Interferon gamma rebalances immunopathological signatures in chronic granulomatous disease through metabolic rewiring

Abstract

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by recurrent life-threatening infections and hyperinflammatory complications. It is caused by mutations in the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex and the consequent loss of reactive oxygen species (ROS) production. Recombinant human interferon gamma (rIFN-γ) prophylaxis reduces the risk of severe infections, but the mechanisms behind its efficacy in CGD are still an open question, as it does not restore NADPH oxidase-dependent ROS production. Here, we demonstrate that the innate immune cells of patients with CGD are transcriptionally and functionally reprogrammed to a hyperactive inflammatory status, displaying an impaired in vitro induction of trained immunity. CGD monocytes have reduced intracellular amino acid concentrations and profound functional metabolic defects, both at the level of glycolysis and mitochondrial respiration. Ex vivo and in vivo treatments with IFN-γ restored these metabolic defects and reduced excessive interleukin 1β (IL-1β) and IL-6 production in response to fungal stimuli in CGD monocytes. These data suggest that prophylactic rIFN-γ modulates the metabolic status of innate immune cells in CGD. These data shed light on the effects of NADPH oxidase-derived ROS deficiency to the metabolic programs of immune cells and pose the basis for targeting this immunometabolic axis, potentially beyond CGD, with IFN-γ immunotherapy.

Graphical abstract

Figure 1.

Patients with CGD display a dysregulated hyperinflammatory status. (A) Uniform manifold approximation and projection (UMAP) visualization of scRNA-seq profiles of 50 959 white blood cells from 5 patients with CGD and 5 age- and sex-matched healthy control (HC) colored according to cell type identity. (B) UMAP visualization of cells colored by condition, HC (green) and CGD (purple), downsampled to 3000 cells per donor. (C) Numbers of DEGs identified as upregulated (red) or downregulated (blue) in neutrophils, CD14⁺ monocytes, and CD16⁺ monocytes. (D) Gene functional enrichment analysis based on DEGs upregulated or downregulated in neutrophils, CD14⁺ monocytes, CD16⁺ monocytes, and neutrophils using HALLMARK gene sets. Illustrated are all significant terms (adjusted P < .05). Dots are colored by directionality of differential expression (red, upregulated DEGs; blue, downregulated DEGs). Dot size depicts the number of DEGs in the respective HALLMARK term. (E) Volcano plot of differential abundance of plasma circulatory proteins in patients with CGD (n = 5) as compared with HC (n = 14). Results are displayed as log₂fold change (FC) of CGD compared with HC, plotted against minus log₁₀ of the adjusted P values. Proteins significantly differentially expressed (with adjusted P < .05 after correction for multiple testing) are displayed in red. (F) IL-8 production after 4-hour stimulation in neutrophils from CGD (n = 5) and HC (n = 12) with either medium (RPMI), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), or PMA (50 ng/mL). (G) IL-6 and IL-1β production after 24-hour stimulation in CGD monocytes (n = 5) and HC (n = 13, except for the stimulations RPMI 10% human pooled serum (HPS) and A fumigatus where n = 4) with either medium (RPMI, with or without 10% HPS) or LPS (10 ng/mL), Pam₃Cys (10 μg/mL), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), live A fumigatus (1 × 10⁶ conidia per mL) in the presence of 10% HPS, S aureus ATCC 29213 (1 × 10⁶ colony-forming units per mL), or palmitic acid (C16:0; 50  μM) in combination with monosodium urate crystals (300  μg/mL). (H-I) Volcano plot of differential protein abundance from 24 hour-unstimulated (H) and A fumigatus–stimulated (I) Peripheral blood mononuclear cells in culture supernatants of CGD and HC monocytes. Results are displayed as log₂-FC of CGD (n = 5) compared with HC (n = 14), plotted against minus log₁₀ of the P values. Proteins with significantly differential abundance (with adjusted P < .05 after correction for multiple testing) are displayed in red. (J) IL-6 production after 24-hour LPS (10 ng/mL)-restimulation at day 6 after BCG (5 μg/mL)-training protocol of monocytes from HC (n = 5) and patients with CGD (n = 6). (F-G, J) Data are presented as the means ± standard error of the mean (SEM). In the bar plots, different colors indicate different mutations in patients with CGD as indicated by the panel legends. For the enzyme-linked immunosorbent assay results, statistical analysis was performed using the Mann-Whitney U test between patients and HC; a 2-sided P value < .05 was considered statistically significant. C16, palmitate; MSU, monosodium urate; mDc, myeloid dendritic cells; NK, natural killer; pDC, plasmacytoid dendritic cells; Plt. act., platelet-activated; Pam₃Cys, (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys₄-OH, trihydrochloride; TNF, tumor necrosis factor.

