Abstract
Alveolar macrophages are responsible for clearance of airborne dust and pathogens. How they recognize and phagocytose a variety of engineered nanomaterials (ENMs) with different properties is an important issue for safety assessment of ENMs. Surfactant-associated proteins, specifically existing in the pulmonary surfactant, are important opsonins for phagocytosis of airborne microorganisms. The purposes of the current study are to understand whether opsonization of ENMs by surfactant-associated proteins promotes phagocytosis of ENMs and cytokine production, and to determine whether a common pathway for phagocytosis of ENMs with different properties exists. For these purposes, four ENMs, MWCNT-7, TiO2, SiO2, and fullerene C60, with different shapes, sizes, chemical compositions, and surface reactivities, were chosen for this study. Short-term pulmonary exposure to MWCNT-7, TiO2, SiO2, and C60 induced inflammation in the rat lung, and most of the administered ENMs were phagocytosed by alveolar macrophages. The ENMs were phagocytosed by isolated primary alveolar macrophages (PAMs) in vitro, and phagocytosis was enhanced by rat bronchioalveolar lavage fluid (BALF), suggesting that proteins in the BALF were associated with phagocytosis. Analysis of proteins bound to the 4 ENMs by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Both BALF and SP-A, but not SP-D, enhanced TNF-α production by MWCNT-7 treated PAMs; BALF, SP-A, and SP-D increased IL-1β production in TiO2 and SiO2 treated PAMs; and BALF, SP-A, and SP-D enhanced IL-6 production in C60 treated PAMs. Knockdown of CD14, a receptor for SP-A/D, significantly reduced phagocytosis of ENMs and SP-A-enhanced cytokine production by PAMs. These results indicate that SP-A/D can opsonize all the test ENMs and enhance phagocytosis of the ENMs by alveolar macrophages through CD14, suggesting that SP-A/D-CD14 is a common pathway mediating phagocytosis of ENMs. Cytokine production induced by ENMs, however, is dependent on the type of ENM that is phagocytosed. Our results demonstrate a dual role for surfactant proteins as opsonins for both microbes and for inhaled dusts and fibers, including ENMs, allowing macrophages to recognize and remove the vast majority of these particles, thereby, greatly lessening their toxicity in the lung.
Highlights
The respiratory tract is the major exposure routine for airborne dusts and pathogens
We addressed two main questions: 1) whether a common spectrum of surfactant proteins was bound to different ENMs? 2) what proteins on Alveolar macrophages (AMs) were responsible for recognizing the bound surfactant proteins and subsequent phagocytosis and cytokine secretion? For these purposes, 4 types of ENMs, differing in size, shape, surface reactivity, and chemical composition, were chosen for analysis of their binding proteins in rat bronchoalveolar lavage fluid (BALF) and cytokine productions
Negative control RNA or Small interfering RNAs (siRNA) for LDL receptor related protein 1 (LRP1), CD14, and signal regulatory protein alpha (SIRPa) were transfected into the cells using Lipofectamine 2000 (Thermo Fisher, USA). 6 hours later, the culture media was changed to X-VIVOTM serum-free medium (Lonza), and treated with 1 mg/ml MWCNT-7, TiO2, SiO2, or C60 suspended in saline containing 0.5% (w/v) PF68; or 1 mg/ml of MWCNT-7, TiO2, SiO2, or C60, pre-incubated for 4 hours with 1 mg/ml of recombinant human SP-A, SP-D or 10% concentrated bronchioalveolar lavage fluid (BALF), and continued culture for 12 hours
Summary
The respiratory tract is the major exposure routine for airborne dusts and pathogens. Coating of MWCNTs with pulmonary surfactant and bronchoalveolar lavage fluids (BALF)-derived protein corona on diesel exhaust nanoparticles enhance the nanoparticle uptake and pro-inflammatory responses in macrophages in vitro [20, 21], and SP-A and SP-D are found to bind the nanoparticles [21]. 4 types of ENMs (fullerene C60, TiO2, SiO2, and MWCNT), differing in size, shape, surface reactivity, and chemical composition, were chosen for analysis of their binding proteins in rat bronchoalveolar lavage fluid (BALF) and cytokine productions. SP-A and SP-D were identified as common binding proteins for all 4 ENMs, and enhanced phagocytosis of the ENMs and cytokine production by AMs in vitro.
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