Bacillus Calmette-Guerin Infection in NADPH Oxidase Deficiency: Defective Mycobacterial Sequestration and Granuloma Formation

Abstract

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.

Susceptibility of Mycobacterium abscessus to Antimycobacterial Drugs in Preclinical Models

“…The immune systems of the following mice were capable of clearing M. abscessus: the beige mice with a dominant Th2 immunity; the Nos2 Ϫ/Ϫ mice lacking NO2; Cybb Ϫ/Ϫ mice, which are devoid of the superoxide-generating enzyme that forms the reactive oxygen species (ROS) associated with chronic granulomatous disease; TNFR Ϫ/Ϫ mice, which lack the tumor necrosis factor receptor; C3HeB/FeJ mice capable of forming tubercular granulomas and GKO Ϫ/Ϫ deficient in IFN-␥; and the MyD88 knockout mice, with an allele that encodes a deletion of exon 3 of the myeloid differentiation primary response deleteriously impacting innate and acquired immunity causing increased susceptibility to bacterial pathogens (19)(20)(21)(22)(23). After 40 days of infection, the C3HeB/FeJ, GKO Ϫ/Ϫ , and MyD88 Ϫ/Ϫ mice had bacterial loads of about 2.0 to 2.5 log 10 persisting in the lungs.…”

“…Mice with M. abscessus infection that have the MyD88 gene knocked out and a loss of NF-B activation were shown to be still capable of bacterial clearance. Additionally, Cybb knockout mice, a mouse model of chronic granulomatous disease (CGD), have a recessive disorder characterized by a defective phagocyte respiratory burst oxidase required for mycobacterial intracellular killing, but the lack of this oxidase does not allow for RGM survival (20). Moreover, targeting more essential genes for the cytokines required for clearance of M. tuberculosis and even Mycobacterium avium, such as the tumor necrosis factor receptor (TNFR) and IFN-␥, two cytokines essential for acquired immune removal of more virulent pathogens when knocked out, allowed for early RGM persistence but eventually resulted in clearance (21).…”

Immunometabolism within the tuberculosis granuloma: amino acids, hypoxia, and cellular respiration

“…Using mouse models of chronic granulomatous disease (CGD, loss of NADPH function) and a high dose intravenous M. bovis BCG infection, Deffert et al described a critical role for macrophage ROS production that could not be compensated by production of these species by neutrophils [92]. The authors selectively rescued ROS production in macrophages in Ncf1 −/− mice (human CD68 driven wild type Ncf1 , encoding the p47 phox subunit of NADPH oxidase), but not in neutrophils, and saw that the rescued mice were protected from BCG-induced weight loss and death observed in the CGD control mice, even though bacterial clearance was not drastically altered.…”

Mycobacterial infection induces a specific human innate immune response

“…The phagocyte NAPDH oxidase is responsible for generating reactive oxygen species, which are utilized to fight intracellular pathogens [36][37][38][39][40][41]. These reactive oxygen species may also serve a signaling role in activating other immune cell types to ensure proper granuloma formation and killing of mycobacteria [42]. Loss-of-function mutations in subunits of the NAPDH oxidase cause chronic granulomatous disease (CGD) [41], which is characterized by the formation of granulomas throughout the body due to the inability of phagocytes to kill the ingested pathogens.…”

“…Loss-of-function mutations in subunits of the NAPDH oxidase cause chronic granulomatous disease (CGD) [41], which is characterized by the formation of granulomas throughout the body due to the inability of phagocytes to kill the ingested pathogens. In contrast to wild type animals, mice with mutations in subunits of the phagocyte NAPDH oxidase develop tuberculosis after infection with the vaccine strain, BCG [42]. Humans who are administered the vaccine before being diagnosed with CGD also develop the disease [41].…”