Figure 1.

Patients with CGD display a dysregulated hyperinflammatory status. (A) Uniform manifold approximation and projection (UMAP) visualization of scRNA-seq profiles of 50 959 white blood cells from 5 patients with CGD and 5 age- and sex-matched healthy control (HC) colored according to cell type identity. (B) UMAP visualization of cells colored by condition, HC (green) and CGD (purple), downsampled to 3000 cells per donor. (C) Numbers of DEGs identified as upregulated (red) or downregulated (blue) in neutrophils, CD14⁺ monocytes, and CD16⁺ monocytes. (D) Gene functional enrichment analysis based on DEGs upregulated or downregulated in neutrophils, CD14⁺ monocytes, CD16⁺ monocytes, and neutrophils using HALLMARK gene sets. Illustrated are all significant terms (adjusted P < .05). Dots are colored by directionality of differential expression (red, upregulated DEGs; blue, downregulated DEGs). Dot size depicts the number of DEGs in the respective HALLMARK term. (E) Volcano plot of differential abundance of plasma circulatory proteins in patients with CGD (n = 5) as compared with HC (n = 14). Results are displayed as log₂fold change (FC) of CGD compared with HC, plotted against minus log₁₀ of the adjusted P values. Proteins significantly differentially expressed (with adjusted P < .05 after correction for multiple testing) are displayed in red. (F) IL-8 production after 4-hour stimulation in neutrophils from CGD (n = 5) and HC (n = 12) with either medium (RPMI), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), or PMA (50 ng/mL). (G) IL-6 and IL-1β production after 24-hour stimulation in CGD monocytes (n = 5) and HC (n = 13, except for the stimulations RPMI 10% human pooled serum (HPS) and A fumigatus where n = 4) with either medium (RPMI, with or without 10% HPS) or LPS (10 ng/mL), Pam₃Cys (10 μg/mL), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), live A fumigatus (1 × 10⁶ conidia per mL) in the presence of 10% HPS, S aureus ATCC 29213 (1 × 10⁶ colony-forming units per mL), or palmitic acid (C16:0; 50  μM) in combination with monosodium urate crystals (300  μg/mL). (H-I) Volcano plot of differential protein abundance from 24 hour-unstimulated (H) and A fumigatus–stimulated (I) Peripheral blood mononuclear cells in culture supernatants of CGD and HC monocytes. Results are displayed as log₂-FC of CGD (n = 5) compared with HC (n = 14), plotted against minus log₁₀ of the P values. Proteins with significantly differential abundance (with adjusted P < .05 after correction for multiple testing) are displayed in red. (J) IL-6 production after 24-hour LPS (10 ng/mL)-restimulation at day 6 after BCG (5 μg/mL)-training protocol of monocytes from HC (n = 5) and patients with CGD (n = 6). (F-G, J) Data are presented as the means ± standard error of the mean (SEM). In the bar plots, different colors indicate different mutations in patients with CGD as indicated by the panel legends. For the enzyme-linked immunosorbent assay results, statistical analysis was performed using the Mann-Whitney U test between patients and HC; a 2-sided P value < .05 was considered statistically significant. C16, palmitate; MSU, monosodium urate; mDc, myeloid dendritic cells; NK, natural killer; pDC, plasmacytoid dendritic cells; Plt. act., platelet-activated; Pam₃Cys, (S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys₄-OH, trihydrochloride; TNF, tumor necrosis factor.

Figure 2.

