Functionality of a Peripheral Blood Derived Microglia-like Cell System for Drug Discovery to Treat Sickle Cell Related Chronic Pain

Abstract

Background: Abnormal microglial activation is emerging as a clinically promising drug target to treat chronic pain in sickle cell disease (SCD). But research involving microglia has been stymied by difficulty in obtaining human CNS microglia and differences between human and animal neurobiology. To address such limitations, we have developed an in vitro culture system to induce human microglia-like cells (MLC) from peripheral blood mononuclear cells. In this study, we further validated the functionality of the cultured peripheral blood derived microglia-like cell (PB-MLC) and evaluate the pathological characteristics captured in the PB-MLC model system, which is an important requirement for disease model systems used in drug discovery. Methods: Human peripheral blood mononuclear cells were cultured with human GM-CSF (10 ng/ml), human IL-34 (100 ng/ml), TGF-β2 (2 ng/mL) and cholesterol (1.5 µg/mL) for 7 days to induce Human peripheral blood derived microglia-like cells (HPB-MLC). HPB-MLC were morphologically analyzed by phase contrast microscopy, phenotyped by immunofluorescence staining with anti-TMEM119, P2RY12, and Iba1 antibodies. To validate that the HPB-MLC culture system recapitulates important pathological features, we compared the microglial morphology, activation states and phagocytic activity of HPB-MLC from individuals with SCD with or without chronic pain, individuals with headache and normal donors without pain: Cells were stimulated with 100 ng/ml LPS for 24 hours, and secretion of TNF-alpha, IL-1beta, and IL-6 was measured by ELISA. The microglial phagocytic function of HPB-MLC was determined by assessing the uptake of fluorescent-labeled Zymosan particles with fluorescent microscopy and imaging flow cytometry. The model system was also validated by comparing cultured PB-MLC to CNS derived microglia cells using Sprague-Dawley rats: Rat brain derived microglia (BDM) and rat peripheral blood monocytes were isolated and then cultured with murine IL-34 (100 ng/ml), murine GM-CSF (10 ng/ml). TGF-β2 (2 ng/mL), and cholesterol (1.5 µg/mL) for 7 days. The cultured cells were morphologically analyzed as above; phenotyped with anti-TMEM119, P2RY12, and Iba1 antibodies. Rat BDM, rat PB-MLC and HPB-MLC were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25, 50 μg/mL) for 24 hours; TNF-alpha secretion was measured by ELISA. Results: Rat PB-MLC resemble BDM morphologically and express microglia specific markers. HPB-MLC express microglia specific markers (TMEM119, P2RY12, Iba1), exhibit microglial phagocytic activity. HPB-MLC retain the patient pain phenotype: Cultures from donors without chronic pain show more ramified morphology and lower phagocytic activity than cultures from individuals with chronic pain (Figure 1); HPB-MLC can be activated by LPS, causing microglial deramification and increased the expression of microglial activation marker CD68. HPB-MLC from patients with pain are more easily activated and secrete more inflammatory cytokines in response to LPS; SCD pain and headache samples exhibit different pro-inflammatory cytokine profile. To evaluate the possibility of using the PB-MLC model system to screen compounds to inhibit microglia activation, we tested PB-MLC cells with the microglial inhibitor, minocycline. The compound significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLC in a dose-dependent manner (Figure 2). Conclusions: We have developed and validated a microglial-like cell culture system from the peripheral blood of patients with sickle cell disease that retains the patient pain phenotype. HPB-MLC are physiologically functional and exhibit mature microglial features, including LPS-induced activation, microglial specific morphological transition, microglial phagocytic activity, and drug response to a well-studied microglia inhibitor, minocycline. As a cost-effective, efficient, and patient-specific microglia model, HPB-MLC culture system can be applied to identify target compounds that normalize microglia activation, and potentially treat chronic pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal