A novel retinoic acid receptor-γ agonist antagonizes immune checkpoint resistance in lung cancers by altering the tumor immune microenvironment.

All-trans-retinoic acid (ATRA), the retinoic acid receptors (RARs) agonist, regulates cell growth, differentiation, immunity, and survival. We report that ATRA-treatment repressed cancer growth in syngeneic immunocompetent, but not immunodeficient mice. The tumor microenvironment was implicated: CD8+ T cell depletion antagonized ATRA's anti-tumorigenic effects in syngeneic mice. ATRA-treatment with checkpoint blockade did not cooperatively inhibit murine lung cancer growth. To augment ATRA's anti-tumorigenicity without promoting its pro-tumorigenic potential, an RARγ agonist (IRX4647) was used since it regulates T cell biology. Treating with IRX4647 in combination with an immune checkpoint (anti-PD-L1) inhibitor resulted in a statistically significant suppression of syngeneic 344SQ lung cancers in mice-a model known for its resistance to checkpoints and characterized by low basal T cell and PD-L1 expression. This combined treatment notably elevated CD4+ T-cell presence within the tumor microenvironment and increased IL-5 and IL-13 tumor levels, while simultaneously decreasing CD38 in the tumor stroma. IL-5 and/or IL-13 treatments increased CD4+ more than CD8+ T-cells in mice. IRX4647-treatment did not appreciably affect in vitro lung cancer growth, despite RARγ expression. Pharmacokinetic analysis found IRX4647 plasma half-life was 6 h in mice. Yet, RARα antagonist (IRX6696)-treatment with anti-PD-L1 did not repress syngeneic lung cancer growth. Together, these findings provide a rationale for a clinical trial investigating an RARγ agonist to augment check point blockade response in cancers.

Structure-guided bifunctional molecules hit a DEUBAD-lacking hRpn13 species upregulated in multiple myeloma.

Proteasome substrate receptor hRpn13 is a promising anti-cancer target. By integrated in silico and biophysical screening, we identified a chemical scaffold that binds hRpn13 with non-covalent interactions that mimic the proteasome and a weak electrophile for Michael addition. hRpn13 Pru domain binds proteasomes and ubiquitin whereas its DEUBAD domain binds deubiquitinating enzyme UCHL5. NMR revealed lead compound XL5 to interdigitate into a hydrophobic pocket created by lateral movement of a Pru ß-hairpin with an exposed end for Proteolysis Targeting Chimeras (PROTACs). Implementing XL5-PROTACs as chemical probes identified a DEUBAD-lacking hRpn13 species (hRpn13Pru) present naturally with cell type-dependent abundance. XL5-PROTACs preferentially target hRpn13Pru, causing its ubiquitination. Gene-editing and rescue experiments established hRpn13 requirement for XL5-PROTAC-triggered apoptosis. These data establish hRpn13 as an anti-cancer target for multiple myeloma and introduce an hRpn13-targeting scaffold that can be optimized for preclinical trials against hRpn13Pru-producing cancer types.

Comparative microsomal proteomics of a model lung cancer cell line NCI-H23 reveals distinct differences between molecular profiles of 3D and 2D cultured cells.

Lung cancer is the leading cause of cancer-related deaths in the USA and worldwide. Yet, about 95% of new drug candidates validated in preclinical phase eventually fail in clinical trials. Such a high attrition rate is attributed mostly to the inability of conventional two-dimensionally (2D) cultured cancer cells to mimic native three-dimensional (3D) growth of malignant cells in human tumors. To ascertain phenotypical differences between these two distinct culture conditions, we carried out a comparative proteomic analysis of a membrane fraction obtained from 3D- and 2D-cultured NSCLC model cell line NCI-H23. This analysis revealed a map of 1,166 (24%) protein species regulated in culture dependent manner, including differential regulation of a subset of cell surface-based CD molecules. We confirmed exclusive expression of CD99, CD146 and CD239 in 3D culture. Furthermore, label-free quantitation, targeting KRas proteoform-specific peptides, revealed upregulation of both wild type and monoallelic KRas4BG12C mutant at the surface of 3D cultured cells. In order to reduce the high attrition rate of new drug candidates, the results of this study strongly suggests exploiting base-line molecular profiling of a large number of patient-derived NSCLC cell lines grown in 2D and 3D culture, prior to actual drug candidate testing.

