Abstract
Purpose :
Diurnal rhythms govern both circulation but infiltration of leukocytes into the tissues. Recent evidence suggests an interplay between metabolism and the circadian clock and diabetes is long been suspected to affect circadian rhythmicity. We hypothesized that Type 1 diabetes affects the diurnal rhythmicity of immune cell infiltration into the retina. Thus, we performed a multicolor immunofluorescence phenotyping of the time of the day at which leukocytes infiltrate the retina in control and diabetic mice.
Methods :
We used the Ins2Akita mouse model as a model of diabetes induced hyperglycemia. Four months old control and diabetic mice were euthanized at certain time points of a ZT cycle (ZT1, ZT5, ZT9, ZT13, ZT17, ZT21, where ZT= Zeitgeber Time, ZT=0, lights on). Mice were perfused with PBS and tissues were isolated and either were prepared for single cell suspensions or snap frozen. Multicolor flow cytometry (antibodies for CD45, CD11b, Ly6G, Ly6C, CD115, F4/80, CD11c, CD19, CD3, NKp46 and CD43) was employed to dissect the composition of immune cells in the retina at each time point. Control gating was drawn based on FMOs and comparison to splenic composition. Cosine function was used to identify if there is a circadian rhythmicity.
Results :
Overall, a complex immune cell landscape of the retinal parenchyma (perfused retinas) was identified with various subtypes of macrophages and dendritic cells that cannot be accurately identified with the markers used in this study. Differences in the subset composition of infiltrating cells into the retina were identified between control and diabetes. Specifically at ZT1 there were statistically significant more (p<0.05) dendritic cells and monocytes in the retinas of diabetic mice but this was normalized at later times. Rhythmic behavior was identified not only in the infiltrating CD45hi cells but also in microglia with peak time around the early morning hours. The only exception was the CD11blo F480hi myeloid cells, whose numbers peaked during the late hours of the day (ZT13).
Conclusions :
These data indicate that the infiltration of immune cells into the retina is not static but changes throughout the day. It also demonstrates that at early hours of the day there is higher immune cell infiltration in the diabetic mice compared to control. Altogether, these data inform us for the optimal timing of therapeutic targeting immune cell activation in the retina.
This is a 2020 ARVO Annual Meeting abstract.