Conclusions and Proposals for Future Work

Gene therapy

The CRISPR/Cas9 system is a prokaryotic defense mechanism that in bacteria confers resistance to foreign DNA by recognizing and cutting specific sequences. CRISPR/Cas9 has also been used in recent research as a low-cost genome editing tool with tremendous potential. Given that CGD is a disease of a single genetic anomaly, it might be an easy target for Cas9 mediated repair. In fact, previous studies have given a proof-of-principle approach to gene therapy using CRISPR/Cas9 in induced pluripotent stem cells, showing that genotypic and phenotypic correction of CGD is possible (Flynn et al. 2015). Future avenues of research must include a delivery mechanism for CRISPR/Cas9 systems into bone marrow for correction of hematopoietic stem cells. Possible research could involve the application of micelles expressing certain bacterial or fungal lipids or antigens such that they are phagocytosed, which would allow the CRISPR/Cas9 system to enter the cell.

crispr-infographic
Figure describing the process of CRISPR/Cas9 genetic engineering in 4 simple steps. Genetic engineering is a promising therapy for monogenic defects such as CGD but is challenged by the difficulty of intracellular delivery. Obtained from Business Insider.

 

Prevention of granuloma formation

A component of CGD pathology is the formation of chronic granulomata. However, translational research into manipulating the underlying mechanisms of granuloma formation is lacking. Yepes et al. recently explored a combination therapy of edelfosine and praziquantel in schistosomiasis, a parasitic disease that stimulates granulomatous reactions by the immune system (Yepes et al. 2015). Praziquantel kills the schistosomes, which treats the parasitic infection. Edelfosine, however, is an oncodrug that targets the Fas5 death receptor. Edelfosine in combination showed a significant size reduction in granulomata and a downregulation of Th1, Th2 and Th17 post-infection responses (Yepes et al. 2015). This research, although seemingly unrelated to CGD, shows a drug effecting the cellular immune response that underlies granuloma development. If a drug were to be developed with a similar mechanism of action and was shown to function in CGD patients, it could be used to reduce the severity of chronic granuloma formation and prevent blockages in hollow organs.

Increasing intracellular peroxide

Thyroid hormones are synthesized in the presence of hydrogen peroxide (Ohye and Sugawara 2010). The synthesis of hydrogen peroxide occurs by the enzyme dual oxidase 2 (DUOX2). The DUOX family is closely related to the NADPH oxidases but is expressed exclusively in the thyroid (Ohye and Sugawara 2010). The formation of superoxide by NADPH oxidase is as a precursor to hydrogen peroxide, which is converted into antimicrobial compounds by myeloperoxidase. Because CGD causes a NADPH oxidase defect that prevents superoxide production, it may be interesting to examine whether expressing DUOX2 in phagocytes would confer antimicrobial activity, since hydrogen peroxide is also a precursor to antimicrobial compounds generated during respiratory burst. If possible, the expression of DUOX2 in CGD-derived phagocytes would likely restore the ability to kill invading pathogens.

Flowchart indicating the various transitions from molecular oxygen (O2) to antimicrobials (HOCl, ONOO) and strong oxidants (OH). Notice that hydrogen peroxide is an intermediate between superoxide and HOCl, and can also be produced from molecular oxygen by the enzyme DUOX. Obtained from Google Images.
Flowchart indicating the various transitions from molecular oxygen (O2) to antimicrobials (HOCl, ONOO) and strong oxidants (OH). Notice that hydrogen peroxide is an intermediate between superoxide and HOCl, and can also be produced from molecular oxygen by the enzyme DUOX. Obtained from Google Images.

10 Replies to “Conclusions and Proposals for Future Work”

  1. Great job on this project Elliot. Your idea of using a alternative molecular oxidant in place of the mutated one is quite creative and interesting! I think the challenge in this would be how to encode the cell to trigger the activation of DUOX in the proper context and at the proper amounts.

    I have a few questions. One, since patients with CGD are on life long daily antibiotic and anti-fungal therapy has there been evidence of bacterial resistance in the patient population? One thought I had would be regarding the formation of the granulomata, I understand that the phagocytes and pathogens accumulate together, however, how is the fibrous coating formed? Is it based off of the tissue surrounding the accumulated cells or by deposition of another material on top of the mass? If so could this be a target for reducing the formation of granulomata?

    1. Thanks Elaine, I agree that expressing DUOX sounds easy but the practical challenges are immense! In response to your questions, I have come across a couple of cases of antibiotic and antifungal resistant infections in CGD patients. These infections are often more severe and require drastic treatment, even surgical intervention.

      With regard to your second question, granuloma formation is the result of a gradual accumulation process, wherein a variety of immune cells can join in the growing mass in addition to the macrophages. Often the cause of a granuloma can be determined by examining the cells comprising it. However, the fibrous coating is generated when the body reacts and tries to repair a site of infection it believes is damaged. This is essentially a dump of excess connective tissue in the form of extracellular matrix proteins. If you are interested, there is recent research linking fibrosis to epigenetic determinants such as DNA methylation: http://fibrogenesis.biomedcentral.com/articles/10.1186/s13069-015-0035-8. Perhaps by modulating this epigenetic pathway we might reduce the fibrosis associated with chronic granulomata.

