Resolvins modulate haematopoietic stem and progenitor cell dysfunction and neutrophil activation in sickle cell disease Free

Chronic inflammation is characteristic of sickle cell disease (SCD), and the resulting neutrophil activation plays a significant role in SCD pathophysiology. Growing evidence suggests that chronic inflammation affects Haematopoietic Stem Cells (HSCs) and that this may contribute to HSC dysfunction and skewed lineage production.

Resolvins are a class of Specialized Pro-resolving Mediators (SPM) derived from omega-3 fatty acids. They are crucial for resolution of inflammation and their dysregulation leads to chronic inflammation. In experimental models of SCD, altered resolvin levels are linked with adverse effects of chronic inflammation, and may also be a remediable cause of HSC dysfunction in SCD. Emerging research from murine experiments highlights the importance of resolvins in maintaining normal haematopoiesis. Defects in resolvin biosynthesis in the bone marrow (BM) are associated with a pro-inflammatory phenotype in myeloid cells. Hence, we hypothesised that neutrophilia and neutrophil activation in SCD may result from dysregulated HSC function brought about by altered resolvin production and increased expression of mediators of chronic inflammation in BM environment. Consequently, restoration of resolvin levels might modify HSC function and ameliorate the abnormalities in neutrophil progeny.

We first explored whether HSC function is altered in SCD resulting in neutrophils that carry a pathogenic phenotype. Here BM derived CD34+ cells from SCD patients (n=7) and healthy donors (n=3) were differentiated to neutrophils in media supplemented with StemSpan™ Myeloid Expansion Supplement. CD34+ cells derived from SCD patients demonstrated increased neutrophil yield with more neutrophils [23%±7of total yield vs 12%±3] when compared with those derived from healthy volunteers. Furthermore, neutrophils generated from SCD CD34+ cells had a higher basal oxidative stress (p=0.048) and augmented response to fmlp, a bacterial formylated peptide mimetic, in generating reactive oxygen species (96% higher mean fluorescence intensity(MFI), p=0.033) compared to control HSCs. Neutrophils derived from SCD HSCs displayed activated phenotype with a higher basal CD11b expression [MFI 4093±2089 vs 2777±441)].

We next explored the role of resolvins in restoring neutrophil functions. Neutrophils differentiated in vitro from HSCs which had been incubated with resolvin (Rv)D1, RvD2 or RvD5_{n-3 DPA}(n-3 docosapentaenoic acid-derived RvD5) showed reduced cellular ROS (27%±11% reduction, with RvD5_{n-3 DPA}, p=0.06), improved phagocytic capability (67% ± 22% improvement, p=0.01 with RvD2), and a reduced expression of CD11b (14.6% reduction, 7% SEM, p=0.03 with RvD2) compared to the neutrophils derived from vehicle treated SCD HSC.

The effect of HSC dysregulation on neutrophil numbers and activation was further studied in vivo by transplanting SCD HSC (n=6) or control HSC (n=3) into sub-lethally irradiated HuNOG-EXL mice. Peripheral blood (PB) collected 10 weeks post-transplantation showed 71% higher proportion of neutrophils among human CD45+ cells in mice transplanted with SCD HSCs compared to those with control HSCs (40%±9% vs 23%±5). Neutrophils in mice transplanted with SCD HSCs demonstrate higher CD11b than those derived from control HSCs (p=0.04).

To study neutrophil function in resolvin-treated recipient mice, HSCs from SCD patients were incubated for 24 hours with resolvins (RvD1, RvD2 and RvD5_{n-3 DPA}) or vehicle and transplanted into HuNOG-EXL mice (6 treated and 6 vehicle). Mice were then treated with either vehicle or resolvins intra-peritoneally for a period of 4 weeks post-transplantation with a 6-week treatment-free period before PB or BM was collected. The treatment resulted in a >40% reduction in the proportion of neutrophils in PB (p=0.03) and a reduction in CD11b expression. There was also a higher proportion of human CD34+ cells in BM samples of mice treated with resolvins compared to vehicle suggesting that resolvins may have a role in resetting increased cycling seen in HSCs in SCD.

These data provide further insights into HSC dysfunction in SCD and the mechanisms by which chronic inflammation leads to neutrophil dysfunction. The effects of resolvin treatment on correcting HSC dysfunction in vitro and in vivo are encouraging and suggest a possible new therapeutic approach to disease modification in SCD targeting chronic inflammatory pathways as well as for optimising HSC health for autologous stem cell therapy.