Neutrophils have a short lifespan that is extended after exposure to granulocyte macrophage colony stimulating factor (GM-CSF) or lipopolysaccharide (LPS)1. While the survival is regulated by BCL-2 family proteins2, it is not known which pro-survival proteins are involved. GM-CSF stimulation in neutrophils upregulates A1, but A1-deficient mice showed no defects in this cell type3. MCL-1 is critical for the survival of quiescent neutrophils4,5, but it is not known whether the same holds true after activation. We hypothesized that A1 and MCL-1 have overlapping roles in the survival of activated neutrophils.

We generated mutant mice deficient for A1 and lacking one allele of Mcl-1 (Mcl-1+/–A1–/–). Mcl-1+/–A1–/– mice are grossly normal in the haematopoietic compartment, with only a small reduction in lymphocyte numbers, similar to Mcl-1+/– mice6 (Supplementary Fig. 1A). Loss of A1 did not cause a survival defect in GM-CSF-stimulated neutrophils. Here, we examined the survival of neutrophils activated with LPS plus GM-CSF from A1–/–, Mcl-1+/–, and Mcl-1+/–A1–/– mice. Without stimulation, Mcl-1+/– neutrophils had a significant survival disadvantage compared to their wild-type and A1–/– counterparts and no further decrease in cell survival was observed in Mcl-1+/–A1–/– neutrophils (Fig. 1a). Presumably, this increased apoptosis observed in Mcl-1+/– neutrophils is due to the in vitro conditions, as we saw normal neutrophil numbers in vivo in Mcl-1+/– or Mcl-1+/–A1–/– mice (Supplementary Fig. 1B). After activation with LPS plus GM-CSF, the A1−/− and Mcl-1+/–A1–/– neutrophils exhibited significantly poorer survival, whilst Mcl-1+/– neutrophils behaved similarly to wild-type cells (Fig. 1b). LPS treatment alone was ineffective at promoting a survival advantage and failed to induce neutrophil blasting or upregulate pro-survival MCL-1 expression (Supplementary Fig. 2A–C). GM-CSF treatment alone promoted survival, blasting, and MCL-1 upregulation in wild-type and A1–/– cells3. GM-CSF is known to induce expression of the TLR4 co-receptor CD147. We observed marked upregulation of CD14 on neutrophils after GM-CSF stimulation, and more so after treatment with GM-CSF plus LPS (Supplementary Fig. 2C). Hence, the survival defect of LPS plus GM-CSF-stimulated A1−/− neutrophils could be due to a lack of increased A1 expression, contributing to the survival of activated neutrophils8,9.

Fig. 1
figure 1

Survival analysis of neutrophils from mice with the indicated genotypes cultured in a simple medium (no added cytokines), b after stimulation with 10 ng/mL GM-CSF plus 10 ng/mL LPS, c after treatment with Fc-FASL (0.6 ng/mL), and d after stimulation with LPS plus GM-CSF (10 ng/mL each) and Fc-FASL (0.6 ng/mL). e FASL-specific apoptosis when compared to survival of cells stimulated with LPS plus GM-CSF. Data are from five combined experiments (WT n = 9, A1–/– n = 9, Mcl-1+/– n = 6, Mcl-1+/–A1–/– n = 7, Bid–/– n = 7, and Bid–/–A1–/– n = 7 mice). Statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001) was determined using Student’s t-test at each timepoint compared to WT (*) or A1–/– (†).

Neutrophils are highly sensitive to FAS-induced apoptosis1, but this death is delayed when they are activated by LPS plus GM-CSF1. We analyzed FASL-induced apoptosis with and without LPS plus GM-CSF stimulation in neutrophils from A1 and Mcl-1 mutant mice. Additionally, FASL-induced apoptosis in neutrophils is dependent on caspase-8-mediated activation of the pro-apoptotic BCL-2 family member BID (called tBID)10, which A1 binds to with high affinity11. We therefore also included Bid–/– mice12 as a control in our experiments and, furthermore, generated Bid–/–A1–/– mice in order to examine whether any effects seen in the A1–/– cells were dependent on A1–tBID interactions.

Mcl-1+/– (and Mcl-1+/–A1–/–) neutrophils died quicker than wild-type cells after FASL treatment (Fig. 1c). FASL-induced apoptosis was greater than basal apoptosis in culture (Supplementary Fig. 3). Bid–/– neutrophils were protected from FASL-induced apoptosis10. LPS plus GM-CSF protected both wild-type and Mcl-1+/– neutrophils against FASL-induced killing (Fig. 1d). In contrast, A1–/– and Mcl-1+/–A1–/– neutrophils exhibited significantly more apoptosis across all time points after treatment with FASL in LPS plus GM-CSF-activated neutrophils. Taking into account the increase in apoptosis after LPS plus GM-CSF stimulation in A1–/– neutrophils. We observed a trend towards more FASL-specific apoptosis in the A1-deficient cells, although this only reached statistical significance at 72 h (Fig. 1e). The amount of FASL-specific apoptosis did not differ between Bid–/– and Bid–/– A1–/– cells, indicating that the increased sensitivity of activated A1–/– neutrophils to FASL killing is mediated by tBID. Bid–/–A1–/– neutrophils displayed lower viability than their Bid–/– counterparts, both after LPS plus GM-CSF stimulation (Supplementary Fig. 4) and with the combination of LPS, GM-CSF, and FASL (Fig. 1d), fitting with the role we showed for A1 in promoting cell survival after LPS plus GM-CSF stimulation alone.

Collectively, we demonstrate that upregulation of A1 after stimulation imparts a survival advantage in neutrophils, including FASL-induced apoptosis. However, A1’s role is relatively small, and other factors must also regulate the survival of activated neutrophils. These results suggest a previously unrecognized role for A1 in promoting neutrophil survival in an inflammatory context.