X-linked Chronic Granulomatous Disease Patients Research Articles

In adult X-CGD patients, regulatory T cells are expanded while activated T cells display a NOX2-independent ROS increase

The X-linked chronic granulomatous disease (X-CGD), a rare genetic disease characterised by recurrent infections, is caused by mutations of NOX2. Significant proportions of X-CGD patients display signs of immune dysregulation. Regulatory T cells (Tregs) are CD4+T lymphocytes that expand in active inflammation and prevent autoimmune disorders. Here we asked whether X-CGD is associated to Treg dysfunctions in adult patients. To this aim, the frequency of Tregs was analysed through intracellular flow cytometry in a cohort of adult X-CGD patients, carriers and controls. We found that Tregs were significantly expanded and activated in blood of adult X-CGD patients, and this was associated with activation of conventional CD4+T cells (Tconvs). T cell activation was characterised by accumulation of intracellular ROS, not derived from NOX2 but likely produced by cellular metabolism. The higher TNF production by Tconvs in X-CGD patients might contribute to the expansion of Tregs through the TNFR2 receptor. In summary, our data indicate that Tregs expand in adult X-CGD in response to immune activation, and that the increase of NOX2-independent ROS content is a feature of activated T cells.

EOSINOPHILIC CYSTITIS IN A PEDIATRIC PATIENT WITH X-LINKED CHRONIC GRANULOMATOUS DISEASE

<h3>Introduction</h3> Pediatric Eosinophilic Cystitis is an uncommon inflammatory disease associated with X-linked Chronic Granulomatous Disease (X-CGD)¹. We present a rare case of a teenage patient with refractory eosinophilic cystitis. <h3>Case Description</h3> Patient is a 14-year-old male with X-CGD, colitis, and complications from chronic steroid use, including bilateral cataracts, osteoporosis, and adrenal insufficiency. His colitis was well-controlled with vedolizumab when he presented for evaluation of new onset dysuria and intermittent scrotal pain that improved with voiding. Infectious workup was unrevealing. CT of abdomen and pelvis demonstrated asymmetric, irregular thickening, nodularity and enhancement of the left bladder wall and bladder base. Pathology from cystoscopy was significant for scattered eosinophils, suspicious for eosinophilic cystitis. He demonstrated mild peripheral eosinophilia (0.63 K/µL). He was started on intravesical dexamethasone 10mg, to minimize complications from systemic steroid use, and oral loratadine 5mg on intravesical treatment days. However, his symptoms did not resolve and intravesical dexamethasone was increased to 25mg then to 50mg. Loratadine was increased to 10mg daily. His bladder ultrasound normalized after 12 doses of intravesical dexamethasone and daily 10mg loratadine. Three weeks after loratadine was weaned to every other day, his urinary symptoms returned. Loratadine was increased to 10mg daily and he remained asymptomatic off steroids. <h3>Discussion</h3> Reported cases of pediatric eosinophilic cystitis are mostly in young children, only requiring a short course of intravesical steroids and oral antihistamine, unlike our patient^2,3. More research is needed to elucidate the history, optimal diagnosis and guide appropriate management of eosinophilic cystitis in pediatric X-CGD patients.

Four novel mutations in the gene encoding gp91-phox of human NADPH oxidase: consequences for oxidase assembly

The superoxide-forming nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase of human phagocytes comprises membrane-bound and cytosolic proteins, which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients with chronic granulomatous disease (CGD) are defective in one of the phagocyte oxidase (phox) components, p47-phox or p67-phox, which reside in the cytosol of resting phagocytes, or gp91-phox or p22-phox, which constitute the membrane-bound cytochrome b(558). In four X-linked CGD patients we have identified novel missense mutations in CYBB, the gene encoding gp91-phox. These mutations were associated with normal amounts of nonfunctional cytochrome b(558) in the patients' neutrophils. In phorbol-myristate-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of p47-phox and p67-phox with the membrane fraction of the cells with Cys369-->Arg, Gly408-->Glu, and Glu568--> Lys substitutions was strongly disturbed. Only a Thr341-->Lys substitution, residing in a region of gp91-phox involved in flavin adenine dinucleotide (FAD) binding, supported a normal translocation. Thus, the introduction or reversal of charge at residues 369, 408, and 568 in gp91-phox destroys the correct binding of p47-phox and p67-phox to cytochrome b(558). Based on mutagenesis studies of structurally related flavin-dependent oxidoreductases, we propose that the Thr341-->Lys substitution results in impaired hydride transfer from NADPH to FAD. Because we found no electron transfer in solubilized neutrophil plasma membranes from any of the four patients, we conclude that all four amino acid replacements are critical for electron transfer. Apparently, an intimate relation exists between domains of gp91-phox involved in electron transfer and in p47/p67-phox binding. (Blood. 2000;95:666-673)

