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Background: Individuals with chronic pain may have hyperactive microglia, which stimulate neurons to send a pain signal with little or no stimuli. Microglia are therefore a potential drug target to treat chronic pain, but drug discovery has been stymied by differences between human and animal neurobiology, and lack of healthy human CNS microglia. We cultured peripheral blood derived monocytes to develop characteristics of CNS derived microglia, termed peripheral blood derived microglia like cells (PB-MLC). We found that lipopolysaccharide (LPS) treated PB-MLCs from patients with chronic pain, from sickle cell disease (SCD) or chronic headaches, secreted more pro-inflammatory cytokines (TNF-alpha, IL-1beta, and IL-6) than PB-MLCs from normal donors, suggesting that patient pain phenotype was preserved in culture; PB-MLC from individuals with chronic pain were hyperactive in vitro as their microglia are in vivo. We hypothesize that PB-MLCs can be developed as a cell-based assay to screen compounds to treat chronic pain. To validate our model system, we compared cultured PB-MLCs to CNS derived microglia cells, using Sprague-Dawley rats, and treated human PB-MLC with microglia activation inhibitors shown to work in vivo in murine models. Methods: We isolated rat brain derived microglia (BDM) and rat peripheral blood monocytes; both were cultured with murine IL-34 (100 ng/ml) and GM-CSF (10 ng/ml). BDM and rPB-MLC were morphologically analyzed by fluorescence imaging microscopy, combined with machine learning, phenotyped by RT-qPCR and indirect immunofluorescence with anti-TMEM119, CD68, and Iba1 antibodies. Cells were treated with LPS for 24 hours, and TNF-alpha, IL-1beta, and IL-6 secretion measured by ELISA. For human PB-MLC studies, monocytes were cultured with GM-CSF (10 ng/ml) and IL-34 (100 ng/ml) for 7 days. PB-MLC morphology was analyzed as above; phenotyped with anti-CX3CR1, TMEM119, CD68, and Iba1 antibodies. PB-MLCs were treated with 100 ng/ml LPS with or without minocycline (2.5, 5, 10, 25 μg/mL), clopidogrel (1, 2, 4 μM) and MRS2395 (1, 5, 10 μM), for 24 h; TNF-alpha and IL-1beta secretion measured by ELISA. Results: We found that rPB-MLC resemble BDM morphologically, express the same microglia specific markers (TMEM119, P2RY12) and can be activated by LPS (Figure 1). Monocytes not cultured with IL-34 and GM-CSF did not express microglia specific genes (Figure 2A). 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 following microglial inhibitors shown to be active in murine models in vivo: minocycline, MRS2395, and clopidogrel. MRS2395 and clopidogrel significantly suppressed the release of proinflammatory cytokine TNF-alpha from LPS-induced activated PB-MLCs in a dose-dependent manner (Figure 2B); minocycline did not. Conclusions: We validated our model system by comparing CNS derived microglia to rPB-MLCs and found they share morphology, similar cytokine secretion in response to LPS, and expression of microglia-specific genes. We confirmed that human PB-MLC expressed microglia specific genes while the original monocytes did not. Since P2Y12 is implicated in chronic pain, we tested two P2Y12 receptor agonists, clopidogrel and MRS2395, in our human PB-MLC system. When challenged with LPS, clopidogrel and MRS2395 inhibited LPS-induced PB-MLC activation in vitro as it had in vivo in a murine chronic pain model. We propose to use our human PB-MLC to screen for compounds that reduce microglia hyperactivity, to identify pharmacologic agents to treat chronic pain. Disclosures No relevant conflicts of interest to declare.

