Neonatal <i>S</i>. <i>pneumoniae</i> infection enhances ovalbumin-induced airway hyperresponsiveness.

<p>Whole-body plethysmography in uninfected, allergic (OVA), neonatal infected, allergic (Neo/OVA), neonatal infected, non-allergic (Neo), and uninfected, non-allergic (control) mice was conducted 24 h following challenge with methacholine. Data are reported as mean ± standard error from three separate experiments (<i>n</i> = 6–8 mice/group); *<i>P</i> < 0.05, **<i>P</i> < 0.01, ***<i>P</i> < 0.001 <i>vs</i>. control; ^#<i>P</i> < 0.05, ^###<i>P</i> < 0.001 <i>vs</i>. OVA.</p

Establishment of neonatal <i>S</i>. <i>pneumoniae</i> infection model and schematic of study protocol.

<p>Neonatal BALB/c mice were divided into the following groups: infected non-allergic (Neo), infected allergic (Neo/OVA), uninfected allergic (OVA), and uninfected non-allergic (control). Mice were infected intranasally with <i>S</i>. <i>pneumoniae</i> or phosphate-buffered saline (PBS) on day 0 (1 week-old). The <i>S</i>. <i>pneumoniae</i> clearance time (A) and the body weight (B) were monitored. Mice were sensitized by an i.p. injection of ovalbumin (OVA) or PBS on days 21 and 28, and challenged with aerosolized OVA or PBS to induce allergic airways disease (AAD) on days 35–42. Key features of AAD were characterized within 24 h after the final challenge (on day 43) (C).</p

Airway inflammation in mice with neonatal <i>S</i>. <i>pneumoniae</i> infection correlates with Th17 cytokine production.

<p>Cytokine levels of interleukin (IL)-17A (A), IL-5 (B), IL-13 (C), interferon (IFN)-γ (D), IL-10 (E), and transforming growth factor (TGF)-β (F) in the bronchoalveolar lavage fluid of uninfected, allergic (OVA), neonatal infected, allergic (Neo/OVA), neonatal infected, non-allergic (Neo), and uninfected, non-allergic (control) mice were measured by ELISA. Data are reported as mean ± standard error from three separate experiments (<i>n</i> = 6–8 mice/group); *<i>P</i> < 0.05; **<i>P</i> < 0.01, ***<i>P</i> < 0.001 <i>vs</i>. controls; ^#<i>P</i> < 0.05, ^##<i>P</i> < 0.01, ^###<i>P</i> < 0.001 <i>vs</i>. OVA.</p

Neonatal <i>S</i>. <i>pneumoniae</i> infection enhances ovalbumin (OVA)-induced leukocyte and neutrophil infiltration of the airways.

<p>Total cells (A), eosinophils (B), and neutrophils (C) were counted from bronchoalveolar lavage fluid (BALF) collected 24 h after the final challenge. OVA, uninfected, allergic; Neo/OVA, neonatal infected, allergic; Neo, neonatal infected, non-allergic; Control, uninfected, non-allergic. Data are shown as mean ± standard error from three separate experiments (<i>n</i> = 6–8 mice/group); ***<i>P</i> < 0.001 <i>vs</i>. controls; ^###<i>P</i> < 0.001 <i>vs</i>. OVA.</p

S. pneumoniae infection-induced interleukin (IL)-17A is responsible for the aggravated allergic airways disease (AAD).

<p>Anti-IL-17A monoclonal antibody was administrated by i.p. injection on days 34 and 36, and features of AAD were assessed on day 43 (A). The influx of total inflammatory cells (B), neutrophils (C), eosinophils (D), airway hyperresponsiveness (E), tissue pathology (F), and the levels of interferon (IFN)-γ (G), IL-5 (H), IL-13 (I) in bronchoalveolar lavage fluid (BALF) were assessed. Data are reported as mean ± standard error from three separate experiments (<i>n</i> = 4–6 mice/group); ***<i>P</i> < 0.001 <i>vs</i>. isotype control; ^<i>###</i><i>P</i> < 0.001 <i>vs</i>. OVA. OVA = uninfected, allergic, Neo/OVA = neonatal infected, allergic.</p

Changes in the percentage of CD8⁺ and CD8⁺TCRγδ⁺ IELs in the rats in the VAD group and in the rats in the four VAS groups in the presence or absence of LPS (n = 8 to 10).

