METABOLIC INFLAMMATION IN CROHN’S DISEASE: EFFECTS OF CARNITINE PAMITOYLTRANSFERASE ACTIVITY ON INFLAMMATORY SIGNALING IN THE INTESTINAL EPITHELIUM

Abstract BACKGROUND Growing evidence indicates that dysfunction in epithelial metabolic pathways may be linked with Crohn’s disease (CD). This suggests that changes in gene expression and cellular behavior affected by these abnormal metabolic pathways could be a mechanism in the pathogenesis of CD. Thus, we hypothesized that by blocking transport of long-chain fatty acids into the mitochondria for oxidation by inhibiting carnitine palmitoyl transferase (CPT1) activity with ST1326, we would observe an effect on inflammatory signaling cascades in epithelial cells. METHODS Organoids were established with crypts isolated from ileal and rectal mucosal biopsies of patients consented at Children’s Healthcare of Atlanta. Organoids grown in Intesticult Human Growth Media (OGM) or in house organoid media (OM) were treated with IFN-γ (1 ng/ml), TNF-α (25 ng/ml), and IL-13 (10 ng/ml), alone or in combination (cytomix) for 48 hrs, along with different doses/combinations of ST1326 (5, 10, 25, and 50 uM). Expression levels of CXCL8, CXCL9, and ICAM1 mRNAs were measured by real-time PCR. Phosphorylated (p) STAT1 (Y701 and S727), pSTAT3 (Y705 and S727), pSTAT6 (Y641), pERK1/2 (T202/Y204) were determined via Western blotting. RESULTS Western blotting of lysates from organoids treated with different inflammatory stimuli showed that IFN-γ increased phosphorylation of STAT1 at Y701 and S727, and that IL-13 increased levels of pSTAT6 (Y641). Similar experiments showed that IL-13 and TNF-α both increased pSTAT3 (Y705) while only IL-13 increased pSTAT3 (S727) levels. pERK1/2 (T202/Y204) phosphorylation increased with IFN-γ and TNF-α stimulation. ST1326 did not affect the levels of STAT1 Y701 phosphorylation that were increased by INF-γ (Figure 1), nor did it significantly affect IL-8 and ICAM-1 levels when co-administered with TNF-α or INF-y alone (Figure 2). However, ST1326 reduced the IL-13-dependent phosphorylation of pSTAT6 (Y641) (Figure 1). CONCLUSION Fatty acid oxidation regulates aspects of the JAK/STAT pathways during inflammatory signaling with inhibition of CPT1 by ST1326 reducing energy production and leading to disruption of cellular metabolism. Changes in cytokine transcript levels can sometimes be seen with the addition of inhibitors like ST1326 to an inflammatory mileu showcasing the heterogeneity of patients’ immune profiles. Ongoing studies will examine the impact of CPT1 inhibition on the secretome with a focus on functional consequences of these differences with effects on the epithelium.