Bacillus Calmette-Guérin (BCG) Infections at High Frequency in Both AR-CGD and X-CGD Patients Following BCG Vaccination.

Patients with chronic granulomatous disease (CGD) develop severe infections, including Bacillus Calmette-Guérin (BCG). Although the autosomal recessive CGD (AR-CGD) patients should hypothetically develop relatively fewer infections compared to the X-linked CGD (X-CGD) patients due to more residual reactive oxygen intermediates, the impacts of BCG vaccination on AR-CGD and X-CGD patients are unclear. Herein, we demonstrated the clinical features of BCG infections, treatments, and genetic factors in CGD patients after BCG vaccination under the Japanese immunization program. We collected data retrospectively from 43 patients with CGD and assessed their history of initial infection, age at diagnosis of CGD, BCG vaccination history, clinical course, treatment for BCG infections, and genetic mutations associated with CGD. Fourteen CGD patients avoided BCG vaccination because of other preceding infections and family history. Of 29 patients with CGD who received BCG vaccination, 20 patients developed BCG infections. Although the age at onset of initial infection in X-CGD patients was significantly younger than that in AR-CGD patients (P < .01), the onset and frequency of BCG infections were similar in X-CGD and AR-CGD patients. In X-CGD patients, BCG infections equally developed in the patients carrying missense, insertion, deletion, nonsense, and splice mutations of CYBB. All CGD patients with BCG infections were successfully treated with anti-tuberculous drugs. Although X-CGD patients develop severe infections at a younger age than AR-CGD patients, our data suggested that BCG infections develop at high frequency in both AR-CGD and X-CGD patients, regardless of genotype and mutant forms.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease

Background: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. Methods: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b₅₅₈ were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. Results: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. Conclusions: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.

Downregulation of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) in a co-culture system with human stimulated X-linked CGD neutrophils.

Phagocytes in patients with chronic granulomatous disease (CGD) do not generate reactive oxidative species (ROS), whereas nitric oxide (NO) production is increased in response to the calcium ionophore A23187 in CGD phagocytes compared with healthy phagocytes. Recently, patients with X-linked CGD (X-CGD) have been reported to show higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production from phagocytes. We studied NOS3 and EDN1 mRNA and protein expression in human umbilical vein endothelial cells (HUVECs) in a co-culture system with neutrophils from X-CGD patients. HUVECs were co-cultured for 30 minutes with human neutrophils from X-CGD or healthy participants in response to A23187 without cell-to-cell contact. The expression of NOS3 and EDN1 mRNA in HUVECs was quantified by real-time polymerase chain reaction. Moreover, we demonstrated the protein expression of eNOS, ET-1, and NFκB p65, including phosphorylation at Ser1177 of eNOS and Ser536 of NFκB p65. Neutrophils from X-CGD patients showed significantly higher NO and lower H2O2 production in response to A23187 than healthy neutrophils in vitro. Compared with healthy neutrophils, X-CGD neutrophils under A23187 stimulation exhibited significantly increased NO and decreased H2O2, and promoted downregulated NOS3 and EDN1 expression in HUVECs. The total expression and phosphorylation at Ser1177 of eNOS and ET-1 expression were significantly decreased in HUVECs co-cultures with stimulated X-CGD neutrophils. Also, phosphorylation at Ser536 of NFκB p65 were significantly decreased. In conclusions, eNOS and ET-1 significantly down-regulated in co-culture with stimulated X-CGD neutrophils through their excessive NO and the lack of ROS production. These findings suggest that ROS generated from neutrophils may mediate arterial tone affecting eNOS and ET-1 expression via their NO and ROS production.

