Mouse Model Of Chronic Granulomatous Disease Research Articles

Survival Factor A (SvfA) Contributes to Aspergillus nidulans Pathogenicity.

Survival factor A (SvfA) in Aspergillus nidulans plays multiple roles in growth and developmental processes. It is a candidate for a novel VeA-dependent protein involved in sexual development. VeA is a key developmental regulator in Aspergillus species that can interact with other velvet-family proteins and enter into the nucleus to function as a transcription factor. In yeast and fungi, SvfA-homologous proteins are required for survival under oxidative and cold-stress conditions. To assess the role of SvfA in virulence in A. nidulans, cell wall components, biofilm formation, and protease activity were evaluated in a svfA-gene-deletion or an AfsvfA-overexpressing strain. The svfA-deletion strain showed decreased production of β-1,3-glucan in conidia, a cell wall pathogen-associated molecular pattern, with a decrease in gene expression for chitin synthases and β-1,3-glucan synthase. The ability to form biofilms and produce proteases was reduced in the svfA-deletion strain. We hypothesized that the svfA-deletion strain was less virulent than the wild-type strain; therefore, we performed in vitro phagocytosis assays using alveolar macrophages and analyzed in vivo survival using two vertebrate animal models. While phagocytosis was reduced in mouse alveolar macrophages challenged with conidia from the svfA-deletion strain, the killing rate showed a significant increase with increased extracellular signal-regulated kinase ERK activation. The svfA-deletion conidia infection reduced host mortality in both T-cell-deficient zebrafish and chronic granulomatous disease mouse models. Taken together, these results indicate that SvfA plays a significant role in the pathogenicity of A. nidulans.

Bacillus calmette-guerin infection in NADPH oxidase deficiency: defective mycobacterial sequestration and granuloma formation.

Patients with chronic granulomatous disease (CGD) lack generation of reactive oxygen species (ROS) through the phagocyte NADPH oxidase NOX2. CGD is an immune deficiency that leads to frequent infections with certain pathogens; this is well documented for S. aureus and A. fumigatus, but less clear for mycobacteria. We therefore performed an extensive literature search which yielded 297 cases of CGD patients with mycobacterial infections; M. bovis BCG was most commonly described (74%). The relationship between NOX2 deficiency and BCG infection however has never been studied in a mouse model. We therefore investigated BCG infection in three different mouse models of CGD: Ncf1 mutants in two different genetic backgrounds and Cybb knock-out mice. In addition, we investigated a macrophage-specific rescue (transgenic expression of Ncf1 under the control of the CD68 promoter). Wild-type mice did not develop severe disease upon BCG injection. In contrast, all three types of CGD mice were highly susceptible to BCG, as witnessed by a severe weight loss, development of hemorrhagic pneumonia, and a high mortality (∼50%). Rescue of NOX2 activity in macrophages restored BCG resistance, similar as seen in wild-type mice. Granulomas from mycobacteria-infected wild-type mice generated ROS, while granulomas from CGD mice did not. Bacterial load in CGD mice was only moderately increased, suggesting that it was not crucial for the observed phenotype. CGD mice responded with massively enhanced cytokine release (TNF-α, IFN-γ, IL-17 and IL-12) early after BCG infection, which might account for severity of the disease. Finally, in wild-type mice, macrophages formed clusters and restricted mycobacteria to granulomas, while macrophages and mycobacteria were diffusely distributed in lung tissue from CGD mice. Our results demonstrate that lack of the NADPH oxidase leads to a markedly increased severity of BCG infection through mechanisms including increased cytokine production and impaired granuloma formation.

Human miR223 Promoter as a Novel Myelo-Specific Promoter for Chronic Granulomatous Disease Gene Therapy

Targeting transgene expression to specific hematopoietic cell lineages could contribute to the safety of retroviral vectors in gene therapeutic applications. Chronic granulomatous disease (CGD), a defect of phagocytic cells, can be managed by gene therapy, using retroviral vectors with targeted expression to myeloid cells. In this context, we analyzed the myelospecificity of the human miR223 promoter, which is known to be strongly upregulated during myeloid differentiation, to drive myeloid-restricted expression of p47(phox) and gp91(phox) in mouse models of CGD and in primary patient-derived cells. The miR223 promoter restricted the expression of p47(phox), gp91(phox), and green fluorescent protein (GFP) within self-inactivating (SIN) gamma- and lentiviral vectors to granulocytes and macrophages, with only marginal expression in lymphocytes or hematopoietic stem and progenitor cells. Furthermore, gene transfer into primary CD34+ cells derived from a p47(phox) patient followed by ex vivo differentiation to neutrophils resulted in restoration of Escherichia coli killing activity by miR223 promoter-mediated p47(phox) expression. These results indicate that the miR223 promoter as an internal promoter within SIN gene therapy vectors is able to efficiently correct the CGD phenotype with negligible activity in hematopoietic progenitors, thereby limiting the risk of insertional oncogenesis and development of clonal dominance.

