MicroRNA-375 regulation of thymic stromal lymphopoietin by diesel exhaust particles and ambient particulate matter in human bronchial epithelial cells

Abstract

Air pollution contributes to acute exacerbations of asthma and the development of asthma in children and adults. Airway epithelial cells interface innate and adaptive immune responses, and have been proposed to regulate much of the response to pollutants. Thymic stromal lymphopoietin (TSLP) is a pivotal cytokine linking innate and Th2 adaptive immune disorders, and is upregulated by environmental pollutants, including ambient particulate matter (PM) and diesel exhaust particles (DEP). We show that DEP and ambient fine PM upregulate TSLP mRNA and human microRNA (hsa-miR)-375 in primary human bronchial epithelial cells (pHBEC). Moreover, transfection of pHBEC with anti-hsa-miR-375 reduced TSLP mRNA in DEP but not TNF-α-treated cells. In silico pathway evaluation suggested the aryl hydrocarbon receptor (AhR) as one possible target of miR-375. DEP and ambient fine PM (3 μg/cm(2)) downregulated AhR mRNA. Transfection of mimic-hsa-miR-375 resulted in a small downregulation of AhR mRNA compared with resting AhR mRNA. AhR mRNA was increased in pHBEC treated with DEP after transfection with anti-hsa-miR-375. Our data show that two pollutants, DEP and ambient PM, upregulate TSLP in human bronchial epithelial cells by a mechanism that includes hsa-miR-375 with complex regulatory effects on AhR mRNA. The absence of this pathway in TNF-α-treated cells suggests multiple regulatory pathways for TSLP expression in these cells.

Figure 1. DEP and ambient fine PM upregulate TSLP and hsa-miR-375 in a dose-dependent manner

Confluent pHBEC were cultured (6h) with increasing concentrations of DEP or ambient fine PM. RNA was isolated and levels of TSLP mRNA and hsa-miR-375 measured (qPCR). Data are expressed as fold increase compared to GAPDH (TSLP) or RNU 6-2 (hsa-miR-375) (2^−ΔΔCt; mean ± SE; n = 3 independent experiments).

Figure 2. DEP and ambient fine PM, but not TNF-α upregulate hsa-miR-375

Confluent pHBEC were cultured (6h, 37°C) with DEP (3 μg/cm²), ambient fine PM (3 μg/cm²), carbon (3μg/cm²) or TNF-α (5 ng/ml). RNA was isolated and levels of hsa-miR-375 measured (qPCR). Data are expressed as fold increase compared to resting (2^−ΔΔCt; mean ± SE; n = 3 independent experiments; * = p < 0.05).

Figure 3. Synthetic mimic hsa-miR-375 induced TSLP expression

pHBEC were transfected with vehicle alone or with mimic-hsa-miR-375 or syn-miR control oligonucleotides (HiPerFect, 18h). Cells were stimulated as depicted (6h, 37°C), RNA was isolated and TSLP mRNA measured (qPCR, GAPDH internal control). Data are expressed as fold increase compared to resting vehicle control (2^−ΔΔCt; mean ± SE; n = 3 independent experiments; * = p <0.05).

Figure 4. Upregulation of TSLP by DEP is dependent on hsa-miR-375

(4A)TSLP mRNA expression in pHBEC transfected with vehicle, anti-hsa-miR-375, or anti-miR control oligonucleotides (HiPerFect, 18h, 37°C). Cells were stimulated with defined agents (6h, 37°C), RNA isolated and TSLP mRNA measured (qPCR, GAPDH internal control). Data are expressed as fold increase compared to resting vehicle control (2^−ΔΔCt; mean ± SE; n = 3 independent experiments; * = p < 0.05 vs. resting, ** = p < 0.05 vs. DEP). (4B) TSLP protein was measured (ELISA) in pHBEC supernates after treatment of pHBEC with defined stimuli (18h, 37°C) in cells transfected with mimic-hsa-miR-375, syn-miR control, anti-hsa-miR-375 or anti-miR control. Data are mean ± SEM (n = 3 independent experiments, * = p <0.05 vs. resting, ** = p < 0.05 vs. DEP.

Figure 5. DEP and ambient fine PM down-regulate AhR expression

pHBEC were cultured (6h, 37°C) with DEP (3 μg/cm²), ambient fine PM (3 μg/cm²), carbon (3 μg/cm²) or TNF-α (5 ng/ml). RNA was isolated and levels of AhR and CYP1a1 measured (qPCR, GAPDH internal control). Data are expressed as fold change compared to resting (2^−ΔΔCt; mean ± SE; n = 3 independent experiments; * = p<0.05 compared to resting AhR; ** = p<0.05 compared to resting CYP1a1).

Figure 6. AhR expression is regulated by hsa-miR-375

pHBEC were transfected with vehicle or with synthetic oligonucleotides (mimic hsa-miR-375, syn-hsa-miR control, anti- hsa-miR-375, anti- hsa-miR control). After 18h, cells were stimulated with DEP as depicted (6h, 37°C), RNA isolated and AhR mRNA measured (qPCR, GAPDH internal control). Data are expressed as fold change compared to resting vehicle control (2^−ΔΔCt; mean ± SE; n=3 independent experiments; * = p < 0.05).

Figure 7. TSLP and AhR mRNA degradation

pHBEC were resting or transfected with mimic-hsa-miR-375 or anti- hsa-miR control and were treated with actinomycin D (10 μg/ml) in either the presence or absence of DEP (3 μg/cm²). Total RNA was isolated after 0, 1, 2, and 4 hours, and TSLP, AhR and GAPDH mRNA determined by qPCR. Data are presented as the percentage of mRNA relative to 0 time point after normalization to GAPDH (mean ± SE, n = 3 independent experiments).