Tryptophan/kynurenine metabolism in human leukocytes is independent of superoxide and is fully maintained in chronic granulomatous disease

Abstract

In chronic granulomatous disease (CGD), defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity causes reduced superoxide anion (O(2)(·)) radical production leading to frequent infections as well as granulomas and impaired wound healing indicative of excessive inflammation. Based on recent mouse studies, the lack of O(2)(·)-dependent interferon γ (IFNγ)-induced synthesis of kynurenine (kyn), an anti-inflammatory tryptophan metabolite produced by indolamine 2,3 deoxygenase (IDO), was proposed as a cause of hyperinflammation in CGD and this pathway has been considered for clinical intervention. Here, we show that IFNγ induces normal levels of kynurenine in cultures of O(2)(·)-deficient monocytes, dendritic cells, and polymorphonuclear leukocytes from gp91(PHOX)- or p47(PHOX)-deficient human CGD donors. Kynurenine accumulation was dose- and time-dependent as was that of a downstream metabolite, anthranilic acid. Furthermore, urinary and serum levels of kynurenine and a variety of other tryptophan metabolites were elevated rather than suppressed in CGD donors. Although we did not specifically evaluate kyn metabolism in local tissue or inflamed sites in humans, our data demonstrates that O(2)(·) anion is dispensable for the rate-limiting step in tryptophan degradation, and CGD patients do not appear to have either hematopoietic cell or systemic deficits in the production of the anti-inflammatory kynurenine molecule.

Figure 1

CGD monocytes and PMN express IDO after IFNγ treatment. Immunoblots of lysates from monocytes (MNC) or PMN treated with indicated doses of IFNγ were probed for indoleamine 2,3-dioxygenase (IDO) expression and β-actin as a loading control. Blots shown are representative of 2 experiments.

Figure 2

LC-ESI-MS detection of metabolites in cell culture supernatants and urine. Representative traces are provided of standards (black line), a normal monocyte culture treated with IFNγ and supplemented with 500μM tryptophan (red trace), and a 24-hour urine sample from a CGD patient (blue trace). The traces are staggered to facilitate comparison.

Figure 3

Dose response to IFNγ of tryptophan metabolism in monocytes. Cells were incubated in media alone or with the indicated amounts of IFNγ for 48 hours before collection and analysis of cell culture supernatants. N.D. indicates that anthranilic acid was not detected in the absence of IFNγ treatment.

Figure 4

Kynurenine production by Normal and CGD monocytes and PMN. Tryptophan and kynurenine concentrations were measured in culture supernatants after 24 (PMNs) or 48 hours (monocytes) of treatment with 1000 IU IFNγ/mL. Data shown represent mean kynurenine or tryptophan concentration (micromolar) ± SE from normal (circles) monocytes (n = 16) or PMNs (n = 14), A-CGD (squares) monocytes (n = 7) or A-CGD PMNs (n = 6), or X-CGD (triangles) monocytes (n = 7) or PMNs (n = 6).

Figure 5

Kinetics of IFNγ-induced tryptophan metabolism of MDDC from normal and CGD donors. MDDC were treated as described for monocytes and tryptophan and kynurenine concentrations measured at the indicated time points. Plotted are means ± SEM of 4 to 8 normal donors and 2 to 6 CGD donors (both genotypes).

Figure 6

Urinary trptophan levels in normal and CGD donors. Twenty-four-hour collections of urine were analyzed as described and the resulting concentrations (micromolar) plotted for each replicate (n = 3 normal, 2 A-CGD, 4 X-CGD). A line is drawn at the mean ± SEM for each set. ND indicates not detected.