Effect of allergy and inflammation on eicosanoid gene expression in CFTR deficiency

Abstract

Background: Allergic bronchopulmonary aspergillosis (ABPA) is a complicating factor in cystic fibrosis (CF), affecting 2-15% of patients. We hypothesized that sensitization/challenge of CFTR(-/-) mice with an Aspergillus fumigatus (Af) extract will affect eicosanoid pathway gene expression, impacting ABPA and CF.

Methods: FABP-hCFTR(+/-)-CFTR(-/-) mice were sensitized/challenged with an Af extract and gene expression of lung mRNA was evaluated for >40 genes, with correlative data in human CF (IB3.1) and CFTR-corrected (S9) bronchoepithelial cell lines.

Results: Pla2g4c, Pla2g2c, Pla2g2d and Pla2g5 were induced in response to Af in CFTR(-/-) mice. Interestingly, PLA2G2D was induced by LPS, IL-2, IL-6, IL-13, and Af only in CFTR-deficient human IB3.1 cells. Prostanoid gene expression was relatively constant, however, several 12/15-lipoxygenase genes were induced in response to Af. Numerous cytokines also caused differential expression of ALOX15 only in IB3.1 cells.

Conclusions: The distinct regulation of PLA2G4C, PLA2G2D and ALOX15 genes in Aspergillus sensitization and/or cystic fibrosis could provide new insights into diagnosis and treatment of ABPA and CF.

Figure 1

Secretory phospholipase A₂ steady state mRNA levels in mouse lungs and human cell lines. (A) CFTR^−/− mice were sensitized/challenged with PBS or Af. Real-time RT-PCR was performed from whole lung mRNA for group II, V, X and XII PLA₂s: Pla2g2c, Pla2g2d, Pla2g2e, Pla2g5, Pla2g10 and Pla2g12b. Data points are the means of 2^−ΔΔCT ± SEM (7≤n≤10). * indicate p≤0.05 as compared to PBS-sensitized mice. (B) Real-time RT-PCR of PLA2G2D following a 12h treatment of S9 and IB3.1 cells with the indicated inflammatory stimuli. Data points are the means of 2^−ΔΔCT ± SEM (n=3). * and + indicate p≤0.05 as compared to IB3.1 control and treated S9 cells, respectively.

Figure 2

Group IV cytosolic phospholipase A₂s steady state mRNA levels in mouse lungs. CFTR^−/− mice were sensitized/challenged with PBS or Af as described in Fig. 1. Real-time RT-PCR was then used to determine the steady state mRNA levels from whole lungs for the group IV PLA₂s, Pla2g4a, Pla2g4b and Pla2g4c. * indicates p≤0.05 as compared to PBS-sensitized mice.

Figure 3

Gene expression analysis of enzymes involved in prostanoid synthesis in mouse lungs. CFTR^−/− mice were sensitized/challenged as described in Fig. 1, and real-time RT-PCR on whole lung mRNA was performed for Ptgs1, Ptgs2, Ptges, Tbxas1, Ptgds, and Ptgis. Data points are the means of 2^−ΔΔCT ± SEM (7≤n≤10).

Figure 4

Gene expression analysis of enzymes in the lipoxygenase pathway in mouse lungs and human cell lines. (A) CFTR^−/− mice were sensitized/challenged as with PBS or Af, and whole lung mRNA was analyzed by real-time RT-PCR for lipoxygenase gene expression (Aloxe3, Alox5, Alox5ap, Alox12, Alox12b, Alox15, Alox8, and Alox12e). Data points are the means of 2^−ΔΔCT ± SEM (7≤n≤10). * indicate p≤0.05 as compared to PBS-sensitized mice. (B) Steady state mRNA levels of human ALOX15 in response to the indicated stimuli in S9 or IB3.1 cells. Data points are the means of 2^−ΔΔCT SEM (n=3). (C) Steady state mRNA levels of human ALOX15B in response to the indicated stimuli in S9 or IB3.1 cells. Data points are the means of 2^−ΔΔCT ± SEM (n=3). * and + indicate p≤0.05 as compared to control and S9 cells, respectively.