Targeting the SDF-1/CXCR4 axis to attenuate pain in sickle cell disease: A preclinical evaluation of AMD3100 as a non-opioid therapeutic strategy Free

Background

Sickle cell disease (SCD) is the most common monogenic disorder, affecting about 100,000 Americans and 20 million people worldwide. Pain is the main feature of SCD; it is often chronic and debilitating, causing serious psychosocial and economic issues. Current pain management methods are not enough, and opioids frequently result in paradoxical hyperalgesia. Emerging evidence implicates the stromal-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis in key pathogenetic pathways of SCD-associated pain, like immune cell migration, angiogenesis, and tissue repair. Hypoxia and hemolysis -central features of SCD - upregulate SDF-1 and CXCR4, promoting inflammatory cytokine release and neuronal hyperexcitability. This study investigates the vascular-neuro-immune crosstalk mediated by SDF-1/CXCR4 and evaluates the therapeutic potential of AMD3100, an FDA-approved CXCR4 antagonist, in mitigating SCD pain.

Methods

Humanized sickle hemoglobin (HbSS) and normal hemoglobin (HbAA) mice were exposed to hypoxia (7% O₂ for 2 hours) or normoxia. SDF-1 and CXCR4 levels were measured in plasma and dorsal root ganglia (DRG) with ELISA and immunoblotting. THP-1 cells were treated with heme (25 µM) or exposed to low oxygen (3% O₂) to check CXCR4 expression. We evaluated leukocyte migration, activation, and survival using Boyden chamber assays, flow cytometry, and cytokine profiling. Pain-related behaviors were assessed via von Frey testing, mouse grimace scale, and grip strength. DRG neurons were analyzed using whole-cell patch-clamp electrophysiology to determine excitability and ion channel dynamics. AMD3100 was administered to evaluate its effect on pain behaviors and neuronal properties.

Results

Hypoxia significantly increased SDF-1 levels in the HbSS mice compared to the HbAA mice, both in the plasma (p<0.05) and DRG (p<0.05). CXCR4 expression was elevated in the DRG of HbSS mice post-hypoxia (p<0.05). Hemolysis, indicated by increased plasma cell-free heme in the HbSS mice, was more prominent in the HbSS mice versus HbAA mice (p<0.05) in vivo. In vitro studies revealed CXCR4 upregulation in THP-1 cells exposed to heme vs DMSO (p<0.05) and hypoxia vs. normoxia (p<0.05). Additionally, hypoxia exposure resulted in elevated TNF-α (p<0.05) and IL-6 (p<0.05) levels that are produced by neutrophils in HbSS mice compared to normoxic status. Neutrophil migration toward SDF-1 was significantly reduced by AMD3100 (p<0.05). Compared to control HbAA mice, DRG neurons from HbSS mice exhibited depolarized resting membrane potential (p<0.05), increased membrane resistance (p<0.01), reduced AP amplitude and width (p<0.05), and increased AP firing (p<0.0001). Hypoxia-induced mechanical allodynia was attenuated by AMD3100 more in the HbSS mice vs. HbAA mice, both at 30 minutes and 3 hours after treatment (p<0.05 for both). AMD3100 also reduced pERK expression and cytokine release, suggesting decreased neuronal sensitization. The mechanical hypersensitivity response to hypoxia was more prominent in older than in younger HbSS mice.

Conclusions

The SDF-1/CXCR4 axis plays a pivotal role in SCD-associated pain through its regulation of inflammatory and neuronal pathways. Hypoxia and hemolysis increase SDF-1/CXCR4, which boosts leukocyte recruitment and activation, leads to cytokine release, and causes sensory neuron hyperexcitability. AMD3100 effectively reduces these effects. It lowers pain behaviors and neuronal excitability in HbSS mice. These findings back the potential of CXCR4 antagonism as a non-opioid option for managing SCD pain. They also emphasize the need to focus on vascular-neuro-immune interactions in precise medicine methods.