Role of Patrolling Monocytes in Diabetic Retinopathy in an animal model of Type 1 Diabetes

Based on their functional and morphological characteristics, monocytes can be divided in inflammatory (CD115+, Ly6C+ in mice; CD14+ CD16– in humans), intermediate (CD115+Ly6Cint in mice; CD14+ CD16+ in humans), and patrolling monocytes (PMo) (CD115+Ly6C– in mice; CD14dimCD16+ in humans). PMo survey blood vessels and have housekeeping functions for the endothelium. We wished to learn how PMo react to diabetes; whether PMo contribute to leukostasis; and whether diabetes alters the functionality of PMo. To test whether diabetes affects PMo in the macrocirculation, we performed flowcytometric analysis on circulating blood of diabetic and non-diabetic mice. We found that diabetes reduced the number of circulating leukocytes, and that this decrease was driven by a decrease in lymphocytes, while granulocytes and total monocytes were unaltered. Diabetes induced a rearrangement of the monocyte subpopulations: PMo decreased, intermediate increased, inflammatory were unchanged. To test whether diabetes affects PMo in the microcirculation, we performed immunofluorescence of whole retinas from intracardially perfused mice. Diabetes induced a ~4-fold increase in the absolute number of PMo. We next tested whether the absence of PMo would affect leukostasis, by taking advantage of an animal model of absence of PMo, the NR4A1-/- mouse. Diabetic NR4A1-/- mice did not show the increase present in wild-type mice, supporting the finding that PMo are key elements in diabetic leukostasis. Next, we assessed retinal microvascular damage in NR4A1-/- mice, by determining the number of acellular capillaries in retinal trypsin digests. We found that healthy aging and 4-months duration of diabetes did not induce any increase in retinal microangiopathy in NR4A1-/- mice. Conversely, we found that 6-month duration of diabetes induced in NR4A1-/- mice more retinal microangiopathy. Notably, we found no difference in vascular permeability between WT or Nr4a1-/- mice, with and without diabetes, indicating that the blood-retinal barrier is intact in Nr4a1-/- mice. These findings led us to inquire whether diabetes induces changes in the transcriptome of PMo by Next Generation Sequencing. We studied wild-type mice with 5 months duration of diabetes, based on the previous observation that diabetic NR4A1-/- mice showed increased retinal microangiopathy after 6, but not 4, months of diabetes. Analyzing the signature of the differentially expressed genes, we found that 5 months of diabetes induced in PMo a comprehensive anti-inflammatory, anti-apoptotic, pro-adhesive/pro-migratory, and vasculo-protective signature. On these data we performed Gene Set Enrichment Analysis, Ingenuity Pathway Analysis, and STRING analysis. Based on their results, we planned the real-time qPCR on PMo sorted from WT mice after 3, 5, and 7 months of diabetes duration. We found that a protective program is activated in mice as early as after 3 months of diabetes, and up to 7 months of diabetes duration. However, CXCR4 is upregulated in mice with 3 and 5 months ̶ but not 7 months ̶ of diabetes duration. This finding, together with the morphological observation that microangiopathy was not apparent before 6 months of diabetes, suggests that CXCR4 may be critical for the delivery of protective/healing activities by PMo on retinal vessels. The first novel concept is the discovery of mechanisms that protect and repair retinal microvessels, thereby indicating that diabetic retinal microangiopathy reflects the balance of damage and repair. The second novel concept is that leukostasis represents an attempt to bring healing influences to damaged microvessels, rather than a pro-inflammatory event as currently proposed, thereby changing the meaning of this phenomenon. The results of this work have the potential to change the clinical management of retinopathy, by fostering the development on the one hand of a biomarker of risk of retinopathy, and on the other hand new interventions.