<p>A) The effects of VAS and LPS challenge on the percentage of CD8⁺ IELs in the rats in the five groups (VAD, VASP, VAS1, VAS2 and VAS3). B) The number of CD8⁺ IELs was significantly higher in the rats in the combined VASP and VAS1 groups compared with the rats in the VAD group. C) The LPS treatment significantly affected the percentage of CD8⁺ IELs after combining data from the five VA treatments. D) The percentages of CD8⁺TCRγδ⁺ IELs were significantly decreased in the rats in the four VAS groups independent of exposure to LPS (n = 8 to 10). The values are the means±SD, and **<i>P</i><0.01, ***<i>P</i><0.001, ^$$$<i>P</i><0.001; n.s. = not significant in post hoc tests.</p

Changes in the number of CD11c⁺ DCs and CD4⁺CD25⁺ T cells in the Peyer's patches of rats in the VAD group and rats of the four VAS groups in the presence or absence of LPS (n = 16)

<p>. A) The effects of VAS and LPS treatment on the percentage of CD11c⁺DCs cells in the PPs of the rats in the five groups. B) The number of CD11c⁺DCs cells was significantly higher in the rats in the combined VAS1 group compared with the rats in the combined VAD group. C) The effects of VAS and LPS treatment on the percentage of CD4⁺CD25⁺ T cells in the PPs of the rats in the five groups. D) No differences were found in the percentage of CD4⁺CD25⁺ T cells between the rats in the combined VAD group and the rats in the four VAS groups. The values are the means±SD, and ***<i>P</i><0.001; n.s. = not significant in post hoc tests.</p

Effects of VAS and LPS challenge on the levels of pIgR mRNA expression in the intestine.

<p>The changes in pIgR mRNA expression levels in the intestine were determined in both the VAS1 and VAD rats with or without LPS challenge using real-time PCR (n = 8). The values are the means±SD, ***<i>P</i><0.001, ^$$$<i>P</i><0.001; and interaction <i>P</i><0.0001 in post hoc tests.</p

Changes in the percentages of CD4⁺CD25⁺ and CD4⁺CD8⁺ T lymphocytes in the spleens of rats in the VAD group and rats in the four groups (VASP, VAS1, VAS2 and VAS3) in the presence or absence of LPS.

<p>A) Effects of VAS and LPS challenge on the percentages of CD4⁺CD25⁺ T cells in the spleens of the rats in the five groups (n = 8 to 10). B) The percentage of CD4⁺CD25⁺ T cells was significantly higher in the rats in the combined VAS1 group compared with the rats in the combined VAD group. C) The LPS challenge significantly enhanced the number of CD4⁺CD25⁺ T cells after the five VA treatments were combined. D) The effects of VAS and LPS challenge on the percentages of CD4⁺CD8⁺ T cells in the spleens of the rats in the five groups (n = 8 to 10). E) There were no significant differences in the percentage of CD4⁺CD8⁺ T lymphocytes among the rats in the combined groups (VAD, VASP, VAS1, VAS2 and VAS3), although the <i>P</i> value of the VA effect was 0.0213. F) Exposure to LPS significantly reduced the number of CD4⁺CD8⁺ T lymphocytes after the five VA treatments were combined. The values are the means±SD, and ***<i>P</i><0.001, n.s. = not significant in post hoc tests.</p

Schematic diagram showing the four time points at which VAS was provided (VASP, VAS1, VAS2 and VAS3).

<p>The rats in the VASP group were supplemented with VA beginning on gestational day 14 (G14). The lactating maternal rats were then fed a VAN diet followed by VAS until the weaning period; the pups of the rats in the VASP group were given the same VAN diet after they were weaned until they reached 6 weeks of age. The pups (VAS1 group) of gestational VAD rats were supplemented with VA on postnatal day 1 (D1), and the nursing maternal rats were fed a VAN diet until the weaning period. After weaning, the pups in the VAS1 group were given the same VAN diet until they reached 6 weeks of age. The pups (VAS2 group) of gestational VAD rats were supplemented with VA on postnatal day 14 (D14), and the nursing maternal rats were fed a VAN diet followed by VAS until the weaning period. After weaning, the pups in the VAS2 group were given the same VAN diet until they were 6 weeks old. The rats in the VAS3 group were supplemented with VA on postnatal day 28 (D28) and fed the VAN diet for 2 weeks following VAS.</p