NEW YORK – After making significant strides in lowering solid organ transplant rejection rates through use of pharmaceuticals and nucleic acid-based organ health monitoring assays, scientists are beginning to forge a path toward even further improvement by leveraging the microbiome.
Acute kidney transplant rejection rates in the US have declined from over 50 percent in the 1970s to between roughly 10 percent and 20 percent today. Lung transplant rejection rates are slightly higher, between 20 and 30 percent, and heart transplant rejection rate estimates vary, with one recent study funded by the National Heart, Lung, and Blood Institute estimating an overall rate of roughly 23 percent.
In an effort to lower these numbers even further, scientists have begun looking at how changes in a recipient's microbiome correlate with –– and possibly directly impact –– acute rejection, the idea being that altering these microbial shifts could lead to better outcomes.
Although no commercial enterprise appears to have begun developing microbiome-targeted allograft rejection tests in earnest, research continues to build evidence for their clinical utility, suggesting they may become commercially viable in the not-too-distant future.
Immune system modulation
Much of the evidence for the importance of the human microbiome –– and the gut microbiome in particular –– comes from studies demonstrating the integral role it plays in activating and modulating the body's immune responses.
The relationship between gut microbiota and the immune system begins early on, with the microbiome playing key roles in the development and modulation of the mucosal innate and adaptive immune system. It also plays an important part in protecting against pathogens by maintaining gut integrity and regulating permeability of the intestinal barrier. When the composition of the gut microbiome is altered, the resulting dysbiosis can change the immune system and increase one's susceptibility to pathogenic organisms.
Researchers are working to identify the biological pathways through which dysbiosis impacts allograft rejection, with the bulk of the investigations focusing on the kidney, which is the most frequently transplanted organ worldwide. Although the study of relationships between the microbiome and allograft rejection remains somewhat niche, it has seen a significant rise in interest over the past 10 years, with fewer than a dozen papers published per year prior to 2015, after which the number of publications per year rose rapidly to close to 200 by 2024, according to PubMed.
"The research, while increasing in its amount, is really still in its infancy," said Sangeeta Bhorade, chief medical officer of organ health at Natera, which sells the Prospera cell-free DNA blood test for kidney transplant rejection.
The high degree of variability between individual microbiomes, Bhorade explained, makes it hard to generalize many findings in a way that enables the development of commercial microbiome-based tests.
Despite the nascent state of research, Bhorade does see some advances pushing the field forward. Where researchers had initially focused almost solely on understanding the composition of the microbiome, cataloging which microbes are more prevalent than others, for example, and measuring the relative amounts of the various taxa, she has observed a shift toward more functional biology. Researchers are increasingly zeroing in on the metabolites produced by commensal microbes, for example, and how those metabolites may impact the immune system.
"In the beginning, it was really about which bugs are more prevalent with decreased rejection," she said. "Now [we're] trying to understand why those bacteria might be associated with less rejection."
Dysbiosis and graft survival
One intriguing line of research aimed at why dysbiosis can impact graft rejection revolves around the effects of immunosuppressive drugs on the microbiome's ability to produce short chain fatty acids (SCFA).
Immune suppressants are integral to the success of organ transplantations. They reduce the risk of the recipient's immune system reacting to the donor organ and causing rejection. They are also often prescribed in conjunction with antibiotics, and both can significantly alter the recipient's microbiome.
These medications significantly disrupt the microbiome by altering the balance of bacteria within the body, often decreasing the proportion of beneficial bacteria and increasing that of potentially harmful microbes. The resulting dysbiosis can further compromise the immune system and correlates with increased post-transplant mortality.
For example, mycophenolic acid (MPA), sold under the brand names Myfortic and CellCept, is one of the most widely used immunosuppressive drugs in transplantation.
MPA is the active metabolite of the prodrug mycophenolate mofetil (MMF) and can significantly impact SCFA production within the gut microbiome. In a mouse model, reduced SCFA production has been observed to contribute to the pathophysiology of mycophenolate-induced gastrointestinal issues, which was reversed by SCFA supplementation.
Because of this crosstalk between immunosuppressants and microbial metabolites, some researchers see the use of these medicines as a double-edged sword, causing extensive gut dysbiosis by damaging the gut microbiome, which weakens the immune system, while also keeping the recipient alive by preventing immune-mediated organ rejection.
Roland Lawson, associate professor of pharmacology at France's University of Limoges, focuses his research on the microbial mechanisms of graft rejection and has identified SCFA as a critical component in this process.
Lawson said that a growing body of largely preclinical evidence implicates reduced microbial SCFA production in certain common transplant comorbidities, such as cardiovascular and metabolic disorders.