NAPDH Oxidase-Specific Flow Cytometry Allows for Rapid Genetic Triage and Classification of Novel Variants in Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is an innate immune deficiency, primarily affecting the phagocytic compartment, and presenting with a diverse phenotypic spectrum ranging from severe childhood infections to monogenic inflammatory bowel disease. Dihydrorhodamine (DHR) flow cytometry is the standard diagnostic test for CGD, and correlates with NADPH oxidase activity. While there may be genotype correlation with the DHR flow pattern in some patients, in several others, there is no correlation. In such patients, assessment by flow cytometric evaluation of NADPH oxidase-specific (NOX) proteins provides a convenient and rapid means of genetic triage, though immunoblotting has long been used for this purpose. We describe the clinical utility of the NOX flow cytometry assay through assessment of X-linked and autosomal recessive CGD patients and their first-degree relatives. The assessment of specific NOX proteins was correlated with overall NADPH oxidase function (DHR flow), clinical phenotype and genotype. NOX-specific protein assessment is a valuable adjunct to DHR assessment and genotyping to classify and characterize CGD patients. The atypical clinical presentation of some CGD patients can make genotype-phenotype correlation with DHR flow data challenging. Genetic testing, while useful for confirmation of diagnosis, can take several weeks, and in some patients does not provide a conclusive answer. However, NADPH-oxidase-specific protein flow assessment offers a rapid alternative to identification of the underlying genetic defect in cellular subsets, and can be utilized as a reflex test to an abnormal DHR flow. Further, it can provide insight into correlation between oxidative burst relative to protein expression in granulocytes and monocytes.

High Efficiency Gene Correction in Hematopoietic Cells By Template-Free Crispr/Cas9 Genome Editing

IntroductionA significant fraction of inherited monogenic disorders is caused by patient-specific mutations dispersed over the entire locus of the affected gene. Correcting these mutations by introducing healthy gene copies into the genome of the diseased cells proved successful in several clinical gene therapy trials. Most of these trials employed retroviral vectors, which by inserting randomly throughout the genome deprived the transduced genes of their endogenous control and caused insertional mutations leading to secondary disease. The development of genome editing tools capable of modifying any prespecified genomic sequence with unprecedented accuracy opened up a wide range of new possibilities in gene manipulation including targeted gene repair. In particular, CRISPR/Cas9, a prokaryotic adaptive immune system, and its swift repurposing for genome editing was widely adopted as the hitherto simplest genome editing tool. CRISPR/Cas9 is an RNA guided endonuclease that uses RNA-DNA base pairing to target genomic DNA. Bound to its target via the guide (g)RNA, Cas9 induces DNA double strand breaks (DBS) at prespecified genomic sites that promptly activate the endogenous DNA repair machinery. DSB repair is accomplished by either non-homologous end joining (NHEJ) or homology directed repair (HDR). Thus far, correction of human mutations in hematopoietic cells relied entirely on HDR requiring gene specific donor templates in addition to the RNA guided endonucleases (RGNs). However, although the HDR offers precision, its efficiency is low and requires positive selection to enrich for gene corrected cell. Because in mammalian cells DSB repair by NHEJ significantly exceeds HDR and even more importantly, is the dominant DSB repair pathway in hematopoietic stem- and progenitor cells (HSPC), we thought to exploit NHEJ for gene therapy because in theory, approximately one third of the indels associated with NHEJ should restore the open reading frame (ORF) disrupted by a disease mutation. This would lead to a significant number of ORF reconstitutions of which some, depending on the position and type of the original mutation, should either completely or partially recover protein function.ResultsTo test gene repair efficiency by NHEJ in human hematopoietic cells, we generated PLB-985 (PLB) reporter cells expressing mutationally inactivated EGFP (mEGFP). Transduction of mEGFP expressing PLBs (mEGFP-PLB) with integrase-deficient lentiviral (IDLV) particles encoding for RGNs targeting the EGFP mutation reconstituted EGFP expression in up to 27% of the mEGFP PLB cells. Indel analysis revealed that 13 out of 28 (46%) restored the EGFP-open reading frame of which 7 (25%) reconstituted EGFP activity. To test whether the donor-template free IDLV strategy would also effectively correct bone fide disease mutations, we performed similar experiments with X-CGD PLB cells expressing transgenes encoding patient-specific frameshift, missense and nonsense CYBB mutations causing X-linked chronic granulomatous disease (X-CGD) which is an inherited, life threatening immunodeficiency disorder. Transduction of the cells with IDLVs carrying RGNs directed against each of these mutations restored CYBB function in up to 10% of cells harboring frameshift mutations which is sufficient to protect X-CGD patients from microbial infections. However, RGNs directed against the nonsense or missense mutations restored CYBB function in only 1-2% of the cells, suggesting that these mutations are less amenable to CRISPR/Cas9 mediated repair. As Cas9 frequently tolerates single nucleotide mismatches, selection against solitary nucleotide substitutions may explain this low efficiency of gene repair.ConclusionsFrameshift mutations can be effectively repaired by NHEJ in hematopoietic cells by CRISPR/Cas9 transducing IDLVs. As about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that about a quarter of patients suffering from monogenic blood disorders could benefit from personalized, template free CRISPR/Cas9 gene therapy. DisclosuresNo relevant conflicts of interest to declare.

