Development of an in vitro cell model for LPS-and TNFα-induced airway inflammation

OBJECTIVE To develop an in vitro airway epithelial cell model suitable for the large-scale studies of compounds that can suppress lipopolysaccharide(LPS)-and tumor necrosis factor alpha(TNFα)-induced airway inflammation.METHODS We have optimized the protocols to culture BEAS-2B,a normal human bronchial epithelial cell line,in glass-bottom 384-well microtiter plates.The cells were stimulated with TNFαand LPS from Pseudomonas aeruginosa,a common lunginfection pathogen in cystic fibrosis and chronic obstructive pulmonary disease.We used ELISA to measure the secretion levels of two pro-inflammatory cytokines,interleukin(IL)-6and-8,after 0,4,8,16,24 hof stimulation;and immunofluorescence microscopy to measure the nuclear translocation of RelA,a subunit of the NF-κB complex,after 0,15,30,60,120 min of stimulation.To suppress the inflammatory response,we pre-treated the cells with a specific IκB kinase-2inhibitor,TPCA-1;the main bioactive component of Andrographis paniculata,andrographolide;and DMSO control for 1h.RESULTS We found that individual stimulant(either TNFα10ng·mL-1 or LPS 10μg·mL-1)increased the IL-6and IL-8 secretion levels by^12-17 foldsas compared to DMSO controls after 8h of stimulation.The combined stimulation(10ng·mL-1 TNFαand 10μg·mL-1 LPS)induced even higher IL-6 and -8 levels(~18-21 folds)at the same time points.Importantly,our imaging study shows that the NF-κB activation is early but transient under TNFαstimulation,late but sustained under LPS stimulation,and early and sustained under the combined stimulation.Finally,we also found that TPCA-1 10μmol·L-1 or andrographolide 30μmol·L-1 drastically reduced the IL-6 and -8 levels down to 4.5-9 folds as compared to the controls.CONCLUSIONThe combined TNFαand LPS stimulations induce faster and more sustained inflammatory responses,which can still be suppressed by anti-inflammatory compounds in our cell model.These more comprehensive activations of inflammatory signaling pathways will enable us to study and distinguish the mechanisms of different anti-inflammatory compounds or natural products.