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

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

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

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

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