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

Immune cell recruitment into the lungs of neonatal and adult mice infected with MRSA.

<p>Neonatal (5 d) and adult mice (6 wks) were infected with 1x10⁷ CFU’s of MRSA and BAL performed at 1 and 3 dpi. The cells recovered from the BALF were counted by differential staining. NSA = neonatal / MRSA infection, ASA = adult / MRSA infection.</p

Survival and bacterial clearance of neonatal mice to MRSA.

<p>(A) C57Bl/6 neonate (5 d) and adult mice (6 wks) were intranasally infected with increasing doses of USA300 (6 mice/group) and survival was determined over a 6 d period. (B) C57Bl/6 pups (5 d) and adult mice (6 wks) were intranasally infected with 1x10⁷ CFU’s of MRSA and the level of bacteria in the lung was measured on 1, 2 3 and 6 dpi. NSA = neonatal / MRSA infection, ASA = adult / MRSA infection.</p

In vivo phagocytosis of MRSA by neonatal and adult mice.

<p>Neonate and adult mice were infected with 2x10⁷ CFU’s of TAMRA-MRSA or unlabeled MRSA. The lungs were removed 30 min pi and intracellular expression of TAMRA was measured by flow cytometry. The level of background fluorescence measured using unlabeled MRSA was 6.7%. NSA = neonatal / MRSA infection, ASA = adult / MRSA infection.</p

Neonatal mice mounted limited Th1 and Th17 response to MRSA infection.

<p>Neonate and adult mice were infected with 1.24 x10⁷ CFU’s of MRSA or vehicle. Lung single cells were isolated at 6 dpi and Th profile was measured by flow cytometry. (A) Th1/Th2 cell percentage of CD4+ T cells. (B) Representative flow plots of Th1/Th2 percentage of CD4+ T cells. (C) Th1/Th17 cell subsets. (D) Representative flow plots of Th1/Th17 subsets. NSA = neonatal / MRSA infection, NS = Neonatal / sham infection, ASA = adult / MRSA infection, AS = Adult sham infection.</p

A Neonatal Murine Model of MRSA Pneumonia

<div><p>Pneumonia due to methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is a significant cause of morbidity and mortality in infants particularly following lower respiratory tract viral infections such as Respiratory Syncytial Virus (RSV). However, the mechanisms by which co-infection of infants by MRSA and RSV cause increased lung pathology are unknown. Because the infant immune system is qualitatively and quantitatively different from adults we developed a model of infant MRSA pneumonia which will allow us to investigate the effects of RSV co-infection on disease severity. We infected neonatal and adult mice with increasing doses of MRSA and demonstrate that neonatal mice have delayed kinetics in clearing the bacteria in comparison to adult mice. There were differences in recruitment of immune cells into the lung following infection. Adult mice exhibited an increase in neutrophil recruitment that coincided with reduced bacterial titers followed by an increase in macrophages. Neonatal mice, however, exhibited an early increase in neutrophils that did not persist despite continued presence of the bacteria. Unlike the adult mice, neonatal mice failed to exhibit an increase in macrophages. Neonates exhibited a decrease in phagocytosis of MRSA suggesting that the decrease in clearance was partially due to deficient phagocytosis of the bacteria. Both neonates and adults responded with an increase in pro-inflammatory cytokines following infection. However, in contrast to the adult mice, neonates did not express constitutive levels of the anti-microbial peptide Reg3γ in the lung. Infection of neonates did not stimulate expression of the co-stimulatory molecule CD86 by dendritic cells and neonates exhibited a diminished T cell response compared to adult mice. Overall, we have developed a neonatal model of MRSA pneumonia that displays a similar delay in bacterial clearance as is observed in the neonatal intensive care unit and will be useful for performing co-infection studies.</p></div

Proinflammatory cytokine and anti-microbial peptide expression in neonatal and adult mice.

<p>Neonate and adult mice were infected with 1x10⁷ CFU’s of MRSA and cytokine gene expression measured at 6 and 24 hr pi (A-F). Expression of mRNA for cytokines and anti-microbial peptides was determined by qRT-PCR on RNA isolated from individual lung lobes (n = 5–6 mice per group) and relative expression is fold induction over WT unexposed mice of the same age. The level of IL6 (G)and IFNγ (H) in the lung homogenates was measured by ELISA. The data represent mean ± SEM of duplicate samples and significance was determined using one-way ANOVA with Tukey post-hoc test. NSA = neonatal / MRSA infection, NS = Neonatal / sham infection, ASA = adult / MRSA infection, AS = Adult sham infection.</p

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

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