Nanoparticle physicochemical properties determine the activation of intracellular complement.

The complement system plays an essential role in both innate and adaptive immunity. The traditional understanding of this system comes from studies investigating complement proteins produced by the liver and present in plasma to "complement" the immune cell-mediated response to invading pathogens. Recently, it has been reported that immune cells including, but not limited to, T-cells and monocytes, express complement proteins. This complement is referred to as intracellular (IC) and implicated in the regulation of T-cell activation. The mechanisms and the structure-activity relationship between nanomaterials and IC, however, are currently unknown. Herein, we describe a structure-activity relationship study demonstrating that under in vitro conditions, only polymeric materials with cationic surfaces activate IC in T-cells. The effect also depends on particle size and occurs through a mechanism involving membrane damage, thereby IC on the cell surface serves as a self-opsonization marker in response to the nanoparticle-triggered danger affecting the cell integrity.

Preparation and Immunoaffinity Depletion of Fresh Frozen Tissue Homogenates for Mass Spectrometry-Based Proteomics in the Context of Drug Target/Biomarker Discovery.

The discovery of novel drug targets and biomarkers via mass spectrometry (MS)-based proteomic analysis of clinical specimens has proven to be challenging. The wide dynamic range of protein concentration in clinical specimens and the high background/noise originating from highly abundant proteins in tissue homogenates and serum/plasma encompass two major analytical obstacles. Immunoaffinity depletion of highly abundant blood-derived proteins from serum/plasma is a well-established approach adopted by numerous researchers; however, the utilization of this technique for immunodepletion of tissue homogenates obtained from fresh frozen clinical specimens is lacking. We first developed immunoaffinity depletion of highly abundant blood-derived proteins from tissue homogenates, using renal cell carcinoma as a model disease, and followed this study by applying it to different tissue types. Tissue homogenate immunoaffinity depletion of highly abundant proteins may be equally important as is the recognized need for depletion of serum/plasma, enabling more sensitive MS-based discovery of novel drug targets, and/or clinical biomarkers from complex clinical samples. Provided is a detailed protocol designed to guide the researcher through the preparation and immunoaffinity depletion of fresh frozen tissue homogenates for two-dimensional liquid chromatography, tandem mass spectrometry (2D-LC-MS/MS)-based molecular profiling of tissue specimens in the context of drug target and/or biomarker discovery.

Discovering Targets of Non-enzymatic Acylation by Thioester Reactivity Profiling.

Non-enzymatic protein modification driven by thioester reactivity is thought to play a major role in the establishment of cellular lysine acylation. However, the specific protein targets of this process are largely unknown. Here we report an experimental strategy to investigate non-enzymatic acylation in cells. Specifically, we develop a chemoproteomic method that separates thioester reactivity from enzymatic utilization, allowing selective enrichment of non-enzymatic acylation targets. Applying this method to cancer cell lines identifies numerous candidate targets of non-enzymatic acylation, including several enzymes in lower glycolysis. Functional studies highlight malonyl-CoA as a reactive thioester metabolite that can modify and inhibit glycolytic enzyme activity. Finally, we show that synthetic thioesters can be used as novel reagents to probe non-enzymatic acylation in living cells. Our studies provide new insights into the targets and drivers of non-enzymatic acylation, and demonstrate the utility of reactivity-based methods to experimentally investigate this phenomenon in biology and disease.

Comparative proteomics of a model MCF10A-KRasG12V cell line reveals a distinct molecular signature of the KRasG12V cell surface.

Oncogenic Ras mutants play a major role in the etiology of most aggressive and deadly carcinomas in humans. In spite of continuous efforts, effective pharmacological treatments targeting oncogenic Ras isoforms have not been developed. Cell-surface proteins represent top therapeutic targets primarily due to their accessibility and susceptibility to different modes of cancer therapy. To expand the treatment options of cancers driven by oncogenic Ras, new targets need to be identified and characterized at the surface of cancer cells expressing oncogenic Ras mutants. Here, we describe a mass spectrometry-based method for molecular profiling of the cell surface using KRasG12V transfected MCF10A (MCF10A-KRasG12V) as a model cell line of constitutively activated KRas and native MCF10A cells transduced with an empty vector (EV) as control. An extensive molecular map of the KRas surface was achieved by applying, in parallel, targeted hydrazide-based cell-surface capturing technology and global shotgun membrane proteomics to identify the proteins on the KRasG12V surface. This method allowed for integrated proteomic analysis that identified more than 500 cell-surface proteins found unique or upregulated on the surface of MCF10A-KRasG12V cells. Multistep bioinformatic processing was employed to elucidate and prioritize targets for cross-validation. Scanning electron microscopy and phenotypic cancer cell assays revealed changes at the cell surface consistent with malignant epithelial-to-mesenchymal transformation secondary to KRasG12V activation. Taken together, this dataset significantly expands the map of the KRasG12V surface and uncovers potential targets involved primarily in cell motility, cellular protrusion formation, and metastasis.

