Figure 2. Reconstitution of Skap2-/- mice with WT bone marrow hematopoietic stem cells confers protection against K. pneumoniae.

(A) Schematic used to generate bone marrow chimeras in (B–D, F). (B–F) Mice were infected with 5 × 10³ cfu K. pneumoniae (red asterisk); 24 hpi mice were sacrificed and lungs harvested. (E–F) WT and Skap2-/- mice were injected intraperitoneally with 50 μg of α-Ly6G (1A8) or 20 μg of α-CCR2 (MC-21) to deplete neutrophils and iMOs, respectively, or PBS 16 hr prior to infection. Bacterial burden (B, E, F) and percent live neutrophils (CD11b⁺ Ly6G^hi) (C), or inflammatory monocytes (CD11b⁺ Ly6C^hi) (D) from K. pneumoniae-infected lungs. Data are compiled from 2 to 4 independent experiments using groups of 2–3 mice/genotype/experiment. Each dot represents a mouse, bars are geometric means (B, E–F) or means (C–D). Statistics were assessed using one-way ANOVA with (C–D) Sidak’s post-test, or (B, E–F) Tukey’s post-test. (D) Percent of iMOs were compiled, and comparison between WT and Skap2-/- irradiated recipient disregarding the donor BM were assessed by two-tailed unpaired Student’s t test.

Figure 2—figure supplement 1. Transplantation efficiency of bone marrow reconstitution into WT and Skap2-/- recipients.

(A) Schematic for gating strategy of flow cytometry data in bone marrow chimeras. Blocks and arrows indicate the cell population used for next gating step. (B–H) Neutrophil (CD11b⁺ Ly6G^hi) (C–E), or inflammatory monocytes (CD11b⁺ Ly6C^hi) (F–H) from K. pneumoniae-infected lungs were unstained or intracellularly stained with SKAP2 antibody or isotype control. (B) SKAP2 expression in CD11b⁺ cells shown as mean fluorescent intensity (MFI). Dotted line indicates average level from cells intracellularly stained with isotype control. Data are compiled from 2 to 4 independent experiments using 2–3 mice/genotype/experiment. Each dot represents a mouse, bar represents means. Statistical significance was assessed using one-way ANOVA with Sidak’s post-test.

Figure 2—figure supplement 2. Evaluation of immune cell populations in depletion studies in WT and Skap2-/- mice.

Analysis of inflammatory monocytes (CD11b⁺ Gr1^lo or CD11b⁺ Ly6C^hi Ly6G^lo) and neutrophils (CD11b⁺ Gr1^hi or CD11b⁺ Ly6C^int Ly6G^hi) from non-depleted (PBS), α-Ly6G (clone 1A8)-depleted, or MC-21-depleted (α-CCR2) K. pneumoniae-infected lungs. (A–B) Example of flow cytometry analysis for depletion experiments. (C–F) Quantification of neutrophils (CD11b⁺ Gr1^hi) (C–D), or inflammatory monocytes (CD11b⁺ Ly6C^hi Ly6G^lo) (E–F) from α-Ly6G, α-CCR2, or PBS-treated, and K. pneumoniae-infected WT or Skap2-/- mice based on flow cytometry analysis. (G–J) Quantification of neutrophils (G–H), or inflammatory monocytes (I–J) from α-Ly6G, α-CCR2, or PBS-treated, K. pneumoniae-infected irradiated Skap2-/- mice based on flow cytometry analysis. Data are compiled from 3 to 4 independent experiments with 2–3 mice/genotype/experiment. Each dot represents a mouse, bars represent geometric means. (D, F, H, J) were log-transformed. Significance was assessed using one-way ANOVA with Tukey’s post-test.