"These comorbidities pave the way for graft rejection," Lawson said. "For example, increased diabetes in the post-transplant period has been linked to an increased risk of rejection."
According to research done largely in preclinical models, Lawson explained, alterations in SCFA-producing bacteria appear to form a common link between these comorbidities and rejection.
SCFAs have anti-inflammatory roles, help regulate glucose and lipid metabolism, help maintain the integrity of the intestinal barrier, and promote the differentiation and function of immune cells with immunosuppressive properties. Because they play a key immunomodulatory role across diverse physiological conditions, their impairment in transplant patients might at least partially counterbalance the effect of immunosuppressive drugs, which could in turn activate biological pathways contributing to graft rejection.
Somewhat on the flip side of the anti-inflammatory SCFAs lie the pro-inflammatory lipopolysaccharides (LPS). Similar to SCFAs, LPS have been linked to graft rejection, mechanistically in preclinical models and by correlation in observational human studies.
Lawson said that he has additional data he hopes to publish this year showing increased LPS in plasma samples taken from transplant patients.
In that study, he and his colleagues examined approximately 20 individuals who had received either a heart, kidney, or lung transplant and compared their data to age-matched healthy volunteers. They observed a threefold increase in LPS across all transplant groups.
Although Lawson's group has yet to correlate these findings with specific clinical outcomes, he said that they are significant, as they highlight a potential link between elevated LPS levels and intestinal permeability in transplant recipients, which warrants further exploration.
Lawson explained that LPS is a major component of Gram-negative bacterial cell walls, serves as a biomarker of microbial translocation, and promotes chronic and systemic inflammation. Elevated LPS levels suggest a compromised intestinal barrier, a condition called "leaky gut syndrome" that is often associated with gut dysbiosis.
This compromised barrier facilitates the translocation of bacterial antigens, such as LPS, from the gut into the bloodstream, as reflected in the elevated blood LPS levels. Clinically, the increase in LPS is significant because it drives chronic systemic inflammation, which could potentially exacerbate complications including graft rejection.
Although preliminary, given the study's small size, Lawson said the finding is novel and he expects his team to likely be the first to publish evidence of increased intestinal permeability in transplant patients. He hopes to strengthen these results with additional biomarkers of intestinal permeability and to include more patients in the near future.
"It's important to understand the link between dysbiosis, leaky gut, [and] systemic inflammation, and how this systemic inflammation can lead to comorbidities that promote graft rejection," Lawson said.
Causal roles of both SCFA and LPS in graft rejection remain to be proven in humans, and the degree to which dysbiosis impacts transplant health and recipient survival remains an open question. Although research into the latter is in a relatively early state, some evidence suggests that dysbiosis could matter significantly.
One observational study, for instance, found that greater intestinal microbial diversity correlated with better survival in over 1,300 allogeneic hematopoietic cell transplant participants across multiple treatment centers.
A separate observational study largely corroborated those results. This study analyzed the microbiome profiles of 1,337 fecal samples provided by 766 kidney, 334 liver, 170 lung, and 67 heart transplant recipients and compared them with profiles of 8,208 non-transplant recipients, all living in the same area in the northern Netherlands.
The investigators examined microbial diversity, the prevalence of antibiotic resistance genes, and virulence factors capable of helping bacteria invade cells and evade immune defenses. They found that the further any of these three profiles diverged between transplant recipients and the general population, the more likely those recipients were to die sooner after their transplants, regardless of which organ they received. In the end, they identified 19 bacterial species that were particularly associated with increased risk of death.
Neither of these observational studies could show causative relationships between the observed dysbiosis and transplant outcomes, however.
A complex ecology
Jack Gilbert, a professor of pediatrics at the University of California San Diego School of Medicine and director of the Microbiome and Metagenomics Center, said that identifying such causal relationships in an ecology as complex as the human microbiome is "mind-bogglingly complicated."
Not only are there no effective or ethical methods for directly accessing most of the microbiome in its native environment, he explained, but it is exceptionally diverse and constantly changing. Besides bacteria, which attract the bulk of attention, the microbiome consists of many species of archaea, fungi, protists, and viruses, and its relative composition changes from one site of the body to another, from one person to another, and from one time point to another.
Given this complexity, even determining how to classify the bugs comprising our microbiomes as commensals, pathogens, or opportunistic pathogens presents a significant obstacle, as these definitions may be context dependent.
To disentangle all these variables, Gilbert said, "you need a study design that will actually identify a trait or a feature of the microbiome that is modifiable or is associated mechanistically with the outcome."
Scientists are making headway in understanding the microbiome, Gilbert said, but even some of the success stories underscore the complexity of the overall task.
As one such success, Gilbert pointed to recent studies in vulvovaginal candidiasis, a relatively common yeast infection that causes vaginal itching and discharge.
