Additional file 1: of Regulatory T cells and IL10 suppress pulmonary host defense during early-life exposure to radical containing combustion derived ultrafine particulate matter

Time-course of TNFα levels after exposure to EPFRs and influenza infection. Lungs were isolated on 1 and 4 dpe and on 1 and 6 dpi and whole lung homogenates were analyzed for TNFα levels using ELISA. TNFα was found to be below the limit of detection (1.5 pg/ml) in Air and DCB exposed lungs at 1 and 4 dpe and in Air/Flu and DCB/Flu mice at 1 dpi. Detectable levels of TNFα were found only in 3 of the DCB/Flu (n = 6) mice at 6 dpi. (TIF 18 kb

Additional file 3: of Regulatory T cells and IL10 suppress pulmonary host defense during early-life exposure to radical containing combustion derived ultrafine particulate matter

Effect of Treg depletion on IL10 levels in the lungs of EPFR exposed and influenza infected neonatal mice. IL10 levels in the lungs of Treg depleted and EPFR exposed neonatal mice at 6 dpi after infection with influenza virus (DCB/PC61/Flu) in comparison to Air/Flu, DCB/Flu, and EPFR exposed neonatal mice treated with rat IgG isotype control and infected with influenza virus (DCB/Isotype/flu). Data are plotted as means ± SEM, *p < 0.05. One-way ANOVA with Holm-Sidak’s multiple comparisons test. (DOCX 12 kb

Exposure to Deepwater Horizon Crude Oil Burnoff Particulate Matter Induces Pulmonary Inflammation and Alters Adaptive Immune Response

The ″<i>in situ</i> burning” of trapped crude oil on the surface of Gulf waters during the 2010 Deepwater Horizon (DWH) oil spill released numerous pollutants, including combustion-generated particulate matter (PM). Limited information is available on the respiratory impact of inhaled <i>in situ</i> burned oil sail particulate matter (OSPM). Here we utilized PM collected from <i>in situ</i> burn plumes of the DWH oil spill to study the acute effects of exposure to OSPM on pulmonary health. OSPM caused dose-and time-dependent cytotoxicity and generated reactive oxygen species and superoxide radicals <i>in vitro.</i> Additionally, mice exposed to OSPM exhibited significant decreases in body weight gain, systemic oxidative stress in the form of increased serum 8-isoprostane (8-IP) levels, and airway inflammation in the form of increased macrophages and eosinophils in bronchoalveolar lavage fluid. Further, in a mouse model of allergic asthma, OSPM caused increased T helper 2 cells (Th2), peribronchiolar inflammation, and increased airway mucus production. These findings demonstrate that acute exposure to OSPM results in pulmonary inflammation and alteration of innate/adaptive immune responses in mice and highlight potential respiratory effects associated with cleaning up an oil spill

Respiratory Syncytial Virus Disease Is Mediated by Age-Variable IL-33

<div><p>Respiratory syncytial virus (RSV) is the most common cause of infant hospitalizations and severe RSV infections are a significant risk factor for childhood asthma. The pathogenic mechanisms responsible for RSV induced immunopathophysiology remain elusive. Using an age-appropriate mouse model of RSV, we show that IL-33 plays a critical role in the immunopathogenesis of severe RSV, which is associated with higher group 2 innate lymphoid cells (ILC2s) specifically in neonates. Infection with RSV induced rapid IL-33 expression and an increase in ILC2 numbers in the lungs of neonatal mice; this was not observed in adult mice. Blocking IL-33 with antibodies or using an IL-33 receptor knockout mouse during infection was sufficient to inhibit RSV immunopathogenesis (i.e., airway hyperresponsiveness, Th2 inflammation, eosinophilia, and mucus hyperproduction); whereas administration of IL-33 to adult mice during RSV infection was sufficient to induce RSV disease. Additionally, elevated IL-33 and IL-13 were observed in nasal aspirates from infants hospitalized with RSV; these cytokines declined during convalescence. In summary, IL-33 is necessary, either directly or indirectly, to induce ILC2s and the Th2 biased immunopathophysiology observed following neonatal RSV infection. This study provides a mechanism involving IL-33 and ILC2s in RSV mediated human asthma.</p></div

Modulation of IL-33 levels during primary RSV infection alters ILC2 numbers and IL-13 production at 1 dpi.

<p>(<b>a</b>) Number of ILC2s (lineage^- CD45⁺ ICOS⁺ ST2⁺) expressed as percentage of total lung cells and MFI of surface ST2 on ILC2s at 1 dpi in neonatal mice pretreated with IL-33 neutralizing antibody (α-IL-33 + NR) or control IgG antibody (Isotype + NR) and adult mice pretreated with recombinant IL-33 (rIL-33 + AR) or vehicle control (Control + AR). (<i>n</i> = 5–7 per group). (<b>b</b>) IL-13 protein levels in whole lung homogenates. (<b>c</b>) Pulmonary viral loads measured at 4 dpi (peak) using the TCID₅₀ method. *<i>P</i> < 0.05 vs. indicated group, (Student’s <i>t</i>-test). Data are representative of two independent experiments (means ± s.e.m).</p

IL-33 levels during primary RSV infection determine disease severity after reinfection.

