Vaso-occlusion is one of the most common complications in sickle cell disease (SCD). Our previous studies have shown that neutrophils play an important role in promoting sickle cell vaso-occlusion by capturing sickle red blood cells (sRBC) through activated αMβ2 integrin (Mac-1). However, high Mac-1 activity was only observed in a small subset of adherent neutrophils (Nat. Med. 2009;15:384). Recent studies have shown that a CD62Llow CXCR4high subset of neutrophils represents the cells that have truly aged in vivo (Cell. 2013;153:1025). Since aged neutrophils may have experienced activation signals when they survey the whole body, we hypothesized that they are over-active cells that promote sickle cell vaso-occlusion. To test this possibility, we first analyzed the correlation between CD62L expression and Mac-1 activity of adherent neutrophils using multichannel fluorescence intravital microscopy and albumin-coated fluosphere beads that specifically bind activated Mac-1. We found a strong inverse correlation where neutrophils with lower CD62L expression showed greater bead binding capacity (P < 0.001). Mice deficient in P-selectin (Selp-/-), an adhesion molecule required for neutrophil clearance from the circulation, showed a dramatic increase in the percentages of CD62Llow CXCR4high aged neutrophils (wild-type / Selp-/-: 9.0 ± 1.0% / 74.7 ± 2.1%, P < 0.01). When purified neutrophils from Selp-/- mice were transferred into wild-type recipients, these aged neutrophils exhibited significantly higher Mac-1 activity compared to those purified from wild-type animals (wild-type / Selp-/-: 0.26 ± 0.06 / 1.52 ± 0.35 beads per adherent neutrophil, P < 0.05). Since microbial products derived from the microbiota could translocate into the system and modulate the innate immunity (Nat. Rev. Microbiol. 2011;9:233), we hypothesized that microbiota-derived signals could regulate the neutrophil aging process. To test this idea, we depleted the microbiota by treating mice with ampicillin, neomycin, metronidazole and vancomycin for 4 weeks. In these mice, the numbers of CD62Llow CXCR4high aged neutrophils were significantly decreased, and this reduction was reversible by intragastric gavage of Lipopolysaccharide (LPS; Control / Antibiotics-treated (ABX) / ABX + LPS: 87.2 ± 20.3 / 12.9 ± 2.0 / 83.9 ± 60.0 cells / μL blood, P < 0.05 between first two groups). To analyze the kinetics of neutrophil aging in vivo, we transferred blood from CD45.1+ mice into CD45.2+ control or antibiotics-treated mice, and monitored the percentages of CD62Llow CXCR4high population in CD45.1+ donor neutrophils. Strikingly, the aging process of donor neutrophils was significantly slower in antibiotics-treated recipients (Control / ABX: 78.2 ± 3.4% / 45.7 ± 7.7%, 5h after transfer, P < 0.01; 95.5 ± 0.7 / 67.1 ± 7.8%, 9h after transfer, P < 0.05). Since MyD88 mediates the signaling of most toll-like receptors (TLRs), we analyzed the neutrophil aging phenotypes in LysM-Cre/MyD88-flox mice, in which MyD88 is specifically deleted in the myeloid lineage. Similarly, we observed a dramatic decrease in the numbers of aged neutrophils in these mice (wild-type / LysM-Cre/MyD88-flox: 70.1 ± 22.2 / 23.8 ± 3.7 cells / μL blood, P = 0.08), and also a significantly slower aging process when we transferred blood from LysM-Cre/MyD88-flox mice into wild-type recipients (wild-type / LysM-Cre/MyD88-flox: 15.0 ± 3.1% / 5.0 ± 1.0%, 10min after transfer, P=0.09; 49.7 ± 5.2% / 12.0 ± 1.5%, 5h after transfer, P < 0.05; 59.6 ± 2.0% / 19.5 ± 3.2%, 9h after transfer, P < 0.01). To test whether neutrophil aging was relevant in SCD, we depleted the microbiota in a humanized SCD mouse model (“Berkeley” mice). Five weeks after depletion, we observed a dramatic decrease in the numbers of aged neutrophils (Control SCD / ABX SCD: 4392 ± 574 / 819 ± 358 cells/ μL blood, P < 0.01), and a significant improvement of splenomegaly (Control SCD / ABX SCD: 691.7 ± 46.4 / 453.5 ± 28.5 mg, P<0.05). Further, preliminary data indicate that microbiota depletion could protect against vaso-occlusive crisis induced by TNF-α and surgical trauma, leading to prolonged survival. Taken together, these data suggest that aged neutrophils, modulated by microbiota-derived signals, represent an over-active subset of neutrophils that promotes sickle cell vaso-occlusion. Gaining understanding of neutrophil aging may lead to novel ways to control the manifestations of the disease and its complications.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.