Surfactant protein A /D-CD14 is Associated with Phagocytosis of Nanomaterials and Cytokine Production by Alveolar Macrophages

Background: 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, specically 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 nd out a common pathway for ENMs with different properties. Results: 4 ENMs including MWCNT-7, TIO 2 , SIO 2 , and fullerene C60, each with different shape, size, chemical composition and surface reactivity, were chosen for the study. Short-term pulmonary exposure of MWCNT-7, TIO 2 , SIO 2 , and C60 induced inammation 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, which was enhanced by the rat bronchioalveolar lavage uid (BALF), suggesting that proteins in BALF were associated with the phagocytosis. Further analysis of the 4 ENMs-bound proteins by LC/MS indicated that surfactant-associated proteins A and D (SP-A, SP-D) were common binding proteins for all the 4 ENMs. Like BALF, SP-A, but not SP-D, enhanced TNF-a production in the MWCNT-7-treated PAMs; both SP-A and SP-D increased IL-b production in the TIO 2 -or SIO 2 stimulated PAMs; while SP-A and SP-D enhanced IL-6 production in the C60-stimulated PAMs. Knockdown CD14, a receptor and ENMs phagocytosis. Conclusions: These results indicate that SP-A/D can opsonize all the studied ENMs to enhance ENMs, engineered nanomaterials; SP-A/D, surfactant-associated proteins A/D; AMs, alveolar macrophages; PAMs, primary alveolar macrophages; PRRs, pattern-recognition receptors; PAMPs, pathogen-associated molecular patterns; BALF, bronchoalveolar lavage uid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; LC-MS, Liquid Chromatography Method with Tandem Mass Spectrometry.


Background
The respiratory tract is the major exposure routine for airborne dusts and pathogens. Alveolar macrophages (AMs) reside in the airway and alveoli, account for 95% of leukocytes in the lower respiratory tract [1], and function mainly in host defense and alveolar homeostasis. Phagocytosis by AMs is a major mechanism for clearance of dusts and microorganisms often encountered in the lung [2]. AMs have many types of pattern-recognition receptors (PRRs) on the plasma membrane, and recognize a variety of pathogen-associated molecular patterns (PAMPs) in microorganisms [3,4]. AMs also express Fc receptors and complement receptors for the Fc portions of IgG antibodies and complements that speci cally bind IgG or complements-coated pathogens [5,6]. The process of coating pathogens to promote phagocytosis is called opsonization. The direct binding of PRRs to their corresponding PAMPs and the binding of Fc receptors and complement receptors to opsonized pathogens induce a number of responses in AMs, including production of cytokines, in ammatory mediators and microbicidal enzymes, and mobilization of cytoskeleton leading to phagocytosis, cell migration and granule exocytosis.
Secreted cytokines and in ammatory mediators then exert their regulatory functions of in ammation and immune responses [7,8]. AMs are also involved in the clearance of apoptotic and necrotic cells and the subsequent regression of pulmonary in ammation [9].
With advance in nanotechnology, engineered nanomaterials (ENMs) are increasingly being developed.
This may lead to increased respiratory exposure of ENMs during their production, consumption and disposal. Numerous studies in animals indicate that respiratory exposed ENMs are taken up and cleared by AMs [10,11]. ENMs have little antigenicity and do not possess molecular structural regions like PAMPs in microbes. How do AMs recognize different ENMs with different size, chemical composition, physical morphology and surface reactivity? It is widely accepted that ENMs are bound with proteins from various biological uids, forming tiers of proteins coating the surface of ENMs, and in turn, the so-called protein corona may promote phagocytosis through interaction of bound complements or IgG with complement receptors and Fc receptors [12,13]. Properties of ENMs can modulate the composition of the protein corona [14]. Such studies on the interaction between ENMs and proteins are mainly based on the serum/plasma. In the lung, the pulmonary surfactant is a complex mixture composed of more than 90% lipids and 5-10% proteins, covers all the inner face of the alveoli, and reduces surface tension at the airwater interface in the alveoli to prevent alveolar collapse at end-expiration [15]. Surfactant-associated proteins (SP-A, SP-B, SP-C and SP-D) differ from one another in their synthesis, oligomerization, and function [16]. SP-B and SP-C are hydrophobic and reduce the surface tension of the distal lung, while SP-A and SP-D are more hydrophilic and have important roles in the regulation of innate immune responses [17]. SP-A and SP-D can bind to many different microbial pathogens and act as opsonins to enhances phagocytosis of microbial pathogens by AMs [18,19].
In the current study, 4 kinds of ENMs (fullerene C60, TiO 2 , SiO 2 and MWCNT) with different size, shape, surface reactivity and chemical composition were chosen for analysis of their binding proteins from rat bronchoalveolar lavage uid (BALF). SP-A and SP-D were identi ed as common binding proteins for all the 4 ENMs, and enhanced phagocytosis of the ENMs and cytokine production by AMs in vitro. Also, the enhancing effects were CD14-dependent. Our results are helpful for understanding of ENMs-induced toxicity and for production of safe ENMs.

