(217) Microglia-Like Cells Derived from Hematopoietic Stem and Progenitor Cells from Patients with Sickle Cell Disease as a Model System to Investigate Chronic Pain

Abstract

Patients with sickle cell disease (SCD) experience severe acute pain episodes, and often progress to chronic pain. In patients with chronic pain, microglia are readily activated, stimulating neurons to send a pain signal; studying microglia may provide important insights into development of chronic pain. Human microglia are difficult to obtain; we proposed to culture microglia-like cells (MLC) from peripheral blood from three individuals from each of the following groups: patients with SCD and chronic pain (SCD CP+, defined as pain for more than 50% of days for at least 3 months), without chronic pain (SCD CP-), and blood donors (WT). When cultured with GM-CSF and IL-34, SCD ± CP and WT PBMCs developed microglial morphology, were CD11bhigh and CD45low by flow cytometry, CX3CR1+ by fluorescence microscopy, consistent with microglia. We treated our MLC with LPS, and found that treated MLC cells had significantly higher CD68 positivity compared to resting microglia cells, and significantly higher level of Iba1 positivity measured by immunofluorescence, indicating activation. MLC differed significantly depending on donor group. SCD CP+ had shorter and fewer branches than WT; branching of MLC from SCD CP- were intermediate in number and length. When treated with 100 ng/ml LPS, nearly 100% of MLC derived from patients with SCD CP+ became activated, compared to approximately 25% WT or SCD CP-. WT cells released 1098 ±136 pg/ml TNF-alpha, 84±18 pg/ml IL-1beta, and 121 ±23 pg/ml IL-10 in response to LPS stimulation; SCD CP- cells released 2959±410 pg/ml TNF-alpha, 209±55 pg/ml IL-1beta, and 401±50 pg/ml IL-10 in response to LPS stimulation. This data suggests that donor characteristics are retained by the MLC developed in culture. We propose to use this model system to derive mechanistic insights into the development of chronic pain in SCD, and to screen pharmacologic agents to treat and prevent chronic pain.