557. Targeted CYBB Minigene Insertion into the CYBB Locus for Correction of X-CGD iPSCs Requires Intronic Elements for Expression

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency characterized by defective phagocyte production of microbicidal reactive oxygen species (ROS), resulting in recurring, life-threatening infections and hyper-inflammation. Mutations causing X-CGD span the entire 13 exons or intronic splice sites of the >30-kb CYBB gene encoding gp91phox, resulting in a loss of gp91phox protein expression. We previously tested a TALEN-mediated targeted gene therapy approach to insert a codon-optimized CYBB minigene into the start site of endogenous CYBB. Although targeted insertion into the endogenous start site was achieved in X-CGD patient iPSCs, little or no gp91phox expression or ROS activity was observed upon granulocyte differentiation, suggesting that downstream intronic or regulatory elements may be necessary for efficient gene expression from the CYBB promoter. To test this hypothesis, we tested CRISPR-mediated targeted insertion of a codon-optimized CYBB cDNA consisting of exons 2 through 13 (CYBB2-13) together with a puromycin-resistance gene cassette into exon 2 of the CYBB locus. In iPSCs from X-CGD patients with a CYBB mutation in exon 5, exon 7, or intron 10, the efficiency of targeted insertion of the CYBB2-13 plasmid donor without random inserts in puromycin-selected clones was 50-66%. Upon granulocyte differentiation of CYBB2-13 corrected X-CGD iPSCs, gp91phox expression and ROS production were restored to levels 64-100% (gp91phox) and 68-76% (DHR) of normal healthy donor controls. As expected for expression from the endogenous CYBB promoter, expression of gp91phox was specific to CD13+ granulocytes, and was undetected in undifferentiated iPSCs. This targeted gene therapy approach should allow correction of ~90% of X-CGD patient mutations (those involving mutations in exons 2 through 13), to restore ROS activity while maintaining normal regulation of CYBB expression. Further, these findings demonstrate a key issue for the design of targeted gene insertion to capture expression from an endogenous promoter: for some endogenous promoters, the inclusion of intronic elements is necessary for efficient expression of the insert.

57. Seamless Targeted Correction of CYBB Exon 5 Mutations Restores Granulocyte Function in X-Linked Chronic Granulomatous Disease iPSCs

