Macrophage phenotype is associated with disease severity in preterm infants with chronic lung disease.

The etiology of persistent lung inflammation in preterm infants with chronic lung disease of prematurity (CLD) is poorly characterized, hampering efforts to stratify prognosis and treatment. Airway macrophages are important innate immune cells with roles in both the induction and resolution of tissue inflammation

Numbers of neutrophils in the airway correlate with numbers of mature macrophages.

<p>Infants were ventilated and lavaged within 24-CD15, anti-CD14, anti-HLA-DR, anti-CD36 or matched isotype antibodies and analysed by flow cytometry. Data are expressed as absolute numbers of CD15+ events plotted against absolute numbers of CD14/CD36+ (A) or CD14/HLADR+ (B). Data are analysed by liner regression (n = 11 of which CLD∶RDS; 8∶3) and show R square and p values.</p

Preterm delivery is associated with increased non-classical macrophages in the airway.

<p>Infants were ventilated and lavaged within 24-CD14, anti-CD16, anti-CD36, anti-HLADR or matched isotype antibodies and analysed by flow cytometry. Non-classical macrophages are defined as CD14+/CD16++ and expressed as percent of all macrophages (A) or absolute numbers of CD14+/CD16++ cells/ml lavage (B). CD14+/HLA-DR+ and CD14+/CD36+ cells are expressed as percent of all macrophages (C, D). Statistical analyses were carried out by Mann Whitney test and compared samples collected from pre-term (n = 10–12 of which CLD∶RDS; 7–8∶3–4) to term (n = 4) infants.</p

Flow cytometry strategy for identifying functionally polarised macrophage populations in BALF.

<p>Infants were ventilated and lavaged within 24-CD15 (A), anti-CD14 (B), anti-CD16 (C), anti-HLADR (D), anti-CD36 (E) or matched isotype antibodies and analysed by flow cytometry. Staining profiles showing isotype controls in blue and target antibody in red. CD14/CD16 staining profile shown in F. Positively stained events are higlighted in FSC and SSC plots in blue for HLADR (G), CD36 (H), CD14/HLADR dual stained (I) and CD14/CD36 dual stained (J). FSC/SSC profiles of CD14+ and: HLADR− (K), HLADR+ (L), CD36− (M) and CD36+ (N) events. Plots illustrate representative data from 3 individual subjects.</p

Morpholocial assessment of BAL cellularity.

<p>Infants were ventilated and lavaged within 24(A,B) and infants who developed CLD (C,D) or RDS (E–H) were studied. Images were viewed under ×400 (A,C,E) or ×1000 (B,D,E–H) lenses. Cell populations indicated as follows: m: macrophage, ec: epithelial cell, n: neutrophil Scale bar indicates 0–7.5 um in length.</p

Early delivery is associated with increased numbers of macrophages and decreased populations of cells with an anti-inflammatory phenotype.

<p>Infants were ventilated and lavaged at 3 days of age and cells were stained with anti-CD14, anti-HLA-DR, anti-CD36 or matched isotype antibodies and analysed by flow cytometry. Data are expressed as percentage (A) or absolute numbers (B) of CD14+ events. Cells dual stained with CD14+/HLADR (C) or CD14+/CD36+ (D) are expressed as a percentage of all CD14+ events. Data are analysed by liner regression (n = 9–11 of which CLD∶RDS; 6–8∶3) and show R square and p values.</p

Preterm delivery is associated with increased neutrophils and monocyte–macrophages in the airway.

<p>Infants were ventilated and lavaged within 24-CD14, anti-CD15 or matched isotype antibodies and analysed by flow cytometry. Neutrophils (A, B) are defined as CD15+ events and macrophages (C, D) are defined as CD14+ve events and expressed as percent of all cells (A, C) or as absolute numbers/ml lavage fluid (B, D). Statistical analyses were carried out by Mann Whitney test and compared samples collected from pre-term (n = 12–16 of which RDS∶CLD 6–8∶8) to term (n = 4) infants.</p

Inflammation resolution is associated with increased populations of anti-inflammatory macrophages.

<p>Infants were ventilated and lavaged within 24-CD14, anti-HLA-DR, anti-CD36 or matched isotype antibodies and analysed by flow cytometry. CD14⁺/HLA-DR⁺ and CD14⁺/CD36⁺ cells are expressed as percent of all macrophages (A, B). Statistical analyses were carried out by Mann Whitney test and compared samples collected from CLD (n = 6–7) to RDS (n = 4) infants.</p