Interleukin-27 ameliorates inflammation-induced chronic pain in sickle cell disease

Abstract Sickle cell disease (SCD) causes debilitating chronic pain in over 50% of adults, highlighting the urgent need for new non-opioid-based therapies. Inflammation is central to SCD and is driven by activated neutrophils producing damage-associated molecular patterns (DAMPs), including neutrophil extracellular traps (NETs) via caspase-dependent gasdermin-D activation. We reported that neutrophil activation could be inhibited by interleukin-27 (IL-27), an anti-inflammatory cytokine shown to have the potential to regulate neuroinflammation (Alagbe et al. 2017). Therefore, we hypothesized that IL-27 could reduce chronic pain in SCD by inhibiting NET formation and neuroinflammation.MethodsWe analyzed stored plasma from adult patients with SCD in steady state and controls enrolled in an IRB-approved clinical trial. Patient were administered the Brief Pain Inventory short form for the assessment of pain severity (O'Brien et al. 2024). We quantified circulating NET levels as triple positive for citrullinated histones, extracellular DNA, and neutrophil elastase using imaging flow cytometry, and explored correlations with the patients' BPI scores. Additionally, fresh blood samples from prospectively enrolled SCD patients in steady state were treated ex vivo with hemin (20µM) ± recombinant human (rh) IL-27 (200 ng/mL) to assess neutrophil inflammatory signaling. Finally, aged Townes SS mice were treated with recombinant mouse (rm) IL-27 (IP 16 mg/Kg body weight in 200 μL saline) or sham for two weeks (3 doses/week); tactile, cold, and heat hypersensitivity were assessed at baseline and weekly. Circulating NETs were quantified at baseline and post-treatment, and circulating neutrophils and dorsal root ganglia (DRGs) were isolated and analyzed for inflammatory signaling and histology, respectively.ResultsThe study included 53 individuals with sickle cell disease (HbSS, HbSC, HbSβ⁺, and HbSβ⁰) and 17 age-matched healthy Black controls (mean age 38.6 ± 12.4 vs. 38.8 ± 10.6 years). Despite being in steady state, a majority of the SCD patients reported pain in the prior 24 hours. Patients also had higher levels of circulating NETs compared to the healthy controls (p = 0.042), and their pain scores positively correlated with circulating NET levels (r = 0.32) and inversely with plasma IL-27 levels (r = - 0.33). Hemin induced caspase-5-dependent GSDMD activation in SCD neutrophils ex vivo, which was inhibited by rhIL-27.Townes SS mice (n = 6) treated with rmIL-27 showed significantly reduced heat and cold hypersensitivity (p < 0.01 for both), with no change in tactile sensitivity compared to sham (n = 5). The rmIL-27-treated mice had a reduction in the circulating NET levels, while the sham mice had an increase in circulating NETs compared to their respective baseline levels (p = 0.08). IL-27 treatment also tended to reduce caspase-5-dependent GSDMD activation in neutrophils, and H&E staining revealed decreased inflammatory cell infiltration in the DRG compared to controls.ConclusionsThese findings suggest that IL-27 may attenuate chronic pain in SCD by suppressing neutrophil activation, NET formation, and neuroinflammation. The observed correlations between pain, circulating NETs, and IL-27 levels in patients, along with the therapeutic effects of IL-27 in a preclinical SCD model, support its potential as a novel, non-opioid therapeutic strategy for SCD-associated pain. Further studies are warranted to explore IL-27–based interventions in clinical settings.