<p>(<b>a</b>) Lung sections obtained from mice treated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005217#ppat.1005217.g004" target="_blank">Fig 4</a>, stained with periodic acid-Schiff (PAS) to observe mucus (bright purple; indicated by black arrowheads). Upper images taken at 100X with inset (black box) magnified underneath to 400X. (<b>b</b>) Quantification of airway mucus in mice treated as in <b>a</b> (methods). *<i>P</i> < 0.05 vs. indicated group (one-way ANOVA with Bonferroni post-hoc tests). Data are representative of two independent experiments (means ± s.e.m).</p

RSV induces robust, rapid IL-33 and IL-13 production in the lungs of neonates.

<p>(<b>a</b>) Cytokine protein levels of IL-33 and IL-13 in whole lung homogenates of RSV-infected neonate (5-day-old; NR) and adult (6-8-week-old; AR) mice (<i>n</i> = 4–10 per group) at different days (0–10) post-infection (dpi). (<b>b</b>) Cytokine protein levels of IL-33 detected in BAL at 1 dpi from NR and AR mice (<i>n</i> = 8–10 per group) compared to controls. (<b>c</b>) Gating strategy for determination of IL-33 expression by median fluorescence intensity (MFI) in epithelial cells (CD45^- EpCam⁺) with representative IL-33 MFI histogram (quantified in inset vs. fluorescence-minus-one (FMO) control (dotted line)). (<b>d</b>) Representative micrographs of <i>in situ</i> hybridization for IL-33 mRNA with red arrows to indicate positive staining cells (top), magnified inset (bottom), and quantification of IL-33 mRNA positive cells per unit area of lung. Scale bar = 100 μm. (<b>e</b>) Cytokine protein levels of IL-33 in whole lung homogenates of neonates infected with RSV or UV-RSV compared to control at 1 dpi. *<i>P</i> < 0.05 (Student’s <i>t</i>-test; <b>a</b>, <b>c</b>, <b>d</b>) (Two-way ANOVA; <b>b</b>) (One-way ANOVA; <b>e</b>). Data are representative of two (<b>a</b>, <b>c</b>) or 2 pooled (<b>b</b>, <b>d</b>, <b>e</b>) independent experiments (means ± s.e.m).</p

The percentage of ILC2s in the lungs of neonatal mice are further increased after RSV infection.

<p>(<b>a</b>) Gating strategy and (<b>b</b>) ILC2 marker expression on ILC2s at baseline and 1 dpi. (<b>c</b>) ILC2s as percent of total lung cells, and (<b>d</b>) MFI of ST2 expression of/on ILC2s at baseline and 1 dpi. ILC2s defined as lineage^- CD45⁺ ICOS⁺ ST2⁺ with gating based on FMO controls. *<i>P</i> < 0.05 (Two-way ANOVA). Data are representative of four independent experiments (means ± s.e.m).</p

IL-33 signaling is required for neonatal RSV immunopathophysiology.

<p>(<b>a</b>) Number of Th1, Th2, and multifunctional mTh cells in the lungs of wild-type (WT) or ST2-deficient (<i>Il1rl1</i>^-/-) mice at 6 dpi following reinfection with RSV (methods) (<i>n</i> = 5–6 per group). (<b>b</b>) Change in airway resistance in response to increasing doses of inhaled methacholine after treatment as in <b>a</b> (<i>n</i> = 6 per group), compared to naïve control mice of similar size and age. (<b>c</b>) Total cells (Total), monocytes/macrophages (Mo/MΦ), lymphocytes (Lymph), neutrophils (Neutro), and eosinophils (Eos) in BAL fluid after treatment as in <b>a</b> (<i>n</i> = 5–8 per group). *<i>P</i> < 0.05 (Student’s <i>t</i>-test (<b>a</b>, <b>c</b>) or two-way ANOVA with Bonferroni post-hoc tests (<b>b</b>)). Data are representative of two independent experiments (means ± s.e.m).</p

IL-33 and IL-13 concentrations are elevated in nasal aspirates from infants hospitalized with RSV infection.

<p>(<b>a</b>) Concentrations of IL-33 and IL-13 in nasal aspirates taken from RSV-infected infants (<i>n</i> = 19) on the first day (d1) of clinical presentation to Le Bonheur Children’s Hospital and again four weeks later (d28). (<b>b</b>) Correlation of IL-33 and IL-13 concentrations for all patients with d1 samples (<i>n</i> = 81). Samples with cytokine concentrations below the LOD of the assay are replaced by a value equal to the LOD divided by the square root of 2, and denoted with a filled circle. *<i>P</i> < 0.05 (Sign test (<b>a</b>) or Kendall’s tau correlation (<b>b</b>)).</p