Mass spectrometry in cancer biomarker research: a case for immunodepletion of abundant blood-derived proteins from clinical tissue specimens.

The discovery of clinically relevant cancer biomarkers using mass spectrometry (MS)-based proteomics has proven difficult, primarily because of the enormous dynamic range of blood-derived protein concentrations and the fact that the 22 most abundant blood-derived proteins constitute approximately 99% of the total plasma protein mass. Immunodepletion of clinical body fluid specimens (e.g., serum/plasma) for the removal of highly abundant proteins is a reasonable and reproducible solution. Often overlooked, clinical tissue specimens also contain a formidable amount of highly abundant blood-derived proteins present in tissue-embedded networks of blood/lymph capillaries and interstitial fluid. Hence, the dynamic range impediment to biomarker discovery remains a formidable obstacle, regardless of clinical sample type (solid tissue and/or body fluid). Thus, we optimized and applied simultaneous immunodepletion of blood-derived proteins from solid tissue and peripheral blood, using clear cell renal cell carcinoma as a model disease. Integrative analysis of data from this approach and genomic data obtained from the same type of tumor revealed concordant key pathways and protein targets germane to clear cell renal cell carcinoma. This includes the activation of the lipogenic pathway characterized by increased expression of adipophilin (PLIN2) along with 'cadherin switching', a phenomenon indicative of transcriptional reprogramming linked to renal epithelial dedifferentiation. We also applied immunodepletion of abundant blood-derived proteins to various tissue types (e.g., adipose tissue and breast tissue) showing unambiguously that the removal of abundant blood-derived proteins represents a powerful tool for the reproducible profiling of tissue proteomes. Herein, we show that the removal of abundant blood-derived proteins from solid tissue specimens is of equal importance to depletion of body fluids and recommend its routine use in the context of biological discovery and/or cancer biomarker research. Finally, this perspective presents the background, rationale and strategy for using tissue-directed high-resolution/accuracy MS-based shotgun proteomics to detect genuine tumor proteins in the peripheral blood of a patient diagnosed with nonmetastatic cancer, employing concurrent liquid chromatography-MS analysis of immunodepleted clinical tissue and blood specimens.

Preparation of the low molecular weight serum proteome for mass spectrometry analysis.

The discovery of viable biomarkers or indicators of disease states is complicated by the inherent complexity of the chosen biological specimen. Every sample, whether it is serum, plasma, urine, tissue, cells, or a host of others, contains thousands of large and small components, each interacting in multiple ways. The need to concentrate on a group of these components to narrow the focus on a potential biomarker candidate becomes, out of necessity, a priority, especially in the search for immune-related low molecular weight serum biomarkers. One such method in the field of proteomics is to divide the sample proteome into groups based on the size of the protein, analyze each group, and mine the data for statistically significant items. This chapter details a portion of this method, concentrating on a method for fractionating and analyzing the low molecular weight proteome of human serum.

Trypsin-mediated ¹8O/¹6O labeling for biomarker discovery.

Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines.

Fractionation of peptides by strong cation-exchange liquid chromatography.

Chromatography in all its formats plays an important role in proteomics research. The search for protein biomarkers for different diseases has been an active area of research. The dynamic concentration range and the large number of proteins in a proteome require the development of multidimensional separation strategies to allow for the identification of the largest number of proteins. Strong cation-exchange chromatography (SCX) has been used extensively for the fractionation of proteins and peptides. This chapter provides a detailed description for the SCX fractionation of complex proteome samples.

Post-digestion ¹8O exchange/labeling for quantitative shotgun proteomics of membrane proteins.

