Characterization of a New Immortalized Human Microglial Cell Line and its Pro-Inflammatory Activation. 31st Meeting of the European Association for the Study of Diabetes Eye Complications Study Group (EASDec) Odense, Denmark, 28th – 30th October 2021

DESIGN. Experimental PURPOSE. The complexity of retinal structure reflects on the difficulty to describe its composite cell interactions and the pathways involved. Microglial cells are the main responsible cell-type for the modulation of immune responses to inflammatory stimuli that occur in the retina in diabetes. The majority of studies on retinal inflammation in diabetic retinopathy in the literature use rodent microglial lines, because of the scarce availability of human cell sources. We carried out a detailed characterization of a new commercially available immortalized human microglial line (Innoprot, Spain), and tested its susceptibility to inflammatory stimuli, to create species-specific cell models to study the multiple interactions between the vascular and neuronal portions of the retina. METHODS. First, we checked the expression of microglial markers, capable of discriminating between resident microglia and peripheral macrophages (TMEM119, Iba-1, CD14, CD16, CD68), and human vs rodent origin (CD11b). Subsequently, we tried LPS stimulation as well as several different pro-inflammatory cocktails to determine the best combination able to induce a significant M1 (inflammatory) response after 24h exposure. We then measured M1 induction through the modulation of the expression of pro- and anti-inflammatory molecules, determined by flow cytometry, Western blotting, and RT-PCR. Finally, we performed morphology and functional assays, measuring viability, apoptosis, migration and ROS production. RESULTS. Marker expression confirmed the microglial derivation of the cells and their human origin. Differently from rodents, LPS is not able to induce an M1 profile in human microglial cells. The best pro-inflammatory combination among those tested was 20ng/ml hIL-1β + 10 ng/ml hTNFα + 50ng/ml hINFγ. This was able to induce changes in morphology, resulting in elongation and ramification, characteristic of activated cells, and to increase proliferation, apoptosis, migration, and ROS production. In addition, M1 conditions was associated with increased expression of inflammatory markers: IL-6, IL-8, MMP2, VCAM-1, TNFα, MHC-II, and pNFKβ/NFKβ, and the pro-inflammatory miR-155 and miR146a. CONCLUSIONS. This immortalized human microglial line proved to be a potential experimental tool to investigate the pathophysiology of the inflammatory component of diabetic retinopathy in species-specific models.