6 research outputs found
Depletion methodologies during VACV infection.
<p>Depletion methodologies during VACV infection.</p
Splenic MZ macrophages are only depleted by conditions that allow systemic spread of VACV.
<p>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 <b>(A-F)</b> were isolated and cells extracted. Flow cytometry was used to count the total number of (<b>A</b>) macrophage/monocytes (F4/80<sup>+</sup> CD11c<sup>-</sup> CD11b<sup>+</sup> Ly6G<sup>-</sup>), (<b>B</b>) inflammatory monocytes (F4/80<sup>-</sup> CD11c<sup>-</sup> CD11b<sup>+</sup> Ly6C<sup>++</sup> Ly6G<sup>-</sup>), (<b>C</b>) neutrophils (F4/80<sup>-</sup> CD11c<sup>-</sup> CD11b<sup>+</sup> Ly6C<sup>+</sup> Ly6G<sup>+</sup>), (<b>D</b>) CD8<sup>+</sup> DC (CD11c<sup>+</sup> F4/80<sup>-</sup> CD8<sup>+</sup> CD11b<sup>-</sup>), (<b>E</b>) DC (CD11c<sup>+</sup> F4/80<sup>-</sup>), and (<b>F</b>) CD11b<sup>+</sup> DC (CD11c<sup>+</sup> F4/80<sup>-</sup> CD11b<sup>+</sup> CD8<sup>-</sup>). (<b>A</b>-<b>F</b>) 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). (<b>G</b>) 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).</p
Systemic macrophages, but not DC, are crucial for blocking VACV dissemination during infection that bypasses the D-LN.
<p>(<b>A–C</b>) Mice were treated as follows and at 5 days post-infection, ovaries were harvested and used in a plaque assay for VACV titer. (<b>A</b>) 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>(B)</b> C57BL/6 or Batf3<sup>-/-</sup> 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) <b>(C)</b> 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.. (<b>D</b>) 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.</p
VACV infects splenic metallophilic MZ and MZ macrophages.
<p><b>(A)</b> 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>(B)</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). <b>(C)</b> 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).</p
Depletion of local myeloid cell populations increases local pathogenesis and tissue damage.
<p>Mice on the C57BL/6 background were infected intradermally with 10,000 pfu VACV in each ear pinna. <b>(A-C)</b> MaFIA mice (n > 8) were injected with CLL i.v. as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006435#ppat.1006435.g001" target="_blank">Fig 1A</a> 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<sup>+</sup> Ly6C<sup>++</sup> Ly6G<sup>-</sup> inflammatory macrophages, CD11b<sup>+</sup> Ly6C<sup>+</sup> Ly6G<sup>+</sup> tissue-protective myeloid cells, and total CD11b<sup>+</sup> myeloid cells. (<b>D-F)</b> 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<sup>+</sup> Ly6C<sup>++</sup> Ly6G<sup>-</sup> inflammatory macrophages, CD11b<sup>+</sup> Ly6C<sup>+</sup> Ly6G<sup>+</sup> tissue-protective myeloid cells, and total CD11b<sup>+</sup> myeloid cells. (<b>A</b>-<b>F</b>) 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). <b>(G-J)</b> Mice were treated as follows and VACV lesion size measured daily. <b>(G)</b> MaFIA mice (n = 5) were injected with AP20187 or vehicle as described in Materials and Methods. <b>(H)</b> 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. <b>(I)</b> 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. <b>(J)</b> CCR2<sup>-/-</sup> or C57BL/6 mice were infected with VACV as above. All VACV pathogenesis data is representative of 3 independent experiments. <b>(K)</b> 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<sup>+</sup> Ly6C<sup>++</sup> Ly6G<sup>-</sup> inflammatory macrophages and CD11b<sup>+</sup> Ly6C<sup>+</sup> Ly6G<sup>+</sup> tissue-protective myeloid cells. (<b>L</b>) 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.</p
Depletion of local myeloid cell populations does not allow systemic spread of VACV, but does affect survival following ECTV infection.
<p>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<sup>-/-</sup>, or CX3CR1<sup>-/-</sup> 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<sup>-/-</sup> mice were left undepleted and survival was monitored. All ECTV graphs represent pooled data from 2 to 4 experiments (n = 10–20).</p