Abstract
Background: Patients with sickle cell disease (SCD) experience acute pain episodes in early childhood; over 50% of adult SCD patients go on to develop chronic pain. In animal models, microglia, derived from hematopoietic stem cells, play an important role in the transition from acute to chronic pain; in patients with chronic pain microglia are often activated with minimal stimuli, releasing TNF-alpha and stimulating neurons. Research involving microglia has been stymied by difficulty in obtaining and culturing microglia, particularly from humans. We therefore propose to culture microglia-like cells from peripheral blood mononuclear cells (PBMC) from pediatric patients with SCD with and without chronic pain (defined as pain for more than 50% of days for at least 3 months) and from normal blood donors.
Objectives: Our goal was to determine if our cultured cells exhibited microglia morphology and surface markers, could be activated with inflammatory stimuli, and if donor characteristics were retained in the culture derived microglia-like cells.
Methods: PBMC were obtained from peripheral blood by Ficoll separation from three unique patients with SCD and no history of chronic pain (SCD CP-), peripheral blood from three unique patients with SCD with history of chronic pain (SCD CP+), or buffy coat from three human blood donors. PBMC were cultured with recombinant human GM-CSF (10 ng/ml; R&D Systems, MN), recombinant human IL-34 (100 ng/ml; R&D Systems), as previously described (Ohgidani et al, 2014) to induce ramified microglia. On day 7 of culture, cells were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry by staining with CD11b and CD45 antibodies, indirect immunofluorescence performed with anti-CX3CR1antibodies and imaging by confocal laser scanning microscope. Microglia-like cells from 3 normal blood donors, 3 SCD CP- and 3 SCD CP+, were stimulated by 100 ng/mL LPS for 24 hours, beginning on day 6 of culture. Deramification (activation) was assessed by immunofluorescence using anti-Iba1, and by flow using anti-CD68.
Results: PBMCs from patients with SCD ± CP and normal blood donors developed microglial morphology, namely, soma bodies with branched collaterals, after incubation with GM-CSF and IL-34. When subjected to flow cytometry, treated cells were CD11bhigh and CD45low; fluorescence microscopy revealed CX3CR1 positivity in the cultured cells, all consistent with accepted microglia phenotype. PBMCs untreated with GM-CSF and IL-34 maintained their round morphology, and were CD11blow, CD45high by FACS.
Microglia-like morphology differed significantly between PBMC sources. Microglia-like cells from SCD CP+ had shorter and fewer branches than normal individuals; branching of microglia-like cells from SCD CP- were intermediate in number and length, greater than to SCD CP+ but less than normal individuals.
When treated with LPS, nearly 100% of microglia-like cells derived from patients with SCD CP+ became amoeboid in shape, indicating microglial deramification, or activation, compared to approximately 25% of microglial-like cells derived from normal human blood donors or SCD CP-.
We also assessed deramification through flow measurement of CD68, a marker found on activated microglia and found that the microglia-like cells derived from HSPCs from SCD CP+ patients had significantly higher CD68 positivity compared to microglia-like cells derived from HSPCs from normal human blood donors or SCD CP-, and significantly higher Iba1 positivity as measured by immunofluorescence.
Conclusions: We established the microglia-like nature of the cultured PBMCs derived from patients with SCD±CP and normal blood donors through morphologic examination and confirmation of characteristic surface markers via flow and fluorescence microscopy, and through demonstration of activation with LPS. Our microglial-like cells derived from patients with SCD CP+ were activated at higher percentages by LPs treatment than microglial-like cells derived from normal human blood donors and SCD CP-, suggesting that donor characteristics are retained by the microglial-like cells 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.
No relevant conflicts of interest to declare.
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