| Background: With the rapid development of nanotechnology,the application of nano-scale material in biomedical field is increasingly extensive.The impact of nanoparticles on environment and human health has attracted increasing attention.The physical and chemical properties of nanomaterials are the basis for determining their fate in the body,such as morphology,particle size,surface chemistry,etc.,which directly determines its interaction with the circulatory system,the phagocytic cells in vivo and the metabolic pathways.The liver is an important detoxification organ and also one of the major accumulation sites of nanomaterials in human body.Therefore,it is important to fully assess the adverse effects of nanoparticles exposure.However,both clinical and laboratory lack clear biological indicators with high sensitivity and high acceptance for evaluating the complex interactions among inflammation of liver cells,inflammatory mediators and the acute immune responses after the exposure of nano-drug,nano-adjuvant or nano-contrast agent.Serum amyloid A(SAA)is a very sensitive acute phase response protein.Liver is the primary site for the synthesis of SAA during acute phase response.It has been reported that SAA,C-reactive protein(CRP)combined with haptoglobin(HP)can be used as biomarkers for the prediction of silica nanomaterials exposure toxicity.So,can SAA be used as a sensitive and reliable biological indicator to evaluate and screen a wide range of widely used GNPs and to explore the association between acute toxicity and its particle size and morphology?In addition to being a sensitive biomarker of nano-hepatotoxicity,SAA may also have immunomodulatory effect.It has been reported that during acute inflammation,elevated SAA in the circulatory system induced macrophages to secrete chemokines which recruiting more leukocytes to the site of inflammation.So whether SAA in the body can control the migration and homing of T cells,thus to enhance the CTL function and heighten its anti-tumor effect? In order to answer these scientific questions,we carry out the research work in this paper.Objective: To investigate the feasibility of SAA as a sensitive and reliable biomarker for nano-hepatotoxicity and its role in immunoregulation.The aim of this study was to assess the acute inflammatory response in liver after GNPs exposure,and to elucidate the mechanisms of inflammatory cascade and signal pathways involved in the process.At the same time,the immunoregulation effect of SAA on the chemotaxis and homing of adoptive T cells in vivo was also been explored as well as the related mechanisms.Methods and Results:(1)The establishment and validation the mouse model of SAA-Luc and STAT3-Luc.The reporter gene plasmids of SAA and STAT3 were delivered and integrated into mouse chromosomes by hydrodynamic transfection technique combined with codon-optimized phage integrase system,which enables real-time,dynamic,and noninvasive monitoring the expression of the above functional proteins via the in vivo fluorescence imaging system.It was found that the results obtained from the mouse model were consistent well with those obtained by the traditional protein detection method.At the same time,the reporter gene can achieve stable expression in the liver parenchyma for a long time due to the introduction of phage integrase system.The SAA-Luc mouse model could still produce strong and rapid response upon lipopolysaccharide(LPS)challenge even after 90 days of modeling.These results showed that we successfully constructed the SAA and STAT3 mouse model,which is particularly suitable for long-term and dynamic monitoring the liver function proteins.(2)The expression of SAA in hepatocytes induced by gold nanoparticles of different morphological and particle size.The acute inflammatory response of liver was evaluated by SAA-Luc mouse model.The order of SAA activation was as follows: GNS50 > GNS30 > GNS10 > GNCs > GNRs > GNS80.Spherical gold with a diameter of about 50 nm,at a critical scale,can cause a stronger acute inflammatory response to the liver than GNPs of other size and topography.It also suggested that the pathway of liver uptake of nanoparticles and the degree of liver inflammatory response with the same surface modification were determined by the geometrical shape,scale and distribution in the organism of gold nanoparticles.In case of gold nanomaterials studied,the strength of liver acute inflammation caused by them is scale-dependent,but the impact is non-linear correlation.