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from lack of production of microbicidal reactive oxygen species (ROS) by phagocytic cells. Mutations causing X-CGD can occur throughout the >30-kb CYBB gene encoding gp91phox, with the majority of patients exhibiting a single causative mutation within one of the 13 exons or adjoining intronic splice sites, resulting in a loss of gp91phox protein expression. We previously demonstrated targeted “safe-harbor” gene therapy of X-CGD in iPSCs through insertion of a codon-optimized CYBB minigene into the AAVS1 locus, resulting in constitutive gp91phox expression from a CAG promoter. Another approach targeting insertion of the codon-optimized minigene or normal CYBB cDNA to the start site of endogenous CYBB resulted in little or no gp91phox expression or ROS activity in iPSC-derived granulocytes, suggesting that regulatory elements downstream of the CYBB promoter may be required for efficient expression at this locus. In order to maintain normal regulation of gp91phox expression with minimal alterations in corrected cells, we now demonstrate a strategy for seamless targeted repair of CYBB exon 5 mutations using an exon 5-specific TALEN pair or CRISPR, along with a donor plasmid containing the corrected 146-bp exon 5 interrupted by a piggyBac transposon cassette containing a PGK promoter and puroDtk gene for positive/negative selection, and flanked by ~900-bp upstream and downstream intron sequences for homologous recombination. iPSCs from X-CGD patients with exon 5 458T>G mutation or 461A deletion were nucleofected with donor and TALEN or CRISPR expression plasmids. Both TALENs and CRISPR enabled efficient targeted insertion in 82-98% of puromycin-resistant clones, 25-65% of which contained off-target inserts, for an overall efficiency of 29-75% of selected clones containing only the targeted insert. Targeted iPSC clones lacking off-target inserts were nucleofected with “excision-only” piggyBac transposase expression plasmid and selected with ganciclovir for removal of the PGK-puroDtk cassette, leaving only a silent codon change to accommodate the TTAA sequence left behind after piggyBac excision, without additional alterations detected beyond correction of the CYBB mutation. Corrected iPSCs maintained pluripotency and upon in vitro granulocyte differentiation exhibited restoration of gp91phox expression and ROS production comparable to normal blood neutrophils (3100% of normal gp91phox expression and 85-99% of normal ROS activity by mean fluorescence intensity in DHR assay). Our findings demonstrate efficient and seamless targeted repair of exon 5 mutations in X-CGD iPSCs with an exon replacement strategy, resulting in normal regulation of gp91phox expression and functional correction of the disease defect in iPSC-derived granulocytes. The development of similar approaches for the other exons of CYBB will enable seamless repair of the majority of causative mutations found in X-CGD patients.

298. Transfection of Neutrophils from X-Linked Chronic Granulomatous Disease Patients with gp91phox mRNA Restores Oxidase Activity with High Efficiency and Viability

Chronic granulomatous disease (CGD) is an inherited immune deficiency due to defective phagocyte NADPH oxidase required for production of antimicrobial oxidants. Fungal or bacterial infections in CGD may persist despite intensive and prolonged antibiotic therapy. In settings where conventional antibiotic therapy and/or surgical debridement do not control infection, use of allogeneic unmatched donor granulocyte transfusions has been therapeutic. However, CGD patients treated with granulocyte transfusions may develop high titers of anti-HLA antibodies, restricting use of allogeneic granulocyte transfusions and further complicating potentially curative bone marrow transplant. Using the CGD patient as an autologous donor for granulocyte transfusions would be a helpful solution.Here we demonstrate highly efficient functional correction of the functional defect in CGD by transfection of patient's own granulocytes with mRNA while retaining viability using a scalable, cGMP-compliant MaxCyte® GT™ electroporation system capable of handling clinically relevant numbers of cells (>1010cells).Freshly collected granulocytes from normal volunteers were electroporated using the MaxCyte electroporation system with eGFP-mRNA on the day of collection. Flow cytometry of demonstrated that >90% of neutrophils expressed eGFP with viability exceeding 90% at 24 hrs after electroporation. Phagocyte NADPH oxidase activity of the normal neutrophils was retained as well as bactericidal activity against Burkolderia cepacia, a CGD bacterial pathogen. Antibacterial activity of electroporated granulocytes was comparable to unmanipulated granulocytes confirming retention of cellular function following the electroporation. Finally, electroporated eGFP positive human neutrophils injected into CGD mice were shown to remain in circulation as well as migrate to the peritoneal cavity following thioglycollate IP injection and there were no adverse effects observed in the mice.Electroporation of gp91phox mRNA into human X-linked CGD (gp91phox-deficient) patient neutrophils resulted in expression of gp91phox protein in >70% of cells with >90% viability at 24 hours after treatment. Using the dihydrorhodamine assay of phagocyte NADPH oxidase activity, >70% of neutrophils remained functionally corrected 2 days after transfection. Other studies of cellular function in vitro and in vivo with electroporated corrected CGD neutrophils are in progress. In summary, we show the feasibility of restoring the function of CGD patient-autologous neutrophils via electroporation of mRNA encoding the defective gene product. This approach may have potential clinical utility in the management of severe chronic bacterial and fungal infections in CGD but also short-term correction of other hematopoietic gene diseases.

