Mouse strains with specific deficiency of given hematopoietic lineages provide invaluable tools for understanding blood cell function in health and disease. Whereas neutrophils are dominant leukocytes in humans and mice, there are no widely useful genetic models of neutrophil deficiency in mice. In this study, we show that myeloid-specific deletion of the Mcl-1 antiapoptotic protein in Lyz2Cre/CreMcl1flox/flox (Mcl1ΔMyelo) mice leads to dramatic reduction of circulating and tissue neutrophil counts without affecting circulating lymphocyte, monocyte, or eosinophil numbers. Surprisingly, Mcl1ΔMyelo mice appeared normally, and their survival was mostly normal both under specific pathogen-free and conventional housing conditions. Mcl1ΔMyelo mice were also able to breed in homozygous form, making them highly useful for in vivo experimental studies. The functional relevance of neutropenia was confirmed by the complete protection of Mcl1ΔMyelo mice from arthritis development in the K/B×N serum-transfer model and from skin inflammation in an autoantibody-induced mouse model of epidermolysis bullosa acquisita. Mcl1ΔMyelo mice were also highly susceptible to systemic Staphylococcus aureus or Candida albicans infection, due to defective clearance of the invading pathogens. Although neutrophil-specific deletion of Mcl-1 in MRP8-CreMcl1flox/flox (Mcl1ΔPMN) mice also led to severe neutropenia, those mice showed an overt wasting phenotype and strongly reduced survival and breeding, limiting their use as an experimental model of neutrophil deficiency. Taken together, our results with the Mcl1ΔMyelo mice indicate that severe neutropenia does not abrogate the viability and fertility of mice, and they provide a useful genetic mouse model for the analysis of the role of neutrophils in health and disease.

Objective Gain‐of‐function mutations and genome‐wide association studies have linked phospholipase Cγ2 (PLCγ2) to various inflammatory diseases, including arthritis in humans and mice. PLCγ2‐deficient (Plcg2–/–) mice are also protected against experimental arthritis. This study was undertaken to test how PLCγ2 triggers autoantibody‐induced arthritis in mice. Methods PLCγ2 was deleted from various mouse cellular lineages. Deletion efficacy and specificity were tested by immunoblotting and intracellular flow cytometry. Autoantibody‐induced arthritis was triggered by K/BxN serum transfer. The role of neutrophil PLCγ2 was further investigated by analysis of the inflammatory exudate, competitive in vivo migration assays, and in vitro functional studies. Results PLCγ2 deficiency in the entire hematopoietic compartment completely blocked autoantibody‐induced arthritis. Arthritis development was abrogated by deletion of PLCγ2 from myeloid cells or neutrophils but not from mast cells or platelets. Neutrophil infiltration was reduced in neutrophil‐specific PLCγ2‐deficient (Plcg2ΔPMN) mice. However, this was not due to an intrinsic migration defect since Plcg2ΔPMN neutrophils accumulated normally when wild‐type cells were also present in mixed bone marrow chimeras. Instead, the Plcg2ΔPMN mutation blocked the accumulation of interleukin‐1β, macrophage inflammatory protein 2 (MIP‐2), and leukotriene B4 (LTB4) in synovial tissues and reduced the secondary infiltration of macrophages. These findings were supported by in vitro studies showing normal chemotactic migration but defective immune complex–induced respiratory burst and MIP‐2 or LTB4 release in PLCγ2‐deficient neutrophils. Conclusion Neutrophil PLCγ2 is critical for arthritis development, supposedly through the generation of the inflammatory microenvironment. PLCγ2‐expressing neutrophils exert complex indirect effects on other inflammatory cells. PLCγ2‐targeted therapies may provide particular benefit in inflammatory diseases with a major neutrophil component.

