AhR Activation at the Air-Blood Barrier Alters Systemic microRNA Release After Inhalation of Particulate Matter Containing Environmentally Persistent Free Radicals

Abstract

Particulate matter containing environmentally persistent free radicals (EPFRs) is formed when organic pollutants are incompletely burned and adsorb to the surface of particles containing redox-active metals. Our prior studies showed that in mice, EPFR inhalation impaired vascular relaxation in a dose- and endothelium-dependent manner. We also observed that activation of the aryl hydrocarbon receptor (AhR) in the alveolar type-II (AT-II) cells that form the air-blood interface stimulates the release of systemic factors that promote endothelial dysfunction in vessels peripheral to the lung. AhR is a recognized regulator of microRNA (miRNA) biogenesis, and miRNA control diverse signaling pathways. We thus hypothesized that systemic EPFR-induced vascular endothelial dysfunction is initiated via AhR activation in AT-II cells, resulting in a systemic release of miRNA. Using a combustion reactor, we generated EPFR of two free radical concentrations-EPFR_lo (10^16-17 radicals/g particles) and EPFR (10^18-19 radicals/g)-and exposed mice by inhalation. EFPR inhalation resulted in changes in a distinct array of miRNA in the plasma, and these miRNAs are linked to multiple systemic effects, including cardiovascular diseases and dysregulation of cellular and molecular pathways associated with cardiovascular dysfunction. We identified 17 miRNA in plasma that were altered dependent upon both AhR activation in AT-II cells and ~ 280 ug/m³ EPFR exposure. Using Ingenuity Pathway Analysis, we found that 5 of these miRNAs have roles in modulating endothelin-1 and endothelial nitric oxide signaling, known regulators of endothelial function. Furthermore, EPFR exposure reduced the expression of lung adherens and gap junction proteins in control mice but not AT-II-AhR deficient mice, and reductions in barrier function may facilitate miRNA release from the lungs. In summary, our findings support that miRNA may be systemic mediators promoting endothelial dysfunction mediated via EPFR-induced AhR activation at the air-blood interface.

Keywords: Air pollution; Alveolar type-II cells; Aryl hydrocarbon receptor; Cardiovascular health; Environmental toxicology; Environmentally persistent free radicals; Inhalation; Particulate matter; microRNA.

Fig. 1

EPFR exposure alters levels of exosomal miRNA in plasma. Exosomal miRNA was extracted from plasma of male C57BL/6J mice (n = 8/group) exposed to EPFR for 5 days. miRNA levels are expressed as fold changes. Data were analyzed using one-way ANOVA with Tukey’s post hoc tests. The heatmap shown was generated from miRNA whose levels were significantly altered in mice exposed to EPFR lo (left) or EPFR (right). *p < 0.05

Fig. 2

Ingenuity pathway analysis (IPA) reveals systemic health effects A as well as molecular and cellular functions B potentially impacted by EPFR-induced alterations in miRNA levels. The graph illustrates category scores, where the threshold represents the minimum significance level (measured as –log (p-value) derived from Fisher’s exact test) set at 1.25

Fig. 3

EPFR-mediated AhR activation produces a distinct miRNA signature. Male (n = 16) and female (n = 20) mice deficient in AhR in alveolar type-II cells (AT-II AhR KO; n = 5–6/group) and littermate control mice (Control; n = 5–6/group) were exposed to EPFR versus filtered air for 5 consecutive days. A heat map was generated for miRNA significantly altered by EPFR exposure in each strain, assessed as a significant interaction detected using two-way ANOVA. *p < 0.05

Fig. 4

AhR-responsive miRNA is linked to target genes known to be important in regulating endothelial function. miRNA whose levels were altered in an EPFR- and AhR-dependent manner were subjected to an IPA microRNA target filter to illuminate potential mRNA targets. Pathway analysis suggested potential gene targets associated with endothelin-1 signaling (A) and endothelial nitric oxide signaling pathways (B). GNAQ guanine nucleotide binding protein alpha q subunit, GNAZ guanine nucleotide binding protein alpha z subunit, ADCY3 adenylate cyclase 3, NRAS neuroblastoma Ras oncogene, PLA2G2D phospholipase A2 group IID, PIK3R3 phosphoinositide-3-kinase regulatory subunit 3, BRAF B-Raf transforming gene, RAP1B RAS related protein 1b, PNPLA2 patatin-like phospholipase domain containing 2, PIK3CB phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta, PIK3CG phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit gamma, PRKCE protein kinase C epsilon, PTGS2 prostaglandin-endoperoxide synthase 2, HMOX1 heme oxygenase 1, PLCG2 phospholipase C gamma 2, RASD2 RASD family member 2, PLA2G10 phospholipase A2 group X, GNAL guanine nucleotide binding protein alpha stimulating olfactory type, PLCB4 phospholipase C beta 4, PLD2 phospholipase D2, CNGB3 cyclic nucleotide gated channel beta 3, CHRNB1 cholinergic receptor nicotinic beta 1 subunit, CAV1 caveolin 1, AQP11 aquaporin 11, LPAR2 lysophosphatidic acid receptor 2, PRKAG3 protein kinase AMP-activated gamma 3 non-catalytic subunit, PRKAR2A protein kinase cAMP-dependent regulatory type II alpha

Fig. 5

EPFR reduced the expression of genes associated with lung adherens and tight junction proteins. AT-II cell-specific AhR knockout and littermate control male (n = 16) and female (n = 20) mice (n = 5–6/group) were exposed to EPFR, EPFR lo, or filtered air for 5 consecutive days. mRNA levels for Tjp1 (A) and Ocel1 (B) were normalized to the Hprt gene. Data represent means ± SEM. *p < 0.05, **p < 0.01