The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. Post-digestion (18)O exchange is a simple and efficient method for differential (18)O/(16)O stable isotope labeling of two biologically distinct specimens, allowing relative quantitation of proteins in complex mixtures when coupled with shotgun MS-based proteomics. Due to minimal sample consumption and unrestricted peptide tagging, (18)O/(16)O stable isotope labeling is particularly suitable for amount-limited protein specimens typically encountered in membrane and clinical proteomics. This chapter describes a protocol that relies on shotgun proteomics for quantitative profiling of the detergent-insoluble membrane proteins isolated from HeLa cells, differentially transfected with plasmids expressing HIV Gag protein and its myristylation-defective N-terminal mutant. Whilst this protocol depicts solubilization of detergent-insoluble membrane proteins coupled with post-digestion (18)O labeling, it is amenable to any complex membrane protein mixture. Described approach relies on solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (v/v) methanol followed by differential (18)O/(16)O labeling of protein digests in 20% (v/v) methanol buffer. After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion (18)O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.

Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary disease.

Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential (16)O/(18)O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: Integrated omics.

Global proteomics and metabolomics in cancer biomarker discovery.

Chromatography and electrophoresis have been used for the last half-century to separate small and large molecules. Advances in MS instrumentation and techniques for sample introduction into the mass analyzer (i.e. matrix-assisted laser desorption/ionization and electrospray ionization), chromatography in all its formats and modes and two-dimensional gel electrophoresis, including two-dimensional difference gel electrophoresis, enabled the separation of complex biological mixtures, such as the proteome and the metabolome, in a biological sample. These advances have made it possible to identify compounds that can be used to discriminate between two samples taken from healthy and diseased individuals. The objective is to find proteins or metabolites that can be used as a clinical test for the early diagnosis, prognosis and monitoring of the disease and the outcome of therapy. In this manuscript, we present an overview of what has been achieved in the search for biomarkers, with emphasis on cancer, using separation science and MS.

Molecular profiling of the human nasal epithelium: A proteomics approach.

A comprehensive proteomic profiling of nasal epithelium (NE) is described. This study relies on simple subcellular fractionation used to obtain soluble- and membrane-enriched fractions followed by 2-dimensional liquid chromatography (2D-LC) separation and tandem mass spectrometry (MS/MS). The cells were collected using a brushing technique applied on NE of clinically evaluated volunteers. Subsequently, the soluble- and the membrane-protein enriched fractions were prepared and analyzed in parallel using 2D-LC-MS/MS. In a set of 1482 identified proteins, 947 (63.9%) proteins were found to be associated to membrane fraction. Grand average hydropathy value index (GRAVY) analysis, the transmembrane protein mapping and annotations of primary location deposited in the Human Protein Reference Database (HPRD) confirmed an enrichment of hydrophobic proteins on this dataset. Ingenuity Pathway Analysis (IPA) of soluble fraction revealed an enrichment of molecular and cellular functions associated with cell death, protein folding and drug metabolism while in membrane fraction showed an enrichment of functions associated with molecular transport, protein trafficking and cell-to-cell signaling and interaction. The IPA showed similar enrichment of functions associated with cellular growth and proliferation in both soluble and membrane subproteomes. This finding was in agreement with protein content analysis using exponentially modified protein abundance index (emPAI). A comparison of our data with previously published studies focusing on respiratory tract epithelium revealed similarities related to identification of proteins associated with physical barrier function and immunological defence. In summary, we extended the NE molecular profile by identifying and characterizing proteins associated to pivotal functions of a respiratory epithelium, including the control of fluid volume and ionic composition at the airways' surface, physical barrier maintenance, detoxification and immunological defence. The extent of similarities supports the applicability of a less invasive analysis of NE to assess prognosis and treatment response of lung diseases such as asthma, cystic fibrosis and chronic obstructive pulmonary disease.

Comparison of fluorescence, laser-induced fluorescence, and ultraviolet absorbance detection for measuring HPLC fractionated protein/peptide mixtures.