Background and objectives Rheumatoid arthritis is a chronic autoimmune disease with a prevalence of 1%. The pathomechanism of the disease is not completely understood. Previous studies from our laboratory showed that phospholipase Cγ2 (PLCγ2) is a critical player of the effector phase of the autoantibody-mediated K/BxN serum transfer arthritis model. Prior studies also showed that myeloid cells such as neutrophils and macrophages are essential for arthritis development in this model. The aim of our experiments was to investigate the role of PLCγ2 expression within myeloid cells during K/BxN serum transfer arthritis. Materials and methods Lineage-specific deletion of PLCγ2 was achieved by crossing LysM and MRP8 promoter-driven Cre recombinase (LysMCre/Cre and MRP8Cre/Cre) transgenic mice with PLCγ2flox/flox animals. PLCγ2 expression in bone marrow neutrophils and bone marrow-derived macrophages was tested by Western blot. Mice were injected with arithrogenic (K/BxN) or control serum intraperitoneally. The development of arthritis was assessed by clinical scoring of the visible signs of the inflammation, measuring ankle thickness and testing articular function by a hanging test. Results As expected, LysM promoter-driven Cre recombinase deleted PLCγ2 from both neutrophils and macrophages whereas MRP8 promoter-driven Cre recombinase deleted PLCγ2 from neutrophils but not macrophages. Although wild type mice injected with arithrogenic serum develop severe arthritis, no sings of the disease was assessed in similarly treated LysMCre/CrePLCγ2flox/floxanimals. MRP8Cre/CrePLCγ2flox/flox mice were also almost completely protected from arthritis development. Conclusions Our results indicate that the expression of PLCγ2 in myeloid cells and in particular neutrophils indispensable for the development of autoantibody-induced arthritis in experimental mice.

Background and objectives Genetic deletion of specific leukocyte lineages strongly contributes to understanding the role of various leukocyte subsets in physiological and pathological conditions. There have been a number of attempts to generate genetically neutrophil-deficient mouse strains. However, all those strains suffer from substantial limitations such as the limited efficiency of neutrophil deletion, the effect of the mutations on other lineages or the limited survival of the mutant mice. We have previously shown that the antiapoptotic Mcl-1 protein is essential for the survival of neutrophils but not macrophages. Therefore, we tested whether myeloid-specific deletion of Mcl-1 could provide a novel and more suitable genetic model of neutrophil deficiency in experimental mice. Materials and methods LysMCre mice expressing Cre recombinase in the myeloid compartment were crossed with animals carrying the Mcl-1flox mutation to obtain LysMCre/CreMcl-1flox/flox (referred to as Mcl-1ΔMyelo) mice characterised by myeloid-specific conditional deletion of Mcl-1. Leukocyte populations in the peripheral blood, spleen and bone marrow of intact Mcl-1ΔMyelo mice and Mcl-1ΔMyelo bone marrow chimaeras were tested by flow cytometry. K/BxN serum-transfer arthritis was used as a neutrophil-dependent in vivo inflammation model. Arthritis severity was followed by clinical scoring, ankle thickness measurement and assessment of the articular function. Results Mcl-1ΔMyelo mice are viable and fertile without any gross abnormalities, even in homozygous form. The mice have a strong (>95%) reduction of circulating neutrophil counts and severe deficiency of splenic and bone marrow neutrophils. However, other leukocytes such as circulating eosinophils, monocytes, T- or B cells, or splenic dendritic cells and macrophages are not affected. Intact Mcl-1ΔMyelo mice and Mcl-1ΔMyelo bone marrow chimaeras are completely protected from all signs of arthritis development in the K/BxN serum-transfer model. Conclusions The Mcl-1ΔMyelo mutation leads to dramatic reduction of the number of circulating and tissue neutrophils without affecting other blood and splenic leukocyte lineages. The Mcl-1ΔMyelo mice are viable and fertile in homozygous form but are completely protected from arthritis development in a known neutrophil-dependent in vivo disease model. Taken together, the Mcl-1ΔMyelo mice may provide a useful novel model of genetically determined neutrophil deficiency.