Figure 1.

Macrophage subset-epithelial cell co-culture and cytokine production determine barrier integrity. Caco-2 epithelial cells were cultured to confluence (intact barrier) in transwell inserts and incubated in co-culture with M1 and M2 Mφ subsets (a,b) or Mφ-derived cytokines in the absence of Mφs (c,d). Co-cultures received LPS in the apical compartment and cytokine production was analysed in the basolateral compartment (a) and the corresponding TEER as a measure of barrier integrity (b). Effect of Mφ-derived TNFα, IL-1β, IL-8 and IL-10 on barrier integrity is presented as TEER in Ω cm² (c) and expression of the tight junction molecule, ZO-1 mRNAexpression (d) is presented as relative expression (RQ, Arbitary Units) compared to GAPDH housekeeping gene expression. Mφ co-culture and effect of inflammatory stimuli is further represented by immunohistochemical characterisation of ZO-1 protein staining in the epithelial barriers incubated in co-cultures (e–k), where (e) Caco-2 epithelial barrier control, (f) Caco-2 + M1 Mφs, (g) Caco-2 + M1 + LPS, (h) Caco-2 + M1 + IL-1β, (i) Caco-2 + M2 Mφs, (j) Caco-2 + M2 + LPS and (k) Caco-2 + M2 + IL-1β. Data displayed is a representative experiment with triplicate samples for n = 4 replicate experiments (a–d) and n = 3 experiments (e–k). Significant effects of Caco-2/Mφ co-culture are compared to Caco-2 control (b) and cytokine effects compared to unstimulated Caco-2 control (c,d) and significance indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.