Background: Patients with sickle cell disease (SCD) often experience severe chronic pain. In chronic pain, microglia are readily activated, stimulating neurons to send a pain signal. Human microglia are difficult to obtain; we have cultured peripheral blood derived microglia-like cells (PBMLC) from human peripheral blood to develop a model system to investigate chronic pain in sickle cell disease. We have shown that our PB-MLC resemble microglia morphologically, have the same surface markers, and can be activated by lipopolysaccharide (LPS). Our PB-MLC are more easily activated and secrete more inflammatory cytokines if they are derived from a SCD patient with chronic pain compared to a HbAA individual without pain or a SCD patient with acute pain only. Now, we seek to assess the suitability of our model system to screen for compounds to treat chronic pain by using of a panel of compounds to reduce microglia and PB-MLC activation and cytokine release in response to LPS. Finally, we propose to validate our model system by comparing the morphology, surface markers, and cytokine release between PB-MLC and true in situ brain derived microglia (BDM), both from Sprague Dawley (SD) rats. Methods: Human peripheral blood mononuclear cells (HPB-MCs) were obtained from three individuals with SCD with chronic pain and three normal donors (WT). HPB-MCs were cultured with human GM-CSF (10 ng/ml) and human IL-34 (100 ng/ml) to induce peripheral blood derived microglia (HPB-MLC). TNF-alpha and IL-1beta secretion by HPB-MLCs in response to LPS with or without a panel of drugs were measured with ELISA. Rat BDM were isolated from fresh rat brain tissues using anti‐rat CD11b/c microbeads and cultured with murine IL-34(100 ng/ml) and murine GM-CSF (10 ng/ml). Rat PB-MLCs were developed from rat PB-MCs by culturing with murine IL-34 (100 ng/ml) and murine GM-CSF (10 ng/ml). On day 7 of culture, rat BDM and PB-MLCs were collected and morphology analyzed by phase contrast microscopy, phenotyped by flow cytometry and indirect immunofluorescence with anti-CX3CR1, TMEM119, CD68, Iba1 antibodies. Microglia morphology was evaluated by quantitative analysis of cell body roundness and branch length. TNF-alpha, IL-1beta, and IL-6 secretion by rat BDM and PB-MLCs in response to LPS with or without piceatannol was measured with ELISA. Results: To evaluate the possibility of using our PB-MLC model system to screen compounds to reduce abnormal microglia activation and treat chronic pain, we tested HPB-MLC cells from patients with SCD and chronic pain with the following drugs: gabapentin, metformin, piceatannol, and resveratrol, chosen based on published reports of their effect on microglia activation. All suppressed the release of proinflammatory cytokine from LPS-induced HPB-MLC in a dose-dependent manner (Figure 1), and reversed the deramification, or rounding of activated PB-MLC upon LPS stimulation by quantitative analysis of cell body roundness and branch length with immunostaining of Iba1. Gabapentin exhibited the smallest effect on reduction of HPB-MLC activation. Rat BDM and PBMLC both exhibited characteristic microglia branched morphology in culture, and rat PBMLC from SD rats expressed microglia specific marker TMEM119. Both PB-MLC and BDM from rats became amoeboid with LPS treatment, and showed increased expression of CD68, and Iba1 (Figure 2). In both BDM and PBMLC from rats, piceatannol reduced LPS activation and TNF-alpha, IL-1beta, and IL-6 secretion (Figure 3). Conclusions: We have established the microglia-like nature of PBMLC from patients with SCD and normal blood donors, and the preservation of the patient pain phenotype in culture. Here, we show that compounds reported to reduce microglia activation reduce the inflammatory cytokine release from HPB-MLCs from patients with SCD. BDM and PB-MLCs from SD rats have similar morphology, both quiescent and activated, and secrete inflammatory cytokines in the same manner in response to LPS. Piceatannol reduces activation and cytokine release in both. This comparison between peripheral blood derived and brain derived microglia supports our assertion that PB-MLC capture significant aspects of true in situ brain microglia biology, particularly that of activation and drug response. We therefore 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 chronic pain. Disclosures No relevant conflicts of interest to declare.

The BABY HUG clinical trial of hydroxyurea in infants and young children with sickle cell disease (SCD) established the safety and clinical benefit of hydroxyurea therapy in this age group. The study did not establish, however, if escalation to maximum tolerated dose (MTD) could be safely accomplished and resulted in increased benefit in this age group. We sought to determine the safety and clinical effectiveness of dose-escalation hydroxyurea therapy in children with SCD under the age of two years. We conducted a retrospective chart review of children with HbSS or HbS β0Thalassemia who initiated hydroxyurea therapy before the age of two years between 2011 and 2017 at the Texas Children's Hematology Center. We assessed the clinical and laboratory response to dose-escalation therapy as well as any adverse events associated with hydroxyurea therapy. We identified 95 patients ranging from 5 to 24 months in age who were initiated on hydroxyurea therapy during the study period. MTD was achieved in 75 patients (76.5%), of whom 51 were still at MTD at end of monitoring. There were significant increases in hemoglobin concentration (Hb) and hemoglobin F (HbF) levels and decline in absolute reticulocyte count (ARC) on hydroxyurea therapy (Table 1). Additionally, the magnitude of increases in Hb and HbF were greater than those seen in the BABY HUG hydroxyurea treatment group both at the time of MTD and at end of assessment. The proportion of patients experiencing neutropenia or thrombocytopenia as well as the number of events per patient-year (PPY) over 193.9 patient-years were similar to those in the BABY HUG hydroxyurea treatment group (Table 2). The incidence of acute chest syndrome (ACS) and transfusions was similar between the two groups, while episodes of splenic sequestration were more common in patients advanced to MTD. In summary, hydroxyurea therapy with dose escalation to MTD appears to be safe in infants and young children and results in more significant and sustained increases in hemoglobin concentration and %HbF than were seen with fixed dosing in the BABY HUG study. In the context of this retrospective study, though, hydroxyurea dose escalation did not clearly result in increased clinical benefit relative to fixed-dose treatment. Disclosures No relevant conflicts of interest to declare.