Short communicationPro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises
Introduction
Sickle cell disease (SCD) is caused by the presence of hemoglobin S (HbS) due to a point mutation in the β-hemoglobin (Hb) gene. Polymerization of deoxygenated-HbS leads to “sickling” of red blood cells, chronic hemolytic anemia and recurrent episodes of acute vaso-occlusive crises (VOC). All patients have progressive end-organ damage and a reduced life expectancy caused by a complex pathophysiology. Increased circulating levels of pro-inflammatory, chemotactic and adhesion products (e.g. TNF-α, IL-6, IL-18, IL-17α, IL-1β, ICAM, P-selectin) are present at both steady state and VOC [1], [2]. Early clinical epidemiological studies have pointed to neutrophils having a major role in promoting SCD pathophysiology. High neutrophil count and their overactivation correlate with poor disease outcome and early death [3], [4]. Activated neutrophils can also produce Neutrophil Extracellular Traps (NETs), the extracellular DNA-scaffolded structures that anchors granules components with potent anti-pathogen properties that allow for a more effective antimicrobial activity. NETs formation has also been described in sterile pathologies with a major inflammatory component, including SCD [5], [6]. Hydroxyurea (HU) administration reduces the frequency and severity, but does not abolish the occurrence of the sickle VOCs, and reduction of neutrophil count due to the HU myelosuppressive effect is thought to contribute to its efficacy [7].
We asked whether under HU therapy, the SCD environment remained pro-inflammatory enough to cause neutrophils to produce harmful NETs, and whether increased NETs formation associated with high levels of known pro-inflammatory products.
Section snippets
Patients
All SCD patients had HbSS genotype; paired plasma samples were obtained from 14 patients, age 22–57 (34.3 ± 9.4) years, all undergoing HU treatment, in crises and then again at steady state. Due to limitations of sample size, only 13 of the 14 pairs were used for both Luminex and indirect NETs assays. A “pain crisis” was defined as an episode of acute pain with no evident cause other than SCD, resulting in hospitalization and treatment with parenteral opioids. The “steady state” was when the
Results and discussion
Due to assay sensitivity giving rise to variability in the number of analytes that could be detected, only 24 of the 35 analytes could be assessed. Across the study cohort, 13 (P-selectin, IL-18, IL-18PBa, IL8/CXCL8, MIP-1β/CCL4, SDF1α/CXCL12, Eotaxin/CCL11, TNF-α, IL-4, IL-7, IL-15, IL-1β, and E-selectin) of the total 24 analytes had statistically significant higher plasma levels in the patients’ samples at both steady state and crisis compared to the healthy controls (Supplemental Table 1).
Capsule summary
Hydroxyurea therapy reduces frequency but does not abolish sickle cell vaso-occlusive crises (VOC). Plasma from SCD patients in VOC whilst on hydroxyurea treatment, can still trigger NETosis, and degree of NETosis appears to correlate with increased levels of IL-6 and IL-1ra, a regulator of the IL-1 family of cytokines, suggesting that the latter might serve as biomarkers of sickle cell crisis episodes.
Authorship
EAB designed and executed experiments, analyzed data and wrote the manuscript. LS and LM executed experimental work, contributed to data analysis and reviewed the manuscript. SLT supervised the project, wrote and reviewed the manuscript.
Funding
This work was supported by NHLBI intramural support to SLT.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
Authors thank Jim Nichols and Darlene Allen for their help with the donors’ recruitment process and all patients and healthy volunteers who participated in our study.
References (15)
- et al.
Neutrophils and NETs in modulating acute and chronic inflammation
Blood
(2019) - et al.
Heme-induced neutrophil extracellular traps contribute to the pathogenesis of sickle cell disease
Blood
(2014) - et al.
P-selectin mediates the adhesion of sickle erythrocytes to the endothelium
Blood
(2001) - et al.
The pathway of neutrophil extracellular traps towards atherosclerosis and thrombosis
Atherosclerosis
(2019) - et al.
Altered levels of pro-inflammatory cytokines in sickle cell disease patients during vaso-occlusive crises and the steady state condition
Eur. Cytokine Network
(2013) - et al.
Altered levels of cytokines and inflammatory mediators in plasma and leukocytes of sickle cell anemia patients and effects of hydroxyurea therapy
J. Leukoc. Biol.
(2009) - et al.
Mortality in sickle cell disease. Life expectancy and risk factors for early death
N. Engl. J. Med.
(1994)
Cited by (0)
- 1
Current address: Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, United States.