Response: Protecting against Aspergillus infection in CGD

We would like to thank Remijsen and colleagues for their comments regarding our recent publication in Blood titled “Restoration of NET formation by gene therapy in CGD controls aspergillosis.”¹ 

We regret that we neglected to cite the work of Romani et al.²  This was because the “Brief Report” format in Blood is restricted in length and number of citations. Romani et al reported that superoxide produced by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase regulates indoleamine 2,3-dioxygenase (IDO), which leads to L-kynurenine formation and eventually to the activation of interleukin-17 (IL-17)–producing T cells, which are involved in the acquired immune response. Indeed, treatment with L-kynurenine and interferon-γ helps resolve Aspergillus infections in p47^phox-deficient mice, a murine model of chronic granulomatous disease (CGD).

We analyzed the level of L-kynurenine and IL-17 in sera collected from the CGD patient described in our publication¹  before, as well as 11, 17, and 98 days after, gene therapy (GT). There were no significant differences in L-kynurenine levels in the sera before and after GT as measured by high-performance liquid chromatography.³  The concentration of IL-17 in sera was less than the detection limit of 30 pg/mL of the enzyme-linked immunosorbent assay (R&D Systems) at all time points analyzed. Because of ethical considerations, we did not analyze local L-kynurenine and IL-17 levels in tissue biopsies, and therefore cannot exclude a local effect of the IDO pathway on Aspergillus infection.

The complex clinical phenotype in CGD patients reflects the pleiotropic functions of the NADPH oxidase in immune defense. It is likely that several mechanisms, including the activity of IDO, contribute to the restoration of immune defense after GT, and it would certainly be important to address this point if the opportunity arises. Treatment with L-kynurenine, however, has not yet been tested in CGD patients because of concerns about its epileptogenic potential.

The high incidence of aspergillosis compared with other opportunistic infections in CGD patients is indeed difficult to explain. It would certainly be interesting to survey how neutrophils isolated from these patients respond to a spectrum of different opportunistic microbes. Interestingly, knockout mice in any of the NADPH oxidase subunits seem susceptible to diverse microbial insults. Regardless, it is clear that the NADPH oxidase, as a part of the innate immune system, is initially important in microbe clearance, through, for example, phagocytosis and neutrophil extracellular trap (NET) formation. This enzyme, as elegantly shown by Romani et al, is also important in the second function of innate immunity, which is to set the stage for an acquired immune response.

We reported the acquisition of in vitro anti-Aspergillus activity of NETs after GT. This is in line with the observations that fungi are more susceptible to NETs than to phagocytic killing.⁴  More importantly, the patient started to get better within few days after engraftment of gene-transduced cells, suggesting an immediate innate response. The role of NETs in the recovery of the patient will remain a correlation since we reported the in vitro function of human cells; in vivo experiments are obviously out of the question. It is likely that the improvement after restoration of NADPH oxidase activity in the CGD patient reported was due to several pathways where this enzyme is involved. Our data, however, suggest that NETs played a prominent role in the clearance of the Aspergillus infection.