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ObjectiveTo explore the effect of activin A on the migration and adhesion of murine breast cancer 4T1 cells. MethodsThe viability of in vitro cultured 4T1 cells was studied using the 3- (4,5-dimethyl-2-thiazyl)-2,5-diphenyl-2h-tetrazolium bromide (MTT) assay. Scratch assay was conducted to evaluate the wound healing capacity of 4T1 cells. The ability of adhesion of 4T1 cells was detected by real time cell analysis (RTCA). The microfluidic chip-based cell patterning was used to assay 4T1 cells migration. Results MTT assay results showed that the optical density (OD) values of culture medium of 4T1 cells of control group, treated with activin A of 1.25, 5, and 20 (ng/mL) for 24 hours were 0.47 ± 0.01, 0.53 ± 0.006, 0.63 ± 0.02, and 0.66 ± 0.02 and those treated for 48 hours were 0.63 ± 0.04, 0.68 ± 0.01, 0.75 ± 0.03, and 0.85 ± 0.03, respectively, indicating significantly increased viability of 4T1 cells treated with activin A of 5 and 20 ng/mL (P < 0.01 for all). Significantly difference was observed in percentage of migration area between 4T1 cells of control (18.3 ± 2.15%) and those treated with activin A (37.0 ± 1.44%) in scratch-wounded test (P < 0.01). The microfluidic observation showed that the number of migrated cells per vision field was 16 ± 3 for 4T1 cells treated with 20 ng/mL activin A for 12 hours, significantly higher than that (8 ± 2) of control cells in microfluidic chip-based cell patterning (P < 0.01). RTCA showed a significant difference in cell index between control 4T1 cells and the 4T1 cells treated with 20 ng/mL activin A for 8 hours (1.06 ± 0.12 vs. 1.43 ± 0.05, P < 0.01). ConclusionIn summary, activin A promotes not only viability, but also migration and adhesion of murine breast cancer 4T1 cells, which may play an important role in metastasis of breast cancer cells.

ABSTRACT Aspergillus fumigatus is an opportunistic pathogen that infects immunocompromised patients and imposes a heavy burden on global health. Searching for key genes in host resistance to A. fumigatus infection can help us understand the mechanisms of A. fumigatus-host interactions and provide new targets for the treatment of fungal infections. TRAF3 is one of the intracellular adapter proteins of the innate immune response that regulates signaling in various cellular processes, including host defense against pathogens. However, the defense mechanism of TRAF3 against A. fumigatus infection remains unknown. In this study, we found that TRAF3 overexpression led to the adhesion and internalization of more spores of A. fumigatus in lung epithelial cells and thus greater host immune surveillance evasion. Meanwhile, TRAF3 was able to regulate the expression of pro-inflammatory cytokines in lung epithelial cells infected with A. fumigatus through the negative regulation of NF-κB and MAPK signaling pathways. In this study, we also examined the effects of TRAF3 in the interaction between lung epithelial cells, macrophages, and A. fumigatus spores, and the results showed that TRAF3-overexpressing lung epithelial cells reduced the migration and activation of macrophages after A. fumigatus infection. In vivo experiments using TRAF3-overexpressing transgenic zebrafish larvae revealed that TRAF3 overexpression increased the fungal load and mortality of zebrafish infected with A. fumigatus. In conclusion, the TRAF3 gene can negatively regulate the resistance of lung epithelial cells to A. fumigatus, which plays an important role in the early infection processes of A. fumigatus. IMPORTANCE Aspergillus fumigatus can infect immunocompromised individuals and cause chronic and fatal invasive fungal infections. A better understanding of the molecular mechanisms of A. fumigatus-host interactions may provide new references for disease treatment. In this study, we demonstrated that the TRAF3 gene plays an important role in the early infection of A. fumigatus by regulating the resistance of lung epithelial cells to A. fumigatus. Macrophages are the most abundant innate immune cells in the alveoli; however, few studies have reported on the interactions between lung epithelial cells and macrophages in response to A. fumigatus invasion. In our study, it was demonstrated that the TRAF3 gene reduces migration to macrophages and cytokine production by negatively regulating lung epithelial cell adhesion and internalization of A. fumigatus spores. Together, our results provide new insights into lung epithelial cell-macrophage interactions during A. fumigatus infection.

Infection with Aspergillus fumigatus can cause life-threatening diseases in immunocompromised patients with an unacceptable mortality rate. Angioinvasion is one of the features of severe invasive aspergillosis. Neutrophils are short-lived immune cells regulated by colony-stimulating factor 3 (CSF3) that play a key role in anti-fungal immune responses. To investigate the interactions between A. fumigatus and the host immune cells, such as neutrophils, we stimulated human umbilical vein endothelial cells (HUVECs) with the conidia of A. fumigatus, and co-cultured them with human neutrophils. Apoptosis and functions of neutrophils were analyzed. Our results showed that HUVECs upregulate the expression of CSF3, which could reduce the apoptosis of neutrophils while enhancing their functions. Lack of CSF3 was associated with enhanced apoptosis of neutrophils with impaired function. This work indicated that the CSF3 is required for neutrophil survival and function, at least in the early stages of A. fumigatus infection.