Table 1.
Depletion methodologies during VACV infection.
Fig 1.
Kinetics of VACV spread and cell populations following intradermal VACV infection.
C57BL/6 mice were infected intradermally with 10,000 pfu VACV in each ear pinna. (A) Mice were given a single injection of CLL i.v. 4 hours pre-infection, and their ear pinnae was surgically removed at the times shown post-infection. 5 days post-infection, ovaries were harvested and used in a plaque assay for VACV titer. (B) Mice were treated with a single injection of CLL i.v. 24 hours pre-infection via intradermal infection or scarification of the ear in the presence of 106 pfu VACV. (C) Mice were given a single injection of CLL i.v. at the time shown pre- or post-infection. 5 days post-infection, ovaries were harvested and used in a plaque assay for VACV titer. (D) Mice were given a single injection of CLL i.v. at the time shown pre-infection. 5 days after infection, ovaries were harvested and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–14).
Fig 2.
Depletion of local myeloid cell populations increases local pathogenesis and tissue damage.
Mice on the C57BL/6 background were infected intradermally with 10,000 pfu VACV in each ear pinna. (A-C) MaFIA mice (n > 8) were injected with CLL i.v. as described in Fig 1A or AP20187 i.p. on Day 0 pre-infection and Days 1, 3 and 4 post-infection. At 5 dpi, ear pinnae were isolated and cells extracted. Flow cytometry was used to count the number of CD11b+ Ly6C++ Ly6G- inflammatory macrophages, CD11b+ Ly6C+ Ly6G+ tissue-protective myeloid cells, and total CD11b+ myeloid cells. (D-F) LysMcre:iDTR mice (n = 4) were injected i.p. with DT (40 ng/g) or vehicle on the day of infection. At 5 dpi, ear pinnae were isolated and cells extracted. Flow cytometry was used to count the number of CD11b+ Ly6C++ Ly6G- inflammatory macrophages, CD11b+ Ly6C+ Ly6G+ tissue-protective myeloid cells, and total CD11b+ myeloid cells. (A-F) In order to compile data across many experiments, data are expressed as % of the mean number of cells in untreated mice. Results include all data from a minimum of 3 independent experiments (n = 8–14). (G-J) Mice were treated as follows and VACV lesion size measured daily. (G) MaFIA mice (n = 5) were injected with AP20187 or vehicle as described in Materials and Methods. (H) LysMcre:iDTR mice (n = 5) were injected with DT (40 ng/g) i.p. or vehicle on Day 0 pre-infection and Days 2, 5, 8, 11 and 14 post-infection. (I) C57BL/6 mice (n = 5) were injected with CLL or vehicle i.v. on Day 0 pre-infection and Days 1, 3, 4, 11 and 18 post-infection. (J) CCR2-/- or C57BL/6 mice were infected with VACV as above. All VACV pathogenesis data is representative of 3 independent experiments. (K) At 5 dpi, ear pinnae were isolated and cells extracted from C57BL/6 or CCR2-/- mice. Flow cytometry was used to count the number of CD11b+ Ly6C++ Ly6G- inflammatory macrophages and CD11b+ Ly6C+ Ly6G+ tissue-protective myeloid cells. (L) C57BL/6 (treated or untreated with CLL), MaFIA or LysMcre:iDTR mice were infected as above. Ears were harvested 5 days post-infection and virus was quantified using a standard plaque assay.
Fig 3.
Depletion of local myeloid cell populations does not allow systemic spread of VACV, but does affect survival following ECTV infection.
Mice (n ≥ 5) on the C57BL/6 background were infected intradermally with 10,000 pfu VACV in each ear pinna. (A) LysMcre:iDTR mice were injected with CLL i.v., DT (40 ng/g) i.p., or vehicle i.p., on day 0 pre-infection and days 1, 3 and 4 post-infection. At 5 or 7 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–14). (B) MaFIA mice were injected with CLL i.v. on Day 0 pre-infection and Days 1, 3 and 4 post-infection, or AP20187 as described in Materials and Methods. At 5 or 7 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–14). (C) Ovaries were harvested from wild-type, CCR2-/-, or CX3CR1-/- mice 3, 5, 7 or 9 dpi and used in a plaque assay for VACV titer. Results include all data from a minimum of 3 independent experiments (n = 8–12). (D) Wild-type C57BL/6 mice were injected with CLL i.v. on Day 0 pre-infection, or the anti-Ly6G antibody 1A8 (50 μg/g) on Days -4, -2 and 0 pre-infection, or the anti-Thy1 antibody T24 (30μg/g) on day -1 pre-infection and 3 dpi. At 5 dpi, ovaries were harvested and used in a plaque assay for VACV titer. Results include data from 3 independent experiments (n = 9). (E-G) Mice were infected in the footpad with 3,000 pfu ECTV, and survival was monitored for 2 weeks post-infection. (E) LysMcre:iDTR or wild-type mice were injected i.p. with 40 ng/g DT or vehicle on days -3, -2, and -1 pre-infection and days 2, 5, 8 and 11 post-infection. (F) MaFIA mice or wild-type mice were injected i.p. with 10 μg/g AP20187 or vehicle as described in Materials and Methods. (G) C57BL/6 or CCR2-/- mice were left undepleted and survival was monitored. All ECTV graphs represent pooled data from 2 to 4 experiments (n = 10–20).
