Cystic Fibrosis Research News № 4

January 2022

How Anaerobic Bacteria Drive Disease Progression

To paint a more complete picture of the CF microbiome, researchers are now investigating its more obscure, less well-studied inhabitants - namely, anaerobic bacteria. Anaerobes thrive in poorly oxygenated regions, which are present in the CF lungs due to mucus obstruction. Following a single patient through a pulmonary exacerbation (PEx) – a period of acute lung function loss (typically) requiring hospitalization – a team led by Cynthia Silveira at the University of Miami has demonstrated that anaerobes may actually be a driving force behind rapid lung function decline.

Understanding interactions between bacteria in the CF lungs during PEx, and what microbes in particular are driving lung function decline, can help clinicians prescribe the best set of antibiotics to improve patient lung function. Such endeavors to understand how the complex collection of CF microbes influence each other – and contribute to CF disease progression – have been buttressed by modern -omics techniques. This study employed genomics, transcriptomics, and metabolomics techniques in tandem to analyze samples from a single patient.

Working closely with clinicians in San Diego, the study took advantage of a new protocol called the CF Rapid Response (CFFR). As the patient underwent IV antibiotic treatment to stabilize lung function (first in the hospital and later at home), sputum samples were collected for analysis, as were the results of the patient’s lung function tests performed in the hospital. The resulting data allowed the team to monitor very precisely the patient’s clinical progression over the course of antibiotic treatment, and then to characterize the microbial community residing in the lungs.

The clinicians observed that the patient’s lung function oscillated wildly between the periods of hospitalization and home-care. The patient entered the hospital with a ppFEV1 (lung function reported as a percentile measure relative to the general population) of 44%, which later improved to 59% following treatment with a cocktail of IV antibiotics. However, after discharge and discontinuation of antibiotic treatment, ppFEV1 fell to 50%. The clinicians then prescribed clindamycin, an oral antibiotic, which ultimately stabilized ppFEV1 around 49%.

Unstable, and ultimately declining lung function, is the unfortunate reality for patients with advanced CF lung disease and chronic, polymicrobial infection. The clinical history of the patient in this study is a case in point. Patients frequently see periods of rapid lung function decline that can be stabilized only by hospitalization. In fact, in just the one year prior to participating in the study, this patient had been hospitalized for rapid lung function decline ten times. In such cases, clinicians can prescribe combinations of antibiotics that they believe will work based upon patient history, but they do not always stabilize lung function effectively.

During the course of treatment, 16S rRNA sequencing and full genome sequencing were used to determine which bacteria were present in the patient’s lungs, identifying bacteria by their genetic signature. Following hospitalization and after discontinuing antibiotics (i.e., in the periods of rapid lung function decline), the clinicians discovered that three anaerobic species dominated: Rothia mucilaginosa, Staphylococcus haemolyticus, and Streptococcus sanguinis. These bacteria are not commonly studied, as most researchers tend to work with better known CF pathogens like Pseudomonas aeruginosa or the Burkholderia species.

The ‘classic CF pathogens’ like Pseudomonas are so well studied because they are frequently cultured during routine visits to the clinic. Decades of research have provided substantial evidence that Pseudomonas and Burkholderia infections are indeed associated with worse clinical outcomes for CF patients. The anaerobes are less abundant and less likely to be noted in the clinical record - yet this new research suggests that they could play a very important role during exacerbations.

Especially interesting was the finding that, after examination of metabolomics and transcriptomics data, Pseudomonas may actually depend on anaerobes for its own biofilm growth and virulence factor production. This is due to the fact that the anaerobes are actually better than Pseudomonas at digesting mucus and extracting its nutrients. The researchers demonstrated that after discontinuing antibiotics, Streptococcus sanguinis and Rhodotorula mucilaginosa produced more free amino acids and short-chain fatty acids. Both of these are taken up by Pseudomonas and enhance its transcription of phenazine, a virulence factor that promotes Pseudomonas biofilm formation. These combined factors were associated with lung function decline, which was only halted upon treatment with an antibiotic (clindamycin) known to attack anaerobic bacteria.

As an n-of-1 study, the findings cannot be generalised to the broader population of CF patients. Nonetheless, the results are compelling, as they highlight a new potential approach to treating pulmonary exacerbations. Further studies of Anaerobic species in the CF lungs may help physicians develop more effective antibiotic regimens to treat pulmonary exacerbations.

