OBJECTIVE: The main function of the human eye is to detect light, motion, and color from our surroundings. This information is then processed and translated in the brain as vision. However, what is less known about the eye is its ability to regulate immune function. It is this ocular immune privilege that maintains the eye's ability collect visual information. The degeneration of immune privilege causes inflammation, which can cause damage to the eye, an increased susceptibility of eye disorders such as autoimmune uveitis (inflammation of the uvea), and may lead to vision impairment. Research in ocular immune privilege can open up potential clinical applications for maladies such as uveitis, septic shock, hypersensitivity, multiple sclerosis and allograft survival. Previous research has shown the importance of the retinal pigment epithelial (RPE) cells for maintenance of ocular immune privilege, and has identified the use of neuropeptides to suppress inflammatory responses in macrophages. This project aims to study the role and mechanism of the RPE cells in phagolysosome activation in macrophages that mediate inflammation.
METHODS: Posterior eyecups were prepared from eyes of healthy, EAU immunized, or post-EAU mice. Eyecups, which consisted of the sclera, choroid, and a single layer of RPE, were cultured in serum-free media (SFM). During a 24-hour incubation period, peritoneal macrophages were collected intraperitoneal (IP) and cultured. Conditioned media (CM) was applied to the collected macrophages along with pHrodo-red opsonized bioparticles and were incubated for 24-hours in 37°C. After incubation, cells were examined by fluorescent microscopy for phagolysosome activation. Also, this was also done with RPE CM depleted of α-MSH, NPY, and α-MSH + NPY. A viability assay was performed on macrophages treated with depleted RPE CM to investigate the possibility that removing the neuropeptides will induce cell death. To examine the cytokines involved in RPE CM--from healthy, EAU and post-EAU mice--modulation of phagolysosome activation, a mouse cytokine array was performed that assessed for twenty different mouse cytokines.
RESULTS: Results from fluorescent microscopy showed that healthy RPE CM caused significant suppression of phagolysosome activity in macrophages. The RPE CM depleted of α-MSH, NPY, and α-MSH + NPY showed a significant suppression of phagolysosome activity in macrophages. However, these results may have been misrepresented as the macrophages treated with depleted RPE CM were non-viable. RPE CM from EAU mice showed the inability to down-regulate phagolysosome activity while RPE CM from post-EAU mice recovered its ability to down-regulate phagolysosome activity. The mouse cytokine array of RPE CM from healthy, EAU and post-EAU mice identified keratinocyte-derived cytokine (KC), high concentrations of interleukin-6 (IL-6), trace amounts of vascular endothelial growth factor (VEGF), and no other pro or anti-inflammatory cytokines.
CONCLUSIONS: Healthy RPE cells suppress phagolysosome activation in activated macrophages. In contrast, RPE cells from mice with active EAU lose its ability to regulate phagolysosome activation, but regain this ability when the disease resolves following α-MSH treatment. RPE CM from post-EAU mice treated with MC5r agonist did not recover suppression of phagolysosome activation which suggests that α-MSH causes suppression of phagolysosome activation through other melanocortin receptors or that this suppression requires other mechanisms in conjunction to MC5r stimulation. Cytokine IL-6 may be involved in RPE suppression of phagolysosome activation; however, further study will have to be done. The results demonstrate that part of the mechanisms of ocular immune privilege is the tight control by RPE of the phagocytic process in macrophages. It is possible that this contributes to ocular immune privilege minimizing the potential of processing and presenting self-proteins, and to allow for clearance of harmful materials while suppressing the activation of inflammation.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/16271 |
| Date | 08 April 2016 |
| Creators | Choe, Yoona |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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