chronic granulomatous disease ( cgd ) is an inherited immunodeficiency characterized by recurrent severe bacterial and fungal infections . the underlying genetic defect of the phagocyte nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase results in failure to produce reactive oxygen intermediates ( roi ) , favoring invasive infections . cgd is also characterized by a hyperinflammatory response following immune activation , which might lead to granuloma formation even in the absence of pathogen . a recent report proposed that impaired activation of the immunoregulatory indoleamine 2,3-dioxygenase ( ido ) pathway [ 57 ] contributed to an amplified inflammatory response in murine cgd . ido , mediating tryptophan metabolism , is a heme - containing enzyme and requires posttranslational activation through the reduction of the ferric ( fe ) to the ferrous ( fe ) form   before the metabolic breakdown of tryptophan along the kynurenine pathway can be initiated . in consistence with this concept , ido activity was found to be abolished in cgd mice . cgd ( p47 phox ) ( phox , phagocytic oxidase ) mice responded to experimental aspergillus infection with hyperinflammation , indicated by severe lung injury and a th17 polarized response , and showed reversal of the hyperinflammatory phenotype upon application of the tryptophan breakdown product kynurenine . as this finding may have significant impact on the understanding of cgd pathophysiology , we investigated whether defective ido activation was also present in human cgd . heparinized blood was collected after written informed consent from four cgd patients , including one with x - linked ( gp91phox ) and three with autosomal recessive ( p47phox ) inheritance ( table  1 ) . all patients were free of infection and presented no clinical or laboratory signs of inflammation at the time of blood collection ( not shown ) . the first diagnosis of cgd was made in all patients by <  10% oxygen radical production , as determined by chemiluminescence , dihydrorhodamine , and/or nitroblue tetrazolium tests in pma - stimulated phagocytes , followed by genetic analyses . immature dendritic cells ( dcs ) were generated from cgd patients and controls in parallel , as described , and were activated with 50  ng lipopolysaccharide ( lps ) ( calbiochem ; escherichia coli o111:b4 ) and 1000  u / ml interferon - gamma ( ifn- ) ( boehringer ingelheim ) for 48  hours . following stimulation , the dc recovery rate was analyzed by flow cytometry using propidium - iodide staining . ido protein expression was determined by immunoblotting , using a monoclonal mouse anti - human ido antibody kindly provided by o. takikawa ( national institute for longevity sciences , national center for geriatrics and gerontology , aichi , japan ) . ido activity was determined measuring the release of the tryptophan breakdown product kynurenine into cell culture supernatants by high performance liquid chromatography . the kynurenine release by patient cells was compared to that of control cells by calculating the mean kynurenine concentration    the standard error of the mean ( sem ) in cell culture supernatants . statistical analyses were performed using the student 's t - test ( paired , two - tailed ) . a p - value <  0.05 was considered to indicate statistical significance . to analyze whether the ido immunoregulatory pathway was impaired in human cgd , we compared the ability of ido activation in monocyte - derived dcs obtained from cgd patients and from healthy controls . this strategy recapitulates to some extent inflammatory stimulation and consistently results in abundant ido activity in human cells . to exclude that any difference in ido activity in cgd patients and controls was related to an altered sensitivity to apoptosis in cgd patients , we first ascertained that the recovery rate of cgd and control dcs after activation with lps / ifn- was within the same range ( fig . likewise , upon activation , patient and control dcs developed a similarly mature phenotype , as assessed by flow cytometric staining for expression of cd83 , cd80 , cd86 , and hla - dr   ( not shown ) . next , immunoblotting of ido protein expression showed that lps / ifn- activation of immature dcs induced comparable ido protein expression in cgd and in control dcs ( fig . strikingly , analyzing ido activity , we found that abundant tryptophan catabolism was consistently inducible in dcs of cgd patients . while immature dcs did not metabolize tryptophan ( fig . 1c ) , activation of dcs obtained from cgd patients resulted in the release of 11.7    4.1  m kynurenine , and this was within the same range as that of control cells , which released 11.2    0.6  m kynurenine ( p  =  0.83 ) ( fig . these findings clearly demonstrate that in humans the induction of ido enzymatic activity is not essentially dependent of roi and that the ido activity in cgd patients is preserved . in accordance with our findings and contrasting the previous concept of ido activation being dependent on superoxide and roi , a recent study reported that in human cells superoxide dismutase mimetics , while being able to decrease intracellular superoxide , failed to significantly alter ido activity . instead , cytochrome b5 , in the presence of cytochrome p450 reductase and nadph , was found to be sufficient for reduction of fe - ido to fe - ido , thus allowing for effective initiation of ido - mediated tryptophan breakdown , indicating that cytochrome b5 rather than superoxide plays a major role in the activation of ido in human cells . yet , other roi - dependent processes such as activation of nuclear factor - erythroid 2-related factor 2 ( nrf2 ) , which serves as an antioxidant response element and restrains the activation of nf-b , might contribute to the hyperinflammatory state in some patients with cgd . in conclusion , this study provides clear evidence that the ido immunoregulatory pathway is intact in human cgd despite defective nadph oxidase function . our findings are consistent with the observation of normal kynurenine levels in the sera of a cgd patient , who suffered from severe aspergillus infection , before as well as after gene therapy when the patient was cured from this infection . the hyperinflammatory state in some cgd patients may be linked to an impaired immunoregulatory activity of roi , e.g. , by insufficient nrf2 activation , or other mechanisms still to be discovered . should kynurenine be considered as a treatment option in cgd at all the rationale for its use could be to augment rather than to replace ido - mediated immunoregulation .
chronic granulomatous disease ( cgd ) is characterized by a disability to produce reactive oxygen intermediates ( roi ) caused by a defect of phagocyte nicotinamide adenine dinucleotide phosphate ( nadph ) oxidase . a hyperinflammatory response to immune activation 
 has been reported to contribute to the pathology of cgd . 
 the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase ( ido ) is considered critical for regulating immune responses and suppression of inflammation . 
 ido is generally believed to require roi for enzymatic activity and was found to be inactive in murine cgd . here , we report that , strikingly , in human cgd ido metabolic activity is intact . 
 monocyte - derived dendritic cells generated from cgd patients , harbouring x - linked and autosomal recessive forms of cgd , and from healthy controls produced similar amounts of the tryptophan metabolite kynurenine upon activation with lipopolysaccharide and interferon-. thus , in humans , roi apparently are dispensable for ido activity . 
 hyperinflammation in human cgd can not be attributed to disabled ido activation .