BCL10 and TLR4-mediated pathway of human colonic epithelial cell inflammation induced by carrageenan

Abstract

Carrageenan (CGN) has been widely used as a food additive in the Western diet, and as an inducer of inflammation in experimental animal models for several decades. However, its molecular mechanism of action in human tissues has been largely unexplored, and investigation of this mechanism was the purpose of our investigation. Using ex vivo human colonic tissue specimens, primary colonic epithelial cells, and the normal colonic epithelial cell line NCM460, we have investigated CGN-induced inflammation with microarrays for cDNA and cytokines, immunoblots, immunohistochemistry, RNA silencing by siRNA, promoter transfection with luciferase reporter, and ELISA techniques. We have discovered a distinct pathway of NFκB and IL-8 activation mediated by BCL10. BCL10 is a caspase-recruitment domain (CARD)-containing gene that is involved in innate immune responses, and its activation in lymphocytes and macrophages is associated with constitutive activation of NFκB. The CARD domain of BCL10 resembles that of NOD2/CARD15, which has been identified as a gene frequently mutated in Crohn's disease. In the human colonic epithelial cells, we have found two-fold increases in IL-8 secretion following exposure to undegraded (MW>1,000,000) κ (kappa), λ (lambda) or ι (iota) CGN at a concentration of 1 μg/ml, for intervals ranging from 1 to 96 hours. The concentration of CGN is considerably less than the concentration of CGN anticipated to arise in association with daily ingestion of CGN. Measurements of dietary content of CGN have varied, ranging from 30 mg/day to 3.5 g/day. In the 1970s, studies by the National Academy of Sciences reported intake by adults in the United States was 100 mg/day. Proposals initiated by the FDA at that time to reduce CGN exposure in the diet were subsequently withdrawn. In our experiments, we have found that CGN exposure leads to increases in nuclear NFκB, phospho-IκBα, and IL-8 promoter activity, as well as Bcl10 mRNA and protein expression. Decline in total IκBα occurs. CAPE (caffeic acid phenyl ester), an inhibitor of NFκB, blocks nuclear NFκB induction of IL-8 promoter activity. Increases in CGN-induced IL-8 are blocked by CAPE and BCL10 siRNA. BCL10 knockdown by siRNA did not lead to decline in dextran-sodium sulfate (DSS) induced secretion of IL-8. Blocking antibody to TLR4 was associated with decline in CGN and lipopolysaccharide (LPS)-induced IL-8 secretion. CGN structure resembles to some extent that of the naturally occurring glycosaminoglycans (GAGs), since it has a sulfated disaccharide backbone. However, CGN contains an α-1,3-galactosidic linkage, not metabolized by the normal human galactosidases. Hence, we conclude that CGN may activate an immune recognition response through TLR4 and BCL10, leading to activation of an inflammatory cascade that is re-activated with recurrent dietary exposure. Elimination of dietary CGN may help to reduce intestinal inflammation. Additional studies are required to examine changes in expression and activity of possible mediators in the CGN-induced pathway of NFκB and IL-8 activation.