2021 American Thoracic Society BEAR Cage Winning Proposal: Microbiome Transplant in Pulmonary Arterial Hypertension.

What Is the Evidence that the Gut Microbiome Plays a Role in Pulmonary and Cardiovascular Disease?

The gut microbiome is integral to host physiology, including metabolism and immunity (1, 2). Interest in how the microbiome impacts chronic diseases, including chronic obstructive pulmonary disease, asthma, heart failure, idiopathic pulmonary fibrosis (3–5), and others, has been growing. Fecal microbiomes in patients with chronic obstructive pulmonary disease differ in relative abundances of several bacterial species and microbial metabolites compared with healthy control subjects (6). Lower fecal microbiota diversity, assessed by 16S rRNA gene sequencing within the first year of life, correlates with asthma development by age 7 years (7), and infants deemed to be at risk for asthma development have lower levels of the bacterial-produced, antiinflammatory, short-chain fatty acid (SCFA) acetate in their feces (8). Mice supplemented with SCFAs by including acetate in their drinking water develop significantly reduced lung inflammatory cellular infiltrates, whereas mice fed a low-fiber diet have reduced levels of SCFAs in their circulation and increased airway hyperreactivity (9). Trimethylamine N-oxide (TMAO), a bacterial-derived metabolite produced during red meat digestion, has been linked to stroke and myocardial infarction, and in vitro studies have shown that TMAO increases platelet activation and aggregation (10). Mice given chow supplemented with TMAO have significantly reduced cardiomyocyte transverse tubule power and left ventricular ejection fraction compared with control mice fed normal chow (11). These studies make a strong argument for the characterization of the gut microbiome to influence the development of novel treatments.

Is There Evidence in Pulmonary Arterial Hypertension for a Gut–Lung Axis?

Pulmonary arterial hypertension (PAH) is characterized by perivascular lung inflammation and pulmonary vascular remodeling, resulting in increased pulmonary vascular resistance. An increase in right ventricular afterload leads to right-sided heart failure and, ultimately, death. Despite the development of pharmacologic therapies, PAH mortality has not significantly improved (12–14). Although inflammation plays a mechanistic role in PAH, the underlying factors causing it remain unclear. Inflammation in PAH could be driven by an imbalance of pro- and antiinflammatory intestinal microbial metabolites, cytokines, and other mediators and/or direct effects of circulating bacteria all stemming from dysbiosis, gut-barrier dysfunction leading to increased permeability of metabolites and/or bacteria, and, possibly, decreased hepatic filtration of inflammatory gut microbial metabolites. There is evidence that PAH development is, in part, linked to the gut microbiome. One possible etiologic mechanism is the direct translocation of bacteria or bacterial components from the gut into the circulation (15). Supporting this is the finding that mice heterozygous deficient for the BMPR2 (bone morphogenetic protein receptor type II) allele develop PAH after acute exposure to LPS, whereas wild-type mice do not (16). BMPR2 germline mutations are found in 20% of idiopathic and 80% of heritable PAH cases (17). Exposure to LPS was associated with increased cytokine secretion (both in the murine model and in vitro with cultured pulmonary artery smooth muscle cells from humans and mice with PAH) (18). Therefore, TLR4 (Toll-like receptor 4), the receptor for LPS, is thought to play a pivotal role in the development of PAH. Supporting this proposal, it was found that TLR4-deficient mice are resistant to developing PAH in a hypoxia model (16). Supporting a role for gut permeability in PAH, rats treated with monocrotaline that develop PAH have increased levels of systemic intestinal fatty acid binding protein (a marker of gut permeability) (19), small intestinal fibrosis (19), and increased Firmicutes-to-Bacteroidetes ratio compared with control rats (19). The sugen-hypoxia rat model of PAH also replicates an increased Firmicutes-to-Bacteroidetes ratio (20, 21). Further strengthening the association of how the gut microbiota may augment the development of PAH, rats given antibiotics before and after sugen injection and during hypoxia develop significantly reduced right-ventricular systolic pressure and reduced vascular remodeling of pulmonary arteries compared with rats only treated with sugen and hypoxia (21). It is unknown if these effects are causal in promoting the development of PAH or are a bystander effect of gut hypoxia or hypoperfusion, and further research is needed to answer this question. However, these studies suggest that the microbiome does play a role in either the development or advancement of PAH. Thus, we hypothesized that PAH is characterized by gut dysbiosis, leading to altered intestinal permeability and an altered burden of circulating microbial metabolic products promoting perivascular inflammation and PAH.

