Mechanisms Of Carrageenan Induced Immune Response In Macrophages

Carrageenan (CGN), a high molecular weight sulphated polysaccharide extracted from red seaweed, iswidely used as stabilizing, emulsifying, or thickening agents in food processing, and also used in cosmeticand pharmaceutical industries. Meanwhile, CGN has stirred an intense controversy and debate consideringits safety. Many International Agencies have declared that CGN is harmless for human consumption. But incontrast, investigators studied the effects of CGN and suspect that CGN posed a carcinogenic risk tohumans. Although the controversy is fierce, there is a consensus that degraded CGN (dCGN, have averagemolecular weight of10-40kDa) exposure significantly causes inflammation. Macrophages are thought tohave a key role in inflammation. Although CGN exposure has been associated with development ofinflammation in animal models and cell-based experiments, there are no studies on macrophage responsesto CGN exposure.In this study, we present the effects of exposure of THP-1derived macrophage cells and RAW264.7cells, tothree commercially most common carrageenans (κ-，ι-，λ-CGN), dCGN with Mw10-40kDa (κ-，ι-，λ-10-40k)and dCGN with Mw less than5000Da (κ-，ι-，λ<5k), respectively. The carrageenan forms that induce the strongeimmune response of macrophages was assured, and its molecular mechanisms were analyzed. Furtherfore, theregulation of CGN on a highly proinflammatory molecule——LPS-stimulated inflammation in macrophage wasobserved.TNF-α was induced in macrophages following different forms of CGN exposure, and the degradedλ-carrageenan with molecular weight of10-40kDa (λ-10-40k) exhibited the strongest effect to up-regulatethe secretion of TNF-α. It could stimulated TNF-α secretion2.2-fold (P<0.01) and5.1-fold (P<0.001)higher than control in THP-1macrophages and RAW264.7cells, repectively. Although the increase ofTNF-α was not so obvious, λ-10-40k could coordinated enhance the production of immune cytokinesinduced by LPS in macrophages. The amount of TNF-α induced by pretreatment with10μg/mL λ-dCGNwas1.5-fold and2.2-flod (P<0.05) higher than the one produced by LPS treatment alone in THP-1derivedmacrophage cells and RAW264.7cells, respectively. However, this enhancement was reached18-fold and30-fold increase in TNF-α production induced by λ-10-40k treatment alone in THP-1derived macrophagecells and RAW264.7cells, respectively. Interestingly, the amount of TNF-α secreted by RAW264.7cellsinduced by λ-10-40k was much larger than the one secreted by THP-1macrophages, and the enhancementof λ-10-40k on LPS-induced TNF-α was also higher than on RAW264.7cells. Therefore, the molecularmechanisms of λ-10-40k induced immune response and enhancement of LPS-induced inflammatoryresponse were analyzed in RAW264.7cells. We found that TLR4-Bcl10-NF-κB and MAPK/ERK-mediatedpathway may involve in λ-10-40k induced TNF-α secretion. Furthermore, λ-10-40k could synergeticallyenhance the immune response of LPS by increasing the expression of TLR4, and enhance the activation ofERK-JNK-AP-1pathway. We demonstrate that degraded carrageenan probably coordinated enhance theproduction of immune cytokines induced by LPS, leading to magnify its inflammatory effect.