Characterisation of the Kynurenine Pathway in Skin‐Derived Fibroblasts and Keratinocytes

Abstract

Acute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon-gamma (IFN-γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD(+) ) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD(+) levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN-γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD(+) production, providing support for involvement of the KP in the de novo synthesis of NAD(+) in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.

Indoleamine 2,3-Dioxygenase 2 Deficiency Exacerbates Imiquimod-Induced Psoriasis-Like Skin Inflammation

“…Our data show that IDO2 might suppress IMQ-induced psoriasis-like skin inflammation. With respect to IDO1 and skin, keratinocytes and fibroblasts can upregulate IDO1 expression and activation in vitro in response to proinflammatory stimuli [ 37 , 38 ]. Nevertheless, myeloid dendritic cells are the major IDO1 -expressing skin cells in vivo, and IDO1 is not expressed in the epidermis [ 39 ].…”

Application of Targeted Mass Spectrometry for the Quantification of Sirtuins in the Central Nervous System

“…For the gene expression studies RNA was extracted from human brain cells using RNeasy mini kits (Qiagen, Germany). The cDNA was prepared using SuperScript III First-Strand Synthesis System and random hexamers (Invitrogen Corporation, USA) as previously described 39 . Detailed protocol and primer sequences are described in Supplementary Protocols and Supplementary Table S7 respectively.…”

EWS‐FLI1 impairs aryl hydrocarbon receptor activation by blocking tryptophan breakdown via the kynurenine pathway

“…The KYN pathway has been implicated in a variety of diseases and disorders such as AIDS, Alzheimer's disease, depression, schizophrenia, Huntington's disease, amyotrophic lateral sclerosis, and neoplasia . Different metabolites of the KYN pathway have been associated with immune active properties , and because of their roles in immunity and the central nervous system, the KYN pathway has emerged as an attractive target for drug development . Several downstream metabolites of the KYN pathway are biologically active in various physiological and pathological processes, including KYN, KYNA, 3‐HK, anthranilic acid, 3‐hydroxyanthranilic acid, and quinolinic acid .…”