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19 + CD27+ memory B cell (p = 0.028 and p = 0.047 respectively), CD45RA cells (with p values of p = 0.000 and 0.033, respectively), the naïve compartment CD3 + CD45RA+ cells percentage and absolute counts (p = 0.005, 0.01respectively), CD3 + CD27 + cells percentage and absolute counts (p = 0.001, 0.012 respectively), CD3 + CD45RA + CD27+ cells percentage and absolute counts (p = 0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. Conclusion: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.

Excessive Nitric Oxide Production of CGD Neutrophils Induces the Down-Regulation of NOS3 and EDN1 Expression in Human Endothelial Cells

Introduction: Chronic granulomatous disease (CGD) is an inherited immunodeficiency due to a mutation in genes that encode the subunits of NADPH oxidase of phagocytes. Phagocytes of CGD can not generate the reactive oxidative species (ROS), whereas nitric oxide (NO) production of CGD phagocytes is increased in response to calcium ionophore, A23187, compared with that of phagocytes from healthy subjects. Recently, X-linked CGD (X-CGD) patients showed lower oxidative stress and higher flow-mediated dilation, suggesting that endothelial cell function is affected by NO production of phagocytes. We studied the effects of NO on the regulation of endothelial gene expression related to dilator and constrictive effect of blood vessels, such as NOS3 and EDN1 using neutrophils from X-CGD patients.Methods: Eighteen X-CGD patients and 18 age-matched healthy male subjects were enrolled in this study from 2009 to 2013. NO production of phagocytes was assessed by flow cytometry using DAF2/DA fluorescent probe. Human umbilical vein endothelial cells (HUVECs) were co-cultured with human neutrophils of X-CGD or healthy subjects in response to calcium ionophore,A23187 without cell to cell contact using the Transwell-permeable support systems. The expression of endothelial NOS3 and EDN1 mRNA gene in HUVECs were quantified by real-time PCR.Results: Neutrophils of X-CGD patients showed significantly higher production of NO in response to A23187 for 30 to 60 minutes than those of normal subjects detected by flow cytometric analyses. NO concentration artificially generated by NO donor was significantly decreased by ROS produced by xanthine and xanthine oxidase. Similarly, neutrophils from healthy subjects, but not from X-CGD stimulated with A23187 induced the decrease in the concentration of NO. These results strongly suggest that the lack of ROS generation in CGD neutrophils leads to the increase of NO production in response to A23187. HUVECs were incubated with NO generation system, ROS generation system alone or with both systems. The expression of NOS3 and EDN1 was significantly decreased depending on the concentration of extracellular NO. In contrast, both NOS3 and EDN1 expression was significantly up-regulated under the ROS generation system without NO. Next, HUVECs were incubated by X-CGD neutrophils or by control neutrophils with or without A23187 for 30 minutes using the wells divided by membrane (0.45μm) to prevent cell to cell contact. Both NOS3 and EDN1 expression of HUVECs incubated with X-CGD neutrophils was significantly down-regulated under A23187 stimulation compared with normal neutrophils (112±117 vs. 34.5±39.9, n=8, p<0.05, 0.94± 0.24 vs. 0.61±0.24, n=5, p<0.05, respectively).Conclusion: This study demonstrated that the stimulated X-CGD neutrophils induced the decreased endothelial NOS3 and EDN1 gene expression through the excessive generation of NO due to the lack of ROS production. These findings suggest that ROS generated by phagocytes may modulate arterial tone affecting the amount of NO, a potent vasodilator molecule produced by endothelial cells. DisclosuresNo relevant conflicts of interest to declare.

