Antioxidants inhibit indoleamine 2,3-dioxygenase in IFN-gamma-activated human macrophages: Posttranslational regulation by pyrrolidine dithiocarbamate

Induction of the home-containing indoleamine 2,3-dioxygenase (IDO) by IFN-g amma is implicated in anti-microbial and pro-inflammatory activities of hum an macrophages. Antioxidants can modulate the expression of immune and infl ammatory genes, and pyrrolidine dithiocarbamate (PDTC) is a frequently used antioxidant to inhibit the transcription factor NF-kappaB. Here we show th at IFN-gamma treatment of human monocyte-derived macrophages (hMDMs) increa sed the proportion of oxidized glutathione. PDTC attenuated this increase a nd inhibited IDO activity, although it increased IDO protein expression and did not affect IDO mRNA expression and enzyme activity directly. Other ant ioxidants, 2-ME, ebselen, and t-butyl hydroquinone, inhibited IDO protein e xpression. Similar to PDTC, the heme biosynthesis inhibitor succinylacetone (SA) and the iron-chelator pyridoxal isonicotinoyl hydrazone inhibited cel lular IDO activity without affecting protein expression, whereas addition o f heroin or the heme precursor delta -aminolevulinic acid increased IDO act ivity. Also, incubation of IFN-gamma -activated hMDM with delta-[C-14]-amin olevulinic acid resulted in the incorporation of label into immunoprecipita ted IDO, a process inhibited by PDTC and SA. Furthermore, supplementation o f lysates from PDTC- or SA-treated hMDM with hemin fully restored IDO activ ity to control levels, and heroin also reversed the inhibitory action of SA but not PDTC in intact cells. Together these results establish a requireme nt for de novo heme synthesis for IDO activity in IFN-gamma -activated hMDM . They show that, similar to other pro-inflammatory proteins, the activity of IDO is modulated by antioxidants though in the case of PDTC this takes p lace posttranslationally, in part by limiting the availability of heme for the formation of holo-IDO.