Neutrophil Aging, Regulated By Microbiota-Derived Signals, Promotes Sickle Cell Vaso-Occlusion

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 α_Mβ₂ 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 CD62L^low CXCR4^high 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 CD62L^low CXCR4^high 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 CD62L^low CXCR4^high 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 CD62L^low CXCR4^high 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.