  2. Very nice and through work in your discussion of CGD. I really liked how you demonstrated how the metabolic basis of disease can result in immunologic consequences as a result of genes associated with CGD.

    A couple of comments/discussion points:
    1. It’s great that you addressed CRISPR/CAS9 to correct an inborn error of metabolism. Although this system may be far off from actual implementation as a therapeutic, the potential of this type of intervention is huge and I commend you for introducing this point.

    1. Although CGD patients clearly have immune-deficiencies, are there any metabolic advantages associated with inability to make radical oxygen species? Are there defects in cell death mechanisms that permit neutrophils (typically a shorter lived population in comparison to other immune cells) and other inflammatory infiltrates persist?

    Very nice job, it was an enjoyable read for this immunologist!

    1. Hello and thank you for your interest in my project! Because ROS are associated with oxidative damage, degenerative diseases, and aging, I agree that there may be some subtle metabolic advantages to a less oxidative cellular environment. However, ROS are also necessary for intracellular signalling, detoxification by cytochrome p450, and apoptosis. In an almost paradoxical way, ROS are potentially carcinogenic by damaging DNA and potentially therapeutic by inducing apoptosis of cancer cells. In this case, I agree with your suspicion that ROS-deficient neutrophils may outlive expectations by a low level of cellular ROS which mediate apoptosis. However, these benefits are clearly outweighed by the lack of immunity associated with CGD.

  3. Well done. This is really interesting. It sounds not too far off from sarcoidosis, a poorly understood autoimmune disease. I wonder if there’s any crosstalk between the two in terms of treatment? Also, granulomas show up occasionally in common variable immunodeficiency (CVID) patients. These patients, who have chronic bacterial infections, suffer an indirect effect of their condition known as T cell exhaustion (http://jem.rupress.org/content/211/10/2033.full), which could make CGD patients even more susceptible to infections down the line. I would also really like to know about CGD patients’ microbiota/gut flora. It’s rare to have a cohort on such a rigorous, long term antibiotic treatment.

    1. Hi Androo,

      Thank you for your interest in my project. I had read a bit about sarcoidosis when learning about granuloma formation as it shares symptomatic granuloma infection with CGD. However I haven’t found any resources on overlapping treatments for these two disorders. With regard to T cell exhaustion, I think it is interesting that your source finds exhaustion of bacteria-specific T cells and no exhaustion of viral-specific T cells, because granulomata do not form in a response to viral infections and because CGD patients can typically clear viral infections just as well without NADPH oxidase.

      When I was researching and writing this project, I remember that a distinct advantage of Co-trimoxazole over other antibiotics was that it generally spared the gut microbiome. However, CGD may also affect the microbiome in a more innate way, as the genotype associated with CGD has been shown to affect the intestinal microbiomic signature of p47phox(-/-) mice. The difference in microbiomic composition has been associated with susceptibility to symptoms of colitis. See this research for more information: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820915/

  4. Great presentation of the disease Elliot. Your explanation of a lot of the immunological aspects of the disease seems to be very strong with a lot of good description! When reading the project, I was very interested in the role of ROS since it is a topic we have covered in class regarding the relationship between ROS and some neurodegenerative disorders. In terms of proposals, you mention the possibility of expressing DUOX2 in phagocytes in an attempt to restore their ability to destroy invading pathogens through the fact that DUOX2 causes for the synthesis of hydrogen peroxide, and in turn ROS and other forms of oxygen antimicrobials. I was curious if there could possibly be any adverse side effects to an experiment like this due to the fact that increased ROS could potentially cause for a lot of oxidative stress and metabolic defects? While you show that CGD results from lower levels of ROS, have you come across any of the literature that shows any negative effects of trying to increase ROS by possibly going too far in terms of trying to balance ROS in the body?

    1. Hey Tyler, you bring up an interesting point. I actually did find a paper quite recently that linked overproduction of ROS to the development of osteoarthritis, specifically ROS from NADPH oxidase and mitochondrial Complex II. The authors first determined that folate deficiency cause increased production of ROS in synoviocytes which correlated with their apoptosis. When treated with NADPH oxidase inhibitors, synoviocytes were protected from apoptosis. These results imply that overproduction of ROS may contribute to osteoarthritis as well as many other diseases. If you want to take a look at the study, you can find it here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4714898/

  5. Congrats on a well-written and interesting project Elliot! My project was on tuberculosis, and I read a lot about the formation of granulomata in regards to that disease. Your proposals for future work were really interesting, and I was curious about any information you found in the literature about decreasing the amount of granulomata, not just in size but in amount. You mention that edelfosine and praziquantel together led to a reduction of granulomata size. Was there any indication that it would be possible to completely get rid of a granuloma, thereby reversing the symptoms of the disease, or even the disease itself?

    1. Hi Nikki, thanks for your comment! I also appreciated the amount of crossover between our projects, if you noticed most of the figures I found depicting granuloma involve latent tuberculosis infection. I believe that the reduction in granuloma size brought on by edelfosine/praziquantel was partially a result of treating the underlying parasitic infection in that study. However, edelfosine targeting of the Fas5 death receptor was interesting because it may target the cellular process underlying granuloma formation. The results imply that it might be possible to prevent the formation of granulomata and thereby reduce their amount.

Leave a Reply

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.