Absolute numbers of inflammation cells in 1.2 ml of NALF and BALF at 24 h after final challenge.

<p>Original magnification was ×400 (A, B). Total inflammation cells and eosinophils in NALF (C) and BALF (D) were significantly inhibited by <i>E. coli</i> infection in AAD mice model. Each bar represents the mean cell number ± standard error of the mean (SEM), n = 10. ^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

Goblet cell metaplasia assessed on alcian blue-periodic acid Stiff (AB-PAS) stained tissue sections of the nasal mucosa and lung.

<p>Original magnification was ×400 for nose and ×200 for lung (A). Goblet cells were counted as the blue cells stained positive by AB-PAS. Percentages of goblet cell metaplasia were calculated from the total numbers of cells counted around the nasal mucosa (B) and the lung (C). Goblet cell metaplasia was relatively minor in mice infected with <i>E. coli</i>. Data is expressed as mean percent ± SEM, n = 10.^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

The experimental protocol and different groups.

<p>Mice were infected with <i>E. coli</i> or PBS, and then sensitized & challenged with OVA or PBS, as described previously in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059174#s2" target="_blank">Materials and methods</a>. Mice were divided into five different groups in this work.</p

The changes of cytokines IL-4, IL-10, IFN-γ and IL-2 in NALF (A) and BALF (B).

<p>As shown above, administration of <i>E. coli</i> exhibited significant inhibition of levels of Th2 cytokines IL-4. Interestingly, the effect was accompanied by high levels of Th1 cytokines IFN-γ and IL-2, as well as the increased production of IL-10 secreted abundantly by Tregs. Additionally, the effects were more significant in neonatal mice infected with 10⁸CFU <i>E. coli</i>. Data is represented as the mean secretion pg/ml ± SEM, n = 8∼10.^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

The changes of allergic symptoms.

<p>The control group showed no or less allergic symptoms, whereas AAD model group had remarkable frequency of nasal rubbing and sneezing. Nevertheless, the frequency was noticeably decreased by <i>E. coli</i> infection. Data is shown by box and whisker plots, with whisker ends indicating minimal and maximal values and horizontal bars representing medians, n = 10. ^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

The changes of serum OVA-specific IgE levels.

<p>OVA-specific IgE levels were apparently higher in AAD model group than that in the control group. However, the levels in <i>E. coli</i> infected mice were significantly inhibited, especially in the (10⁸infN+OVA) group. Bars indicate the mean secretion ng/ml ± SEM, n = 8∼10.^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

Tregs accumulated in PTLN of <i>E. coli</i> infected mice, especially in the (10⁸infN+OVA) group.

<p>Representative scatter plots denoted the fraction of CD4⁺ cells that were CD4⁺ CD25⁺ FoxP3⁺ Tregs (A). Ratios of Tregs were calculated per mouse (B). Percentages of Tregs in AAD model group were increased, compared to the control group. Interestingly, mice infected with <i>E. coli</i> present a more significant up-regulation in numbers of Tregs. Additionally, numbers of Tregs in the (10⁶infN+OVA) and (10⁸infA+OVA) group were lower than that in the (10⁸infN+OVA) group. Data is expressed as mean ± SEM, n = 8.^*<i>p</i><0.05, ^**<i>p</i><0.01 as conducted.</p

Macrophage autophagy induced by drugs and mTOR-siRNA alleviated vulnerability index.

<p>The vulnerability index can be calculated as: (macrophage staining %+ lipid staining %)/(smooth muscle cell %+ collagen fiber %). So we conducted Sirius red (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and oil red O staining together with IH for macrophages (RAM-11) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090563#pone-0090563-g005" target="_blank">Fig. 5</a>) and smooth muscle cells (α-SMA). (A, B) Oil Red O Staining was taken for lipid content evaluation and was expressed as a percentage verse the whole vessel lumen. Our result revealed much smaller plaque in aorta abdominals in group A2, B2 and C2 compared to group D2. (C) IH analysis of α-SMA amount did not detect difference in plaque area among the four groups. L represents for vascular lumen, P represents for plaque. (D) Vulnerability was calculated by macrophage, lipid, α-SMA and collagen. It was significantly lower in the experimental group than the control group. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control.</p

Effect of Pharmacological triggers in the experiment showed the rupture of plaque incidence higher in control group.

<p>IH staining and transmission electron microscope showed mTOR decreased and autophagy increased in experimental group. Pharmacological triggers were done by 0.15·kg⁻¹ of Chinese Russell's viper venom injecting intraperitoneally, 30 min later, 0.02 mg·kg⁻¹ histamine was injected intravenously. (A) Haematoxylin and eosin staining of the cross section of the abdominal aorta in a rabbit of group D2 showing plaque rupture and thrombosis. (B, C) IH staining showed mTOR decreased obviously in group A2, B2 and C2 than D2. (D) Transmission electron microscope analysis showed macrophage autophagy was enhanced in group A2, B2 and C2 compared to group D2. Arrows in picture B2, C2 and d2 represent vacuoles with cytoplasmic inclusions and myeline figure. The “N” represents the nuclear. (E) IH analysis of Atg5-Atg12 conjugation found percentage of positive-stain cells was significantly increased in group A2, B2 and C2 in comparison to group D2. A2: triciribine; B2: rapamycin; C2: mTOR-siRNA; D2: control; ^*<i>P</i><0.01 vs control, ^#<i>P</i><0.05 vs control.</p

Table Serum lipid Profiles of the rabbits in the four groups at week 16.

<p>Group A2 is Akt inhibitor triciribine treatment group, Group B2 is mTOR inhibitor rapamycin treatment group, Group C2 is mTOR-siRNA group and Group D2 is the control group.</p><p>TC, total cholesterol; TG, triglyceride; HDL, high density lipoprotein; LDL, low density lipoprotein.</p