Interstitial lung disease with multiple microgranulomas in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease that is characterized by susceptibility to bacterial and fungal infections. CGD patients also suffer from immune regulatory disorders, such as CGD-associated bowel inflammation with granuloma, which could be caused by excessive inflammation without demonstrable infection. We investigated the clinical manifestation of interstitial lung disease (ILD) resulting from excessive inflammation in X-linked CGD patients. Pulmonary CT images and testing of serum KL-6 levels were performed to assess ILD in the patients. For this study, patients with pulmonary lesions due to demonstrable infections were excluded from among ILD patients. Among 33 CGD patients, four developed ILD; they had increased reticulo-nodular opacities on CT images and elevated serum KL-6 levels. Histopathological examinations revealed multiple homogeneous microgranulomas in the lesions of inflammatory cell infiltration. Mononuclear cells obtained from their pulmonary lesions produced higher amounts of inflammatory cytokines than the peripheral blood mononuclear cells of CGD patients, suggesting that the only infiltrating cells in the pulmonary lesions were activated and produced large amounts of inflammatory cytokines in ILD patients. Interestingly, an anti-inflammatory drug, such as a corticosteroid or thalidomide, but not anti-bacterial or anti-fungal drugs, improved CT image findings and reduced their KL-6 levels. CGD patients' daily exposures to inhaled antigens may induce excessive reactions with the production of inflammatory cytokines leading to the development of ILD with multiple microgranulomas, which could be due to an inadequate production of reactive oxygen species in CGD.

Rare Duplication or Deletion of Exons 6, 7 and 8 in CYBB Leading to X-Linked Chronic Granulomatous Disease in Two Patients from Different Families

Chronic granulomatous disease (CGD) is a rare congenital disorder in which phagocytes cannot generate superoxide (O(2)(-)) and other microbicidal oxidants due to mutations in one of the five components of the O(2)(-)-generating NADPH oxidase complex. The most common form is caused by mutations in CYBB on the X chromosome, encoding gp91phox, the enzymatic subunit of the phagocyte NADPH oxidase. Here, we report two rare cases of male X-linked CGD patients, one caused by a 5.7-kb duplication of a region containing CYBB exons 6 to 8 and the other caused by a deletion of this same region. We found both the duplication in patient 1 and the deletion in patient 2 to be bordered by a GT repeat. Indeed, in control DNA, the 3' part of CYBB intron 5 contains a GT repeat and the 5' part of intron 8 also contains such a repeat. Duplication of exons 6, 7 and 8 in patient 1 was probably caused by a non-homologous crossing over between the two GT repeats. The deletion found in patient 2 probably arose from a similar misalignment. The results found in these patients were confirmed by multiplex ligation-dependent probe amplification. The clinical profile of XCGD is severe in both patients.

Corticosteroid Therapy for Liver Abscess in Chronic Granulomatous Disease

Liver abscesses in chronic granulomatous disease (CGD) are typically difficult to treat and often require surgery. We describe 9 X-linked CGD patients with staphylococcal liver abscesses refractory to conventional therapy successfully treated with corticosteroids and antibiotics. Corticosteroids may have a role in treatment of Staphylococcus aureus liver abscesses in CGD.

Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91(phox) that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47(phox). Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients' neutrophils were measured, and genetic analysis was performed. We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91(phox). Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13-30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91(phox) to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production. X-linked CGD patients with mutations that alter the gp91(phox) protein in regions involved in docking of the cytosolic NADPH oxidase component p47(phox) may have higher than expected hydrogen peroxide generation capability.

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

BackgroundChronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.Methodology/Principal FindingsWe found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47phox−/− mice and gp91phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.Conclusions/SignificanceThese studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.

Retroviral Gene Therapy for X-Linked Chronic Granulomatous Disease: Results from Phase I/II Trial.

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency disease caused by a defect in the gp91phox gene encoding one of the subunits of the NADPH oxidase complex. NADPH oxidase plays an important role in eradicating the pathogen engulfed by the phagocytes. Therefore, CGD patients suffer from recurring life-threatening infection by bacteria or fungi, and die before 30 in most cases. In an effort to treat this life-threatening disease, we initiated a phase I/II gene therapy trial in 2007. Two X-CGD patients were enrolled in the trial. The retroviral vector used for gene delivery was the MLV-based MT vector containing gp91 phox cDNA (Yu et al., Gene Ther 2000; 7: 797, Hong et al., J Gene Med 2004; 6: 724). Viral vectors have been produced from PG13 packaging cells in compliance with GMP. The clinical protocol was approved by the Korean FDA. G-CSF mobilized peripheral blood CD34+ cells were obtained from patients, and transduced in retronectin-coated gas-permeable bags containing SCGM media supplemented with SCF, FLT3L, TPO, and IL-3. The transduction efficiency was 10.5% for patient #1 and 28.5% for patient #2 when assessed by gp91 FACS analysis. Before receiving transduced cells, patients were treated with a conditioning regimen consisting of busulfan (3.2 mg/kg/day for 2 days) and fludarabine (40 mg/m2/day for 3 days). No adverse effects were observed from the use of busulfan and fludarabine. The percentage of superoxide-producing cells, as determined by DHR assay, was 6.4% and 14.5% at day 17, and decreased to less than 0.1% (after 1 year) and 0.4% (after 7 months). Thus far, abnormal cell expansion has not been observed.