"We now know [that] the way specific strains of Candida manage their metabolic pathways are directly associated with disease risk," he said. "So, it's not what species Candida you have, it's how that species is regulating its metabolism," which increases the level of the complexity of the research.
Gilbert said that a key factor in moving microbiome research from the lab bench to clinical trials is more quantitative data, which will require more longitudinal sampling, as well as more effective ways for understanding how microbial strains vary across different contexts.
"If I take one species, and I put it in you, and I take [that] species and I put it in me, it's going to behave quite differently because of the metabolic context [and] the immune context in which it finds itself," he said.
Returning to the vulvovaginal candidiasis example, Gilbert noted that simply having that strain of Candida doesn't mean you'll necessarily get sick.
A structured path forward
Lawson agreed that a path to clinical-stage microbiome research will require a more structured approach, particularly with respect to pharmacomicrobiomics, which he said the field currently lacks.
"We need to find a path that will help us to go from preclinical models to clinical stages to identify interactions by which the gut microbiome could affect the efficacy of a specific drug, before that drug's complete development," he said.
To that end, Lawson and researchers from six other institutions across France recently launched the SPORE (Structured Pharmacomicrobiomics for Precision Medicine) consortium, aimed at establishing such a structured approach to pharmacomicrobiomics. The group hopes to eventually quantify the risk of any drug having a negative interaction with an individual's gut microbiome. In addition to the University of Limoges, consortium members are Limoges University Hospital, two separate schools within University Clermont Auvergne, Paris-Saclay University, and University Toulouse III Paul Sabatier.
The SPORE consortium is in its very early days and does not yet have a website, which Lawson said he hopes to launch in the very near future.
Although Gilbert largely echoed Lawson's call for structured improvements in the field of microbiome research, whether in the organ transplant space or beyond, he cautioned that care needs to be taken in getting there. Standardization can certainly help scientists to better interpret studies, he said, but in a field of research as complex and understudied as the microbiome, it also carries risks.
"If you try and say that everybody has to adhere to this particular standard," he said, "you end up saying that any deviation from that is not appropriate, [thereby] limiting the opportunity for innovation. We can't do that in this context because we don't know what's the best way of analyzing [the microbiome]."
Such standards are already in the works. Late last year, an international group of scientists, led by researchers at Italy's Fondazione Policlinico Gemelli and the Università Cattolica del Sacro Cuore, published a set of recommended best practices for the clinical implementation of microbiome testing. The group said that establishing such guidelines is necessary, owing to the rapidly growing interest in microbiome-based tests among the population and an industry already responding to that interest with commercial tests in the absence of solid scientific evidence.
You are what you eat
The bulk of commercially available microbiome assays test for dysbiosis, even if not specifically related to solid organ transplantation and in spite of the lack of a precise definition of dysbiosis. Interest in these tests among the general public is certainly growing.
Gilbert said that recently, the mother of a woman preparing for a heart transplant contacted him for dietary advice on how to manage her daughter's gut microbiome in order to minimize any potential interference with her immunosuppressants.
"I'm like, no, I'm really, really sorry, but I can't," Gilbert said. "Even if it was my own child, we do not have data to support an intervention which would be explicitly associated with that outcome."
Nevertheless, he said research to produce such data is progressing. Proof-of-concept work in mice by researchers at the Fred Hutchison Cancer Center that was published last year in the journal Immunity, for example, demonstrated that changes to a recipient animal's microbiome prior to transplantation modulated the likelihood and severity of graft-versus-host disease (GVHD).
Their results were largely replicated last year by a group from Charité Berlin Medical University that published its findings as a preprint in MedRxiv. The study showed that decreases in microbial diversity, particularly in bugs limiting the production of SCFA, preceded kidney rejection in human transplant recipients, suggesting that microbiome composition might be used as both a biomarker for rejection and as a therapeutic target for future interventions.
Several other recent studies in preclinical models further support these findings by showing that pre-transplantation treatment with probiotic bacteria and post-transplant SCFA supplementation might reduce dysbiosis and graft rejection.
Despite the growing body of preclinical studies into dietary and antibiotic interventions for mitigating dysbiosis, though, a recent Cochrane review determined that the overall evidence for these interventions remains poor.
The science of how the human microbiome works and how it can be leveraged to improve solid organ transplant outcomes remains something of a Wild West, rife with obstacles and opportunities for misinformation. Fortunately, the relatively small group of scientists dedicated to untangling its myriad webs are taking the challenges in stride and see success as an eventual inevitability.
"I'm convinced that one day we can have biomarkers derived from the gut microbiome that will tell us if a person is at risk of rejecting a graft or not," Lawson said.