Epigenetic landscape and metabolic profile of CGD monocytes. (A) Principal component analysis of the accessible peaks of unstimulated monocytes from HC (n = 5) and CGD (n = 5) detected with ATAC-seq. The first 2 principal components are visualized, where PC1 accounts for 47.976% of the total variance epigenetic profile. (B) Volcano plot of differentially accessible regions mapping in promoter or genic loci of monocytes from HC (n = 5) and patients with CGD (n = 5) assessed by ATAC-seq. Peaks with an adjusted IHW P value <.05 and a mean log₂FC > 1 or < −1 are plotted in dark purple. (C-D) Volcano plots of differentially abundant metabolites between neutrophils (C) and monocytes (D) from HC (n = 3) and patients with CGD (n = 5) assessed by targeted metabolomics. The plots represent log₂FC and −log₁₀ of the corrected P values compared with HC. Metabolites with an adjusted P value < .05 and a mean log₂FC > 1 or < −1 are plotted in dark purple and were considered as differentially regulated. (E-F) Intracellular quantification of triglycerides (F) and free fatty acid (G) in unstimulated monocytes from HC (n = 5) and patients with CGD (n = 4). (E-F) Data are presented as the means ± SEM and statistical analysis was performed using the Mann-Whitney U test between patients and HC; a 2-sided P value <.05 was considered statistically significant. FFA, free fatty acid; IHW, independent hypothesis weighting; kbp, kilobase pairs; ns, not significant; TG, triglyceride; TSS, transcription start site; TTS, transcription termination site.

Figure 3.

CGD neutrophils and monocytes present functional metabolic defects. (A) ECAR on Glyco Stress Test in monocytes from HC (n = 13) and CGD (n = 5) and relative metabolic glycolytic parameters. (B) OCR on Mito Stress Test in monocytes from HC (n = 13) and patients with CGD (n = 5) relative metabolic mitochondrial parameters. (C) ECAR on Glyco Stress Test in neutrophils from patients with CGD (n = 5) and HC (n = 3) and relative metabolic glycolytic parameters. (D) Lactate production after 4 hours in unstimulated neutrophils from HC (n = 12) and patients with CGD. (E) Intracellular NADPH concentration (pmol/1 000 000 cells) in monocytes of HC (n = 6) incubated for 6 hours with NADPH oxidase complex inhibitor apocynin (1 mM) or its vehicle and stimulated for zymosan for 2 hours. (F) ECAR and OCR after preincubation of monocytes with apocynin (1 mM) for 6 hours followed by stimulation by zymosan (80 μg/mL) during measurements. Data were corrected for background signal of each condition. In the bar plots of panels A-C, different colors indicate different mutations in patients with CGD as indicated by the panel legends. Data are presented as the means ± SEM. Statistical analysis was performed using the Mann-Whitney U test between patients and HC; a 2-sided P value < .05 was considered statistically significant. DMSO, dimethyl sulfoxide.

Figure 3.

CGD neutrophils and monocytes present functional metabolic defects. (A) ECAR on Glyco Stress Test in monocytes from HC (n = 13) and CGD (n = 5) and relative metabolic glycolytic parameters. (B) OCR on Mito Stress Test in monocytes from HC (n = 13) and patients with CGD (n = 5) relative metabolic mitochondrial parameters. (C) ECAR on Glyco Stress Test in neutrophils from patients with CGD (n = 5) and HC (n = 3) and relative metabolic glycolytic parameters. (D) Lactate production after 4 hours in unstimulated neutrophils from HC (n = 12) and patients with CGD. (E) Intracellular NADPH concentration (pmol/1 000 000 cells) in monocytes of HC (n = 6) incubated for 6 hours with NADPH oxidase complex inhibitor apocynin (1 mM) or its vehicle and stimulated for zymosan for 2 hours. (F) ECAR and OCR after preincubation of monocytes with apocynin (1 mM) for 6 hours followed by stimulation by zymosan (80 μg/mL) during measurements. Data were corrected for background signal of each condition. In the bar plots of panels A-C, different colors indicate different mutations in patients with CGD as indicated by the panel legends. Data are presented as the means ± SEM. Statistical analysis was performed using the Mann-Whitney U test between patients and HC; a 2-sided P value < .05 was considered statistically significant. DMSO, dimethyl sulfoxide.

