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VITAMIN D MODULATES CYTOKINE RESPONSES INDUCED BY TLR LIGANDS IN PBMC AND MONOCYTE-DERIVED DENDRITIC CELLS IN CROHN'S DISEASE
S Dionne, M Yona, D Levesque, E SeidmanResearch Institute, McGill University Health Centre, Montreal, QC
Aims: Vitamin D modulates innate and adaptive immunity, and was demonstrated by our group (Wang et al J Biol Chem 2010) to induce NOD2, the most common susceptibility gene for Crohn's disease (CD). The aim fo the present stusy was to examine the effect of 1,25D (50 nM) on the immune response to bacterial antigens in CD.
Methods: PBMC from 61 CD patients were stimulated with PAM3CSK4, LPS, flagellin, R848 and muramyldipeptide (MDP), ligands for TLR2, TLR4, TLR5, TLR7/8 and NOD2, respectively. Monocyte-derived dendritic cells (Mo-DC) were prepared from CD14+ cells and stimulated with LPS, R848 and MDP.
Results: TLR ligands induced PBMC TNFalpha, IL-12/IL-23p40 and IL-10. IL-23 was induced by LPS+R848. Combining LPS+R848 increased levels 1.9 to 7.5 fold vs LPS alone, especially IL-23. 1,25D decreased IL-12/IL-23p40 and IL-23 levels induced by TLR ligands (-44% to -78%, p<0.01); IL-10 decreased -4% to -44%; TNFalpha was 0 %to -52% lower. 1,25D decreased IL-10, but less than IL-12/IL-23p40. The IL-10 to IL-12/IL-23p40 ratio increased with 1,25D (1.5x to 3.8x, p<0.05). MDP alone induced low IL-10 and IL-12/IL-23p40, but synergized with LPS and LPS+R848 to induce cytokine(p<0.01). Cytokines increased 2.2- to 6.6 -fold with MDP+LPS. MDP alone or with LPS decreased IL-12/IL-23p40 levels, but the other cytokines were increased. When combined with LPS+R848, cytokines levels decreased (-26% to -49%, p<0.05) but to a lesser extend than without MDP. The anti-inflammatory effects of 1,25D were then examined in Mo-DC. Single TLR triggering resulted in very low cytokine levels. LPS+R848 induced high levels of IL-23 and TNFalpha (8300,4250 pg/ml). Moderate amounts of IL-10 and IL-23 were produced (280 pg/ml and 583 pg/ml, respectively). In addition, IL-12p70 was released (79 pg/ml). Addition of MDP significantly increased all cytokines (>1.5-fold, p<0.05) except for TNFalpha. Cytokines levels induced by LPS+R848 were decreased by 54% to 91% by 1,25D (p<0.01). When MDP was also present, the decrease was more moderate (-17% for IL-10, -46% for TNFalpha 88%). IL-12p70 was similarly decreased (-91% and -88%). IL-23 levels were not decreased in the presence of MDP.
Conclusions: 1,25D decreased TLR-induced inflammatory cytokines TNFalpha, IL-12/23p40 and IL-23 in PBMC from CD. 1,25D lowered the pro-inflammatory profile (increased IL-10 to IL-12/IL-23p40 ratio). 1,25D decreased Mo-DC TLR-induced cytokines, particularly IL-12p70. NOD2 signaling by MDP combined with TLR stimulation resulted in preservation of IL-23 and IL-10 compared to TNFalpha and IL-12. APC production of IL-12p70 and IL-23 promotes Th1 and Th17 cells, respectively. Our results suggest that 1,25D decreased inflammatory cytokines and inhibits the IL-12/23 inflammation pathway and may help to restore the aberrant inflammatory response in CD. Supported by a CIHR Team Grant in Clinical Autoimmunity.