Figure 3. R848-induced monocytosis is driven by TLR7 activation of myeloid cells.

(A, B) Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, monocyte subpopulation ratio, and lymphocytes at baseline and 24 hr after the indicated treatment. (A) C57BL/6 mice (n = 5, black triangles) and Rag2^-/- mice (n = 6, blue circles) received four topical treatments with R848. (B) C57BL/6 mice (n = 6, black rhombi) and Tlr7^fl × Lyz2^Cre/+ mice (n = 5, red triangles) received six topical treatments with R848. (C) C57BL/6 mice (n = 3 per group) were treated once or twice with topical R848 or acetone. Treated ear skin was harvested at 24 hr post-treatment and anal- ysed by flow cytometry. Proportion of monocytes (CD11b+Ly6C+Ly6G-low) and neutrophils (CD11b+Ly6G-high- Ly6C-low) among total live cells (left panels). Representative flow cytometry plots gated on CD11b+ cells (right panels). Data represent a single experiment (A, C) or two experiments (B). Two-way ANOVA, with Tukey’s multiple-comparison for time-course experiments (A, B); unpaired t-test (C). Data are the mean ± SEM; only significant p-values are indicated; *p<0.05; **p<0.01.

Figure 3—figure supplement 1. The role of myeloid cell populations in R848-driven monocytosis.

(A) Lethally irradiated WT or Tlr7^-/- host mice were reconstituted with bone marrow (BM) from WT or Tlr7^-/- donor mice as follows: WT BM into WT host (n = 5, open circles), WT BM into Tlr7^-/- host (n = 5, red triangles), Tlr7^-/- BM into WT host (n = 5, crosses), and Tlr7^-/- BM into Tlr7^-/- host (n = 5, red squares). Each group received 4× topical treatments with R848. Total blood monocytes, Ly6C-high monocytes, and Ly6C-low monocytes are shown as a proportion of CD45+ cells, at baseline and 24 hr after the indicated treatments. (B–D) Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and 24 hr after the indicated R848 treatments. (B) BALB/c (n = 4, black triangles) or ΔdblGATA (n = 6, red squares) mice received six topical treatments with R848. (C) BALB/c mice were either treated with topical R848 (n = 4, black squares) alongside a group of Cpa3-Cre^-/+ mice (n = 4, blue triangles). (D) BALB/c mice were treated with combinations of topical R848 and intraperitoneally (IP) anti-Ly6G or anti-FITC isotype control, with the antibody injected 4 hr prior to the first R848 treatment. Treatments were in the following groups: isotype control and R848 (n = 5, red triangles), anti-Ly6G and R848 (n = 4, blue squares), and anti-Ly6G alone (n = 5, black circles). Statistics compare anti-Ly6G+R848 to anti-FITC+R848. Time-course experiments analysed using two-way ANOVA with Tukey’s multiple-comparisons test to compare between groups at a given time point (A–D); one-way ANOVA with Bonferroni’s multiple-comparison test for comparing >2 groups at a single time point; unpaired t-test for comparing two groups at a single time point. Data are the mean ± SEM; only significant p-values are indicated; * p<0.05, **p<0.01, ***p<0.001.