miRNA Alterations in Lung Macrophages after Nitrogen Mustard Exposure

Activated macrophages have been implicated in lung injury and fibrosis induced by the cytotoxic alkylating agent, nitrogen mustard (NM). Herein, we determined if macrophage activation is associated with altered miRNA expression. Treatment of rats with NM (0.125 mg/kg, i.t.) resulted in significant increases in miRNA expression 1, 3, 7, and 28 d post exposure as determined by PCR array analysis of miRNAs (miR)s involved in inflammation and fibrosis. Expression of many miRNAs were significantly increased at multiple time-points while one miRNA was uniquely expressed 1 d post-NM, one at 3 d, two at 7 d, and five at 28 d. An IPA Core Analysis of predicted mRNA targets of differentially expressed miRNAs identified significant enrichment of Diseases and Functions related to cell cycle arrest, apoptosis, cell movement, cell adhesion, and inflammation 1 d and 28 d post NM. Significant enrichment of pathways related to lipid metabolism is consistent with previous studies reporting the development of macrophage foam cells after NM exposure. miRNA-mRNA interaction network analysis identified highly connected miRNAs representing key upstream regulators of mRNAs involved in significantly enriched Diseases and Functions including miR-34c-5p and miR-27a-3p at 1 d post NM and miR-125b-5p, miR-16-5p, miR-30c-5p, miR-19b-3p, and miR-148b-3p at 28 d post NM. Collectively, these data show that NM promotes histone remodeling and alterations in miRNA expression linked to lung macrophage responses during inflammatory injury and fibrosis. Male Wistar rats (8 wk, 225–250 g) were anesthetized with 2.5% isoflurane, and then administered PBS or NM (0.125 mg/kg, mechlorethamine hydrochloride, Sigma-Aldrich, St. Louis, MO) intratracheally. Animals were euthanized by intraperitoneal injection of pentobarbital (Sleepaway, 50 mg/kg, Fort Dodge Animal Health, Fort Dodge, IA) 1 d, 3 d, 7 d and 28 d after administration of PBS or NM. The lung was removed, and 10 mL of ice-cold PBS slowly instilled and withdrawn through a cannula in the trachea while gently massaging the tissue; this procedure was repeated 4 times. The cells were then centrifuged (300 x g, 8 min), resuspended in 10 mL PBS and viable cells enumerated using a hemocytometer with trypan blue. Total mRNA was collected from lung macrophages by phenol-chloroform extraction. Samples were enriched for miRNAs using an RNeasy MinElute Cleanup kit (QIAGEN Inc., Valencia, CA). Purified miRNA was reverse transcribed using a QIAGEN QuantiTect Reverse Transcription kit and analyzed using a QIAGEN miScript Rat Inflammatory Response and Autoimmunity miRNA PCR Array or by RT-qPCR using a QIAGEN miScript SYBR Green kit according to the manufacturer’s protocol. Amplification was performed using a 7300HT Real Time PCR system (Applied Biosystems, Grand Island, NY). Fold changes were calculated using the ∆∆Ct method. miRNA expression was normalized to Snord72 as its expression not affected by NM administration.