The effect of BPIFA1/SPLUNC1 genetic variation on its expression and function in asthmatic airway epithelium

Bacterial permeability family member A1 (BPIFA1), also known as short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is a protein involved in the antiinflammatory response. The goal of this study was to determine whether BPIFA1 expression in asthmatic airways is regulated by genetic variations, altering epithelial responses to type 2 cytokines (e.g., IL-13). Nasal epithelial cells from patients with mild to severe asthma were collected from the National Heart, Lung. and Blood Institute Severe Asthma Research Program centers, genotyped for rs750064, and measured for BPIFA1. To determine the function of rs750064, cells were cultured at air-liquid interface and treated with 11-13 with or without recombinant human BPIFA1 (rhBPIFA1). Noncultured nasal cells with the rs750064 CC genotype had significantly less BPIFA1 mRNA expression than the CT and TT genotypes. Cultured CC versus CT and TT cells without stimulation maintained less BPIFA1 expression. With IL-13 treatment, CC genotype cells secreted more eotaxin-3 than CT and TT genotype cells. Also, rhBPIFA1 reduced IL-13-mediated eotaxin-3. BPIFA1 mRNA levels negatively correlated with serum IgE and fractional exhaled nitric oxide. Baseline FEV1% levels were lower in the asthma patients with the CC genotype (n = 1,016). Our data suggest that less BPIFA1 in asthma patients with the CC allele may predispose them to greater eosinophilic inflammation, which could be attenuated by rhBPIFA1 protein therapy.NIH/NHLBI [R01HL125128, U10HL109257, UL1TR00448, U10HL109168]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Increased antiviral gene expression in Muc18 knockout (KO) mice infected with human rhinovirus 1B (HRV-1B).

<p>Muc18 WT (n = 9) and KO (n = 13) were intranasally infected with HRV-1B or PBS as a control (WT: n = 7; KO: n = 7) and sacrificed 24 hours post treatment. In lung tissue, KO mice had significantly higher expression of the antiviral genes Mx1 <b>(A)</b> and IP-10 <b>(B)</b> compared to WT mice. Dots represent data from individual of the three independent experiments and the horizontal bars indicate median. A single star (*) indicates p<0.05 and two stars (**) indicates p<0.001.</p

Fluorescent Green Dye to indicate endosome acidity in human tracheobronchial epithelial cells infected with rhinovirus 1B.

<p>Tracheobronchial epithelial cells from donors were seeded onto cover slips and transfected with control or MUC18 siRNA and infected with culture medium (–) or HRV-1B for 2 hours. 30 minutes prior to the end of infection, LysoSensor Dye was added to the cultures. Cover slips were removed and stained with DAPI to indicate the nucleus and mounted onto microscope slides. Control siRNA cells were more green, indicating a lower pH <b>(A)</b> and cells transfected with MUC18 siRNA were less green, indicating a higher pH <b>(B)</b>.</p

Reduced viral load and IL-8 induction in human airway epithelial cells with MUC18 knockdown (KD).

<p>Tracheobronchial epithelial cells from three donors (n = 3 replicates per donor) were transfected with control or MUC18 siRNA and then treated with culture medium (–) or HRV for 2 hours. Cells were harvested 24 hours post infection. Viral load was significantly lower in MUC18 KD cells compared to control <b>(A).</b> Expression of the antiviral gene MX1 was similar between KD and control cells <b>(B).</b> There was less induction of the pro-inflammatory cytokine, IL-8, in MUC18 KD samples compared to control <b>(C)</b>, but was not statistically significant. Successful knockdown of MUC18 was confirmed via Western Blot and densitometry <b>(D)</b> in cells after 24 hours of MUC18 or control siRNA transfection. Data are presented as means ± SEMs. A single star (*) indicates p<0.05 and two stars (**) indicates p<0.001.</p

Reduced neutrophils in Muc18 KO mice infected with human rhinovirus 1B (HRV-1B).

<p>Muc18 WT (n = 9) and KO (n = 13) were intranasally infected with HRV-1B or PBS as a control (WT: n = 7; KO: n = 7) and sacrificed 24 hours post treatment. Muc18 KO mice had significantly lower levels of neutrophils in bronchoalveolar lavage (BAL) than WT mice. This was seen in both percent neutrophils <b>(A)</b> and in absolute number of neutrophils <b>(B)</b>. <b>(C)</b> HRV infection significantly increased KC levels in WT and KO mice, but there was no significant difference in KC levels between WT and KO mice. Dots represent data from individual of the three independent experiments and the horizontal bars indicate median. The Wilcoxon test was used to compare the difference between groups. A single star (*) indicates p<0.05 and two stars (**) indicates p<0.001.</p

Viral load is significantly reduced in BAL fluid and lung tissue of Muc18 KO mice.

<p>Muc18 WT (n = 9) and KO (n = 13) were intranasally infected with HRV-1B or PBS as a control (WT: n = 7; KO: n = 7) and sacrificed 24 hours post treatment. KO mice had significantly lower viral load in BAL fluid than WT mice <b>(A).</b> This trend continued in lung tissue <b>(B)</b>. Dots represent data from individual of the three independent experiments and the horizontal bars indicate median. A single star (*) indicates p<0.05 and two stars (**) indicates p<0.001.</p

Tracheal epithelial cells of Muc18 KO mice have less KC, less viral load, and greater antiviral gene expression.

<p>Muc18 WT (n = 3 replicates) and KO (n = 3 replicates) tracheal epithelial cells were cultured in air-liquid interface and treated with culture medium (–) or infected with HRV-1B for 24 hours. KO cells had significantly less KC <b>(A)</b> and viral load <b>(B)</b> than WT cells. In KO cells, expression of the antiviral genes Mx1 and IP-10 was significantly greater than WT cells, in both infected and non-infected conditions (p = 0.0495)<b>(C, D).</b> IRF-3 gene expression was significantly greater in non-infected KO cells compared to WT (p = 0.0495), but unchanged in infected conditions <b>(E).</b> Data are presented as means ± SEMs. A single star (*) indicates p<0.05 and two stars (**) indicates p<0.001.</p

The effect of BPIFA1/SPLUNC1 genetic variation on its expression and function in asthmatic airway epithelium

Bacterial permeability family member A1 (BPIFA1), also known as short palate, lung, and nasal epithelium clone 1 (SPLUNC1), is a protein involved in the antiinflammatory response. The goal of this study was to determine whether BPIFA1 expression in asthmatic airways is regulated by genetic variations, altering epithelial responses to type 2 cytokines (e.g., IL-13). Nasal epithelial cells from patients with mild to severe asthma were collected from the National Heart, Lung, and Blood Institute Severe Asthma Research Program centers, genotyped for rs750064, and measured for BPIFA1. To determine the function of rs750064, cells were cultured at air-liquid interface and treated with IL-13 with or without recombinant human BPIFA1 (rhBPIFA1). Noncultured nasal cells with the rs750064 CC genotype had significantly less BPIFA1 mRNA expression than the CT and TT genotypes. Cultured CC versus CT and TT cells without stimulation maintained less BPIFA1 expression. With IL-13 treatment, CC genotype cells secreted more eotaxin-3 than CT and TT genotype cells. Also, rhBPIFA1 reduced IL-13-mediated eotaxin-3. BPIFA1 mRNA levels negatively correlated with serum IgE and fractional exhaled nitric oxide. Baseline FEV1% levels were lower in the asthma patients with the CC genotype (n = 1,016). Our data suggest that less BPIFA1 in asthma patients with the CC allele may predispose them to greater eosinophilic inflammation, which could be attenuated by rhBPIFA1 protein therapy