Distal respiratory tract viral infections in young children trigger a marked increase in alveolar mast cells

Abstract

Viral infections predispose to the development of childhood asthma, a disease associated with increased lung mast cells (MCs). This study investigated whether viral lower respiratory tract infections (LRTIs) can already evoke a MC response during childhood. Lung tissue from young children who died following LRTIs were processed for immunohistochemical identification of MCs. Children who died from nonrespiratory causes served as controls. MCs were examined in relation to sensitisation in infant mice exposed to allergen during influenza A infection. Increased numbers of MCs were observed in the alveolar parenchyma of children infected with LRTIs (median (range) 12.5 (0-78) MCs per mm²) compared to controls (0.63 (0-4) MCs per mm², p=0.0005). The alveolar MC expansion was associated with a higher proportion of CD34⁺ tryptase⁺ progenitors (controls: 0% (0-1%); LRTIs: 0.9% (0-3%) CD34⁺ MCs (p=0.01)) and an increased expression of the vascular cell adhesion molecule (VCAM)-1 (controls: 0.2 (0.07-0.3); LRTIs: 0.3 (0.02-2) VCAM-1 per mm² (p=0.04)). Similarly, infant mice infected with H1N1 alone or together with house dust mite (HDM) developed an increase in alveolar MCs (saline: 0.4 (0.3-0.5); HDM: 0.6 (0.4-0.9); H1N1: 1.4 (0.4-2.0); HDM+H1N1: 2.2 (1.2-4.4) MCs per mm² (p<0.0001)). Alveolar MCs continued to increase and remained significantly higher into adulthood when exposed to H1N1+HDM (day 36: 2.2 (1.2-4.4); day 57: 4.6 (1.6-15) MCs per mm² (p=0.01)) but not when infected with H1N1 alone. Our data demonstrate that distal viral infections in young children evoke a rapid accumulation of alveolar MCs. Apart from revealing a novel immune response to distal infections, our data may have important implications for the link between viral infections during early childhood and subsequent asthma development.

FIGURE 1

Experimental plan. a) 8-day-old mice were infected with influenza A virus (H1N1) or given PBS alone. 7 days later, PBS- and H1N1-infected groups were exposed either to 3 weeks of house dust mite (HDM) or saline and sacrificed 72 h after the last HDM exposure (day 36). b) Separate groups of 8-day-old mice were infected with influenza virus or given PBS alone. 7 days later, PBS- and H1N1-infected groups were exposed either to 3 weeks of HDM or saline, then, after the last HDM exposure, mice were rested for 3 weeks and sacrificed at 8 weeks of age (day 57). Mast cell (MC) densities in alveolar parenchyma in mice exposed to HDM or H1N1 alone or in combination compared to saline-treated animals: c) day 36 and d) day 57. e) MC densities at day 36 compared to day 57 in all groups. Toluidine blue stained slides (MCs shown in purple) picturing alveolar parenchyma from f and j) saline-, g and k) HDM-, h and l) H1N1-, and i and m) HDM+H1N1-treated animals. Scale bars=200 µm. Overall significance, using Kruskal–Wallis test, is denoted in each panel. Results were considered significant at p≤0.05, and individual differences between groups using Kruskal–Wallis test with Dunn's post hoc test. alv: alveolus. **: p≤0.01 versus controls; ***: p≤0.001 versus controls; ****: p≤0.0001 versus controls; ^#: p≤0.05 between HDM+H1N1 and HDM; ^##: p≤0.01 between HDM+H1N1 and HDM.

FIGURE 2

Mast cell (MC) densities in a) small airways, b) pulmonary vessels and c) alveolar parenchyma. Double immunohistochemical staining for tryptase (red) and chymase (brown). Representative photograph of d) a small airway and pulmonary vessel in a control lung from a child and e) alveolar parenchyma. f) Small airway in a lung from a child infected with influenza A. Alveolar parenchyma from young children who have died following g) respiratory syncytial virus (RSV), h) adenovirus (ADV) and i) influenza infections. Immunohistochemical staining for influenza A (red) in the epithelium with j and k) fluorescence and l) 3,3′-diaminobenzidine. k) Tryptase-positive MCs are shown in the subepithelium (green). m) RSV positivity in the alveolar parenchyma. Scale bars: d, f and g) 500 µm; e) 200 µm; h and k) 100 µm; i and m) 70 µm; j and l) 50 µm. Results were considered significant at p≤0.05. ns: not significant; LRTI: lower respiratory tract infection; SA: small airway; V: vessel; alv: alveolus; lu: lumen; ep: epithelium. ***: p≤0.001 using Mann–Whitney rank sum test, in comparison to controls.

FIGURE 3

a) Proportion of CD34⁺ mast cells (MCs) in small airways, pulmonary vessels and alveolar parenchyma. b) CD34⁺ (red) MCs (green) in a pulmonary vessel wall. Scale bar=30 µm). c and d) Influenza A-infected lung stained for tryptase (green) and proliferation marker Ki-67 (red). Arrows show Ki-67⁺ cells and * denotes tryptase⁺ cells. e) Alveolar parenchyma stained for tryptase (green) and IgE (red). Inset shows neighbouring IgE⁺ cell and MC. f–h) Alveolar parenchyma stained for tryptase (green) and integrin β₁ (ITGB1) (red). i–k) Tryptase (green) integrin α₄ (ITGA4) (red). Integrin⁺ MCs are denoted with arrows. Results were considered significant at p≤0.05. lu: lumen. **: p≤0.01 using Mann–Whitney rank sum test, in comparison to controls.

FIGURE 4

Vascular cell adhesion molecule (VCAM)-1 expression in a) small airway and b) pulmonary vessel walls, and c) alveolar parenchyma. d) Immunohistochemical staining of VCAM-1 in a pulmonary vessel from a child with lower respiratory tract infection (LRTI). VCAM-1 expression in alveolar parenchyma from e) a control child and f) a child with LRTI, respectively. Scale bars: d) 50 µm; e) 200 µm; f) 100 µm. Results were considered significant at p≤0.05. ns: not significant; ADV: adenovirus; RSV: respiratory syncytial virus; V: vessel; alv: alveolus. *: p≤0.05 using Mann–Whitney rank sum test, in comparison to controls; **: p≤0.01 using Mann–Whitney rank sum test, in comparison to controls.