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. 2022 Oct 25;119(43):e2121077119.
doi: 10.1073/pnas.2121077119. Epub 2022 Oct 21.

Human neutrophil development and functionality are enabled in a humanized mouse model

Yunjiang Zheng  1 , Esen Sefik  1 , John Astle  1   2 , Kutay Karatepe  3   4 , Hasan H Öz  5 , Angel G Solis  1   6 , Ruaidhrí Jackson  1   7 , Hongbo R Luo  8   9 , Emanuela M Bruscia  5 , Stephanie Halene  10   11 , Liang Shan  1   12 , Richard A Flavell  1   13
Affiliations

Human neutrophil development and functionality are enabled in a humanized mouse model

Yunjiang Zheng et al. Proc Natl Acad Sci U S A. .

Abstract

Mice with a functional human immune system serve as an invaluable tool to study the development and function of the human immune system in vivo. A major technological limitation of all current humanized mouse models is the lack of mature and functional human neutrophils in circulation and tissues. To overcome this, we generated a humanized mouse model named MISTRGGR, in which the mouse granulocyte colony-stimulating factor (G-CSF) was replaced with human G-CSF and the mouse G-CSF receptor gene was deleted in existing MISTRG mice. By targeting the G-CSF cytokine-receptor axis, we dramatically improved the reconstitution of mature circulating and tissue-infiltrating human neutrophils in MISTRGGR mice. Moreover, these functional human neutrophils in MISTRGGR are recruited upon inflammatory and infectious challenges and help reduce bacterial burden. MISTRGGR mice represent a unique mouse model that finally permits the study of human neutrophils in health and disease.

Keywords: bacterial infection; humanized mouse; innate immunity; neutrophils.

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Conflict of interest statement

Competing interest statement: R.A.F. is an advisor to Glaxo Smith Kline, Zai Labs, and Ventus Therapeutics.

