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. 2023 Aug 11;12:e85647. doi: 10.7554/eLife.85647

Figure 2. R848-induced monocytosis is specific to TLR7 activation.

Blood counts are shown for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, monocyte subpopulation ratio, and lymphocytes 24 hr after the indicated treatment. (A) BALB/c mice (n = 4 per group) received eight treatments with topical R848 (100 µg, black line), LPS (100 µg, blue line), or Poly l:C (100 µg, red line). (B) C57BL/6 mice received six treatments with topical R848 (100 µg, n = 5, black line) or CpG (100 µg, n = 3, red line). (C) BALB/c mice (n = 4 per group) received four topical treatments with 2.5 nmol TPA. (D) C57BL/6 mice (n = 5, black triangles) or Tlr7-/- mice (n = 5, red circles) received four treatments with topical R848. Data representative of two independent experiments (except A and C). Two-way ANOVA with Tukey’s multiple-comparison for time-course experiments (A, B); unpaired t-test (C, D). Data are the mean ± SEM; only significant p-values are indicated; *p<0.05; **p<0.01; ***p<0.001.

Figure 2—source data 1. FACS raw data.

Figure 2.

Figure 2—figure supplement 1. IFNs and cytokines are not involved in the R848-induced monocytosis.

Figure 2—figure supplement 1.

(A) C57BL/6 (black triangles) or Ifng-/- mice (red squares) were either left untreated (n = 4 WT, n = 2 Ifng-/-) or given 4× topical treatments with R848 (n = 3 per group). Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and at 24 hr after the indicated treatment. Data representative of two independent experiments. (B) C57BL/6 (n = 4, black squares) or Ifnar1-/- (n = 5, blue triangles) mice received eight topical treatments with R848. Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and 24 hr after the indicated treatments. (C) BALB/c mice were treated daily intraperitoneally (IP) with etanercept (anti-TNFα) (n = 6, blue squares) or PBS (n = 6, open circles) and then received 4× topical R848 treatments. Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and at 24 hr after the indicated treatment. (D) BALB/c mice received the following combinations: topical R848 and anakinra daily IP (n = 4, open circles), topical R848 and daily PBS IP (n = 4, black triangles), and topical acetone and anakinra daily IP (n = 4, red squares). Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and at 24 hr after the fourth treatment of R848. (E) BALB/c mice were treated IP with PBS (n = 6, open circles) or anti-IL6R (n = 6, blue squares) or the isotype control (n = 6, red triangle) every 3 d and received 4× topical R848 applications. Blood counts for total monocytes, Ly6C-high monocytes, Ly6C-low monocytes, and the monocyte subpopulation ratio at baseline and at 24 hr after the fourth treatment with R848. Time-course experiments analysed using two-way ANOVA with Tukey’s multiple-comparison test to compare between time points (A–C); one-way ANOVA with Bonferroni’s multiple-comparison test for comparing >2 groups at a single time point calculated (D, E). Data are the mean ± SEM; only significant p-values are indicated; *p<0.05, **p<0.01, ***p<0.001.