CNP-miR146a Decreases Inflammation in Murine Acute Infectious Lung Injury

Abstract

Acute respiratory distress syndrome (ARDS) has approximately 40% in-hospital mortality, and treatment is limited to supportive care. Pneumonia is the underlying etiology in many cases with unrestrained inflammation central to the pathophysiology. We have previously shown that CNP-miR146a, a radical scavenging cerium oxide nanoparticle (CNP) conjugated to the anti-inflammatory microRNA(miR)-146a, reduces bleomycin- and endotoxin-induced acute lung injury (ALI) by decreasing inflammation. We therefore hypothesized that CNP-miR146a would decrease inflammation in murine infectious ALI. Mice were injured with intratracheal (IT) MRSA or saline followed by treatment with IT CNP-miR146a or saline control. Twenty-four hours post-infection, bronchoalveolar lavage fluid (BALF) and whole lungs were analyzed for various markers of inflammation. Compared to controls, MRSA infection significantly increased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; p < 0.05), BALF proinflammatory cytokines (IL-6, IL-8, TNFα, IL-1β; p < 0.01), and inflammatory cell infiltrate (p = 0.03). CNP-miR146a treatment significantly decreased proinflammatory gene expression (IL-6, IL-8, TNFα, IL-1β; p < 0.05), bronchoalveolar proinflammatory protein leak (IL-6, IL-8, TNFα; p < 0.05), and inflammatory infiltrate (p = 0.01). CNP-miR146a decreases inflammation and improves alveolar-capillary barrier integrity in the MRSA-infected lung and has significant promise as a potential therapeutic for ARDS.

Keywords: acute respiratory distress syndrome; bioactive nanoparticle therapeutic; cerium oxide nanoparticles (CNP); infectious lung injury model; microRNA-146a (miR146a).

Figure 1

Relative gene expression of miR146a among study groups demonstrating significantly increased expression of miR146a in the CNP-miR146a treated group compared to both MRSA-infected lungs (p = 0.0001) and control (PBS) lungs (p < 0.0001).

Figure 2

CNP-miR146a treatment four hours after infection significantly lowers proinflammatory gene expression. Relative gene expression compared to GAPDH of TRAF6 (A), IRAK1 (B), NF-κB (C), IL-6 (D), MIP-2 (E), IL-1β (F), and TNFα (G) 24 h after injury. MRSA infection resulted in significantly higher TRAF6, NF-κB, IL-6, MIP-2, IL-1β, and TNFα gene expression 24 h after injury. CNP-miR146a significantly lowered TRAF6, NF-κB, IL-6, MIP-2, IL-1β, and TNFα compared to untreated, MRSA-infected lungs. Control mice in blue, MRSA-infected and untreated mice in red, and MRSA-infected and CNP-miR146a treated mice in green.

Figure 3

CNP-miR146a treatment of MRSA-induced ALI significantly lowers bronchoalveolar lavage fluid (BALF) proinflammatory protein concentration. BALF total protein concentration (A), and proinflammatory cytokines IL-6 (B), MIP-2 (C), IL-1β (D), and TNFα (E) were measured by ELISA 24 h after injury. MRSA infection resulted in significantly higher total protein, IL-6, MIP-2, IL-1β, and TNFα BALF protein concentration. CNP-miR146a significantly lowered total protein, IL-6, MIP-2, and TNFα compared to untreated, MRSA-infected lungs. Control mice in blue, MRSA-infected and untreated mice in red, and MRSA-infected and CNP-miR146a treated mice in green.

Figure 4

Treatment with CNP-miR146a significantly lowers histologic quantification of leukocyte infiltrate in the MRSA-infected lung. (A–C) 20× magnification slides of lung tissue stained for CD45 collected 24 h post-infection. Representative images of leukocyte infiltrate of (B) Control, (C) MRSA, (D) MRSA + CNP-miR146a. (D) Quantitative analysis of CD45+ cells per high-powered field (HPF). Control lungs in blue, MRSA-infected and untreated lungs in red, MRSA-infected and CNP-miR146a treated lungs in green.