(3)The detection of SAA activation detected by SAA-Luc mouse model is more sensitive to that detected by ELISA.The expression of SAA was respectively detected by SAA-Luc mouse model and ELISA upon GNS50 exposure at dose of 0.02,0.1,0.5 and 2.5 mg / kg.The results showed that expression of SAA can be detected in SAA-Luc mouse model under 0.1 mg/kg GNS50 exposure,but the serum SAA concentration can't be detected at the same case by ELISA.It was also found that the SAA expression peak detected by SAA-Luc mouse model was earlier than that obtained by ELISA(4 h vs.12 h).These results indicate that the SAA-Luc mouse model based on BLI is not only more sensitive than that detected by the ELISA method,but also can achieve early warning of nanoparticles exposure.(4)GNPs exposure induced the cascade of the M1 polarization of liver kupffer cells,the secretion of series of proinflammatory factors and the initiation of SAA expression.ELISA was used to detect the levels of IL-1?,IL-6 and TNF-? in the liver homogenate after 2 h of GNS50 exposure.The results showed that these proinflammatory factors were significantly elevated at 1 h after exposure to GNPs.SAA-Luc mouse model was used to dynamically evaluate the activation of SAA between 1 h and 2 h after the exposure to GNS50.It was found that the initiation of SAA in mouse liver started at 80 min after GNS50 exposure,which was later than the time where liver proinflammatory cytokines began to elevate.To investigate the sources of these cytokines,we examined the phenotype of kupffer cells and bone marrow-derived macrophages after exposure to GNPs.The results showed that GNPs could induce the M1 polarization of kupffer cells,and the M1 macrophages could secrete proinflammatory cytokines such as IL-1?,IL-6 and TNF-?.Besides,liver kupffer cells were removed to determine whether they were the primary source of proinflammatory cytokines at the early stage of liver SAA activation.Results showed that the initiation of proinflammatory cytokines was significantly inhibited by the removal of kupffer cells,and the expression of SAA in the liver was also significantly delayed.These above results indicate that gold nanoparticles that enter the liver are firstly internalized by kupffer cells and induce the M1 polarization of them,and then the polarized kupffer cells secreted a series of proinflammatory factors which initiated the SAA expression.(5)The expression of SAA was activated by NF-?B signaling pathway.The activation of NF-?B and STAT3 signaling pathways were detected by the method of NF-?B-Luc mouse and STAT3-Luc mouse model combined with EMSA and Western Blot.Results showed that the expression of SAA was significantly inhibited by the NF-?B inhibitor PDTC,but not by the ASD1480 and cryptotanshinone treatment,suggesting the activation of gold nanopatticles was not through the STAT3 signaling pathway.These results suggest that GNPs induced liver SAA expression via NF-?B signaling pathway.(6)SAA regulates the migration and homing of adoptive T cells.The intratumoral injection of SAA could significantly improve the anti-tumor effect of antigen-specific adoptive CD8 + T cells.Further studies showed that SAA can induce M1 repolarization of tumor-associated macrophages(TAM).Then the M1-polarized TAM secreted a series of proinflammatory factors allowing the reversion of the immunosuppressive state of tumor microenvironment,and the secretion of the chemokines such as CCL3,CCL5 and CXCL9 could promote the migration of adoptive CD8 + T cells to the tumor site,thereby enhancing its inhibition of tumor growth.Conclusion: The SAA-Luc and STAT3-Luc mouse models were constructed by the combination of hydrodynamic gene delivery and codon-optimized phage integrase system,which enables real-time,dynamic,and long-term monitoring of the activation of SAA and STAT3 on the same cohort of mice.The above-mentioned mouse model was used to evaluate the liver inflammatory response caused by the exposure of the GNPs,and the relevant mechanism was also elucidated,which provides a sensitive and reliable animal model system for the evaluation of nanomaterials as drug carriers,photothermal therapeutic agents or vaccine adjuvants in biomedical applications.We also found that SAA could regulate the migration and homing of adoptive T cells,reverse the immunosuppressive state of tumor microenvironment,and finally enhance its ability to inhibit tumor growth. |
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