Figure 5. TGFβ and CSF2 drive EGR2 expression.

A. Normalised expression (by DESeq2) of Egr2 by the indicated populations (data obtained from the ImmGen Consortium).

B. Representative expression of Ly6C and CD64 by live CD45⁺CD3^–CD19^–Ly6G^– cells from the lungs of unmanipulated newborn Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice. Data are from one of two independent experiments performed.

C. Histograms show representative expression of EGR2 by CD64⁺ ‘pre-alveolar macrophages’ and Ly6C^hi monocytes from the lungs of unmanipulated newborn Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice and bar chart shows the mean fluorescent intensity (MFI) of EGR2 expression by these cells. Data are from one of two independent experiments performed with 2 (Egr2 ^fl/fl) or 5 (Lyz2 ^Cre/+.Egr2 ^fl/fl) mice per group.

D. Frequency and absolute number of CD64⁺ ‘pre-alveolar macrophages’ from mice in B. Data are pooled from two independent experiments with 4 (Egr2 ^fl/fl) or 8 (Lyz2 ^Cre/+.Egr2 ^fl/fl) mice per group.

E. FACS plots show representative expression of CD11c and SiglecF by CD64⁺ ‘pre-alveolar macrophages’ from mice in B and bar chart shows the MFI of CD11c, SiglecF and CD11b expression by these cells. Data are pooled from two independent experiments with 4 (Egr2 ^fl/fl) or 8 (Lyz2 ^Cre/+.Egr2 ^fl/fl) mice per group.

F. MFI of CD11c and CD11b and relative MFI of SiglecF and EpCAM (relative to cells from d5 old Egr2 ^fl/fl mice) expression by CD11c^hiCD11b^lo alveolar macrophages obtained from unmanipulated Egr2 ^fl/fl or Lyz2 ^Cre/+.Egr2 ^fl/fl mice at the indicated ages. Data are pooled from two independent experiments with 4-9 mice per group. Coloured * denote significance between d5 and 3 and 5 weeks within the Egr2 ^fl/fl (blue) and Lyz2 ^Cre/+.Egr2 ^fl/fl (red) data. **p<0.01, ***p<0.001, ****p<0.0001 (Two-way ANOVA with Tukey’s multiple comparisons test).

G. Representative expression of EGR2 by alveolar macrophages from adult WT (C57BL/6) and Il4ra ^–/– adult mice. Data from one experiment with 4 mice per group.

H. Representative expression of EGR2 and SiglecF by CD11c^hiCD11b^lo alveolar macrophages obtained from lungs of neonatal (d8) Fcgr1 ^iCre/+.Tgfbr2 ^fl/fl and littermate controls. Bar charts show the absolute numbers of CD11c^hiCD11b^lo alveolar macrophages (upper) and the mean frequency of EGR2⁺ cells amongst CD11c^hiCD11b^lo alveolar macrophages (lower). Data are pooled from two independent experiments with 3-7 mice per group. **** p<0.0001 (One-way ANOVA followed by Tukey’s multiple comparisons post-test).

I. Representative expression of EGR2 (left) and MFI of EGR2 (right) by FACS-purified Ly6C^hi monocytes cultured in vitro with recombinant CSF-1 (20ng/ml) or GM-CSF (20ng/ml) for five days. Symbols represent monocytes isolated from individual mice. Data are from 6 Egr2 ^fl/fl (Cre^–) or 3 Lyz2 ^Cre.Egr2 ^fl/fl (Cre⁺) mice per group pooled from two independents experiment. **** p<0.0001 (Two-way ANOVA followed by Tukey’s multiple comparisons post-test). Coloured * denote significance between CSF-1 and GM-CSF within the Egr2 ^fl/fl (blue) and Lyz2 ^Cre.Egr2 ^fl/fl (red) data.

J. Relative expression of Egr2 by alveolar macrophages obtained from Pparg ^fl/fl or Itgax ^Cre.Pparg ^fl/fl mice from the ImmGen Consortium.

K. qPCR analysis of Pparg and Cebpb mRNA by BAL cells from unmanipulated adult Egr2 ^fl/fl or Lyz2 ^Cre.Egr2 ^fl/fl mice. Data represent 2 Egr2 ^fl/fl or 4 Lyz2 ^Cre.Egr2 ^fl/fl mice per group.

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