Surfactant Proteins A/D–CD14 on Alveolar Macrophages Is a Common Pathway Associated With Phagocytosis of Nanomaterials and Cytokine Production

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.


Figure S2 Chemical composition of MWCNT-7, TIO2, SIO2, and C60s
Suspensions of MWCNT-7, TiO2, SiO2, and C60 (500 g/ml) were diluted with ddH2O 1:200 and placed on an aluminum plate. After air-drying, the samples were observed and analyzed by FIB-SEM (Helios G4 UC, Aztec) connected with an EBSD analysis system (Thermo Scientific). A, B, C, and D represent the spectra of MWCNT-7, TiO2, SiO2, and C60, respectively. The table under the spectra is the element composition and ratio of each of the ENMs.
Supplementary Material 4 Figure S3 Size distribution of MWCNT-7, TIO2, SIO2, and C60 in the suspension 1.6 ml of the four ENM suspensions (equal to 800 g of each ENM) were added with 1 ml of saline or the 20-fold concentrated rat BALF, and incubated at 37℃ in a shaker at 200 rpm for 4-6 hours.
Size distribution in 500 g/ml suspensions of MWCNT-7, TiO2, SiO2 and C60 in the absence (A, C, E and G) or presence (B, D, F and H) of BALF were analyzed with a NanoSight 300 analyzer (Malvern). The representative size distribution curves are shown in figures A and B for MWCNT-7, C and D for TiO2, E and F for SiO2, and G and H for C60, respectively. The mean size and standard deviation (s.d.) for each kind of the ENMs are displayed in the table (E) Figure S4 Intratracheal spraying 1.0 ml of black ink was sprayed into the lung of a 6-week-old SD rat using an intratracheal aerosolizer (series IA-1B, Penn-century, Philadelphia, USA). A. the intratracheal aerosolizer; B. the sprayed ink was evenly distributed in each lobe of the lung; and C. light microscopy showed the sprayed ink in the lung tissue.

Table S1 LC-MS analysis of the nanomaterial-bound proteins
The bound proteins in each group were analyzed by LC/MS. The top 120 proteins in the table are listed by values of IBAQ (intensity-based absolute-protein-quantification) indicating abundance of each bound protein.

Figure S5 Immunostaining of primary alveolar macrophages
Isolated primary alveolar macrophages were immunostained with CD68, a macrophage marker, to confirm their identity. The nucleus was stained with DAPI. The imagines were observed and captured using a florescence microscope (ZEISS LSM880+Airyscan, Germany)

Figure S6 Uptake of ENMs by PAMs via phagocytosis
5◊10 5 primary alveolar macrophages (PAMs) in a 6-well plate were treated with or without 5g/ml cytochalasin B (Cyt B, an inhibitor of actin polymerization, Solarbio, Beijing, China) for 1h and then exposed with MWCNT-7, TiO2, or SiO2 pre-incubated with BALF for another 12h. The cells were fixed, eosin-stained and observed under an ECLIPSE polarizing microscope (LV100NPOL, Nikon, Japan). Percentages of the PAMs with burden particles (arrows) were calculated. Total RNAs were isolated for qRT-PCR analysis of IL-1, TNF- and IL-6, using GAPDH as an internal reference. Two-tailed students' t-test was used for statistical comparison.

Figure S9 Knockdown of CD14, LRP1, and SIRPα expression in PAMs
CD14, LRP1, and SIRPα expression in primary alveolar macrophages was knocked down by genespecific siRNAs. The silencing efficacy for LRP1, CD14, and SIRPα was analyzed by qPCR (A) and confirmed by western blotting (B).

Figure S10 Effects of SP-A, SD-D, BALF and ENMs on expression of cytokines in the receptors-knocked down PAMs
1×10 6 rat PAMs in a 6-well plate was transfected with negative control RNA, or gene-specific siRNAs for LRP1, CD14, and SIRPα and cultured at 37℃ for 6h (A); and after the culture media was changed to X-VIVO TM serum-free medium (Lonza), the cells were treated with saline, 1g/ml of SP-A or SP-D and cultured for 12h (B), or treated with saline containing 0.5% (NC), 1g/ml of MWCNT-7, TiO2, SiO2, or C60 suspended in saline containing 0.5% for 12h. Total RNAs were isolated for qRT-PCR analysis of IL-1, TNF- and IL-6, using GAPDH as an internal reference.
Two-tailed students' t-test was used for statistical comparison.

Supplementary Material
18 Figure 11 Summarized results