Featured Article: Silveira CB, Cobián-Güemes AG, Uranga C, et al. Multi-Omics Study of Keystone Species in a Cystic Fibrosis Microbiome. Int J Mol Sci.2021;22(21):12050. Published 2021 Nov 7. doi:10.3390/ijms222112050.

Immune Cells Become Aggressors in the CF Lung

The immune system is impeccably well-suited to defend the human body from infection. Unfortunately, this also makes it prone to over-action. In the CF lungs, immune cells respond earnestly to infection yet are unable to fully eradicate bacteria like Pseudomonas aeruginosa, which form biofilms that are resistant to the immune system and drug treatment. Adding insult to injury, the immune cells (especially neutrophils, which secrete proteolytic enzymes) work so hard to fight infection that they wind up damaging the lungs themselves.

A recent study has shown that there may be something innately different about the CF lungs that helps engender immune dysfunction even prior to bacterial infection. Researchers at the German Cancer Research Center in Heidelberg have found that high levels of abnormal mucus in the lungs of CF patients, and patients with other muco-obstructive diseases like COPD, can alter immune cell activity.

The Heidelberg team focused on a certain set of immune cells called airway macrophages. Prior research on lung tumor-associated macrophages had shown that the tumor environment causes a dramatic shift in the gene expression of these macrophages, severely limiting their capacity to fight cancer. Inspired by this cancer research, the Heidelberg study sought to determine if an abnormal, mucus-obstructed lung environment can also have an effect on macrophages.

The scientists sought to determine the impact of obstructive mucus by extracting macrophages from the lungs of Scnn1b-Tg mice, which is a lab-grown organism beset by mucus plugging and chronic airway inflammation, similar to a human with CF or COPD. These Scnn1b-Tg macrophages were subjected to various forms of genetic sequencing and compared to macrophages from normal, wild type mice.

The researchers first examined epigenetic markers, which are molecular tags that modify gene expression patterns and can change throughout an animal’s lifetime. These tags either help or hinder the underlying genes in DNA from being expressed. Epigenetic analysis has the power to explain how certain phenotypes are driven by environmental factors, either external (like an individual’s diet) or internal (in this case, the abundance of mucus in the lungs).

Two approaches were made to assess the macrophage epigenetic profile. Differential methylation analysis showed how the methyl group, which tends to repress gene expression, was distributed along the DNA of Scnn1b-Tg vs. WT mice. In the DNA of Scnn1b-Tg macrophages, certain regions were discovered that lacked methyl tags (compared to WT mice), and these regions are transcription factor sites that promote an inflammatory response: IRF2, IRF3, and STAT6.

ATAQseq, the second form of epigenetic analysis, was used to look at the compactness of DNA. In the cell, DNA is well-organized, wound like thread around a spool in a complex called chromatin. Using ATAQseq to see how tightly wound, or not, the DNA is indicates how accessible DNA is to transcription (less tightly wound means more accessible, and vice versa). The research team found that genes encoding the inflammatory cytokines TGF-beta, TNF, IFN-gamma, and IL-4, were all more accessible in Scnn1b-Tg mice, further indicating that the macrophages of these mice were predisposed to cause inflammation.

These findings have several important implications: evidence that mucus obstruction makes macrophages more proinflammatory potentially underscores the value of mucolytic (mucus-thinning) drugs such as hypertonic saline and pulmozyme, which are commonly prescribed to CF patients. It also demonstrates the potential value in developing more targeted therapies that modulate macrophage gene expression and make immune cells less destructive to the lungs.

Featured Article: Hey, J., Paulsen, M., Toth, R. et al. Epigenetic reprogramming of airway macrophages promotes polarization and inflammation in muco-obstructive lung disease. Nat Commun. 12, 6520 (2021).

Food Security Impacts CF Patient Health Outcomes

The determinants of health are a mix of personal and environmental factors. The most fundamental personal factor is genetics: CF is caused by a mutation in the CFTR gene, and different types of mutations in CFTR are associated with better or worse outcomes. The genetic determinants of CF health outcomes have been and continue to be well studied, for good reason. The impact of environmental factors, such as home life, neighborhood, or community on CF patient outcomes, however, is less well known.

Food insecurity is one such environmental factor, which according to the US Department of Agriculture (USDA), is a situation in which households have a “limited or uncertain access to adequate food.” Regions where a substantial number of households suffer food insecurity are known as “food deserts”.

A retrospective study conducted by a team of researchers at the University of Pittsburgh has examined the correlation between food insecurity and health outcomes for CF patients under 18 years old. Existing evidence suggests that patients with CF may be more prone than the general US population to food insecurity. This study examined the actual clinical effects of food insecurity, demonstrating that children with CF living in and around “food deserts” are at much greater risk of having a suboptimal (low) BMI and low lung function relative to CF patients of similar age and socioeconomic status who have greater access to food.

