Frontline Science: Coincidental null mutation of Csf2rα in a colony of PI3Kγ-/- mice causes alveolar macrophage deficiency and fatal respiratory viral infection

Abstract

PI3Ks have been identified as key signaling proteins involved in many basic biologic processes in health and disease. Transgenic animals have been essential tools to study the underlying molecular mechanisms in this context and therefore, have been widely used to elucidate the role of these factors in many different settings. More specifically, PI3Kγ, a subunit highly expressed in the hematopoietic system, has been implicated to play an important role in many inflammatory diseases as well as cancer. Here, we report identification of multiple, additional, previously unknown mutations in the genome of a widely used PI3Kγ-deficient (PI3Kγ^-/-) mouse colony. These include a STOP mutation in the GM-CSFRα chain, leading to a complete and specific deficiency in GM-CSF signaling. PI3Kγ^-/- animals consequently lacked alveolar macrophages (AMs) and succumbed rapidly to influenza virus infection. Furthermore, PI3Kγ^-/- mice carried an additional mutation that affects mucin 2 (Muc2) transcripts. This protein is strongly involved in the regulation of colorectal cancer, and indeed, conflicting reports have indicated that PI3Kγ^-/- animals spontaneously develop colorectal tumors. Thus, we uncover previously unknown, confounding factors present in a strain of PI3Kγ^-/- mice, leading to additional deficiencies in important signaling pathways with potentially wide-ranging implications for the interpretation of previous studies. By separating the mutations, we established a unique Csf2ra^-/- mouse model that allows us to study the role of cell intrinsic GM-CSFR signaling in vivo without confounding variables introduced by defective IL-5R and IL-3R signaling in mice lacking the common β chain (Csf2rb).

Keywords: GM-CSF receptor; PI3K; influenza; mucin 2.

Figure 1. Pik3cg^–/– mice are hypersusceptible to influenza virus infection.

(A and B) Pik3cg^tm1Pen deficient and WT control mice were infected i.t. with 50 PFU influenza A virus PR8 and their survival (A) and weight loss (B) was monitored. (C and D) Lung viral titers were determined by plaque-assay in MDCK cells. (E and F) BAL cells from mice at day 9 p.i. were analyzed by FACS. Plots show numbers of (E) NP34-specific (left panel) and total CD8+ T cells (right panel). (F) Serum from mice at day 9 p.i. was assessed for virus-specific IgG2c and IgG1 by ELISA. (G) AM in the BAL were quantified in naïve and infected mice at d3 using flow cytometry. (H) Crisscross chimeras were generated by transfer of bone marrow (BM) from either WT or Pik3cg^tm1Pen mice to lethally irradiated WT and KO recipients. FACS plots in the upper panel show CD11c and CD11b expression of BAL cells from naïve chimeric mice with gated cells indicating AM. Lower panels depict AM derived from WT (CD45.1+) versus Pik3cg^tm1Pen (CD45.2) expression of gated cells. (I) Irradiated WT and Pik3cg^tm1Pen recipients were reconstituted with a mixture of WT and Pik3cg^tm1Pen BM in a 50:50 ratio. BAL cells from naïve mixed chimeric were analyzed by FACS. Histograms show gating strategy for CD45.1 (WT) expression, FACS plots on the right side illustrate CD11c versus CD11b expression of CD45.1-gated cells. N= 3-5 per group. All experiments have been performed at least twice.

Figure 2. AM deficiency is independent of PI3Kγ but caused by mutated Csf2ra gene.

(A) Flow cytometry of AMs in BAL fluid and lungs of C57BL/6, Pik3cg^–/– (Pik3cg^tm1Pen and Pik3cg^tm1Wym) and Pik3cg^KD (Pik3cg^tm1Ehi) mice, gated on CD45+ cells. Numbers adjacent to outlined areas indicate percent CD11c⁺CD11b^int AMs. (B) WT, Pik3cg^–/– (Pik3cg^tm1Wym) were infected i.t. with 50 PFU influenza A virus PR8 and their survival was monitored. (C) Pik3cg^tm1Pen-/- mice were backcrossed once onto C57BL/6 (F1). Shown is a table containing the F2 offspring of the F1xF1 intercross with their Pik3cg genotype and AM phenotype for all analyzed mice. (D) The frequency of homozygous variants shared between three alveolar macrophage-deficient mice. (E) Filtering pipeline for variants detected by whole-genome sequencing. (F) IGV view of the homozygous Csf2ra 2bp deletion. A neighboring point mutation is indicated in green. (G) Density of shared homozygous variants (plotted as a red line) on the distal end of chromosome 19. The region of high density (indicated by a dashed line) represents a haplotype derived from the 129P2/OlaHsd background. The Csf2ra locus is indicated in blue. (H) The last 5 exons of the Csf2ra locus are depicted. The mutations in the Csf2ra^m1Kopf mouse strain are displayed in red and the resulting changes in the amino acid sequence are indicated (WS, WSXWS motif; TM, transmembrane domain). (I) Sanger sequencing results of the Csf2ra region encoding the mutation.

Figure 3. Csf2ra^m1Kopf mice are functional knockouts for the GM-CSFRα.

(A) Flow cytometry analysis of GM-CSFRα on neutrophils in the bone marrow (BM) or blood and monocytes in the blood of WT and Csf2ra^m1Kopf mice using anti-GM-CSFRα or secondary anti-ratIgG2a control. (B) In vitro differentiation of DCs from BM cells of mice as in (A) using GM-CSF or Flt3L. Expression of CD11c and MHCII on live cells is depicted. (C) Total numbers of CD11c⁺MHCII⁺ cells from in vitro DC cultures as in (B). (D) Flow cytometry of eosinophils and neutrophils in the blood after hydrodynamic injection of an IL-5 expression plasmid in WT, Csf2ra^m1Kopf and Csf2rb^–/– mice. Numbers adjacent to outlined areas indicate percent Siglec-F⁺ eosinophils and Ly-6G⁺ neutrophils gated on CD11b⁺ cells. (E) Frequency of eosinophils as gated in (D).

Figure 4. Csf2ra^m1Kopf develop fatal pulmonary alveolar proteinosis during the course of influenza infection.

Groups of mice were infected with 50 PFU influenza virus PR8 before mice were sacrificed for analysis at the time points indicated. (A) BAL cells were harvested at day 9 p.i. before being analyzed by cytospins (left panel, 10x) and FACS. (B) Panels show H&E-stained histological sections of naïve and day 9-infected mice (10x). (C) BAL fluid was obtained at the time points indicated. Total protein content in BAL fluid was determined by BCA Protein assay (Thermo Scientific) according to the manufacturer’s instructions. Cholesterol levels were measured as described [15] All experiments have been performed at least twice with n = 4 per group.

Figure 5. Reconstitution of Csf2ra^m1Kopf cells with GM-CSFRα restores GM-CSF signaling.

(A) Bone marrow cells from Csf2ra^m1Kopf mice were infected with retrovirus encoding Csf2ra followed by IRES-GFP (Csf2ra-IRES-GFP) and stimulated in vitro with GM-CSF. Total numbers of CD11c⁺MHCII⁺ cells on d5 from the culture of Csf2ra-transfected Csf2ra^m1Kopf cells and untransfected WT and Csf2ra^m1Kopf cells. (B) Flow cytometry analysis on d8 of the GM-CSF culture of cells as in (A).