Proteomics is the study of all proteins in a biological sample. High-pressure liquid chromatography coupled online with mass spectrometry (HPLC/MS) is currently the method of choice for proteomic analysis. Proteins are extracted, separated at the protein or peptide level (after enzymatic digestion), and fractions are analyzed by HPLC/MS. Detection during off-line fractionation is generally conducted using UV-vis, which is not sensitive enough to distinguish fractions having the largest concentration of proteins/peptides and should not be combined prior to HPLC/MS. To overcome this deficiency, we utilize fluorescence or UV-laser induced fluorescence (UV-LIF) detection for measuring proteins/peptides during the off-line fractionation. Fluorescence detection allows low-abundance proteins/peptides that contain aromatic amino acids to be measured. In this study, peptide/protein samples fractionated using ion-exchange chromatography were detected using UV absorbance, fluorescence, and UV-LIF. The results indicated that fluorescence and UV-LIF were able to detect the lower abundance proteins/peptides to give a more representative chromatogram, allowing the analyst to decide which fractions should be combined prior to HPLC/tandem mass spectrometry (MS/MS) analysis.

Quantitation of nanoparticles in serum matrix by capillary electrophoresis.

Sensitive and fast analytical techniques are needed to determine the concentration of nanoparticles in biological samples (e.g., blood and tissues) for biodistribution and toxicity studies. This chapter describes a method for the use of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) for the quantitation of fullerene nanoparticles in human serum matrix. Data on the fullerene-based nanoparticle carboxyfullerene (C3 fullerene) in human serum is presented as an example.

Comparison of strong cation exchange and SDS-PAGE fractionation for analysis of multiprotein complexes.

Affinity purification of protein complexes followed by identification using liquid chromatography/mass spectrometry (LC-MS/MS) is a robust method to study the fundamental process of protein interaction. Although affinity isolation reduces the complexity of the sample, fractionation prior to LC-MS/MS analysis is still necessary to maximize protein coverage. In this study, we compared the protein coverage obtained via LC-MS/MS analysis of protein complexes prefractionated using two commonly employed methods, SDS-PAGE and strong cation exchange chromatography (SCX). The two complexes analyzed focused on the nuclear proteins Bmi-1 and GATA3 that were expressed within the cells at low and high levels, respectively. Prefractionation of the complexes at the peptide level using SCX consistently resulted in the identification of approximately 3-fold more proteins compared to separation at the protein level using SDS-PAGE. The increase in the number of identified proteins was especially pronounced for the Bmi-1 complex, where the target protein was expressed at a low level. The data show that prefractionation of affinity isolated protein complexes using SCX prior to LC-MS/MS analysis significantly increases the number of identified proteins and individual protein coverage, particularly for target proteins expressed at low levels.

Optimized method for computing (18)O/(16)O ratios of differentially stable-isotope labeled peptides in the context of postdigestion (18)O exchange/labeling.

Differential (18)O/(16)O stable isotope labeling of peptides that relies on enzyme-catalyzed oxygen exchange at their carboxyl termini in the presence of H(2)(18)O has been widely used for relative quantitation of peptides/proteins. The role of tryptic proteolysis in bottom-up shotgun proteomics and low reagent costs have made trypsin-catalyzed (18)O postdigestion exchange a convenient and affordable stable isotope labeling approach. However, it is known that trypsin-catalyzed (18)O exchange at the carboxyl terminus is in many instances inhomogeneous/incomplete. The extent of the (18)O exchange/incorporation fluctuates from peptide to peptide mostly due to variable enzyme-substrate affinity. Thus, accurate calculation and interpretation of peptide ratios are analytically complicated and in some regard deficient. Therefore, a computational approach capable of improved measurement of actual (18)O incorporation for each differentially labeled peptide pair is needed. In this regard, we have developed an algorithmic method that relies on the trapezoidal rule to integrate peak intensities of all detected isotopic species across a particular peptide ion over the retention time, which fits the isotopic manifold to Poisson distributions. Optimal values for manifold fitting were calculated and then (18)O/(16)O ratios derived via evolutionary programming. The algorithm is tested using trypsin-catalyzed (18)O postdigestion exchange to differentially label bovine serum albumin (BSA) at a priori determined ratios. Both accuracy and precision are improved utilizing this rigorous mathematical approach. We further demonstrate the effectiveness of this method to accurately calculate (18)O/(16)O ratios in a large scale proteomic quantitation of detergent resistant membrane microdomains (DRMMs) isolated from cells expressing wild-type HIV-1 Gag and its nonmyristylated mutant.

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

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.