Results
Most of the intratracheally exposed nanomaterials were phagocytosed by alveolar macrophages For comparison of toxic effects in the lung, 4 kinds of ENMs with different shape, chemical composition, size and surface property were chosen for this study. MWCNT-7 is a carbon-based nanotube with a hydrophobic surface; TiO 2 is a rod-like particle with a hydrophilic surface; SiO 2 and C60 are round nanoparticles with a hydrophilic and hydrophobic surface, respectively. They formed aggregates in the suspensions with different size. Characterizations concerning their shape, chemical composition and size distribution are shown in Figure S1, S2 and S3.
Short-term administered MWCNT-7, TiO 2 , SiO 2 , and C60 suspensions to the rat lung induced pulmonary in ammation to a different extent. As shown in the left panel of Fig. 1, MWCNT-7 elicited a strong lung in ammation, with accumulation of immune cells and thickening of the alveolar epithelium (Fig. 1A); while TiO 2 and SiO 2 ( Fig. 1B & C) had little effect on the alveolar epithelium, although particle-burden alveolar macrophages were often observed. Similar to MWCNT-7, C60 caused a strong lung in ammation (Fig. 1D).
Light microscopic and electron microscopic observation revealed that most of the administered ENM aggregates were found within alveolar macrophages (Fig. 1E, F, G and H corresponding to MWCNT-7, TiO 2 , SiO 2 , and C60, respectively), indicating that phagocytosis by alveolar macrophages is a main mechanism for clearance of the invading particles.

BALF enhanced the uptake of nanomaterials by alveolar macrophages
Previous studies have demonstrated that ENMs are bound with proteins in many types of biological uids, forming tiers of proteins surrounding the nanomaterials. This so-called protein corona affects biological behaviors of the nanomaterials, including phagocytosis by macrophages [20]. For understanding whether secretory uid in the respiratory tract and alveoli in uences phagocytosis of the studied 4 ENMs by alveolar macrophages, we rst prepared BALF and primary alveolar macrophages (PAMs). Immuno uorescence staining indicated that more than 95% of isolated cells were positive for CD68, a macrophage marker ( Figure S4). The 4 ENMs were then exposed in vitro to the isolated PAMs in the absence or presence of BALF, and the rates of ENMs-burden PAMs were compared under polarized microscopy. The results indicated that addition of BALF increased the uptakes of MWCNT-7, TiO 2 , and SiO 2 by PAMs ( Fig. 2A 2I). Because of C60 was not brightening under the polarized microscope, its phagocytosis could not be observed ( Fig. 2G & H). These results suggested that proteins in the BALF were likely to bind to the ENMs to promote the phagocytosis by PAMs.

SP-A, SP-D and SP-B shared common proteins bound to ENMs
For understanding which proteins from BALF were bound to each of the 4 ENMs, the binding proteins were dissociated, and then subjected for SDS-PAGE and LC-MS. SDS-PAGE and silver staining revealed that kinds and abundance of the binding proteins were different between the 4 ENMs (Fig. 3A). Further analysis with LC-MS showed that total 892 proteins in the BALF were checked out, with 332 proteins being common bound proteins for all the 4 ENMs (Fig. 3B). Scrutinizing top 50 abundant proteins indicating that albumin, complements, immunoglobulins, apolipoproteins and other serum-derived proteins had the highest ratio, and respiratory tract-or lung-derived proteins and other proteins of unknown origin were also common (Table S1). The

