Numbers of B lineage cells in BM following treatment with RU486 and/or Leptin.

a<p>Following treatment with RU486 (R), RU486 plus leptin (RL), leptin (L), vehicle respectively, mice were infected i.n. with 10⁴ PFU of V (V-) or PBS (Mock-), respectively. The right femurs from six mice per group were collected at day 5. BM cells were prepared and stained with anti-CD45/B220, anti-CD43, anti-IgD and anti-IgM, and analyzed by flow cytometer. The numbers of different B cells populations were calculated by multiplying the percentage of each cell type by the total number of BM viable cells. Data is representative of means of six mice ± SD for three independent experiments..</p>b<p>Indicates statistically significant differences (<i>p</i><0.05) comparing to data in V-vehicle group.</p

Alternation of BM B cells populations following virus infection.

<p>Following infected with V and Ts virus, the BM cells from three mice per group per time point was prepared on day 3, 5 and 7 post-infection and the phenotypic FACS analysis of B220, IgM, IgD, CD43 expressions within BM cells was performed. (A) The numbers of B220⁺ cells within BM; (B) the percentages of BM B lineage cells within B220 gated cells populations. B220^lowIgM⁻ cells define pro-B (B220⁺CD43^high) and pre-B (B220⁺CD43^low/−) cells, B220^lowIgM⁺cells are immature B cells and B220^highIgM⁺ cells are mainly mature naive recirculating B cells. Percentages shown are calculated from total B220⁺ cells (erythrocyte-depleted). The numbers of pro- (C), pre- (D), immature B cells (E) and mature naïve B cells (F) were calculated by multiplying the percentage of each cell type by the total number of BM viable cells. Data shown in graph A, C, D, E, F represents mean ± SD for three independent experiments. Data shown in graph B are two-color dot plots from individual mouse, but are representative of three independent experiments. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V and Ts.</p

Role of sympathetic nervous system in the production of GCs and depletion of spleen and BM B cells.

<p>Following 6-OHDA treatment, six mice were infected i.n. with 10⁴ PFU of V (V-6-OHDA) and another six mice were infected i.n. with PBS (mcok-6-OHDA). Another two groups (6 mice/group) treated with vehicle were infected i.n. with 10⁴ PFU of V (V-alone) or PBS (Mock-vehicle), respectively. (A) GC levels in plasma of each group were measured using ELISA. After infected with V virus, spleen and BM cells were prepared on day 5 post-infection. The proportions (B) and numbers (C) of splenic B220⁺ B cells were determined. The numbers of pro-, pre-, immature B cells and mature naïve B cells from BM (D) were calculated by multiplying the percentage by number of total BM cells. Data bars represent means of 6 mice from each infection group ± SD. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V-vehicle and V-6-OHDA. Ns mean no statistical difference between both.</p

The depletion of B220⁺ B cells in spleen is due to the increased apoptosis and arrested cell cycle.

<p>(A) Splenic cells from mice 5 days after infection with V and Ts were stained with PE-conjugated anti-B220 mAb, annexin V and propidium iodide and analyzed by flow cytometer. Numbers indicate the percentages of early apoptosis cells (Annexin-V⁺) and late apoptosis cells (Annexin-V⁺PI⁺) within B220 gated cells populations. The number was estimated by collecting 10,000 events. (B) After challenging, mice (n = 3/group) were euthanized on 5 days p.i. and the histological paraffin sections of spleens were prepared. Apoptotic cells in histological spleen sections were identified using the TUNEL assay. Magnification, ×200. (C) Following infection, spleen cells were prepared and stained with PE-Cy5-anti-FITC and PI and analyzed by flow cytometer. The results were analyzed using MultiCycle AV software. Each graph is from an individual mouse, but is representative of three mice per group and three independent experiments.</p

Comparison of spleen and BM B cells from individual and both pathways blockade mice.

<p>For this experiment, five groups mice (n = 6/group) were prepared. One group (V-6) mice was administrated with 6-OHDA before infection; Second group (V-R) was administrated with RU486 daily during infection; Third group (V-R6) was administrated with 6-OHDA before infection and RU486 daily during infection; Forth group (V-vehicle) was administrated with vehicle (2% ethyl alcohol ethyl alcohol). All the four groups were received with 104 PFU of V virus. The fifth group (Mock-vehicle) was administrated with vehicle and received with PBS as mock group. At day 5 post-infection, spleen and BM cells were prepared. The proportions (A) and numbers (B) of splenic B220⁺ B cells were determined. The numbers of pro-, pre-, immature B cells and mature naïve B cells from BM (C) were calculated by multiplying the percentage by number of total BM cells. Data bars represent means ± SD. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V-vehicle and V-R/V-6/V-R6.</p

The depletion of B220⁺ B cells in BM is associated with arrested cell cycle.

