Figure 4. EGR2 controls distinct functional characteristics of alveolar macrophages.

A. Representative LipidTox staining of alveolar macrophages from the BAL fluid of unmanipulated adult Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice (left). Graph shows the mean fluorescence intensity (MFI) of LipidTox in alveolar macrophages from Lyz2 ^Cre/+.Egr2 ^fl/fl mice relative to those from Egr2 ^fl/fl mice. Data are from 7 (Egr2 ^fl/fl) and 10 (Lyz2 ^Cre/+.Egr2 ^fl/fl) mice per group pooled from three independent experiments. **p<0.01 (unpaired Student’s t-test).

B. Protein levels in the BAL fluid of Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice at 4 or 9-12 months of age. Data are from 6-9 mice per group pooled from two independent cohorts of aged mice.

C. Frequency of 7-AAD⁺ (dead) cells in the BAL fluid of Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice at 9-12 months of age. Data are from 6-9 mice per group pooled from two independent cohorts of aged mice.

D. Representative expression of SiglecF and EGR2 by CD11c^hiCD11b^lo macrophages and MFI of EGR2 by SiglecF-defined CD11c^hiCD11b^lo macrophages obtained from 11-12 month old Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice. Data are from 5 mice per group pooled from two independent cohorts of aged mice. ** p<0.01, *** p<0.001 (One-way ANOVA followed by Tukey’s multiple comparisons post-test.)

E. Frequency (left) and absolute number (right) of SiglecF⁺ cells amongst CD11c^hiCD11b^lo macrophages obtained from 11-12 month old Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice. Data are from 7 mice per group pooled from three independent cohorts of aged mice. **p<0.01 (unpaired Student’s t-test).

F. Frequency of Ki67⁺ cells amongst SiglecF-defined CD11c^hiCD11b^lo macrophages obtained from 11-12 month old Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice. Data are from 5 mice per group pooled from two independent experiments.

G. Log₂-fold expression of genes that are implicated in lipid uptake or metabolism in alveolar macrophages as defined by (8). Expression of Egr2 is included as a reference.

H. Bacterial levels (colony forming units, CFU) in the BAL fluid of Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice 14hrs after infection. Data are from 10 (Lyz2 ^Cre/+.Egr2 ^fl/fl) or 12 (Egr2 ^fl/fl) mice per group pooled from three independent experiments. *p<0.05 (Mann Whitney test).

I. Frequency (left) and absolute number (right) of CD11c^hiCD11b^lo alveolar macrophages and Ly6G⁺ neutrophils in the BAL fluid of Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice 14hrs after infection. Data represent 10 (Lyz2 ^Cre/+.Egr2 ^fl/fl) or 11 (Egr2 ^fl/fl) mice per group pooled from three independent experiments.

J. Expression of Marco, Wfdc10, Colec12 and C3 from the RNA-seq dataset (left panels). *p<0.05, ***p<0.001, ****p<0.0001 (unpaired Student’s t-test).

Symbols represent individual mice in all graphs and error bars represent the standard deviation.