CF patients living in a food desert are highly vulnerable, given the tendency of CF patients towards malabsorption of nutrients, a difficulty gaining weight, and consequent higher caloric requirements. A recently published study of 1,426 healthy infants (born in Tucson, AZ and followed from the 1980s to the 2010s), found that poor nutrition in childhood predicted diminished lung function in adulthood. For people with CF, who struggle with diminished lung function for a number of reasons (chronic lung infection, systemic inflammation, etc.), poor weight gain in childhood is likely to make this already bad situation even worse.

The results of the Pittsburgh study found that people with CF living in or within 600 feet of a food desert were four times more likely to have a low BMI and 3 times more likely to have significantly poorer lung function (FEV1).

This study focused on a single region of the US, with patients located in Pennsylvania, West Virginia, or Ohio. Researchers hope to learn more from other geographic locations in the future. This study demonstrates the value of closely monitoring patient nutrition and providing early intervention when access to food is insecure. To this end, there are already funds that provide CF patients with nutritional support, such as the Cystic Fibrosis Relief Fund, but increased awareness of food insecurity might further help the most vulnerable members of the CF community.

Featured Article: Corbera-Hincapie MA, Kurland KS, Hincapie MR, et al. Geospatial Analysis of Food Deserts and Their Impact on Health Outcomes in Children with Cystic Fibrosis. Nutrients. 2021;13(11):3996. Published 2021 Nov 10.

Featured Article: Voraphani N, Stern DA, Zhai J, et al. The role of growth and nutrition in the early origins of spirometric restriction in adult life: a longitudinal, multicohort, population-based study. Lancet Respir Med. 2021;S2213-2600(21)00355-6.

Featured Five CF Stories

It’s impossible to list all of the amazing research that is on-going for CF. Below is a quick list of a few fascinating articles that seem to show significant promise.

CF Cellular Pathways: A new app allows researchers to explore virtual models of human cells and trace their way through pathways that impact the translation, modification, and cell surface localization of the CFTR protein. The app will be updated continuously as the contours of CFTR-related pathways are further illuminated by new research. (Nature Scientific Reports).

Epidemiology of an Emerging CF Pathogen: In France a study reveals the physical and economic costs of infection with nontuberculous mycobacteria (NTM). The researchers examined data retrospectively from seven years of patient treatment (2010-2017) and concluded that NTM infection is associated with multiple comorbidities including malnutrition and gastro-esophageal reflux. Furthermore, individuals infected with NTM incurred roughly 8x more medical expenses in the year following infection compared to the rest of the population. (BMC Infectious Disease).

Trikafta: A Drug That Shows PROMISE: Researchers have published results from the PROMISE study, and confirmed what was pretty clear already from anecdotal evidence: patients treated with Trikafta see significant improvements in lung function as well as an improved (greater) BMI. (American Journal of Respiratory and Critical Care Medicine).

Lung Function Testing in the Home: The past two years have seen many new clinical developments – with new forms of diagnosis, monitoring, and treatment emerging. In a recent study, 74 people with CF were instructed to perform in-home spirometry (lung function testing) on a small device without supervision. The researchers wanted to know if the CF subjects could produce consistent, high-quality results… and they did – the vast majority of study received a score of ‘excellent’ or ‘very good’ for spirometry quality without supervision. (Journal of Cystic Fibrosis).

CF and Mental Health: The results of a recent survey reveal that for people with CF, the ability to practice self-compassion (kindness towards oneself and away from self-judgment) is associated positively with individual quality of life. Now, researchers and clinicians may develop psychological interventions that can help people with CF learn to improve their capacity for self-compassion. (Journal of Clinical Psychology in Medical Settings).

Clinical Trial Watch

The latest news on CF drug development and clinical trials.

Recruiting: Pulmonary exacerbations for people with CF are defined by rapid lung function decline and tend to spark long-term lung damage. Even after antibiotic treatment, lung function often does not recover to baseline values. A new treatment for hospitalized CF patients may offer a better prognosis for recovery – researchers are testing ketone supplementation in a new trial, with the hopes that it helps stem inflammation and improve lung function more substantially during pulmonary exacerbations. (University of Alabama at Birmingham).

