Figure 4

Blockade of PD-L1 prevents TNF-α/IFN-γ-induced barrier dysfunction of Caco2 monolayer. (A) PD-L1 is constitutively expressed in nontreated Caco2 cells, and human recombinant TNF-α/IFN-γ treatment further elevates PD-L1 expression, measured by flow cytometry. To assess the impact of PD-L1 on Caco-2 monolayer barrier dysfunction, cell monolayers were established in transwell plates or glass coverslips for 21 d, pretreated with anti-PD-L1 or IgG control antibodies, then stimulated with TNF-α/IFN-γ (10 ng/mL each). (B) Treatment with TNF-α/IFN-γ induces increased monolayer permeability (increased FD4 concentration in basal chambers), while antibody blockade of PD-L1 prevents the effect of cytokines on barrier dysfunction (negative control levels of FD4 was set as 100%). Data represent the average of three independent experiments, in triplicate for each treatment. * P < 0.05 versus control; # P < 0.05 versus anti-PD-L1. Oneway ANOVA and Student-Newman-Keuls test. Representative image of immunofluorescent staining for TJ protein ZO-1 (C) and occludin (D) in Caco-2 monolayers. When compared with negative control (Neg Cont), the monolayers treated with TNF-α/IFN-γ (Cyt) showed obvious disruptions in distribution and significantly decreased intensity of ZO-1 (C) and occludin (D) protein staining, calculated in percentage intensity of the fix area. Pretreatment with PD-L1 antibody (Cyt + α-PD-L1) preserved the integrity of monolayers closer to nontreated control, compared with cytokine treated or IgG + cytokine treated monolayers (C and D). Original magnifications × 400.