Improved mouse models and advanced genetic and genomic technologies for the study of neutrophils

Abstract

Mice have been excellent surrogates for studying neutrophil biology and, furthermore, murine models of human disease have provided fundamental insights into the roles of human neutrophils in innate immunity. The emergence of novel humanized mice and high-diversity mouse populations offers the research community innovative and powerful platforms for better understanding, respectively, the mechanisms by which human neutrophils drive pathogenicity, and how genetic differences underpin the variation in neutrophil biology observed among humans. Here, we review key examples of these new resources. Additionally, we provide an overview of advanced genetic engineering tools available to further improve such murine model systems, of sophisticated neutrophil-profiling technologies, and of multifunctional nanoparticle (NP)-based neutrophil-targeting strategies.

Figure 1.

(a) Humanization of immunodeficient IL-2 receptor common γ-chain (Il2rg)^null mice can be achieved by generating the (i) Hu-SRC-SCID model, through injection of human CD34+ hematopoietic stem cells (HSCs) obtained from bone marrow, or granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood, or fetal liver, or umbilical cord blood, into newborn or adult NSG mice, which results in the development of a complete functional human immune system; (ii) Hu-PBL-SCID model, through injection of human peripheral blood leukocytes (PBLs) into adult NSG mice, which results in engraftment and expansion of human T cells within one week. While engrafted mice succumb to xenogeneic graft-versus-host disease (GVHD) within 4–8 weeks, lack of murine major histocompatibility complex (MHC) class I or class II molecules can significantly delay GVHD development; or (iii) bone marrow/liver/thymus (BLT) model, through transplantation of fragments of human fetal liver and thymus under the kidney capsule, followed by intravenous injection of autologous fetal liver CD34+ HSCs. (b) Patient-derived xenograft (PDX) models: PDX models are generated by implanting patient-derived tumors in severely immunodeficient NSG mice. Utilizing humanized mice for human tumor implantation enables the study of human tumor–immune cell interactions and the testing of immunotherapies. Abbreviation: NK cells, natural killer cells.

Figure 2.

Knock-in of large transgenes into the mouse genome using site-specific recombinases: phiC31 integrase utilizes the ~50-bp DNA attachment sites attP and attB to integrate exogenous DNA. Use of CRISPR/Cas9-mediated oligonucleotide homology-directed repair (HDR) placement of the attachment site attP (e.g., attP selectively placed in the Rosa26 locus) using electroporation into NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) zygotes (Step 1), followed by the use of a site-specific serine integrase phiC31 mRNA and donor DNA plasmid or minicircle (transgene) containing the attachment site attB can mediate highly efficient recombination between the attP and attB sequences to catalyze efficient precision transgenesis of large donor constructs (Step 2). Successful recombination results in irreversible unidirectional insertion of the transgene, flanked by attL (left) and attR (right) sites. Abbreviation: sgRNA, small guide RNA.

Figure 3.

The Collaborative Cross (CC) recombinant inbred mouse strains are generated by an eight-way funnel breeding design involving eight genetically diverse parental inbred strains, which include five common laboratory strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ, and NZO/HiLtJ) and three wild-derived strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ). The Diversity Outbred (DO) mouse population is derived from the same eight parental inbred strains. Whereas CC mice are recombinant inbred strains and can be phenotyped multiple times, DO mice are genetically unique and cannot be replicated.

Figure 4.

Mouse models of human disease continue to be invaluable tools for advancing our understanding of human immunology. Embracing new resources, such as humanized mice, patient-derived xenograft (PDX) mice, recombinant inbred strains, and highly diverse outbred mice, can overcome the limitations of either a lack of the genetic diversity that is observed in human populations, or a conducive environment for the study of human tumor–immune interactions. In addition, harnessing new genetic tools, such as site-specific recombinases for knock-in of large transgenes, single-cell technologies to profile the molecular and functional heterogeneity of neutrophils, and systems biology approaches, enables the rapid identification and development of novel therapeutic strategies for targeting of neutrophils in inflammation and cancer. Furthermore, the various mouse resources and the application of computational tools will be essential for testing hypotheses generated from large-scale human data sets. Abbreviations: NSG, NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ.