Epidemiologic studies have demonstrated that some bacteria colonization or infections in early-life increased the risk for subsequent asthma development. However, little is known about the mechanisms by which early-life bacterial infection increases this risk. The aim of this study was to investigate the effect of neonatal Streptococcus pneumoniae infection on the development of adulthood asthma, and to explore the possible mechanism. A non-lethal S. pneumoniae lung infection was established by intranasal inoculation of neonatal (1-week-old) female mice with D39. Mice were sensitized and challenged with ovalbumin in adulthood to induce allergic airways disease (AAD). Twenty-four hours later, the lungs and bronchoalveolar lavage fluid (BALF) were collected to assess AAD. Neonatal S. pneumoniae infection exacerbated adulthood hallmark features of AAD, with enhanced airway hyperresponsiveness and increased neutrophil recruitment into the airways, increased Th17 cells and interleukin (IL)-17A productions. Depletion of IL-17A by i.p. injection of a neutralizing monoclonal antibody reduced neutrophil recruitment into the airways, alleviated airway inflammation and decreased airway hyperresponsiveness. Furthermore, IL-17A depletion partially restored levels of inteferon-γ, but had no effect on the release of IL-5 or IL-13. Our data suggest that neonatal S. pneumoniae infection may promote the development of adulthood asthma in association with increased IL-17A production.
Citation: Yang B, Liu R, Yang T, Jiang X, Zhang L, Wang L, et al. (2015) Neonatal Streptococcus pneumoniae Infection May Aggravate Adulthood Allergic Airways Disease in Association with IL-17A. PLoS ONE 10(3): e0123010. https://doi.org/10.1371/journal.pone.0123010
Academic Editor: Bernhard Ryffel, French National Centre for Scientific Research, FRANCE
Received: May 22, 2013; Accepted: February 26, 2015; Published: March 27, 2015
Copyright: © 2015 Yang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Funding: This research was supported by the National Natural Science Foundation of China (81070015, 81270086). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Asthma is a common disease in children and the origin of the majority of adult cases, indicating that childhood events have an important role in asthma pathogenesis [1–3]. Childhood is an important period for the maturation of the immune system, and specific infections may alter immunologic programming, which plays a critical role in the progression of allergic airways disease (AAD) in adulthood.
Despite remarkable progress in our understanding of the pathogenesis of asthma, the initiating events have not been elucidated. Studies have demonstrated that viral infections in childhood promote subsequent development of asthma [4–7]. Recent studies suggest some bacterial infection may also have an important role in asthma pathogenesis [8,9]. Bronchial microbial florae in asthmatic patients are disturbed compared to healthy controls . Some young children with acute episodes of wheezing have bacterial infections that are closely associated . Neonates colonized with Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, or a combination of these three, have an increased risk for recurrent wheezing and asthma , and these bacteria are consistently associated with exacerbations of asthma in children . S. pneumoniae is the most common bacterial pathogen of community-acquired pneumonia, meningitis and sepsis, especially in young children [14,15]. Preston et al.  showed that S. pneumoniae infection in adult mice induces Tregs cells and suppresses AAD. Whether S. pneumoniae infection in neonatal mice can induce different immune responses is unclear.
Interleukin (IL)-17A is an important mediator of neutrophilic inflammation, and is elevated in the sputum of asthmatic patients with increased neutrophilia . IL-17A also plays a critical role in host protection against bacterial infections, indicating its potential role in the pathogenesis of bacteria-induced neutrophilic asthma [18,19]. In this study, we investigated the effect of neonatal S. pneumoniae infection on AAD in an adult mouse model with or without IL-17A depletion.
Materials and Methods
Pathogen-free pregnant BALB/c dams (6–8 wk of age) were housed in individually filtered cages, maintained on a 12-h light/dark cycle with an ovalbumin (OVA)-free diet, under a constant room temperature (24°C). Adequate amounts of sterile animal food and water were provided. Cages, food, bedding, and water were sterilized before use. The Institutional Animal Care and Research Advisory Committee at Chongqing Medical University approved this study. The use of animals in these experiments was in accordance with the guidelines issued by the Chinese Council on Animal Care.
Non-lethal neonatal S. pneumoniae infection and AAD induction
Non-lethal neonatal S. pneumoniae infection was established according to the procedures described in our previous study . Briefly, S. pneumoniae (D39) were plated onto tryptic soy broth (Pangtong, China), cultured for 8–10 h at 37°C in a 5% CO2 atmosphere, washed, and suspended in sterile phosphate-buffered saline (PBS). Conscious neonatal (Neo, 1-wk-old) female BALB/c mice were infected intranasally with 2 × 107 colony-forming units (CFU) of S. pneumoniae (D39) in 5 μL of PBS. The S. pneumoniae clearance time and the body weight were monitored. S. pneumoniae in lungs were cleared away within 7 days (Fig. 1A), and the body weight was recovered within 14 days (Fig. 1B). Mice were divided into the following groups: infected non-allergic (Neo), infected allergic (Neo/OVA), uninfected allergic (OVA), and uninfected non-allergic (control). To induce AAD, mice in the Neo/OVA and OVA groups were sensitized with i.p. injections of 100 μg OVA (Sigma-Aldrich, St. Louis, MO, USA) diluted in 50% aluminum hydroxide gel (Sigma-Aldrich) for a total volume of 200 μL on days 21 and 28 following inoculation. From days 35–42, mice were exposed to 1% OVA aerosols for 30 min/d. Neo mice and controls were simultaneously sensitized and challenged with sterile PBS. AAD was assessed within 24 h after the final challenge (Fig. 1C). Each experiment was repeated three times for a total of four to eight mice per group.
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 S. pneumoniae or phosphate-buffered saline (PBS) on day 0 (1 week-old). The S. pneumoniae 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).
Airway hyperresponsiveness (AHR)
AHR to methacholine was assessed using a mouse plethysmograph as previously described [21–23]. Briefly, 24 h after the final challenge, AHR was assessed in conscious, spontaneously breathing mice by means of whole-body plethysmography (Emca Technologies, Allmedicus, France). Each mouse was exposed to nebulized PBS followed by incremental doses of nebulized methacholine (3.125, 6.250, 12.500, 25.000, and 50.000 mg/mL; Sigma-Aldrich) for 3 min, and mean Penh values were recorded 5 min after administration of each dose; Penh = peak expiratory flow / peak inspiratory flow × pulmonary airflow resistance.
Histopathology of the lungs
Twenty-four hours after the final OVA challenge, mice were sacrificed by a lethal dose of 10% chloral hydrate (0.3 mL/100 g, i.p.) to harvest the lungs. Formalin-fixed lungs were dissected and embedded in paraffin. Four-micron-thick sections were cut and stained with hematoxylin and eosin (Sigma-Aldrich) according to the manufacturer’s instructions. Images were captured under a Nikon Eclipse E200 microscope connected to a Nikon Coolpix 995 camera (Nikon, Tokyo, Japan). The degree of airway inflammatory cell infiltration was scored in a single-blind fashion to reduce evaluator bias. Lung lesions were scored semi-quantitatively as previously described .
Bronchoalveolar lavage and cell counting
Within 24 h after the final challenge, mice were anesthetized with 10% chloral hydrate (0.1 mL/100 g, i.p.). The trachea was cannulated, and bronchoalveolar lavage fluid (BALF) was obtained by flushing the lungs twice with 1 mL PBS. Total cell numbers in the BALF were counted using microscopy. Differential cell counts were performed under Wright-Giemsa staining and based on standard morphologic and staining characteristics of at least 250 cells per sample. The supernatant was stored at −80°C. All slides were characterized by a single-blinded examiner to reduce evaluator bias.
BALF cytokine measurements
Cytokine concentrations in BALF were measured with commercial enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer's instructions. ELISA kits were used to measure interferon (IFN)-γ (Xinbosheng, Shenzhen, China), IL-5, IL-10 and transforming growth factor (TGF)-β (Sizhengbai, Beijing, China), and IL-17A and IL-13 (eBioscience Inc., San Diego, CA, USA).
Flow cytometric analysis of lung CD4+ T cells
The lungs were minced and incubated for 20 min at 37°C in 1 mL of sterile PBS containing 0.2% collagenase I (Sigma-Aldrich). Single pulmonary cell suspensions were obtained by forcing tissue through a 70 μm cell filter (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Erythrocytes were lysed, and the remaining cells were resuspended in RPMI 1640 medium containing 10% fetal bovine serum. A single-cell suspension from the lung (2 × 106 cells/mL) was incubated for 4–6 h at 37°C and 5% CO2 in six-well flat-bottom plates (Nalgene of Thermo Fisher Scientific, Waltham, MA, USA) in 1 mL medium containing phorbol 12-myristate 13-acetate (50 ng/mL; Sigma-Aldrich), ionomycin (500 ng/mL; Sigma-Aldrich) and GolgiPlug-containing brefeldin A (Becton, Dickinson and Company). The cells were then harvested, washed, pretreated with an Fc blocker, and subsequently stained for surface-associated CD4 (anti–CD4-FITC; Pharmingen of Becton, Dickinson and Company), CD3 (anti–CD3-Cy7; Pharmingen), or CD25 (anti–CD25-PE; eBioscience Inc.). To detect the subsets of CD4+ T cells in lungs, the cells were stained for intracellular IFN-γ (anti–IFN-γ-PerCP-Cy5.5; Pharmingen), IL-17A (anti–IL-17A-PE; Pharmingen), IL-4 (anti–IL-4-APC; Pharmingen), and FOXP3 (anti–FOXP3-PE-Cy5; eBioscience Inc.), and detected by flow cytometry (FACS Canto; Becton, Dickinson and Company). The data were analyzed with CellQuest software (Becton, Dickinson and Company).
IL-17A blockade during OVA-induced AAD
In some experiments, IL-17A was blocked according to the method previously described by Essilfie et al. . Briefly, IL-17A was depleted on days 34 and 36 by i.p. injection of a monoclonal anti-IL-17A neutralizing antibody (clone 50104, rat IgG2a, 100 μg/mouse; R&D Systems, Inc., Minneapolis, MN, USA). Features of AAD were assessed on day 43. Control groups received an IgG2a isotype control antibody (R&D Systems, Inc.).
Results were analyzed using GraphPad Prism (version 5.0; GraphPad, La Jolla, CA, USA) and expressed as mean ± the standard error. Results were interpreted using either one-way analysis of variance (ANOVA) and Tukey’s post-test or a two-way ANOVA with a Bonferroni’s post-test. Differences were considered statistically significant when P < 0.05.
Neonatal S. pneumoniae infection promotes OVA-induced neutrophilic inflammation in a BALB/c mouse model
To determine the effect of neonatal S. pneumoniae lung infection on adulthood airway inflammation, the number of inflammatory cells in BALF was counted. Total numbers of inflammatory cells (Fig. 2A) and eosinophils (Fig. 2B) in the OVA group were elevated > 10-fold compared to controls (13.13 ± 1.49 vs. 1.42 ± 0.11 and 14.41 ± 1.09 vs. 0.03 ± 0.016, respectively; Ps < 0.001). Notably, the neonatal S. pneumoniae infection in the Neo/OVA mice enhanced total numbers of inflammatory cells and neutrophils (Fig. 2A,C) compared with the uninfected OVA group (26.71 ± 1.17 vs. 13.13 ± 1.49 and 120.10 ± 6.43 vs. 37.52 ± 3.34, respectively; Ps < 0.001), while the number of eosinophils did not differ (Fig. 2B).
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 (n = 6–8 mice/group); ***P < 0.001 vs. controls; ###P < 0.001 vs. OVA.
Neonatal S. pneumoniae infection aggravates OVA-induced lung pathology
We next examined the lung pathology following OVA sensitization and challenge. OVA challenge led to a dense peribronchiolar and perivascular infiltrate of inflammatory cells. In the Neo/OVA group, lung tissue inflammation was more severe with greater infiltration of inflammatory cells compared with the OVA group (Fig. 3A). The inflammation scores for pulmonary peribronchiolitis, pulmonary perivasculitis, and pulmonary alveolitis in the Neo/OVA group mice were almost double those in the OVA group (Ps < 0.01) (Fig. 3B-D).
Hematoxylin and eosin staining of lung samples from uninfected, allergic (OVA), neonatal infected, allergic (Neo/OVA), neonatal infected, non-allergic (Neo), and uninfected, non-allergic (control) mice (A) (200×). Histologic scores of pulmonary peribronchiolitis (B), pulmonary perivasculitis (C), and pulmonary alveolitis (D). Data are reported as mean ± standard error from three separate experiments (n = 6–8 mice/group). **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. OVA.
Neonatal S. pneumoniae infection enhances AHR during AAD
AHR was evaluated 24 h after the final challenge by the calculation of Penh values (i.e., enhanced respiratory pausing). OVA sensitization and challenge resulted in increased AHR (Fig. 4). The Penh values for the OVA group were higher than those for controls at methacholine concentrations between 25.0 and 50.0 mg/mL (Ps < 0.001). Furthermore, the Neo/OVA group had a significantly higher Penh value than the OVA and control groups (Ps < 0.001).
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 (n = 6–8 mice/group); *P < 0.05, **P < 0.01, ***P < 0.001 vs. control; #P < 0.05, ###P < 0.001 vs. OVA.
Neonatal S. pneumoniae infection promotes IL-17A production during AAD
We first investigated the effects of neonatal S. pneumoniae lung infection on CD4+ T cell production during ADD. Interestingly, the production of Th17 cells in the Neo/OVA group was significantly higher than in the OVA group (P < 0.001) (Fig. 5A). However, there were no differences in the production of Th2, Th1, and FOXP3+ Treg between the Neo/OVA and the OVA groups (Fig. 5B-D).
Th17 (A), Th2 (B), Th1 (C), and FOXP3+ Treg (D) subsets of CD4+ T cells were measured in the lungs of uninfected, allergic (OVA), neonatal infected, allergic (Neo/OVA), neonatal infected, non-allergic (Neo), and uninfected, non-allergic (control) mice by flow cytometry. Data are reported as mean ± standard error from three separate experiments (n = 6–8 mice/group); *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls; ##P < 0.01 vs. OVA.
Next, we detected cytokine concentrations in BALF. As expected, IL-17A production in the Neo/OVA group was significantly higher (two-fold) than in the OVA group (P < 0.001) (Fig. 6A), though the levels of IL-5 and IL-13 were similar (Fig. 6B,C). The levels of IFN-γ and IL-10 (Fig. 6D,E) in the Neo/OVA group were half of those observed in the OVA group (Ps < 0.001), while TGF-β was similar (Fig. 6F).
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 (n = 6–8 mice/group); *P < 0.05; **P < 0.01, ***P < 0.001 vs. controls; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. OVA.
Neonatal S. pneumoniae infection exacerbates OVA-induced AAD in association with an IL-17A response
To determine whether IL-17A mediates neonatal S. pneumoniae-induced airway inflammation, IL-17A was depleted in the Neo/OVA and OVA mice during AAD (Fig. 7A). As expected, IL-17A depletion in the Neo/OVA mice with an IL-17A-blocking monoclonal antibody significantly reduced total inflammatory cell and neutrophil recruitment into the BALF as compared to isotype antibody treatment (Ps < 0.001) (Fig. 7B,C). Consistent with our results that eosinophils were unaffected in neonatal S. pneumoniae infection, eosinophil numbers were similar in mice treated with either anti-IL-17A or isotype antibody (Fig. 7D). Anti-IL-17A treatment decreased AHR (Fig. 7E) and alleviated airway inflammation (Fig. 7F). Anti-IL-17A treatment also partially restored IFN-γ (Fig. 7G) production, but had no effect on IL-5 (Fig. 7H) or IL-13 production (Fig. 7I).
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 (n = 4–6 mice/group); ***P < 0.001 vs. isotype control; ###P < 0.001 vs. OVA. OVA = uninfected, allergic, Neo/OVA = neonatal infected, allergic.
In this study, we investigated the effect of non-lethal neonatal S. pneumoniae lung infection on the development of allergic asthma in adulthood. We demonstrate that neonatal S. pneumoniae lung infection promotes the recruitment of OVA-induced neutrophils into the airways, aggravates airway inflammation and increases AHR in adulthood. Furthermore, IL-17A depletion alleviates airway inflammation, decreases AHR, and reduces airway neutrophil recruitment. Thus, neonatal S. pneumoniae lung infection exacerbates the hallmark features of AAD in adulthood, which may be associated with increased IL-17A production.
S. pneumoniae has been used to investigate S. pneumoniae-induced infection and host-bacteria-allergen interactions in susceptible BALB/c mice . Studies have found that adulthood S. pneumoniae infection, or treatment with components of or killed organisms, can suppress the hallmark features of AAD by inducing Tregs cells in mice [16,26]. Consistent with these results, we found that adulthood S. pneumoniae infection can suppress Th2 cells and AAD by inducing Tregs cells (data not shown), while neonatal S. pneumoniae infection promotes subsequent adulthood allergic asthma development, characterized by neutrophil recruitment into the airways and increased Th17 and IL-17A production. Larsen et al.  showed an abnormal immune response to airway-colonizing pathogenic bacteria in early-life may lead to chronic airway inflammation and childhood asthma. Horvat et al.  stated early-life, but not adult, chlamydial infection promotes subsequent development of allergic asthma. These data indicate that the immune response to some pathogens in childhood may be different from that in adulthood. Thus, the age of infection may have a crucial role in determining the nature of the effects, particularly in the development of subsequent allergic asthma.
In this study, Th17 cells in the lung, IL-17A and neutrophils in BALF were significantly higher in infected compared to uninfected allergic mice, while Th2 cells and their associated cytokines (IL-5, IL-13) and the number of eosinophils were similar. IL-17A, mainly produced by Th17 cells, is implicated in the pathogenesis of several inflammatory conditions . Lu etal.  stated that IL-17A plays critical role in host protection against S. pneumoniae colonization and infection. Additional studies indicate that enhanced IL-17A levels correlate with increased AHR in asthmatics and allergic asthma in mice [31,17]. IL-17A can induce structural lung cells to secrete proinflammatory cytokines and neutrophil chemotactic proteins, thereby inducing neutrophil infiltration . Thus, IL-17A has a pivotal role in the pathogenesis of asthma. Consistent with this notion, depletion of IL-17A reduced the aggravation features of AAD by neonatal S. pneumoniae infection. IL-17A depletion significantly inhibited neutrophil recruitment into the airways, decreased airway inflammation and AHR, with no significant effect on eosinophil recruitment or IL-5 and IL-13 production, suggesting that IL-17A-mediated neutrophilic inflammation plays an important role in the promotion of asthma development in adulthood by neonatal S. pneumoniae lung infection. These findings are consistent with those of Essilfie et al. , who demonstrated that H. influenzae infection drives IL-17-mediated development of neutrophilic AAD.
Neonatal S. pneumoniae infection also inhibited IFN-γ production, which was partially restored by IL-17A blockade. Newcomb et al.  showed that IFN-γ is a negative regulator of IL-17A expression in a model of respiratory syncytial virus infection during allergic airway inflammation. Whether IFN-γ is involved in regulating IL-17A production in neonatal S. pneumoniae infection-induced AAD in adults requires further investigation.
Neonatal S. pneumoniae lung infection may promote the development of allergic asthma in adulthood in association with enhanced IL-17A production.
We thank Qibo Zhang (Liverpool University, UK) for providing advice throughout the course of this work. We thank the Department of Laboratory Medicine, Key Laboratory of Diagnostic Medicine, Chongqing Medical University for offering the S. pneumoniae strain D39. We also thank the Experimental Animal Center at Chongqing Medical University for providing the BALB/c mice.
Conceived and designed the experiments: ZL. Performed the experiments: BY RL TY XJ LZ. Analyzed the data: BY RL TY ZL EL ZF. Contributed reagents/materials/analysis tools: LW QW ZL. Wrote the paper: BY ZL.
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