Neutrophil TLR4 expression is reduced in the airways of infants with severe bronchiolitis

Abstract

Background: In respiratory syncytial virus (RSV) bronchiolitis, neutrophils account for >80% of cells recovered from the airways in bronchoalveolar lavage (BAL) fluid. This study investigated neutrophil activation and Toll-like receptor (TLR) expression in the blood and lungs of infants with severe RSV bronchiolitis.

Methods: BAL fluid and (blood) samples were collected from 24 (16) preterm and 23 (15) term infants ventilated with RSV bronchiolitis, and 12 (8) control infants. Protein levels and mRNA expression of CD11b, myeloperoxidase (MPO) and TLRs 2, 4, 7, 8 and 9 were measured in neutrophils.

Results: Blood neutrophils had more CD11b in preterm and term infants with RSV bronchiolitis than control infants (p<0.025) but similar amounts of MPO. BAL fluid neutrophils from infants with RSV bronchiolitis had greater amounts of CD11b and MPO than blood neutrophils and BAL fluid neutrophils from controls (p<0.01). Blood neutrophils from term infants with RSV bronchiolitis had less total TLR4 protein than preterm infants with RSV bronchiolitis (p = 0.005), and both had less than controls (p<0.04). Total TLR4 for each group was greater in BAL fluid neutrophils than in blood neutrophils. Blood neutrophils from preterm infants with RSV bronchiolitis had greater TLR4 mRNA expression than term infants with RSV bronchiolitis (p = 0.005) who had similar expression to controls (p = 0.625).

Conclusions: In infants with severe RSV bronchiolitis, neutrophil activation starts in the blood and progresses as they are recruited into the airways. Total neutrophil TLR4 remains low in both compartments. TLR4 mRNA expression is unimpaired. This suggests that neutrophil TLR4 expression is deficient in these infants, which may explain why they develop severe RSV bronchiolitis.

Figure 1

Protein expression of neutrophil activation markers is upregulated in respiratory syncytial virus (RSV) disease. Data are presented as mean (SEM) arbitrary units of antigen binding capacity (ABC) ×10⁶. (A) Histograms of flow cytometry data from two representative patients, an infant with RSV bronchiolitis (left) and a control infant (right), showing the mean protein expression of CD11b in the bronchoalveolar lavage (BAL) fluid compared with isotype control (shaded) (B) To determine mean cell surface CD11b expression, blood and airway neutrophils were stained, fixed and analysed by flow cytometry. CD11b expression was similar for term (n = 15) and preterm infants (n = 16) with RSV bronchiolitis, and both were significantly greater than controls (n = 8). There was significantly greater cell surface CD11b protein expression in the BAL neutrophils than in the blood neutrophils. (C) To determine mean total myeloperoxidase (MPO) expression, neutrophils from blood and BAL fluid were fixed and permeabilised before staining and analysed by flow cytometry. For blood neutrophils there was no difference in protein expression of MPO between infants with bronchiolitis (n = 31) and controls (n = 8). For the BAL fluid neutrophils, both term (n = 15) and preterm infants (n = 16) with RSV bronchiolitis had significantly more MPO expression than control infants (n = 8). There was significantly more MPO in BAL fluid than in blood. *p<0.05.

Figure 2

Toll-like receptor (TLR) 2 protein expression is unaltered in infants with severe respiratory syncytial virus (RSV) bronchiolitis. All data presented as mean (SEM) arbitrary units of antigen binding capacity (ABC) ×10⁶. (A) Representative histograms from flow cytometric staining of TLR2 on bronchoalveolar lavage (BAL) fluid neutrophils from three infants, a preterm infant with RSV bronchiolitis, a term infant with RSV bronchiolitis and a control infant. (B) The mean expression of cell surface TLR2 was determined by staining and fixing neutrophils prior to analysis by flow cytometry. The mean expression of cell surface TLR2 was similar between all three patient groups for airway and blood neutrophils (preterm infants, n = 16; term infants, n = 15; controls, n = 8) and for each group between blood and BAL fluid neutrophils. (C) To determine total TLR2, the neutrophils were fixed and permeabilised prior to analysis by flow cytometry. Total neutrophil TLR2 was similar between all three patient groups in both blood and BAL fluid (preterm RSV, n = 16; term RSV, n = 15; control, n = 8) and between blood and BAL fluid for each group.

Figure 3

Toll-like receptor (TLR) 4 protein expression is downregulated in infants with severe respiratory syncytial virus (RSV) bronchiolitis. Data presented as mean (SEM) arbitrary units of antigen binding capacity (ABC) ×10⁶. (A) Representative histograms from flow cytometric staining of TLR4 on bronchoalveolar lavage (BAL) fluid neutrophils from three infants, a preterm and a term infant with RSV bronchiolitis and a control infant. TLR4 expression was greater than TLR2. For TLR4, term infants with bronchiolitis had less total TLR4 than preterm infants with bronchiolitis and control infants. Shaded histograms are isotype controls. (B) Expression of cell surface TLR4 was analysed by flow cytometry after staining and fixing neutrophils. There was no significant difference between patient groups for cell surface expression of neutrophil TLR4 for either BAL fluid neutrophils (preterm RSV, n = 24; term RSV, n = 23; controls, n = 12) or blood neutrophils (preterm RSV, n = 16; term RSV, n = 15; controls, n = 8) or between blood and BAL fluid neutrophils for each patient group. (C) To determine total neutrophil TLR4 expression, cells were fixed and permeabilised before analysis by flow cytometry. In both blood and BAL fluid neutrophils, term and preterm infants with RSV bronchiolitis had significantly less TLR4 expression than control infants. Term infants with RSV bronchiolitis had significantly less total TLR4 than preterm infants with bronchiolitis (p = 0.03). *p<0.05. Total neutrophil TLR4 in BAL fluid was significantly greater (p<0.05) than in blood for all three groups (not shown).

Figure 4

Neutrophil TLR4 mRNA expression predominantly occurs in the blood compared with the bronchoalveolar lavage (BAL) fluid and is similar in term and control infants. Data presented as mean (SEM) logfold ratio after expression of mRNA assessed by RT-PCR for all Toll-like receptors (TLRs) standardised as a ratio to mRNA for the housekeeping gene L32. (A) Neutrophil TLR4 mRNA was significantly increased in blood neutrophils, mRNA expression was greater in preterm infants (n = 16) than in term infants with respiratory syncytial virus (RSV) bronchiolitis (n = 15), but there was no significant difference between term infants with RSV bronchiolitis and controls (n = 8). (B) Neutrophil TLR4 mRNA was significantly increased in BAL fluid neutrophils from preterm infants with RSV bronchiolitis (n = 16) compared with term infants with RSV bronchiolitis (n = 15), which was similar to control infants (n = 8). There was considerably less mRNA TLR4 for each patient group in the BAL fluid than in the blood (not shown). (C) As the blood seemed to be the main site of TLR4 mRNA production, neutrophil mRNA expression of TLR 2, 7, 8 and 9 was determined for blood neutrophils for preterm (n = 16) and term infants with RSV bronchiolitis (n = 15) and for controls (n = 8). There was no significant difference in the expression of blood neutrophil mRNA TLR2 between the three patient groups. Preterm infants had significantly greater expression of TLR7 and 9 in blood neutrophils than term or control infants (p<0.05). The low levels of expression of neutrophil TLR8 in term infants with RSV bronchiolitis were similar to the levels in control infants; levels of TLR8 in preterm infants with RSV bronchiolitis were undetectable. *p<0.05.