Fig 4.
Draining lymph node macrophage populations do not prevent systemic spread of VACV.
Naïve LysMcre:iDTR mice (n = 8–14 from >3 experiments) were given a single injection of CLL i.v., DT i.p., or vehicle i.v.. At 1 day post-depletion, cervical LN (A-F) were isolated and cells extracted. Flow cytometry was used to count the total number of (A) macrophage/monocytes (F4/80+ CD11c- CD11b+ Ly6G-), (B) inflammatory monocytes (F4/80- CD11c- CD11b+ Ly6C++ Ly6G-), (C) neutrophils (F4/80- CD11c- CD11b+ Ly6C+ Ly6G+), (D) CD8+ DC (CD11c+ F4/80- CD8+ CD11b-), (E) DC (CD11c+ F4/80-), and (F) CD11b+ DC (CD11c+ F4/80- CD11b+ CD8-). (A-F) In order to compile data across many experiments data are expressed as % of the mean number of cells in untreated mice. Results include all data from a minimum of 3 independent experiments (n = 8–14). (G) Naïve LysMcre:iDTR mice (n = 3–4) were given a single injection of CLL i.v. or i.d., DT (100 ng/g) i.p., or vehicle i.v. or i.d.. At 1 day post-depletion, LN were isolated and flash-frozen in OCT compound. Then 10–12 micron sections were fixed using acetone and stained with antibodies to CD169 (red) and SIGNR1 (green). Results are representative of those from 3 independent experiments (n = 6). (H) C57BL/6 mice were infected intradermally with 10,000 pfu VACV in each ear pinna. Mice were given intradermal injections of CLL (25 μl/ear) or intravenous injections of CLL (250 μl/mouse) at the time shown pre- or post-infection. 5 days after infection, ovaries were harvested and used in a plaque assay for VACV titer. (I) Mice were given a single injection of CLL i.v. or vehicle and infected intradermally with 10,000 pfu VACV in each ear pinna. 5 days after infection, D-LN were harvested and used in a plaque assay for VACV titer. Results include all data from 3 independent experiments (n = 6–8).
Fig 5.
VACV infects splenic metallophilic MZ and MZ macrophages.
(A) C57BL/6 mice were infected i.v. with 100–100,000 pfu VACV and ovaries were harvested and titered at 5 days post-infection. (B) C57BL/6 mice were infected i.v. with 10,000 pfu VACV-GFP. Spleens were harvested at 6 hours post infection and flash-frozen in OCT compound. Then 10–12μm sections were fixed and stained with antibodies to CD169 (purple) and SIGNR1 (red). (C) Quantification of fluorescent microscopy of infected cell populations in the spleen at 6hr post infection with VACV-GFP. Results are representative of those from 3 independent experiments (n = 6).
Fig 6.
Splenic MZ macrophages are only depleted by conditions that allow systemic spread of VACV.
Naïve LysMcre:iDTR mice (n = 8–12 from >3 experiments) were given a single injection of CLL i.v., DT i.p., or vehicle i.v.. At 1 day post-depletion, spleens (A-F) were isolated and cells extracted. Flow cytometry was used to count the total number of (A) macrophage/monocytes (F4/80+ CD11c- CD11b+ Ly6G-), (B) inflammatory monocytes (F4/80- CD11c- CD11b+ Ly6C++ Ly6G-), (C) neutrophils (F4/80- CD11c- CD11b+ Ly6C+ Ly6G+), (D) CD8+ DC (CD11c+ F4/80- CD8+ CD11b-), (E) DC (CD11c+ F4/80-), and (F) CD11b+ DC (CD11c+ F4/80- CD11b+ CD8-). (A-F) In order to compile data across many experiments data are expressed as % of the mean number of cells in untreated mice. Results include all data from a minimum of 3 independent experiments (n = 8–14). (G) Naïve LysMcre:iDTR mice (n = 3–4) were given a single injection of CLL i.v. or i.d., DT i.p., or vehicle i.v. or i.d.. At 1 day post-depletion, LN were isolated and flash-frozen in OCT compound. Then 10–12 micron sections were fixed using acetone and stained with antibodies to CD169 (red) and SIGNR1 (green). Results are representative of those from 3 independent experiments (n = 8).
Fig 7.
Systemic macrophages, but not DC, are crucial for blocking VACV dissemination during infection that bypasses the D-LN.
(A–C) Mice were treated as follows and at 5 days post-infection, ovaries were harvested and used in a plaque assay for VACV titer. (A) Naïve CD11ccre:iDTR mice (n = 8–10) were given a single injection of CLL i.v., DT (20 ng/g) i.p., or vehicle i.v. 24 hours pre-infection. (B) C57BL/6 or Batf3-/- mice were infected i.v. with 1000 pfu VACV. As a positive control, half the C57BL/6 mice were pre-depleted with CLL i.v. (n = 6–10) (C) C57BL/6 mice were infected with 1000, 10,000 or 100,000 pfu VACV i.v. or i.d.. 24 hours pre-infection, mice were injected with 250 μl CLL or HBSS i.v.. (D) Mice were pre-depleted with CLL i.v. and infected i.v. with 1000 pfu VACV, then spleen or ovaries were harvested at 0, 2 or 5 days post infection and a plaque assay used to determine VACV titer.