How Is the Human Gut Microbiome in PAH Different from that of Healthy Control Subjects?

An analysis of fecal microbiome compositions of patients with PAH versus a reference cohort using shotgun metagenomics sequencing has shown distinct differences when ecological metrics are measured, including decreases in both richness and diversity as measured by the Shannon and Simpson indices, decreases in evenness (less equal distribution in relative abundances of all species), and distinction in relative abundances of the specific species present (22). Providing further evidence that the microbiome influences disease in PAH, this study found that the PAH cohort had increases in species that are positively correlated with TMAO production and decreases in species that are positively correlated with SCFA secretion compared with the reference cohort (22).

How Can This Knowledge Be Applied to Treat PAH?

We plan to determine if microbiota transplant therapy (MTT), also known as fecal microbiota transplant, can treat PAH. This therapy uses purified bacteria from fecal samples from rigorously screened healthy donors that are stringently tested for infection-causing pathogens and then lyophilized and placed in capsule form to be taken by mouth. MTT is a safe treatment for Clostridioides difficile colitis (23) and is safe in immunocompromised patients (24). The oral route of administration will allow for repeated dosing, which we anticipate will be important in the absence of antibiotic conditioning that creates an ecological space for engraftment.

To test MTT, we aim to determine its safety and feasibility in PAH. Our group has a phase I safety and feasibility trial for MTT in PAH approved by the U.S. Food and Drug Administration. In this open-label trial, 12 patients diagnosed with PAH will receive once-daily MTT capsules for 7 days. Our primary endpoints will be safety and feasibility. To address these endpoints, we will be monitoring patients for success in taking capsules and occurrence of adverse events. Patients will be monitored daily for 2 weeks and then monthly for 6 months. A timeline of this study is shown in Figure 1. Although we will not be selecting patients for the study based on baseline microbiome characteristics or on specific PAH treatments, if safe and feasible, future randomized placebo-controlled trials can help determine how MTT impacts disease based on baseline microbiome characteristics and how various pharmacologic therapies may be impacted or may impact the efficacy of MTT (as bacterial metabolism can affect metabolism of pharmacologic therapies [25]). Future, randomized placebo-controlled trials can also determine how MTT can impact concurrent pharmacologic therapeutic effects on secondary hemodynamic endpoints.

Figure 1.

Timeline of open-label phase I clinical trial of microbiota transplant therapy in pulmonary arterial hypertension. ECHO = echocardiogram; MTT = microbiota transplant therapy; QOL = quality of life.

What Would Success Look Like?

We aim to show that MTT is both safe and feasible in PAH. If achieved, future clinical studies can test the efficacy of MTT in PAH, using a randomized, placebo-controlled, double-blind trial. Our initial safety and feasibility trial will allow for power calculations for our randomization through the collection of exploratory secondary endpoints. These include changes in PAH disease markers, such as right-ventricular function and pulmonary artery systolic pressure measured by echocardiography, exercise tolerance measured by a 6-minute-walk test, and a quality-of-life survey (validated emPHasis-10) (26) before and 6 months after MTT.

Stool samples from patients with PAH undergoing MTT in our phase I safety and feasibility trial will be collected before MTT and at Months 1 and 6 after MTT to study engraftment of donor microbiota. Engraftment measures how much of the donor bacteria remain in the microbiomes of patients with PAH to inform pharmacokinetics and dosing for our efficacy trial.

Conclusions

Though strides have been made in understanding the pathogenesis of PAH, and pharmacologic therapies have been developed that improve morbidity and mortality, PAH remains incurable. Therefore, novel approaches to characterize and treat this disease are needed. Our study is the first step to investigating MTT as a treatment for PAH. Additionally, future studies may also allow for the identification of a specific microbiome signature that can be used as a predictive biomarker or outcome biomarker for PAH. In future studies, if MTT succeeds or improves the efficacy of established pharmacologic therapies, MTT may become a cornerstone for an easy, nontoxic therapy to improve outcomes in PAH.

The BEAR Cage Innovation Award

Currently in its seventh year, the BEAR Cage (Building Education to Advance Research) Competition, sponsored by the American Thoracic Society Drug Device Discovery and Development Committee, provides early career investigators with the opportunity to pitch a new technology as an innovative solution to a pressing human health need. Finalists receive feedback from members of academia and industry, opening doors for further project development and collaborations. The winner of the BEAR Cage Innovation Award benefits from dedicated mentorship with the goal of accelerating clinical translation and, ultimately, impacting patient care.