Abstract
Chitinase 3-like 1 (CHI3L1) is a protein believed to play a role in activating immune cells and tissue remodelling. CHI3L1 is increased in several inflammatory conditions, such as inflammatory bowel disease (IBD). Accordingly, measurement of CHI3L1 levels shows potential as a novel biomarker or possible drug target. This study investigated CHI3L1 gene expression and protein localisation in two cell lines, Caco-2 and THP-1, to further determine what drives CHI3L1 in patients with IBD.
Caco-2 (epithelial) and THP-1 (macrophage) cell lines were cultured for 48 hours before stimulating with tumour necrosis factor (TNF)-α, interleukin (IL)-1ꞵ and IL-6 at 10 or 100 ng/ml, for 1, 3, 6 and 24 hours. Changes in CHI3L1 gene and protein expression were measured by quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA), respectively. To confirm cellular activation, the expression of IL-8 (gene and protein) were measured in the same samples. Additionally, localisation of cellular CHI3L1 was investigated by western blot and immunofluorescent staining.
In Caco-2 cells, CHI3L1 gene expression increased after 3 hours of exposure to IL-1ꞵ and TNF-α, but not IL-6, when compared to the control. Surprisingly, this was not followed by a specific increase in CHI3L1 levels in the same cell culture supernatants or cell lysates. In contrast, an increase in IL-8 gene expression was observed to precede an increase in IL-8 protein in the same samples. In THP-1 cells, CHI3L1 gene expression was only marginally increased upon stimulation with TNF-α, IL-1ꞵ and IL- 6, with no cytokine increasing CHI3L1 over the other. No increase in secreted protein was observed upon stimulation with any of these cytokines. Only TNF-α induced a response in both IL-8 gene expression and protein release in THP-1 cells. Staining of cells demonstrated CHI3L1 localised primarily on the cell membrane, although this was only detected in THP-1 and not Caco-2 cells. Western blotting of Caco-2 cell lysates was unable to detect CHI3L1 protein.
These results suggest that epithelial cells may not be the primary source of the high levels of CHI3L1 reported in individuals with IBD. Instead, it remains a possibility that macrophages responding to pro-inflammatory cytokines may underpin this response, an observation that fits well with the literature that shows increased CHI3L1 expression is a marker of active as opposed to quiescent inflammation. Although the current model did not detect significant alterations in CHI3L1 expression or secretion following cytokine stimulation, evidence of macrophage-associated CHI3L1 suggests further optimisation of the model used here may provide evidence to show that it is these cells that drive the increased production of CHI3L1 in patients with IBD.