Figure 4.

IFN-γ treatment restores functional metabolic defect in CGD monocytes. (A-B) Calculated glycolytic metabolic parameters on the Glyco Stress Test (A) and calculated mitochondrial parameters on Mito Stress Test (B) of monocytes from HC (n = 5 for Glyco Stress Test, n = 7 for Mito Stress Test) and patients with CGD (n = 7 for Glyco Stress Test, n = 8 for Mito Stress Test) after 12 hours treatment with medium (RPMI) or IFN-γ (50 ng/mL) measured by Seahorse XF technology. (C) Mean normalized expression of genes associated with NAD⁺ salvage pathway differentially expressed in CD14⁺ monocytes on 4 hours ex vivo whole blood stimulations with IFN-γ vs unstimulated within the group of HC (n = 4) and patients with CGD (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (D-E) IL-6 (D) and IL-1β (E) production in CGD monocytes (n = 6) and HC (n = 5) preincubated for 4 hours with or without IFN-γ (50 ng/mL) and then stimulated for 24 hours with either medium (RPMI), live A fumigatus (1 × 10⁷ conidia per mL), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), Pam₃Cys (10 μg/mL), or LPS (10 ng/mL). (F) A fumigatus outgrowth in monocytes preincubated for 4 hours with or without IFN-γ (50 ng/mL) after the in vitro killing assay, expressed as percentage killing in respect to the total inoculum (CGD n = 2, HC n = 2). (G) Monocytes from HC (n = 1) and patients with CGD (n = 2) were preincubated with IFN-γ (50 ng/mL) and then trained for 24 hours by incubation with 5 μg/mL BCG in the presence of 10% HPS. Thereafter, the stimulus was removed, and cells were kept in RPMI with 10% HPS (regular medium) or in a medium containing 10% human serum and IFN-γ (50 ng/mL). On day 6, a second stimulation with LPS (10 ng/mL) was performed for an additional 24 hours. IL-6 levels were measured in cell culture supernatants after the second stimulation. (H) Mean normalized expression of selected differentially expressed genes related to glutamine metabolism in CD14⁺ monocytes on 4 hours of ex vivo whole blood stimulation with IFN-γ vs unstimulated within the group of patients with CGD (n = 4) and HC (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (I) Mean normalized gene expression of CXCL8 in neutrophils on 4 hours of in vitro whole blood stimulation with IFN-γ vs unstimulated demonstrated differential expression in each group (CGD n = 4, HC n = 4) comparing in vitro IFN-γ treated vs untreated cells after 4 hours of incubation. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (J) IL-8 production of HC (n = 3) and CGD neutrophils (n = 2) preincubated for 30 minutes with or without IFN-γ (50 ng/mL) and then stimulated with either medium (RPMI) or PMA (50 ng/mL) for 4 hours. (A, B, D-G, J) Data are illustrated as mean ± SEM, and statistical analysis was performed using the Wilcoxon signed-rank test comparing within the patient group (or within the control group) the IFN-γ–treated condition with the untreated condition.

Figure 4.

