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Conor Murphy, Micheal O'Rourke, Mary Connolly, Cheryl Sweeney, Ursula Fearon; The role of peripheral dendritic cells in immune activation in anterior uveitis. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2504.
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Pathogens, such as microbial components express pathogen associated molecular pathogens. These can interact with innate pattern recognition receptors via toll like receptors (TLRs) on antigen presenting cells (APCs). TLRs play a critical role linking innate and adaptive immunity promoting the maturation of APCs through the production of pro-inflammatory cytokines and the up-regulation of co-stimulatory molecules. This interaction also allows APCs to become efficient in the presentation of specific antigens to naïve T cells initiating adaptive immunity. It is evident that these processes can be altered by a class of small non-coding RNAs or microRNA (miR) which exert their biological function through suppression of their target genes, abnormal expression of which has been demonstrated in chronic inflammatory diseases.
Peripheral blood mononuclear cells were isolated from active AU patients and healthy controls (n=5). CD14+ monocytes (purity >97%) isolated by positive selection using magnetic bead separation were differentiated into imDCs by culturing for 7 days with IL-4 and GM-CSF and matured in media for 24 hours. The effects of TLR activation and pro-inflammatory stimuli were examined by culturing moDC with Pam3CSK4 (1μg/ml), Poly I:C (25μg/ml), LPS (1μg/ml), IL-1β (10ng/ml) and TNFα (10ng/ml). Cell surface expression of CD83 (maturity) and CD86 (activation) on DC were quantified by flow cytometry. Cytokine analysis was performed on supernatant by ELISA. Total RNA from the mature DC was isolated using the miRneasy isolation kit. Quantification of miR expression was analyzed by real-time PCR, using miRNA-let-7a as an endogenous control.
CD83 and CD86 expression on AU DC was increased in response to all agonists compared to basal, most notably for TLR3 and IL1b. A significant increase in CD83 mean fluorescent intensity (MFI) in response to IL1b was demonstrated in AU moDC compared to HC (p=0.05). This was paralled by an increase in IL12 (p=0.05) and IL23 expression in cultured supernatants. Furthermore, expression of miR155 was also increased in response to Poly I:C, LPS and Il1b in AU DC.
These results support peripheral activation of DC by TLR3 and Il1b in AU patients suggesting a possible role in AU. Altered miR155 expression in DC from AU patients suggests that this miR may contribute to the pathogenesis of AU by mediating TLR3 activated pro-inflammatory pathways.
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