Correction of Human X-Linked Chronic Granulomatous Disease (X-CGD) Following Transduction of X-CGD CD34+ Cells with a Modified RD114-Pseudotyped Simian Immunodeficiency Virus Lentiviral Vector in a NOD/SCID Mouse Xenograft Model.

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency that results from mutations in the gp91phox subunit of the phagocyte NADPH-oxidase. We have developed a modified RD114-pseudotyped simian immunodeficiency virus (SIVmac) vector encoding human gp91phox and report here that this vector efficiently targets peripheral blood-mobilized CD34+ hematopoietic stem cells (PBSCs) from X-CGD patients, correcting the oxidase deficient phenotype. The majority of lentiviral gene transfer studies have relied on human immunodeficiency virus (HIV) 1-derived vectors pseudotyped with the vesicular stomatitis virus G protein (VSV-G) envelope. Because HIV is a human pathogen, there are theoretical benefits to considering other types of lentivectors engineered from viruses less pathogenic for humans such as SIV-based vectors. In addition, the VSV-G envelope has some toxicity for cells, limiting the titers of vector that can be used. RD114/TR-pseudotyped SIV gp91phox encoding vector particles were transiently produced following transfection of 293T cells and concentrated by high-speed centrifugation achieving stock titers of 1.5 × 10E7 infectious units/ml. PBSCs from two patients with X-CGD (P1 and P2) were cultured and transduced (multiplicity of infection 2–3) overnight four times before transplantation into sublethally irradiated NOD/SCID mice on day 5 of culture. Ex vivo transduction efficiency was 40.5% and 46%, respectively. Cells maintained in culture showed a progressive decrease in marking over the first 9 days, after which marking stabilized at 22.7% (P1) and 27.1% (P2) out to day 26 of culture with an averaged copy number of 2.2–2.4 per transduced cell at that time. Compared to healthy control, 18–19% of myeloid colonies derived from patient CD34+ cells stained positive in the nitroblue tetrazolium (NBT) test. Liquid cell cultures at 20 days were analyzed for production of reactive oxidative species using the flow cytometry dihydrorhodamine 123 (DHR) assay. Cells from P1 demonstrated 15.3% and from P2 9.1% of the activity of healthy cells. At 6 weeks after transplant, analysis of mouse bone marrow (mBM) samples revealed human cell engraftment levels of 36.8–74% (CD45+ cells). Compared to analysis of mice transplanted with healthy human PBSCs, 10.5% (P1) and 7.3% (P2) of the myeloid cells (CD13+) developing from X-CGD CD34+ cells in the chimeric mBM expressed gp91phox transgene. Human CD34+ cells were isolated from mBM and grown in liquid culture or plated for colony assays. 2.1% (P1) and 1.2% (P2) of cultured cells were gp91phox positive at day 14 with an averaged copy number of 2.3–5.2 per marked cell. 4.8% and 3.6% of colonies derived from the same cells were positive in the NBT test. DHR assay at day 21 of culture demonstrated 2.7% (P1) and 2.0% (P2) of the activity of healthy cells. Linear amplification-mediated PCR retrieved multiple unique vector insertion sites in CD34+ cells from both patients isolated from chimeric mBM, without predominance of any one clone suggesting polyclonal marking. In conclusion, these data suggest that modified RD114-pseudotyped SIVmac-based vectors may be suitable for hematopoietic gene therapy for CGD and represent an alternative to HIV-1 based vectors. Studies are ongoing to further improve vector production titers to achieve higher transduction levels of PBSCs.