Although there are over 1,600 Engineered Nanomaterials (ENMs)-containing consumer products available, our understanding of ENM safety is still limited. Airborne ENMs can readily enter the human body through inhalation potentially leading to many adverse health effects such as cardiovascular and pulmonary diseases. The conventional in vitro submerged cell culture method was developed decades ago and has been widely used as a fast screening method to elucidate cellular toxicity upon exposure to hazardous materials; however, it has many limitations compared with the in vivo models. Our group has previously utilized and validated an integrated low flow system capable of generating and depositing airborne nanoparticles (NPs) directly onto cells at an air-liquid interface (ALI) condition, and our results confirmed that this exposure system produced reproducible toxicological data for ENMs including gold (Au), 16% silver coated onto silica (16% Ag-SiO2), and copper oxide (CuO). To further improve this ALI method for an even closer representation of the in vivo model, a co-culture model containing three cell lines (A549, THP-1 differentiated macrophages, and EA.hy 926) was established and validated for testing ENMs toxicity. The co-culture model was exposed to 16% Ag-SiO2 and CuO NPs under the same protocol (4 h ALI exposure with a concentration of 3.5 mg/m3) as monoculture (A549 only) for comparison. Toxicity was assessed by measuring cell viability, reactive oxygen species (ROS) production, lactate dehydrogenase (LDH) release, and interleukin (IL) 8 level. Results showed that 16% Ag-SiO2 NPs induced higher ROS generation, and CuO NPs produced a significant level of proinflammatory response compared with monoculture. In addition, the co-culture model exhibited a similar response with the primary human bronchial epithelia cell line (HBEC) in terms of ROS and IL-8 responses after CuO NPs exposure, suggesting a more advanced refinement of the conventional model for in vitro inhalation study.

Chlamydia trachomatis infections represent the predominant cause of bacterial sexually transmitted infections. As an obligate intracellular bacterium, C. trachomatis is dependent on the host cell for survival, propagation, and transmission. Thus, factors that affect the host cell, including nutrition, cell cycle, and environmental signals, have the potential to impact chlamydial development. Previous studies have demonstrated that activation of Wnt/β-catenin signaling benefits C. trachomatis infections in fallopian tube epithelia. In cervical epithelial cells chlamydiae sequester β-catenin within the inclusion. These data indicate that chlamydiae interact with the Wnt signaling pathway in both the upper and lower female genital tract (FGT). However, hormonal activation of canonical and non-canonical Wnt signaling pathways is an essential component of cyclic remodeling in another prominent area of the FGT, the endometrium. Given this information, we hypothesized that Wnt signaling would impact chlamydial infection in endometrial epithelial cells. To investigate this hypothesis, we analyzed the effect of Wnt inhibition on chlamydial inclusion development and elementary body (EB) production in two endometrial cell lines, Ishikawa (IK) and Hec-1B, in nonpolarized cell culture and in a polarized endometrial epithelial (IK)/stromal (SHT-290) cell co-culture model. Inhibition of Wnt by the small molecule inhibitor (IWP2) significantly decreased inclusion size in IK and IK/SHT-290 cultures (p < 0.005) and chlamydial infectivity (p ≤ 0.01) in both IK and Hec-1B cells. Confocal and electron microscopy analysis of chlamydial inclusions revealed that Wnt inhibition caused chlamydiae to become aberrant in morphology. EB formation was also impaired in IK, Hec-1B and IK/SHT-290 cultures regardless of whether Wnt inhibition occurred throughout, in the middle (24 hpi) or late (36 hpi) during the development cycle. Overall, these data lead us to conclude that Wnt signaling in the endometrium is a key host pathway for the proper development of C. trachomatis.

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