<p>Following infection, BM cells from day 1, 3, 5, 7 post-infection were prepared and stained with PE-Cy5-anti-FITC and PI and analyzed by flow cytometer. The results were analyzed using MultiCycle AV software. Each graph is from an individual mouse, but is representative of three mice per group and three independent experiments.</p

The infection with V led to the atrophy of spleen and the reduced B220⁺ B cells in spleen, MLN and peripheral blood.

<p>Following infection with V and Ts, the spleen (A), MLN and blood from three mice per group per time point were collected, and the numbers of spleen cells (B) were calculated the percent of B220⁺ B cells were analyzed by flow cytometry. The proportion (C) and amount (D) of B220⁺ B cells in spleen were analyzed on day 3, 5 and 7 post-infection. The amount of B220⁺ B cells in MLN (E) and proportion in peripheral blood; (F) were analyzed on day 1, 3, 5 and 7 post-infection. Baseline from PBS inoculated mice (n = 5) is shown as a dashed line in each graph. The numbers of B220⁺ B cells in spleen and MLN were calculated by multiplying the percentage of each cell type by the total number of their viable cells. The data shown represents mean ± SD for three independent experiments. <b>*</b><i>p</i><0.05 and <b>**</b><i>p</i><0.01 between V and Ts.</p

GCs level in plasma and RU486 treatment.

<p>(A) Mice (n = 12/group) were infected i.n. with 10⁴ PFU of V_K627 (▪), rV_K627E (□), Ts_E627 (▾), and rTs_E627K (∇). The plasma from three mice per virus group per time point was prepared and GCs level in plasma was measured by ELISA. Baseline GCs levels from PBS inoculated mice (n = 5) are shown as a dashed line in graph. <b>*</b><i>p</i><0.05 between V_K627 and rV_K627E; ^Λ<i>p</i><0.05 between Ts_E627 and rTs_E627K; (B–D) Following RU486 treatment, mice (n = 6/group) were infected i.n. with 10⁴ PFU of V_K627 (V_K627-RU) and rTs_E627K (rTs_E627K-RU). The control groups were named with V_K627-C and rTs_E627K-C. All the mice were sacrificed at day 5 p.i. Lungs and thymuses from three mice of each group were removed and single cell suspensions were prepared. The percentages of apoptosis (B) and CD4⁺CD8⁺ thymocytes (C) in thymus and T cells and inflammatory cells in lung were measured by flow cytometry. The numbers of T cells and inflammatory cells (D) were calculated by multiplying the percentage of each cell type by the total number of viable lung cells. Lungs from another three mice in each group were removed and homogenized in 1 ml of PBS. Cytokines levels (D) were measured individually and in duplicate. The data shown in B and C represents three independent experiments and the data shown in A and D represents mean ± SD for three independent experiments. <b>*</b><i>p</i><0.05 between V_K627-RU and V_K627-C; ˆ<i>p</i><0.05 between rTs_E627K-RU and rTs_E627K-C.</p

Weight change and viral burden following RU486 treatment.

<p>Following RU486 treatment, mice (n = 11/group) were infected i.n. with 10⁴ PFU of V_K627 (V_K627-RU) and rTs_E627K (rTs_E627K-RU). (A) Mice (n = 8/group) were weighed daily from 0 day p.i. to 7 day p.i. (B) At 5 day p.i., the lungs from three mice per group were removed and homogenized in 1 ml of PBS, and virus titer was determined by plaque assay. The data shown in A and B represents mean ± SD for three independent experiments. <b>*</b><i>p</i><0.05 between V_K627-RU and V_K627-C; <b>ˆ</b><i>p</i><0.05 between rTs_E627K-RU and rTs_E627K-C.</p

Histopathology and CD4⁺CD8⁺ thymocytes in the thymus.

<p>Mice (n = 6/group) were infected i.n. with 10⁴ PFU of V_K627, rV_K627E, Ts_E627, rTs_E627K, and PBS (mock mice). At 5 day p.i. the thymuses from each group were removed. (A) Three thymuses per group were processed for hematoxylin and eosin. Five sections per tissue were analyzed. Magnification, ×50. (B) Another three thymuses per group were prepared for single cell suspension. Following staining, the percentages of CD4⁺CD8⁺ thymocytes were examined by flow cytometry. Three independent experiments yielded consistent results.</p