CD4+ T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease

Aspergillus fumigatus is a model fungal pathogen and a common cause of infection in individuals with the primary immunodeficiency chronic granulomatous disease (CGD). Although primarily considered a deficiency of innate immunity, CGD is also linked to dysfunctional T cell reactivity. Both CD4(+) and CD8(+) T cells mediate vaccine-induced protection from experimental aspergillosis, but the molecular mechanisms leading to the generation of protective immunity and whether these mechanisms are dysregulated in individuals with CGD have not been determined. Here, we show that activation of either T cell subset in a mouse model of CGD is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing and presentation in DCs. Aspergillus conidia activated CD8(+) T cells upon sorting to the Rab14(+) endosomal compartment required for alternative MHC class I presentation. Cross-priming of CD8(+) T cells failed to occur in mice with CGD due to defective DC endosomal alkalinization and autophagy. However, long-lasting antifungal protection and disease control were successfully achieved upon vaccination with purified fungal antigens that activated CD4(+) T cells through the endosome/lysosome pathway. Our study thus indicates that distinct intracellular pathways are exploited for the priming of CD4(+) and CD8(+) T cells to A. fumigatus and suggests that CD4(+) T cell vaccination may be able to overcome defective antifungal CD8(+) T cell memory in individuals with CGD.

Chronic Granulomatous Disease: Lessons from a Rare Disorder

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency with X-linked or autosomal recessive inheritance involving defects in genes encoding phox proteins, which are the subunits of the phagocyte NADPH oxidase. This results in failure to produce superoxide anion and downstream antimicrobial oxidant metabolites and to activate antimicrobial proteases. Affected patients are susceptible to severe, life-threatening bacterial and fungal infections and excessive inflammation characterized by granulomatous enteritis resembling Crohn's disease and genitourinary obstruction. Early diagnosis of CGD and rapid treatment of infections are critical. Prophylaxis with antibacterial and mold-active antifungal agents and the administration of interferon-γ has significantly improved the natural history of CGD. Currently, the only cure is allogeneic hematopoietic cell transplant (HCT), although there remains controversy as to which patients with CGD should get a transplant. Allele-based HLA typing of alternative donors, improved supportive care measures, and use of reduced toxicity conditioning have resulted in event-free survival (EFS) of at least 80% even with an unrelated donor and even better when the patient has no active infections/inflammation. Gene correction of CGD would eliminate the risks of graft-versus-host disease (GVHD) and the immunoablative chemotherapy required for allogeneic HCT. Based on gene therapy trials in patients with SCID-X1, ADA-SCID, and the early experience with CGD, it is clear that at least some degree of myeloablation will be necessary for CGD as there is no inherent selective growth advantage for gene-corrected cells. Current efforts for gene therapy focus on use of lentivector constructs, which are thought to be safer from the standpoint of insertional mutagenesis and more efficient in transducing hematopoietic stem cells (HSCs).

Virulence and Cellular Interactions ofBurkholderia multivoransin Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1beta. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.

Hyperinflammation in chronic granulomatous disease and anti-inflammatory role of the phagocyte NADPH oxidase

Chronic granulomatous disease (CGD) is an immunodeficiency caused by the lack of the superoxide-producing phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, CGD patients not only suffer from recurrent infections, but also present with inflammatory, non-infectious conditions. Among the latter, granulomas figure prominently, which gave the name to the disease, and colitis, which is frequent and leads to a substantial morbidity. In this paper, we systematically review the inflammatory lesions in different organs of CGD patients and compare them to observations in CGD mouse models. In addition to the more classical inflammatory lesions, CGD patients and their relatives have increased frequency of autoimmune diseases, and CGD mice are arthritis-prone. Possible mechanisms involved in CGD hyperinflammation include decreased degradation of phagocytosed material, redox-dependent termination of proinflammatory mediators and/or signaling, as well as redox-dependent cross-talk between phagocytes and lymphocytes (e.g. defective tryptophan catabolism). As a conclusion from this review, we propose the existence of ROS high and ROS low inflammatory responses, which are triggered as a function of the level of reactive oxygen species and have specific characteristics in terms of physiology and pathophysiology.

