Figure 2. Twist1 and Twist2 expression is increased in MDMs after chronic NOD2 stimulation in an IL-10- and TGFβ-dependent manner whereas this increase is impaired in MDMs from Crohn's disease NOD2 risk carriers.

(A) MDMs (n=4) were left untreated or pre-treated with 100μg/ml MDP for 48h, then stimulated with 100μg/ml MDP for 4h (acute). Fold mRNA expression of Twist1 and Twist2 normalized to untreated cells + SEM. (B) MDMs were left untreated or pre-treated with 100 μg/ml MDP for 48h, then treated with 100μg/ml MDP for 8h and assessed for Twist1/2 expression by Western blot. Representative Western blot from 1 of 3 individuals. GAPDH was used as a loading control. (C) MDMs were cultured with isotype control, neutralizing TGFβ (25μg/ml) or neutralizing IL-10 (5μg/ml) antibodies, alone or in combination, for 1h, then left untreated (for acute) or pre-treated with 100μg/ml MDP for 48h, and then treated with 100μg/ml MDP for an additional 4h (acute). Fold mRNA induction of Twist1 and Twist2 normalized to untreated cells (n=4) + SEM. Statistical significance above the neutralizing antibody sample bars is compared to its corresponding isotype treated sample. (D) MDMs from WT NOD2 healthy controls (WT HC, n=8), WT NOD2 Crohn's disease patients (WT CD, n=8) or Crohn's disease NOD2 risk carriers (Risk SNP, n=3; as per Supplemental Fig 1E) were left untreated or pre-treated with 100μg/ml MDP for 48h, then stimulated with 100μg/ml MDP for 4h (acute). Included is pre-treatment and acute stimulation with 0.1μg/ml lipid A to ensure that MDMs from NOD2 risk patients are responsive to other stimuli. Fold Twist1 and Twist2 mRNA expression is represented by normalizing treated samples to untreated samples + SEM. *, p<0.05; **, p<0.01; †, p<1×10⁻⁴. Tx, treatment.