IFN-γ treatment restores functional metabolic defect in CGD monocytes. (A-B) Calculated glycolytic metabolic parameters on the Glyco Stress Test (A) and calculated mitochondrial parameters on Mito Stress Test (B) of monocytes from HC (n = 5 for Glyco Stress Test, n = 7 for Mito Stress Test) and patients with CGD (n = 7 for Glyco Stress Test, n = 8 for Mito Stress Test) after 12 hours treatment with medium (RPMI) or IFN-γ (50 ng/mL) measured by Seahorse XF technology. (C) Mean normalized expression of genes associated with NAD⁺ salvage pathway differentially expressed in CD14⁺ monocytes on 4 hours ex vivo whole blood stimulations with IFN-γ vs unstimulated within the group of HC (n = 4) and patients with CGD (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (D-E) IL-6 (D) and IL-1β (E) production in CGD monocytes (n = 6) and HC (n = 5) preincubated for 4 hours with or without IFN-γ (50 ng/mL) and then stimulated for 24 hours with either medium (RPMI), live A fumigatus (1 × 10⁷ conidia per mL), heat-killed C albicans UC820 (1 × 10⁶ yeast per mL), Pam₃Cys (10 μg/mL), or LPS (10 ng/mL). (F) A fumigatus outgrowth in monocytes preincubated for 4 hours with or without IFN-γ (50 ng/mL) after the in vitro killing assay, expressed as percentage killing in respect to the total inoculum (CGD n = 2, HC n = 2). (G) Monocytes from HC (n = 1) and patients with CGD (n = 2) were preincubated with IFN-γ (50 ng/mL) and then trained for 24 hours by incubation with 5 μg/mL BCG in the presence of 10% HPS. Thereafter, the stimulus was removed, and cells were kept in RPMI with 10% HPS (regular medium) or in a medium containing 10% human serum and IFN-γ (50 ng/mL). On day 6, a second stimulation with LPS (10 ng/mL) was performed for an additional 24 hours. IL-6 levels were measured in cell culture supernatants after the second stimulation. (H) Mean normalized expression of selected differentially expressed genes related to glutamine metabolism in CD14⁺ monocytes on 4 hours of ex vivo whole blood stimulation with IFN-γ vs unstimulated within the group of patients with CGD (n = 4) and HC (n = 4), respectively. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (I) Mean normalized gene expression of CXCL8 in neutrophils on 4 hours of in vitro whole blood stimulation with IFN-γ vs unstimulated demonstrated differential expression in each group (CGD n = 4, HC n = 4) comparing in vitro IFN-γ treated vs untreated cells after 4 hours of incubation. Dots are colored by donor identity, and dot size is scaled to the percentage of cells expressing the respective gene. (J) IL-8 production of HC (n = 3) and CGD neutrophils (n = 2) preincubated for 30 minutes with or without IFN-γ (50 ng/mL) and then stimulated with either medium (RPMI) or PMA (50 ng/mL) for 4 hours. (A, B, D-G, J) Data are illustrated as mean ± SEM, and statistical analysis was performed using the Wilcoxon signed-rank test comparing within the patient group (or within the control group) the IFN-γ–treated condition with the untreated condition.

Figure 5.

IFN-γ in vivo treatment leads to transcriptional reprogramming of cells and restores functional immunometabolic defects in monocytes from 1 patient with CGD. (A) uniform manifold approximation and projection (UMAP) visualization of 12 136 whole blood cells of 1 patient with CGD before and 4 hours after in vivo Imukine (rIFN-γ) subcutaneous treatment (50 mg/m² body surface area, with a maximum of 100 mg) profiled with scRNA-seq and colored by cell type. (B) UMAP visualization of cells colored by condition, before (light purple) and 4 hours after (dark purple) in vivo Imukine (rIFN-γ) treatment. (C) Numbers of differentially expressed genes identified as upregulated (red) or downregulated (blue) in neutrophils and CD14⁺ monocytes. (D) Module score of the HALLMARK gene set “interferon gamma response” in neutrophils and CD14⁺ monocytes before and after rIFN-γ treatment. (E) Violin plots of selected differentially expressed genes in CD14⁺ monocytes isolated before and after in vivo rIFN-γ treatment. (F) IL-8 production after 4 hours of stimulation with either medium or PMA (50 ng/mL) in neutrophils from a patient with CGD before and 4 hours after receiving in vivo Imukine (rIFN-γ) subcutaneously. (G) OCR after Mito Stress Test of monocytes isolated from a patient with CGD before and 4 hours after receiving in vivo Imukine (rIFN-γ) subcutaneously. (H) IL-6 production from monocytes after LPS restimulation at day 6 after BCG-training protocol. Monocytes were isolated from 1 patient with CGD before and 4 hours after receiving Imukine (rIFN-γ). Owing to the low number of subjects, statistical analysis could not be performed for this set of experiments. DCs, dendritic cells; DMSO, dimethyl sulfoxide; NK, natural killer; Plt.act., platelet-activated; QC, quality control.