SP-A and SP-D enhanced nanomaterials-cytokine production
For further clarifying whether surfactant-associated proteins affect macrophage activation, TNF-α, IL-1β and IL-6, the main pro-in ammatory cytokines produced upon macrophage activation, were used as judging parameters. Preliminary experiments showed that SP-B had little effects on production of cytokines (data not shown), therefore, we focused on the effects of SP-A and SP-D in the subsequent experiments. Pre-addition of 1µg/ml human recombinant SP-A or SP-D to the serum-culture medium of PAMs signi cantly increased TNF-α expression in the MWCNT-7-treated PAMs at the mRNA level, while an increase in IL-1β and IL-6 mRNA expression was not found (Fig. 4A). Similar results were also observed by addition of BALF (Fig. 4A). ELISA examination of the culture media indicated that SP-A, SP-D and BALF enhanced MWCNT-7-induced TNF-α secretion (Fig. 4E). In the presence of SP-A, SP-D or BALF, IL-1β expression, but not the TNF-α nor IL-6, was especially elevated in the TiO 2 -or SiO 2 -stimulated PAMs at both the mRNA and protein levels ( Fig. 4B, C, F and G). C60 speci cally stimulated IL-6 production, which was promoted by addition of SP-A, SP-D or BALF ( Fig. 4D and H). In brief, like BALF, SP-A and SP-D enhanced ENMs-induced cytokine production of PAMs, although the in ammatory cytokines differed from one another by the stimulation of different ENMs.
Knockdown of CD14 expression in alveolar macrophages reduced cytokine production and ENMs uptake It has been reported that LRP1, CD14, and SIRPα are potential receptors for SP-A and SP-D [21][22][23]. Thus, we tried to nd out if SP-A/D-enhanced the cytokine production was mediated by these receptors at rst.
The expressions of these receptors in PAMs were knocked down by siRNAs. The genes-speci c siRNAs obviously decreased the mRNA and protein expressions of LRP1, CD14, and SIRPα ( Figure S5).
Knockdown of CD14, but not LRP1 and SIRPα, signi cantly reduced the TNF-α expression enhanced by SP-A in the MWCNT-7-stimulated PAMs ( Fig. 5A and E), indicating that the SP-A/CD14 axis was involved in MWCNT-7 induced TNF-α production. Contrary to SP-A, the SP-D-enhanced TNF-α expression were increased by CD14 silencing (Fig. 5A). TNF-α expression was elevated by knockdown of LRP1 or SIRPα ( Fig. 5A), suggesting that LRP1 and SIRPα induce inhibitory signaling in the TNF-α production. Similarly, qPCR revealed that knockdown of CD14 decreased the IL-1β mRNA expression which was enhanced by SP-A or SP-D in the TiO 2 -or SiO 2 -stimulated PAMs ( Fig. 5B & C). The declined IL-1β protein secretion by CD14 silencing was con rmed by ELISA (Fig. 5F & G). In the SP-A-promoted IL-6 expression of the C60stimulated PAMs, silencing of CD14 and SIRPα had a remarkable downregulation effect (Fig. 5D & H). Taken together, these results indicated that SP-A/D-CD14 axis was a common pathway for ENMs-induced cytokine production, although the ENMs are different from each other in their properties.
Next, we investigated whether the SP-A/D-CD14 axis is associated with phagocytosis of the ENMs by PAMs. Compared with the control, knockdown of CD14 reduced the phagocytosis rate in the MWCNT-7stimulated PAMs (Fig. 6A, B & K) in the presence of SP-A (65.3% vs 12.9%), while the SP-A/D-enhanced phagocytosis of TiO 2 or SiO 2 was decreased by CD14 silencing (Fig. 6C, D,

