Durable and controlled depletion of neutrophils in mice

Abstract

Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.

Fig. 1. Intracellular Ly6G staining overcomes surface Ly6G unavailability.

a Schematic representation of the antigen masking related issue. Upon anti-Ly6G treatment, the in vivo delivered antibody remains bound to the surface antigen even after sampling, impairing fluorochrome-labeled additional binding. In contrast, the intracellular antigen remains available. b (top) Representative image acquired with ImageStream of a neutrophil where the Ly6G antigen has been successively and specifically stained on the membrane then in the intracellular compartment. (down) Representative flow cytometry analysis emphasizing the specificity and sensitivity of the intracellular Ly6G staining. c Mice were treated for 24 h with an isotype control (left) or anti-Ly6G (right) antibody, after which the bone marrows were sampled. CD45⁺CD11b⁺Ly6G^intra+ neutrophils are shown in red. In the anti-Ly6G-treated group, cells were not detectable using the extracellular staining because of the antigen masking.

Fig. 2. Newly synthesized neutrophils upon anti-Ly6G have membrane Ly6G paucity.

a Old (>20 weeks) C57BL/6 mice were treated with anti-Ly6G 50 μg daily for 9 days. After sampling, bone marrow and blood cells were re-incubated with 10 μg of anti-Ly6G to saturate the Ly6G antigen, and subsequently stained with a donkey anti-rat PE-labelled antibody to measure the whole amount of Ly6G antigen at the surface of neutrophils. MFI, mean fluorescence intensity. RNA levels for Ly6g on purified neutrophils from the same mice is also shown in the right panel. n = 4 control (Ctr) and 5 anti-Ly6G mice. b (left) Setting for experiments performed on C57BL/6 mice of >20 weeks. (right) Prevalence of CD45⁺CD11b⁺Ly6G^intra+ neutrophils among total CD45⁺ immune cells and their respective BrdU-60h incorporation. c Neutrophil segmentation, which correlates with neutrophil aging, was analyzed on Giemsa-stained blood smears. n = 5 control (Ctr) and 4 treated mice. All the results from (b, c) were acquired from the same blood samples. *p < 0.05, from Mann–Whitney test; error bars represent s.d. Source data are provided as a Source Data file.

Fig. 3. Rationale for an optimized anti-Ly6G-based “Combo” depletion strategy.

a Scheme introducing the “isotype switch” strategy to enhance the anti-Ly6G killing celerity. b Experimental plan to evaluate the impact of anti-antibodies on the ability of anti-Ly6G to bind the Ly6G antigen. Neutrophils from blood were gated as CD45⁺CD11b⁺Ly6G^intra+. The MFI of Ly6G extracellular was then measured. One can see that both anti-IgG immunized mice or mice treated with anti-Ly6G + anti-rat (MAR 18.5) antibodies have more Ly6G antigens available 1.5 days after anti-Ly6G injection. c According to the data obtained in (b), the ideal scheme for double antibody-based neutrophil depletion requires a daily injection of anti-Ly6G. *p < 0.05, **p < 0.01 from Mann–Whitney test; error bars represent s.d. 5–7 control (Ctr) and 4–5 treated mice were used per group. Source data are provided as a Source Data file.

Fig. 4. Combo induces neutrophil turnover and reduces their prevalence.

a Old C57BL/6 mice (>20 weeks) were treated with isotype control antibody (Ctr), anti-Ly6G or the combination strategy (Combo) for 18 days. 60 h before sampling, mice were injected once with 1 mg of BrdU intraperitoneally. b Neutrophil prevalence among all immune cells was assessed in bone marrow, blood, spleen and lung while the impact on their dynamic flow rate was measured with BrdU-60h labelling. Both anti-Ly6G and Combo treatment elicit a shift of BrdU neutrophils from the bone marrow stock to the peripheral tissue, but only the Combo treatment is “fast” enough to also reduce neutrophil prevalence. BM, bone marrow. c,d Validation of protocol efficacy with Trucount flow cytometry analysis in (c) blood and (d) lung tissue. e Scheme representing neutrophil counts in the circulation from a dynamic perspective, with the impact of increased flow rate on the global prevalence of neutrophils depending on the killing celerity of the depletion strategy. *p < 0.05, **p < 0.01 from Mann–Whitney test; error bars represent s.d. b n = 7 control (Ctr) and 5 treated mice per group. c,d n = 5 mice per group. Source data are provided as a Source Data file.