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Juan-Hua Quan,1,* Fei Fei Gao,2,* Hassan Ahmed Hassan Ahmed Ismail,3,* Jae-Min Yuk,2 Guang-Ho Cha,2 Jia-Qi Chu,4 Young-Ha Lee2 1Department of Gastroenterology, The Affiliated Hospital of Guangdong Medical University, Zhanjiang 524-001, People’s Republic of China; 2Department of Infection Biology and Department of Medical Science, Chungnam National University College of Medicine, Daejeon 301-131, Korea; 3Communicable and Non-Communicable Diseases Control Directorate, Federal Ministry of Health, Khartoum, Sudan; 4Stem Cell Research and Cellular Therapy Center, Affiliated Hospital of Guangdong Medical University, Zhanjiang, Guangdong Province 524-001, People’s Republic of China*These authors contributed equally to this workCorrespondence: Young-Ha LeeDepartment of Infection Biology and Department of Medical Science, Chungnam National University College of Medicine 6 Munhwa-Dong, Jung-Gu, Daejeon 35015, KoreaTel +82-42-580-8273Fax +82-42-583-8216Email yhalee@cnu.ac.krPurpose: External and internal stimuli easily affect the retina. Studies have shown that cells infected with Toxoplasma gondii are resistant to multiple inducers of apoptosis. Nanoparticles (NPs) have been widely used in biomedical fields; however, little is known about cytotoxicity caused by NPs in the retina and the modulators that inhibit nanotoxicity.Materials and Methods: ARPE-19 cells from human retinal pigment epithelium were treated with silver nanoparticles (AgNPs) alone or in combination with T. gondii. Then, the cellular toxicity, apoptosis, cell cycle analysis, autophagy, ROS generation, NOX4 expression, and MAPK/mTOR signaling pathways were investigated. To confirm the AgNP-induced cytotoxicity in ARPE-19 cells and its modulatory effects caused by T. gondii infection, the major experiments carried out in ARPE-19 cells were performed again using human foreskin fibroblast (HFF) cells and bone marrow-derived macrophages (BMDMs) from NOX4−/− mice.Results: AgNPs dose-dependently induced cytotoxicity and cell death in ARPE-19 cells. Apoptosis, sub-G1 phase cell accumulation, autophagy, JNK phosphorylation, and mitochondrial apoptotic features, such as caspase-3 and PARP cleavages, mitochondrial membrane potential depolarization, and cytochrome c release into the cytosol were observed in AgNP-treated cells. AgNP treatment also increased the Bax, Bik, and Bim protein levels as well as NOX4-dependent ROS generation. However, T. gondii-infected ARPE-19 cells inhibited AgNP-induced apoptosis, JNK phosphorylation, sub-G1 phase cell accumulation, autophagy, NOX4-mediated ROS production, and mitochondrial apoptosis. Furthermore, mitochondrial apoptosis was found in AgNP-treated HFF cells and BMDMs, and AgNP-induced mitochondrial apoptosis inhibition via NOX4-dependent ROS suppression in T. gondii pre-infected HFF cells and BMDMs was also confirmed.Conclusion: AgNPs induced mitochondrial apoptosis in human RPE cells combined with cell cycle dysregulation and autophagy; however, these effects were significantly inhibited by T. gondii pre-infection by suppression of NOX4-mediated ROS production, suggesting that T. gondii is a strong inhibitory modulator of nanotoxicity in in vitro models.Keywords: silver nanoparticles, Toxoplasma gondii, mitochondrial apoptosis, human retinal pigment epithelium, reactive oxygen species, NADPH oxidase 4

Additional file 1: Figure S1. Trichomonas vaginalis-induced cell cytotoxicity and ER stress in SiHa cells. SiHa cells were infected with T. vaginalis at various MOIs (1, 2, 5 and 10) for the indicated times (0, 0.5, 2, 6, 12 and 24). a The percentages of LDH-dependent cytotoxicity in the medium was measured by LDH assay. The data represent the mean value �� standard deviation (SD) of at least three independent experiments. Asterisks indicate significant difference (**P

Additional file 3: Figure S3. Trichomonas vaginalis-induced ROS production was suppressed with NAC pretreatment in SiHa cells. SiHa cells were pretreated with different concentrations of NAC and then infected with T. vaginalis at MOI 5 for 6 h. a Cellular ROS production was detected by CellROX. Percentages of CellROX-positive cells, as determined by densitometric analysis of fluorescence images. b Mitochondrial ROS production was detected by MitoSOX reagent. Percentages of MitoSOX positive cells, as determined by densitometric analysis of fluorescence images. Asterisks indicate significant differences (*P < 0.05, **P < 0.01, ***P

Additional file 2: Figure S2. Effects of various concentrations of NAC (ROS scavenger), 4-PBA (ER stress inhibitor) and SP600125 (JNK1/2 inhibitor) on the viability of human cervical cancer SiHa cells. SiHa cells treated with 0.2, 1 and 5 mM of NAC (a), 0.2, 1 and 2 mM of 4-PBA (b) and 0.3, 3 and 30 ��M of SP600125 (c), in 5% CO2 at 37��C for 0, 2, 6, 12 and 24 h. Cell viability was checked by the MTS assay. The data represent the mean �� SD of at least three independent experiments