Figure 4

Effects of gliadin, butyrate, PSA and lactate on barrier function and pro-inflammatory cytokines. (a) Absolute fluorescent intensity of FITC-dextran 4,000 Da recovered in the basolateral side of confluent monolayers after 4 h incubation with 1 mg/mL pepsin-trypsin digested-gliadin (PTG) or media control (Unt). Data represent average ± SEM. ***p < 0.001; Mann-Whitney test; 21 to 30 replicates for n = 5 non-celiac (NC) and n = 6 celiac (CD) patients. NS: not-significant. (b) Secreted cytokines measured by ultra-sensitive multiplex electrochemiluminescence in the basolateral supernatants of non-celiac (NC) and celiac (CD) monolayers upon 4 h challenge with 1 mg/mL PTG. Fold change calculated relative to untreated monolayers. Data represent average ± SEM. *p < 0.05, **p < 0.01, two-side unpaired t-test. ^††p < 0.01, Mann-Whitney test; 10 to 15 replicates for n = 5 NC and n = 6 CD patients. (c) Fold change of transepithelial electrical resistance (TEER) measured in non-celiac (NC) and celiac (CD) confluent monolayers after 48 h treatment with butyrate (But), polysaccharide A (PSA) or lactate (Lac), expressed as fold change of TEER reading at time 0. Unt: untreated. Data represent average ± SEM. *p < 0.05, **p < 0.01, Mann-Whitney test; 44 to 102 replicates for n = 5 NC and n = 6 CD patients. (d) Expression of genes related to gut barrier function assessed by quantitative RT-PCR in epithelial organoids derived from non-celiac (NC) and celiac (CD) patients, after 48 h treatment with butyrate (But), polysaccharide A (PSA) or lactate (Lac). Data represent average expression relative to untreated ± SEM. *p < 0.05, **p < 0.01, two-side unpaired t-test; ^†p < 0.05, Mann-Whitney test. 8 to 12 replicates for n = 4 NC and n = 4 CD patients. (e) Secreted cytokines measured by ultra-sensitive multiplex electrochemiluminescence in the basolateral supernatants of celiac (CD) monolayers. Monolayers were 48 h pre-treated with microbiota derived-bioproducts butyrate (But), polysaccharide A (PSA) or lactate (Lac) and subsequently challenged with 1 mg/mL PT-gliadin (PTG) for 4 h. Fold change (FC) calculated relative to untreated monolayers. Data represent average ± SEM. *p < 0.05, Mann-Whitney test (PTG vs. PTG pretreated with the bioproducts); 12 to 15 replicates for n = 6 (CD) patients.