FIGURE 4.

Schematic representation of the four scenarios (approaches 1 to 4) of LPS and MP applications to the multicellular models. Multicellular human lung models, composed of alveolar epithelial cells (A549; red) and human monocyte-derived macrophages (MDMs; blue) and dendritic cells (MDDCs; green), were cultivated at the air-liquid interface conditions (ALI) for 24 h. Subsequently, the models were challenged with LPS (at time 0 h), followed by treatment with MP following four distinct approaches. Approach 1: LPS was applied in the basal compartment (1 μg/mL in 3 ml cRPMI), and after the first 24 h, MP was added to the apical side as a thin layer of liquid (i.e., at pseudo-ALI) at 10 or 100 μM, for an additional 24 h. Approach 2: LPS was added to the apical compartment at the pseudo-ALI for the first 24 h, and then MP was added in co-exposure from the basal compartment for the next 24 h. Approach 3: LPS, applied apically at the pseudo-ALI, was removed after 24 h, and the models were then exposed to the pre-conditioned medium during the MP treatment for an additional 24 h. Approach 4: Multicellular models were challenged with LPS both from the apical and basal compartment for 24 h. Then, LPS was removed from both the compartments, the model was washed, and a fresh cell-culture medium was applied during the MP treatment. Abbreviations: LPS, lipopolysaccharide; MP, methylprednisolone.