods :
The Manf-het/WT mice (n=3) were injected with 5nM NMDA for 24h to check the inflammatory status compared to control (PBS). The enucleated eyes were used to check the inflammatory marker expression through tissue immunohistochemistry (IHC), and for gene expression analysis through scRNAseq, and PCR, and flow cytometry. The flow cytometry enriched immune population was used for scRNAseq. The morphological & functional changes in undamaged aging eyes (14-17 Months old) were periodically checked by OCT & ERG, and later processed in a similar way as above. Two-tailed Student's t-test was used for statistical analysis.
Results :
The MANF-het mice had higher infiltrating monocytes/macrophages after NMDA damage as revealed by IHC and flow cytometry. The scRNAseq data from CD11b enriched population also displayed higher proinflammatory genes (such as CD68, CD86) and suppression of anti-inflammatory genes. Furthermore, the lipid metabolism genes such as Elovl2, Lpl, Fth1 were dysregulated. In the aging retinas, we observed drusen like structures on OCT in MANF-het mice. The flow cytometry data shows higher Isolectin B4 and CD86 expression in MANF-het compared to control aging mice.
Conclusions :
Our results revealed that reduction in MANF level modulates several pro/anti-inflammatory genes and causes changes in lipid metabolism pathways. Further analysis will help in elucidating specific pathways and key regulators involved in retinal regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.