Secondhand smoke alters arachidonic acid metabolism and inflammation in infants and children with cystic fibrosis

Abstract

Background: Mechanisms that facilitate early infection and inflammation in cystic fibrosis (CF) are unclear. We previously demonstrated that children with CF and parental-reported secondhand smoke exposure (SHSe) have increased susceptibility to bacterial infections. SHSe hinders arachidonic acid (AA) metabolites that mediate immune function in patients without CF, and may influence CF immune dysfunction. We aimed to define SHSe's impact on inflammation mediators and infection in children with CF.

Methods: Seventy-seven children with CF <10 years of age (35 infants <1 year; 42 children 1-10 years) were enrolled and hair nicotine concentrations measured as an objective surrogate of SHSe. AA signalling by serum and macrophage lipidomics, inflammation using blood transcriptional profiles and in vitro macrophage responses to bacterial infection after SHSe were assessed.

Results: Hair nicotine concentrations were elevated in 63% of patients. Of the AA metabolites measured by plasma lipidomics, prostaglandin D₂ (PGD₂) concentrations were decreased in children with CF exposed to SHSe, and associated with more frequent hospitalisations (p=0.007) and worsened weight z scores (p=0.008). Children with CF exposed to SHSe demonstrated decreased expression of the prostaglandin genes PTGES3 and PTGR2 and overexpression of inflammatory pathways. These findings were confirmed using an in vitro model, where SHSe was associated with a dose-dependent decrease in PGD₂ and increased methicillin-resistant Staphylococcus aureus survival in human CF macrophages.

Conclusions: Infants and young children with CF and SHSe have altered AA metabolism and dysregulated inflammatory gene expression resulting in impaired bacterial clearance. Our findings identified potential therapeutic targets to halt early disease progression associated with SHSe in the young population with CF.

Keywords: cystic fibrosis; macrophage biology; paediatric lung disaese; tobacco and the lung.

Figure 1

Infants with cystic fibrosis (CF) have disproportionately high secondhand smoke exposure. CF hair nicotine concentrations were determined by high-performance liquid chromatography-tandem mass spectrometry and plotted by age grouping (infants<1 year, children 1–5 years, children≥5 years). P=0.006 for infants vs children 1–5 years, p=0.002 for infants vs children≥5 years, via t-tests. Analysis of variance p=0.002. Nicotine concentrations were inversely correlated with age (r=−0.33, p=0.011, Pearson’s correlation coefficient), n=57. As a reference, active smokers have been reported to have median hair nicotine concentrations of 16.2 ng/mg hair.

Figure 2

Secondhand smoke exposure (SHSe) alters serum arachidonic acid (AA) metabolism in cystic fibrosis (CF). Serum lipidomics were measured by high-performance liquid chromatography-tandem mass spectrometry in a combined group of infants and children with CF. (A) SHSe was associated with a significant decrease in serum AA metabolites, specifically: prostaglandin D₂ (PGD₂), leukotriene C₄, 8S-hydroxy-5Z, 9E, 11Z, 14Z-eicosatetraenoic acid (8(S)-HETE), 9S-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid (9(S)-HETE), 12(S)-hydroperoxy tetraenoic eicosatetraenoic acid (12(S)-HETE) and 14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14(15)-EET). (B) Decreased PGD₂ concentrations were associated with increased hospitalisations (p=0.007, Wilcoxon rank-sum test) and (C) decreased weight z score (r=0.34, p=0.008). (D) PGD₂ was not associated with FEV₁ (r=0.19). Associations between other AA metabolite concentrations and hospitalisations, weight z score and FEV₁ were not significant. PGD₂ and other AA metabolite concentrations also did not correlate with height z score or oropharyngeal/sputum bacterial cultures (not shown). Correlations were performed using Pearson’s correlation coefficient.

Figure 3

Secondhand smoke exposure (SHSe) alters whole blood transcriptional profiles in cystic fibrosis (CF). (A) Class comparisons using linear models adjusted for age and race (p<0.01; Benjamini-Hochberg corrected flase discovery rate (FDR) and ≥1.25-fold change), in children with CF and SHSe (n=18) vs age-matched healthy controls (n=12) and children with CF and non-SHSe (n=10) vs the same healthy controls, identified 2188 differentially expressed transcripts that were present in either of these conditions. Infants were not analysed. Transcripts are organised in a heatmap format where each row represents a single transcript and each column represents a subject sample. Red colour indicates overexpression and blue colour underexpression of a transcript compared with the median expression of healthy controls (yellow). (B) Two prostaglandin transcripts (PTGR2 and PTGES3) were found to be significantly underexpressed in children with CF exposed to SHSe as compared with those not exposed to SHSe. (C) Modular expression in children with CF exposed to SHSe (n=18) and not exposed to SHSe (n=10), each group was compared with matched healthy controls (n=12). Children with CF exposed to SHSe demonstrated significant overexpression of inflammation genes and greater suppression of protein synthesis and plasma cell-related transcripts. The intensity of the modules (dots) indicates the proportion of overexpressed (in red) or underexpressed (in blue) transcripts within each module. Numeric values indicate the exact percentage of transcripts expressed in each specific module. A blank dot indicates that <10% of the genes in the module were differentially expressed.

Figure 4

Secondhand smoke exposure (SHSe) alters cystic fibrosis (CF) macrophage prostaglandin D₂(PGD₂) production. (A) Cytotoxicity as %LDH release was measured in child and adult CF monocyte-derived macrophage supernatants in response to increasing concentrations of cigarette smoke extract (CSE), 24 hours challenge, n=3. (B) Changes in the arachidonic acid metabolite PGD₂ were measured in CF macrophage supernatants in response to increasing concentrations of CSE during a 24 hours challenge. (C) PGD₂ expression was determined by western blot analysis in CF and non-CF macrophages in response to exposure to a 0% or 15% CSE, n=3, representative image shown.

Figure 5

Secondhand smoke exposure alters arachidonic acid (AA) metabolism in cystic fibrosis (CF) macrophages. Lipidomics for AA metabolites was performed via high-performance liquid chromatography-tandem mass spectrometry in CF and non-CF macrophage supernatants from children and adults after exposure to either 0% or 15% cigarette smoke extract (CSE), n=3, 24 hours challenge. *p<0.05, t-test. LTE₄, leukotriene E₄; LXA4, lipoxin A₄; PGE2, prostaglandin E₂; PGD₂, prostaglandin D₂; 8(S)-HETE, 8S-hydroxy-5Z,9E,11Z,14Z-eicosatetraenoic acid; (9(S)-HETE, 9S-hydroxy-5Z,7E,11Z,14Z-eicosatetraenoic acid; 12(S)-HETE, 12(S)-hydroperoxy tetraenoic eicosatetraenoic acid.

Figure 6

Secondhand smoke exposure decreases methicillin-resistant Staphylococcus aureus (MRSA) killing in cystic fibrosis (CF) macrophages. Human CF and non-CF macrophages from children and adults were subacutely (72 hours) treated with 15% cigarette smoke extract (CSE) and infected with a clinical isolate of either (A) MRSA, (B) Pseudomonas aeruginosa or (C) Burkholderia cenocepacia, n=6. Bacterial counts were measured via colony-forming unit (CFU) assay and presented as CFU/mL of media.