Figures

Fig. 1.
Fig. 1.
G-CSF humanization enhances neutrophil commitment in the bone marrow (BM). (A) Schematic design of human G-CSF knockin replacement. The entire mouse ORF (in blue) is replaced by human ORF (in orange). Both 5′ and 3′ UTR sequences are primarily murine. (B) Relative expression of human and mouse G-CSF mRNA in the bone marrow at 3 h after LPS administration (50 μg, intraperitoneally; n = 5 mice). (C) Human and mouse G-CSF proteins measured in plasma at 3 h after LPS administration (50 μg, intraperitoneally) (MISTRG, n = 12; MISTRGG, n = 11 mice). Data pooled from two independent experiments. (D) Frequencies of human hematopoietic cells (hCD45+), human neutrophils (hCD66b+ SSChi), and mouse neutrophils (mCD45+ SSChi) in the blood (MISTRG, n = 21; MISTRGG, n = 38 mice) and bone marrow (MISTRG, n = 5; MISTRGG, n = 6 mice). (E) Frequencies of human lineages in the blood (MISTRG, n = 14; MISTRGG, n = 20 mice) and the bone marrow (MISTRG, n = 5; MISTRGG, n = 6 mice) at 7 wk postengraftment. (D and E) Data pooled from at least three independent experiments. Mice were irradiated with 150 Rads and intrahepatically injected with 20,000 human CD34+ cells at 1 to 3 d after birth. (B–E) Data are shown as mean ± SEM. P values determined by two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Each dot represents an individual mouse.
Fig. 2.
Fig. 2.
Depletion of murine neutrophils by G-CSFR ablation improves engraftment of human neutrophils in circulation. (A) Schematic design of mouse G-CSFR deletion. The entire G-CSFR ORF and first ∼300 bp of 3′ UTR are deleted. (B) Relative expression of G-CSFR in bone marrow of MISTRG (n = 4), MISTRGG (n = 5), MISTRGGR−/− (n = 6) mice. Hprt was used as a housekeeping gene. (C) Frequencies and numbers of mouse granulocytes (Ly6G+) in blood (Left), bone marrow (Right), and lung (Center) of MISTRG (n = 17 [blood], n = 4 [BM], n = 9 [lung]), MISTRGG (n = 12 [blood], n = 5 [BM], n = 5 [lung]), MISTRGGR+/− (n = 8 [blood], n = 2 [lung]) and MISTRGGR−/− (n = 8 [blood], n = 6 [BM], n = 8 [lung]) mice. Data were representative of at least three independent experiments. Whole lung tissues were analyzed without perfusion. (D–F) Characterization of human engraftment in the blood of MISTRG, MISTRGG, MISTRGGR+/− and MISTRGGR−/− mice at 7 wk postengraftment. Data pooled from at least eight independent experiments. Mice were irradiated with 150 Rads and intrahepatically injected with 15,000∼30,000 human fetal liver CD34+ cells at 1 to 3 d after birth. (D) Frequencies of human hematopoietic cells (hCD45+), human neutrophils (hCD66b+ SSChi), and mouse neutrophils (Ly6G+) (MISTRG, n = 58; MISTRGG, n = 54; MISTRGGR+/−, n = 72; MISTRGGR−/−, n = 58). (E) Numbers of human and mouse neutrophils (MISTRGG, n = 15; MISTRGGR+/−, n = 53; MISTRGGR−/−, n = 35). (F) Frequencies of human immune lineages (MISTRG, n = 58; MISTRGGR+/−, n = 38; MISTRGGR−/−, n = 48). (G) MGG staining of engrafted MISTRGGR−/− blood smears. Neutrophils from two representative mice are shown in the enlarged box. (B–F) Data are shown as mean ± SEM. P values determined by two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Each dot represents an individual mouse.
Fig. 3.
Fig. 3.
Functionalities of reconstituted human neutrophils in MISTRGGR. (A) Representative flow cytometry analysis of phagocytosis by human blood polymorphonuclear leukocytes (PMNs) (Left), reconstituted human neutrophils from the blood of engrafted MISTRGGR−/− (Center Left), mouse neutrophils from the blood of engrafted MISTRG (Center Right), and unengrafted BALB/c (Right). Blood samples were incubated with pHrodo Green E. coli BioParticles Conjugate (10 µg per 50 µL blood) for the indicated times and fluorescent signals were analyzed by flow cytometry. (Top) 0 min; (Middle) 20 min; (Bottom) 90 min. Data are representative of at least two independent experiments. (B) Phagocytosis of P. aeruginosa by human and mouse neutrophils in vitro. Equal numbers of isolated human and mouse neutrophils (100,000 cells per well) from bone marrow of humanized MISTRGGR−/− and human neutrophils from human healthy donors were cultured with live P. aeruginosa at a multiplicity of infection of 1:10 in 37 °C for 30 min. Bacterial counts (Left) and frequencies of internalized bacteria (Right) were measured from culture supernatant and cell lysates separately. (C) Human neutrophils isolated from the bone marrow of MISTRGGR were stimulated with PMA (10 ng/mL) for 1 or 2 h at 37 °C and stained with CellROX Green. ROS production was analyzed by flow cytometry and MFI was calculated. (D) Quantification of NETs-forming neutrophils. Isolated human and mouse neutrophils from engrafted bone marrow were cultured unstimulated or with 20 nM PMA for 4 h at 37 °C. MPO and histone H3 were detected by flow cytometry. Each point represents cells isolated from one individual mouse. Data were pooled from two independent experiments. (E) Ex vivo Chemotactic ability of human neutrophils isolated from engrafted MISTRGGR mice and fresh human bone marrow control were compared using the EZ-taxiscan chamber. Directionality and speed of human neutrophils were measured as they migrated toward IL-8 (1 μM).