Branched fungal β‐glucan causes hyperinflammation and necrosis in phagocyte NADPH oxidase‐deficient mice

Chronic granulomatous disease (CGD), a genetic disorder characterized by the absence of a functional phagocyte NADPH oxidase, is a severe immune deficiency. However, non-infectious hyperinflammation is a second hallmark of the disease. In CGD mouse models, sterile hyperinflammation can be induced by A. fumigatus cell wall preparations. In this study, we used subcutaneous injection of microbial cell walls and cell wall components to identify causes of CGD hyperinflammation and to characterize its histological features. Sterile cell wall preparations from fungi (A. fumigatus, C. albicans, S. cerevisiae), but not from bacteria (S. aureus, P. aeruginosa, E. coli), caused prolonged and severe skin inflammation in CGD mice. To identify fungal cell wall elements responsible for this process, we investigated microbial cell wall-derived monosubstances. Injection of beta(1-3)(1-6)-glucan induced severe hyperinflammation in CGD mice, while other fungal cell components [mannan, (1-3) beta-glucan] or bacterial cell wall components (lipopolysaccharide, lipoteichoic acid) caused no or only moderate inflammation. beta-glucan-induced hyperinflammation was predominantly due to a defect in termination of inflammation, as in the initial stage (2 days), the severity of inflammation and the extent of cell death were comparable in wild-type and CGD mice. At later stages (7 days), beta(1-3)(1-6)-glucan-induced inflammation had subsided in wild-type mice. In contrast, CGD mice showed persistent severe inflammation with central necrosis, containing abundant apoptotic and necrotic cells. In summary, branched fungal beta-glucan induces a severe inflammatory reaction in the absence of phagocyte NADPH oxidase. As opposed to the commonly perceived notion that reactive oxygen species are the cause of cell death, our results demonstrate that tissue necrosis can be caused by the absence of a superoxide-producing enzyme.

A Novel Bacterium Associated with Lymphadenitis in a Patient with Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91phox −/− (X-linked) and p47 phox −/− (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.

The Acetobacteraceae: Extending the Spectrum of Human Pathogens

The Acetobacteraceae: Extending the Spectrum of Human Pathogens

Catalase negative Staphylococcus aureus retain virulence in mouse model of chronic granulomatous disease

Myeloperoxidase-mediated chlorination is thought to be a necessary microbicidal mechanism. The H 2O 2 required for this process is generated by the NADPH oxidase. Staphylococcus aureus can also produce H 2O 2, which is not broken down by catalase negative organisms. It has been thought that this bacterial H 2O 2 can substitute for cellular H 2O 2 in the halogenation reaction in chronic granulomatous disease (CGD) where neutrophils are lacking the NADPH oxidase. We have readdressed this issue in a mouse model of CGD using clinical isolates of catalase positive and negative strains of S. aureus. The results showed these organisms to be equally virulent and that the H 2O 2 they produced is insufficient to cause significant iodination, a marker for chlorination, thereby contradicting the accepted views on this subject.

Gene therapy for chronic granulomatous disease

Recent progress in the development of gene therapy for chronic granulomatous disease (CGD), an inherited immunodeficiency syndrome, is reviewed. This disorder results from defects in any of the four genes encoding essential subunits of respiratory burst oxidase, the superoxide-generating enzyme complex in phagocytic leukocytes. The absence of respiratory burst oxidants results in recurrent bacterial and fungal infections and can also be complicated by the formation of inflammatory granulomas. Although current management, including prophylactic use of antimicrobial agents and interferon-γ, has significantly improved its prognosis, CGD continues to be associated with significant morbidity and mortality from life-threatening infections and complications. Allogeneic bone marrow transplantation can provide a life-long cure of the disease, but difficulty in finding suitable donors and risks associated with this procedure have limited its application. Recently CGD has emerged as a promising candidate for gene therapy targeted at the hematopoietic system. CGD mouse models have been developed with gene targeting technology, and preclinical studies in these animals with recombinant retroviral vectors have demonstrated the appearance of functionally normal neutrophils and increased resistance against pathogens such as Aspergillus. Although the murine studies have provided a promise of long-term cure of patients by gene transfer, phase I clinical studies in a limited number of patients with CGD with such vectors have yet to produce a clinically relevant number of corrected neutrophils for extended time periods. Efforts are ongoing to improve gene transfer efficiency into human hematopoietic stem/progenitor cells and to achieve better engraftment of the gene-corrected stem cells. (J Lab Clin Med 2000;135:122-8)

The p47phox mouse knock-out model of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is caused by a congenital defect in phagocyte reduced nicotinamide dinucleotide phosphate (NADPH) oxidase production of superoxide and related species. It is characterized by recurrent life-threatening bacterial and fungal infections and tissue granuloma formation. We have created a mouse model of CGD by targeted disruption of p47phox, one of the genes in which mutations cause human CGD. Identical to the case in human CGD, leukocytes from p47phox-/- mice produced no superoxide and killed staphylococci ineffectively. p47phox-/- mice developed lethal infections and granulomatous inflammation similar to those encountered in human CGD patients. This model mirrors human CGD and confirms a critical role for the phagocyte NADPH oxidase in mammalian host defense.