Regulation of the diurnal circulation of monocytes in type 1 diabetic mice: implications for diabetic retinopathy (DR).

Abstract

Purpose: In diabetes, a disrupted circadian clock in combination with bone marrow neuropathy leads to altered release of reparative progenitor cells contributing to development of DR. Because inflammatory cells play a role in early DR, we examined whether the diurnal circulation of monocytes is similarly altered and, if so, what effects this might have on early stages of diabetes before the onsets of retinal pathology.

Methods: STZ diabetic mice with 4 month duration of T1D and age matched controls were euthanized every 4 hours during a 24 hour light/dark cycle. Cells from bone marrow (BM), spleen, retina, and blood were analyzed for monocyte subsets using flow cytometry. Fixed bones and spleens were analyzed for loss of neuronal innervation by immunohistochemistry. Plasma was analyzed for multiple endocrine biomarkers using Assaygate multiplex assays. Isolated RNA from tissues was analyzed for gene expression by RT-PCR.

Results: In controls mice, at ZT1 to ZT5 (Zeitgeber time) monocytes reached highest levels in the blood and lowest in the other tissues. Diabetic mice had more monocytes in the blood, but the circadian pattern was similar in BM, blood and retina but not in the spleen. In the spleen, monocytes were in a total anti-phase compared to controls. These effects of T1D were only observed for the inflammatory Ly6C^hi but not for the patrolling Ly6C^lo monocytes. Denervation could not explain these observations in monocyte levels as both BM and spleen had similar tyrosine hydroxylase and neurofilament 200 immunostaining. However, diabetes led to a disruption of the hypothalamic pituitary adrenal axis (HPA) demonstrated by phase advancement of the circadian pattern of adrenocorticotropic hormone release, noradrenaline, and dopamine and an increased in corticosterone levels. Moreover, diabetes led to a significant increased expression of the clock genes Bmal-1 and Per-2 in the spleen, accompanied by a significant shift of the phase of Bmal-1 expression.

Conclusions: At early stages of T1D, alteration in the diurnal circulation of monocytes is observed only for the inflammatory Ly6C^hi monocytes and is detected in the spleen and peripheral blood but not the BM and retinas. These early changes could be regulated by the HPA axis and the disrupted clock gene expression locally in the spleen and adversely could impact the inflammatory environment that leads to DR.