The increased expression of IL-23 in inflammatory bowel disease promotes intraepithelial and lamina propria lymphocyte inflammatory responses and cytotoxicity

Abstract

This study analyzed IL-23p19 expression in inflamed mucosa of IBD and the role in the induction of IEL and NK cell activation as well as Th17 cell differentiation. Expression of IL-23p19 was performed by immunohistochemistry and quantitative real-time PCR. Expression of IL-23R was assessed by flow cytometry. Cytolytic activities of IEL and NK cells by IL-23 were determined by a standard (51)Cr-release assay. Cytokine levels were analyzed by ELISA and quantitative real-time PCR. Expression of IL-23p19 was increased significantly in inflamed mucosa of CD compared with that in UC and healthy controls. Double-staining confirmed that IL-23p19(+) cells were mainly CD68(+) macrophages/DCs. IL-23R(+) cells were increased significantly in PB- and LP-CD4(+) and -CD8(+) T and NK cells. IL-23 markedly promoted IBD IEL and NK cell activation and cytotoxicity and triggered IBD PB- and LP-T cells to secrete significantly higher levels of IFN-γ, TNF, IL-2, and IL-17A compared with controls. Importantly, IL-23 promoted IBD PB- or LP-CD4(+) T cells to differentiate into Th17 cells, characterized by increased expression of IL-17A and RORC. Anti-TNF treatment could markedly reduce IL-23 expression and Th17 cell infiltration in inflamed mucosa of CD patients. These data indicate that IL-23 is highly expressed in inflamed mucosa of IBD and plays an important role in the induction of IEL, NK, and T cell activation, proinflammatory cytokine secretion, and Th17 cell differentiation. Targeted therapy directed against IL-23p19 may have a therapeutic role in treatment of IBD.

Anti-tumour necrosis factor therapy enhances mucosal healing through down-regulation of interleukin-21 expression and T helper type 17 cell infiltration in Crohn's disease

“…After 48 h of culture, supernatants were harvested and analysed for TNF, IFN-g, IL-17A and IL-21 production by sandwich enzyme-linked immunosorbent assay (ELISA) kits according to our previous reports [15,23]. The sensitivity of each assay was 10 pg/ml.…”

“…The positive cells and total stromal cells were counted in the LP. The proportion of positive cells was calculated as follows: [(positive cells)/(total cell number)] ¥ 100 [23][24][25].…”

“…The sensitivity of each assay was 10 pg/ml. Cultured cells were then collected followed by extraction of total RNA; mRNA levels were analysed for evaluation of TNF, IFN-g, IL-17A, IL-21 and RORC, as described previously [15,[23][24][25].…”

“…+ T cells PBMC and LPMC were isolated as described previously [15,23], and peripheral blood (PB)-or LP-CD4 + T cells were then purified by negative selection using the Dynal untouched human CD4 + T cells isolation kit (Dynal AS; Invitrogen). Cell purity was evaluated routinely by flow cytometry and ranged from 95 to 99%.…”

“…All PCR reactions were run in triplicate and performed with 40 cycles using the following conditions: 95°C for 1 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. The relative target gene expression levels were calculated as a ratio relative to the b-actin reference mRNA. Quantitative real-time PCR analysis was carried out using the 2-᭝᭝Ct method [15,23,24].…”