Fig. 4.
Fig. 4.
Enhanced bone marrow granulopoiesis in MISTRGGR mice. Bone marrow cells were analyzed at 8 wk postengraftment. (A) Frequencies of human hematopoietic cells (hCD45+) (Left) (MISTRG, n = 10; MISTRGG, n = 12; MISTRGGR+/−, n = 8; MISTRGGR−/−, n = 15 mice), human lineages (Right) (MISTRG, n = 5; MISTRGG, n = 12; MISTRGGR+/−, n = 8; MISTRGGR−/−, n = 8 mice). Data pooled from three independent experiments. (B) Frequencies (Left) and numbers (Right) of human neutrophils (hCD66b+ SSChi), Pre-Neu (hCD49d+ CD101−), and Neu (hCD101+) in the bone marrow of MISTRGG (n = 6 mice), MISTRGGR+/− (n = 8 mice), and MISTRGGR−/− (n = 8 mice). Data pooled from at least two independent experiments. (C) MGG staining of sorted bone marrow Pre-Neu (Left) and Neu (Center and Right) cells. Enlarged boxes highlight various stages of human neutrophil development. (A and B) Data are shown as mean ± SEM. P values determined by two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001). Each dot represents an individual mouse.
Fig. 5.
Fig. 5.
Robust human neutrophil recruitment to the lung upon inflammatory stimuli in MISTRGGR mice. (A) Experimental timeline of LPS nebulization. Seven- to 8-wk-old engrafted mice were treated with LPS nebulization (12.5 mg administered over 15 min). BALF and lung tissues were harvested at 24 h after nebulization. Prior to being killed, mice were injected with anti-human CD45 antibody intravenously to label circulating human cells. Interstitial neutrophils and intravascular neutrophils were distinguished by gating on in vivo versus in vitro hCD45 fluorochrome signals. (B) Quantifications of intravascular and interstitial human neutrophils (hCD66b+ SSChi) (CTRL MISTRG: n = 12; CTRL MISTRGGR−/−, n = 7; LPS MISTRG, n = 14; LPS MISTRGGR−/−, n = 15 mice) and mouse neutrophils (Ly6G+) (CTRL MISTRG: n = 10; CTRL MISTRGGR−/−, n = 7; LPS MISTRG, n = 10; LPS MISTRGGR−/−, n = 10 mice) in lungs of MISTRG and MISTRGGR−/− mice at control steady state or 24 h after LPS nebulization. Data pooled from at least three independent experiments. (C) Frequencies, and numbers of human neutrophils (CTRL MISTRG: n = 12; CTRL MISTRGGR−/−, n = 8; LPS MISTRG, n = 14; LPS MISTRGGR−/−, n = 13 mice) and mouse neutrophils (CTRL MISTRG: n = 9; CTRL MISTRGGR−/−, n = 5; LPS MISTRG, n = 10; LPS MISTRGGR−/−, n = 10 mice) in BAL at control steady state or 24 h after LPS nebulization. Data pooled from at least three independent experiments. (D) MGG staining of cells isolated from BAL at 24 h after LPS nebulization. Arrows: human neutrophils. (E) Frequencies (Top) (CTRL MISTRG: n = 9; CTRL MISTRGGR−/−, n = 20; LPS MISTRG, n = 8; LPS MISTRGGR−/−, n = 9 mice) and numbers (Bottom) (CTRL MISTRG: n = 4; CTRL MISTRGGR−/−, n = 13; LPS MISTRG, n = 8; LPS MISTRGGR−/−, n = 9 mice) of human neutrophils (hCD66b+ SSChi), preNeu (hCD49d+ CD101−), and Neu (hCD101+) in the bone marrow of engrafted mice at steady state or 24 h after LPS nebulization. Data pooled from at least two independent experiments. (B–D and F) Data are shown as mean ± SEM. P values determined by two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Each dot represents an individual mouse.
Fig. 6.
Fig. 6.
Human neutrophils in MISTRGGR mice provide protection against P. aeruginosa infection. (A) Experimental timeline of intranasal P. aeruginosa infection. Engrafted 7- to 8-wk-old MISTRG and MISTRGGR−/− mice were pretreated with anti-mouse Ly6G antibody (1A8) (two doses of 100 µg, intravenously) for depletion of murine neutrophils, and then intranasally infected with 500 or 5000 CFU of P. aeruginosa. Lungs and livers were harvested for CFU quantification and flow cytometric characterization at 18 h after infection. (B and C) Frequencies and numbers of human neutrophils in the lung (B) and BAL (C) of infected mice (500 CFU, 18 h postinfection) (MISTRG, n = 6; MISTRGGR−/−, n = 5 mice). Data pooled from at least two independent experiments. (D) Frequencies of human lineages (MISTRG, n = 6; MISTRGGR−/−, n = 5 mice) in intravascular and interstitial lung postinfection (500 CFU, 18 h postinfection). (E) Bacterial load in lung (Left) and liver (Right) at 18 h after infection (5,000 CFU MISTRG, n = 12; MISTRGGR−/−, n = 7; 500 CFU MISTRG, n = 13; MISTRGGR−/−, n = 9 mice). Data pooled from at least three independent experiments. (B–E) Data are shown as mean ± SEM. P values determined by two-tailed Mann–Whitney test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Each dot represents an individual mouse.
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