Discussion
As a major type of innate immune cells, macrophages exert a variety of actions both in the innate and the adaptive immunity. AMs reside in the low respiratory tract and alveoli, clear airborne dusts and microbial pathogens often encountered in the lung by phagocytosis and release pro-in ammatory cytokines. Unlike microorganisms composed of biological macromolecules, most kinds of ENMs are usually formed by substances with relatively simple chemical composition, which lack immunogenicity and are di cult to stimulate adaptive immune responses. Therefore, the immune responses against ENMs mainly mediated by innate immune cells such as macrophages and by soluble molecules such as cytokines. It is plausible that ENMs are opsonized and then phagocytosed by AMs in the lung.
The deep lung is composed of approximately 3 million alveolar sacs, forming a broad surface area through which oxygen and carbon dioxide are exchanged in the blood of alveolar capillaries [21]. SP-A and SP-D are lung-speci c proteins existing in the pulmonary surfactant. Except for their functions in the homeostasis of the pulmonary surfactant [24,25], they are also involved in the host defense against various pulmonary pathogens, such as respiratory syncytial virus, mycobacterium tuberculosis, bacteria, viruses and fungi [26,27]. Structurally, SP-A and SP-D belong to the collectin family with a C-terminal carbohydrate recognition domain (CRD) and an N-terminal collagen like domain, [28] and act as opsonins by interaction via the CRD with various microorganisms and their derived components to enhance phagocytic function of AMs through CD14, Toll-like receptors and other receptors expressing on the surface of AMs [29,30].
In the current study, we identi ed SP-A/D-CD14 as a common pathway that mediated cytokine production and/or phagocytosis by PAMs in all the studied ENMs (summarized in the Figure S6). Obviously, in the low respiratory tract and alveoli, ENMs are coated by SP-A and/or SP-D, and in turn, interact with CD14 on the surface of AMs to enhance phagocytosis of the ENMs. This opsonization effect of SP-A/D are often found in the defense of microbial invasion, as mentioned above. Consistent with our observations, SP-A and SP-D are found in the corona of PEG-, PLGA-, or Lipid-modi ed nanoparticles [31] and SP-A increases cellular binding and uptake of nanoparticles with modi cation of different polymers by alveolar macrophages [32]. These observations, combined with our current results, indicate that opsonization by SP-A and/or SP-D is an important defense mechanism both in elimination of invading microbes and in the clearance of inhaled ENMs and other dusts. Of course, plasma-derived opsonins such as IgG antibodies and complements may also be involved in the clearance of ENMs in the lung, since they are found to abundantly bind to MWCNT-7, TiO 2 , SiO 2 and C60. Another nding in the current study is that different ENMs induce different cytokine production, i.e., increased TNF-α by MWCNT-7, elevated IL-1β by TiO 2 , SiO 2 , and enhanced IL-6 expression by C60. The difference in the induction of these cytokines, probably, and other cytokines, contributes to the difference and extent of the pulmonary in ammation by the ENMs. It is likely that expression pro le is ENM-speci c, although the SP-A/D-CD14 axis enhanced phagocytosis is the same. Detailed molecular mechanisms for the ENM-speci c cytokine induction need further investigation. Also, it should be noted that without BALF, SP-A, or SP-D, AMs are still able to phagocytose the studied ENMs ( Fig. 2 and Fig. 6), suggesting that AMs have additional mechanisms for the recognition and phagocytosis of the ENMs other than opsonization.
Conclusions SP-A/D can opsonize all the studied ENMs to 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 the ENMs, however, is dependent on what an ENM is phagocytosed. Our results are helpful for the understanding of clearance of ENMs by alveolar macrophages and mechanisms of different ENMs-induced lung toxicity. Further studies, for example, roles of CD14 in the ENMs-induced lung in ammation and molecular mechanisms for the ENM-speci c cytokine induction, are required for safety assessment of ENMs.

Preparation of Nanomaterial Suspensions
MWCNTs (MWCNT-7) were obtained from Mitsui Chemicals Co., Ltd. Tokyo, Japan; TiO 2 (rutile type, with a mean primary size of 20 nm) was provided by Japan Cosmetic Association, Tokyo, Japan; SiO 2 (with a primary size of 10-20 nm) was purchased from Sigma-Aldrich, USA; and Fullerene C60 (with a mean primary size of 1 nm) was provided by Frontier Carbon Corporation, Japan. 10 mg of these 4 types of ENMs was suspended in saline containing 0.5% (w/v) Pluronic® F-68 (a non-ionic detergent from Sigma-Aldrich, USA) to a nal concentration of 500 µg/ml. The MWCNT-7 suspension was homogenized four times, with 1 minute each time, using the Polytron PT1600E bench-top homogenizer (Kinematica, Switzerland) at a speed of 3000 rpm. The prepared four nanomaterial suspensions were sonicated at 600W for 30 minutes using the JY92-2 sonicator (Scientz Co., Ltd, Ningbo, China), 5 minutes for 6 times with 2 minute-interval rests. To ensure the dispersion and suspension of the nanomaterials, the suspensions were further sonicated for 5 minutes for 4 times just before use. Characterization of the suspended 4 nanomaterials, including shape, size distribution and element analysis, was shown in the Figure S1, S2 and S3.

Animals
Eight weeks-old female wild-type Sprague-Dawley (SD) rats were obtained from and housed in the Animal Center of Anhui Medical University, and received Oriental MF basal diet and water ad libitum. The animal experiment protocols were approved by the Institutional Animal Care and Use Committee.

Intratracheal spraying of nanomaterial suspensions
Twenty-ve female SD rats were divided in to 5 groups and 0.5 ml of the suspensions were intratracheally sprayed with 0.5 ml of the vehicle, 500 µg/ml MWCNT-7, TiO 2 , SiO2, or C60 suspensions, using an intratracheal aerosolizer (series IA-1B, Penn-century, Philadelphia, USA), as previously described [33], 2 times per week for 2 weeks. The total amount of the administered nanomaterials was 1 mg per rat. Three days after the last spraying, the animals were sacri ced under iso urane anesthesia, and the lung was excised, and then xed in 4% paraformaldehyde solution in phosphate-buffered saline (PBS) adjusted to pH 7.3 and processed for light microscopic examination and transmission electron microscopy (TEM) or scanning electron microscopy.

