Surfactant A/D Mediates Different Nanomaterials Uptake And Cytokine Production Through Different Receptors In Alveolar Macrophages

Background & Objective Due to their small size and recognized as foreign bodies in the body,nanomaterials?NM?may have potential hazards to health,and are particularly prone to cause respiratory diseases.Many animal experiments have shown that short-term exposure different nanomaterials to rat can cause lung damage,such as lung inflammation and fibrosis,and long-term exposure of certain nanomaterials can lead to chronic pulmonary fibrosis and malignancy.The phagocytosis of alveolar macrophages is a main mechanism for clearance of NM in the lungs,but alveolar macrophages are also involved in the NM-induced lung pathogenesis.The inflammatory cytokines produced by NM-burden alveolar macrophages are closely correlated with the occurrence of these pulmonary diseases.It has been reported that NM adsorbs a variety of proteins in biological fluids such as plasma to form a protein corona on the NM surface,mediatinge NM phagocytosis.However,what sets of proteins are adsorbed on the NMs with different chemical composition,shape and surface properties in the respiratory tract? Which proteins and their receptors mediate the recognition and phagocytosis by alveolar macrophages? Whether the phagocytosis has any effect on the production of inflammatory cytokines? In this study,we chose four nanomaterials with different chemical composition,shape and surface properties(C₆₀,MWCNT,TiO₂ and SiO₂) to answer these questions.Methods 1)Preparation of bronchoalveolar lavage fluid?BALF?and primary alveolar macrophages from SD rats;2)Stimulation of primary alveolar macrophages with four nanomaterials in vitro to observe cytokine production;3)SDS-PAGE and LC-MS mass spectrometry analysis to identify NM-bound proteins;4)Knockdown of SP-A / D receptors on alveolar macrophages by si RNA to understand the effects of the receptors on cytokine production and phagocytosis;5)Western blotting,RT-QPCR,ELISA and other technologies to detect the production of inflammatory factors;6)Polarizing microscopy to observe uptake of nanomaterials by alveolar macrophages.Results 1)In the presence of BALF,alveolar macrophages increased the uptake of C₆₀,MWCNT,TiO₂ and SiO₂;2)SDS-PAGE and LC-MS mass spectrometry revealed that 4 NMs shared SP-A and SP-D as common bound proteins;3)When SP-A,SP-D or BALF were added,MWCNT-7 stimulated alveolar macrophages to increase TNF-? expression,TiO₂ and SiO₂ induced IL-1b production,while C₆₀ caused elevated IL-6 expression.These results demonstrated that SP-A and SP-D were the major proteins that mediated the NM uptake and produce cytokines;4)Knock down the corresponding receptors of SP-A /D?CD14,LRP1 or SIRPa?on the surface of alveolar macrophages indicated that the TNF-? expression stimulated by MWCNT-7 was mediated by SP-A-CD14;IL-1b expression induced by TiO₂ was mediated by SP-A/D-CD14;IL-1b expression promoted by SiO₂ was via SP-A / D-CD14;while IL-6 expression caused by C₆₀ involved in SP-A-CD14 / LRP1.These results indicate that NM-specific cytokine production is not only dependent on SP-A / D in the protein corona and the corresponding receptors on the alveolar macrophages,but also on the properties of NM themselves.Conclusion Although SP-A / D are common bound proteins in the corona of all the 4 tested nanomaterials,the uptake of nanomaterials and specific cytokine production by alveolar macrophages are mediated by different protein-receptor axes.The roles of phagocytosis and cytokine production by alveolar macrophage in NM-induced pulmonary diseases need further investigation.