Elsevier

Cytokine

Volume 127, March 2020, 154933
Cytokine

Short communication
Pro-inflammatory cytokines associate with NETosis during sickle cell vaso-occlusive crises

https://doi.org/10.1016/j.cyto.2019.154933Get rights and content

Highlights

  • Sickle cell environment remains highly pro-inflammatory even during hydroxyurea treatment.

  • Numerous pro-inflammatory cytokine levels remain elevated at steady state and crises.

  • Plasma from SCD patients in VOC whilst on hydroxyurea treatment, can trigger NETs formation.

  • Increased NETs production at crises appears to correlate with increased levels of IL-6 and IL-1ra.

Abstract

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy.

Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs.

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.

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Current address: Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, United States.

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