Abstract
Introduction
Sickle cell disease (SCD) impacts millions globally. Acute chest syndrome (ACS) is the leading cause of admission to ICU and death among SCD patients. Our preliminary patient data demonstrate that proximal and alternative complement activation is evident during ACS. Corroborating this, a murine model of SCD revealed that complement activation plays a role in driving ACS and is associated with lung neutrophil extracellular trap (NET) formation (Chonat et al., STM 2025). Given these findings, we hypothesized that during ACS, exaggerated hemolysis-mediated complement activation primes neutrophils to generate NETs, resulting in endothelial damage and ACS.
Methods
To investigate how complement modifies neutrophil phenotype, blood collected from SCD patients experiencing ACS (n=16) and those without ACS (baseline; n=10) was used to phenotype and measure complement deposition on neutrophils using high-dimensional flow cytometry. Complement plasma levels were also measured for component 3a (C3a), C5a and C5b-9. Lastly, novel sickle cell (SS) mice deficient in C3 (SSC3KO) were treated with hemin and tested for complement activation and NET formation. Statistics were generated using an unpaired Mann-Whitney test. Significance was defined as p<0.05.
Results
Consistent with the established role of complement in neutrophil activation in SCD mice, Unifold Multiparameter Approximation and Projection (UMAP) analysis demonstrated increased C3 deposition on neutrophils from patients during ACS episodes compared to baseline (2% ACS vs. 0.25% baseline; p<0.01). ACS episodes also displayed increased immature neutrophils compared to baseline (3% ACS vs. 0.2% baseline; p<0.001), and C3 deposition occurred on ~14% of immature neutrophils during ACS. Of those, increased C3 deposition was found on both naïve (4.7% ACS vs. 1.9% baseline; p<0.05) and activated (18% ACS vs. 11% baseline; p<0.05) cells. Moreover, immature neutrophils during ACS episodes also had increased expression of CD88 than at baseline (66% ACS vs. 27% baseline; p<0.001). CD88 is also known as C5aR1 and has been shown to play a crucial role in neutrophil chemotaxis. Among immature neutrophils during ACS episodes, 58% showed a phenotype typical of reverse migration (having entered tissue but migrated back into circulation), with 26% of these cells showing surface C3 deposition. While C3 deposition on mature neutrophils (3.2% ACS vs. 1% baseline; p<0.05) did not associate with altered complement regulatory protein expression, the majority of immature neutrophils in ACS episodes had decreased complement regulatory protein expression. Notably, plasma levels of C3a (median 106 ng/mL ACS vs. 47 ng/mL baseline; p<0.001), C5a (median 16.5 ng/mL ACS vs. 10 ng/mL baseline; p<0.05) and C5b-9 (median 184 ng/mL ACS vs. 118 ng/mL baseline; p<0.001) were all significantly elevated during ACS episodes, directly linking increased complement activation with a shift toward a pro-inflammatory, activated neutrophil phenotype during ACS. Consistent with this, hemin-treated SSC3KO mice failed to develop ACS, and lacked NET formation in the lungs compared to hemin-treated wild-type mice.
ConclusionsThe new findings presented here suggest that systemic complement activation plays a pivotal role in driving neutrophil activation and lung infiltration during ACS. Patients with ACS display high levels of immature and reverse migration phenotypes, a neutrophil subset that is linked to tissue damage. Ongoing follow-up studies leveraging biochemical and genomic approaches to dissect how complement components orchestrate recruitment and priming of neutrophils for NET formation and endothelial dysfunction will generate new insight into the underlying mechanisms of ACS.