Light microscopy and electron microscopy
Hematoxylin-eosin (HE) stained sections of the lung tissues treated with the 4 ENM suspensions were used to observe lung in ammation and localization of the nanomaterials. For transmission electron microscopic (TEM) observation of TiO 2 , SiO 2 and C60, para n blocks were depara nized and small pieces of the lung tissues were embedded in epon resin and processed for nanomaterial observation using the JEM-2100 transmission electron microscope (JEOL Co. Ltd, Tokyo, Japan). Since MWCNT-7 are hard to be cut by the electronic microtome, scanning electron microscopy (SEM) was used to observe the MWCNT-7-treated lung tissues. Brie y, the HE-stained slides of the lung tissues were immersed in xylene for 3 days to remove the cover glass, immersed in 100% ethanol for 10 min to remove the xylene, and then air-dried for 2 hours at room temperature. The slides were then coated with platinum for observation using Model S-4700 Field Emission SEM (Hitachi High Technologies Corporation, Tokyo, Japan) at 5-10 kV.

Preparation of BALF and isolation of rat Alveolar Macrophages
Eight weeks-old female wild-type SD rats were sacri ced under anesthesia with intraperitoneally injected sodium pentobarbital, the lung was excised under aseptic conditions and injected with 5 ml of saline through the trachea. After gently shaking the lung, the uid in the lung was taken out. Repeat the washing steps for another 2 times. The collected uid was centrifuged at 1800g for 5min at 4℃, and the supernatant was BALF, concentrated with a concentrator tube (Millipore), and then stored at -80℃ for later use.
The cell pellet was resuspended in RPMI 1640 medium containing 10% fetal bovine serum (Gibco, USA), seeded in a six-well plate and cultured at 37℃ for 90 minutes. The cells were washed with PBS three times to remove red blood cells, other cells and cell debris, and the remaining adherent cells were stained with immuno uorescence for CD68, a macrophage marker, to con rm their identity. Brie y, the adherent cells were xed in 4% paraformaldehyde and treated with 0.2% Triton X-100 containing 10% fetal bovine serum (Gibco)/1% bovine serum albumin in PBS at room temperature for 15 minutes, and then incubated with rabbit anti-CD68 (1:50 dilution, Bioss, Beijing, China) overnight at 4°C and added with Cy3 labelled anti-rabbit IgG (1:100 dilution, Proteintech, Wuhan, China). After washing, the cells were counter-stanning with DAPI (Sigma-Aldrich). Images were captured with a orescence microscope (ZEISS LSM880 + Airyscan, Germany). As shown in Figure S4, more than 95% of the adherent cells were positive for CD68.
Binding of nanomaterials to BALF proteins, SDS-PAGE and LC-MS Exposure of ENMs to primary alveolar macrophages in vitro siRNAs with the best silencing e cacy determined by preliminary experiments were chosen for further use. The siRNA sequences are as follow: GCUAAACUCGCUCAAUCUATT/UAGAUUGAGCGAGUU-UAGCTT for CD14; CCAUCAAACGGGCAUUCAUTT/AUGAAUGCCCGUUUGAU-GGTT for LRP1; and GCUCUAUGUACUCGCCAAATT/UUUGGCGAGUACAUAG-AGCTT for SIRPα.
Brie y, 1×10 6 rat PAMs were seeded in each well of a 6-well plate and cultured at 37℃ overnight. Negative control RNA or siRNAs for LRP1, CD14 and SIRPα were transfected into the cells using Lipofectamine 2000 (Thermo Fisher, USA). 6 hours later, the culture media were changed with the X-VIVO™ serum-free medium (Lonza) containing 1 µg/ml of recombinant human SP-A or SP-D, or 10% concentrated BALF, and the cells were treated with 1 µg/ml MWCNT-7, TiO 2 , SiO2, or C60 and continuously cultured for 12 hours. The cells were harvested for RNA isolation, qPCR analysis of silencing e cacy and cytokine expression, and western blotting; the culture supernatants were collected for ELISA.
In uence of knockdown of LRP1, CD14 or SIRPα on IL-1β, IL-6 and TNF-α production was analyzed by qPCR and ELISA, and its effect on ENMs phagocytosis was assessed by the polarized microscopy, as described above.

Statistical Analysis
Statistical analysis was performed using SSPS17 software. The statistical signi cance was analyzed using two tailed Student's t test. A p value of < 0.05 was considered to be signi cant.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
Available from the corresponding author on reasonable request.