Recruiting: Infection with the CF pathogen Mycobacterium abscessus (a type of NTM bacteria) is on the rise, and studies suggest that M. abscessus can do a lot of damage to the CF lungs. A new trial is seeking to test a nebulized nitric oxide treatment for those with hard-to-treat M. abscessus infection. The study will establish if the nebulized therapy is well tolerated by patients and whether or not it may impact the amount of bacteria growing in the lungs. (Papworth Hospital NHS Foundation Trust, Cambridge, UK).

Recruiting: Aside from lung disease, CF-related diabetes (CFRD) is perhaps the most pressing clinical challenge for CF patients. Researchers want to know what factors predict the onset of CFRD in the years leading up to its diagnosis. Standard procedure for screening is to conduct a simple finger-prick test every year and monitor patient blood sugar. This new trial is recruiting patients to test a more sensitive screening approach known as continuous glucose monitoring. Once each year over a period of three years, patients will wear a continuous glucose monitor for 14 days, providing valuable data and potential clinical biomarkers for the onset of CFRD. (Institut de Recherches Cliniques de Montreal, Canada).

Recruiting: Airway clearance can be quite cumbersome, requiring people with CF to stay immobilized for an hour or more each day. A new trial will show whether or not a more convenient, portable airway clearance device (one that a person with CF would breathe into rather than wear) is just as effective as traditional airway clearance techniques. (University of Florida).

Funding News

Recent funding for CF researchers or companies.

Cystic Fibrosis Foundation awards $21.5m for CF research: CFF awarded $766,000 to Carmine Inc for non-viral gene therapy research. GenexGen Inc received $595,000 for their therapy that may blocm immune response to adeno-associated virus. Specific Biologics Inc was awarded $527,000 to test their gene editing technique on the most common nonsense mutations, and Pulmocide received an investment of $3.5 million for development of Novel antifungal treatments. (Cystic Fibrosis Foundation).

Polyphor completes merger with EnBiotix, renamed Spexis: Polyphor AG and EnBiotix Inc. announced the completion of the merger of the two companies and the new combined company will be called Spexis AG. The combined pipeline includes front-line therapy ColiFin, and an inhaled antibiotic specifically targeting Pseudomonas aeruginosa. (Spexis AG).

Biofilm Matrices for Novel Antibiofilm Strategies: Cornell University researcher John W Brady was awarded $284,262 by the NIH for research in methods to characterize the biofilms formed by opportunistic pathogen species belonging to the Burkholderia cepacia Complex (BCC) and by Klebsiella pneumoniae. (PMC).

Selenocyanate as a novel CF treatment: Brian J Day, an investigator at National Jewish Heath in Denver, was awarded a grant of $358,087 for research on the mechanism(s) by which infection-mediated inflammatory processes drive progressive lung disease, and may be treated by novel targeting of prokaryotic Cys-TrxR by HOSCN/HOSeCN. (PubMed).

A Call to Action

Cystic fibrosis (CF) research is very much dependent on the strength of the CF community. It’s not simply an effort carried out by scientists in white lab coats - although there are many of them, and their work has enormous impact. Advances in research also depend on the technicians and engineers who operate the laboratory equipment that enables drug discovery, and the industrial machinery that allows drug development. Research depends on both business and marketing professionals, those who make biopharma companies viable and promote clinical trials. Successful research further depends on clinical trial coordinators, who carry out studies and work tirelessly to recruit and support patients throughout the complicated trial process. Particularly for rare diseases like cystic fibrosis, research depends on the work of foundations and patient advocates, which includes in the United States organizations such as the CF Foundation, Emily’s Entourage, CFRI, and the Boomer Esiason Foundation, as well as countless other across the globe, and hundreds of committed clinicians and researchers. Most importantly, research depends on people with CF and their devoted families and friends.

There can be no progress in CF research without patients willing to participate in clinical trials: not only to test new drugs, but also to provide, quite literally, their flesh and blood. It is with the help of patient samples that scientists can understand the damage that CF inflicts upon the human body, and also how drugs developed by the research community can remedy these damages.

This newsletter aims to pull all of these threads together; allowing the CF community to more fully appreciate how well the aims of its many members are aligned (and it extends an invitation to all readers not yet a part of the CF community, to embrace the cause and take up the task of pushing CF research forward). There’s something here for everyone - those interested in the clinical side of CF care, or in drug development, or the technical work performed in CF-centered laboratories. The newsletter also has as its objective to showcase new clinical trials; an opportunity for patients and clinicians to take part. Wherever and whoever you are in the world, you too may push CF research forward - either by direct participation, or simply by reading and sharing this newsletter with others.