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Microbe meets cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Microbes Meet Cancer

Understanding cancer’s relationship with the human microbiome could transform immune-modulating therapies.

By Kate Yandell | April 1, 2016  http://www.the-scientist.com/?articles.view/articleNo/45616/title/Microbes-Meet-Cancer

 © ISTOCK.COM/KATEJA_FN; © ISTOCK.COM/FRANK RAMSPOTT  http://www.the-scientist.com/images/April2016/feature1.jpg

In 2013, two independent teams of scientists, one in Maryland and one in France, made a surprising observation: both germ-free mice and mice treated with a heavy dose of antibiotics responded poorly to a variety of cancer therapies typically effective in rodents. The Maryland team, led by Romina Goldszmidand Giorgio Trinchieri of the National Cancer Institute, showed that both an investigational immunotherapy and an approved platinum chemotherapy shrank a variety of implanted tumor types and improved survival to a far greater extent in mice with intact microbiomes.1 The French group, led by INSERM’s Laurence Zitvogel, got similar results when testing the long-standing chemotherapeutic agent cyclophosphamide in cancer-implanted mice, as well as in mice genetically engineered to develop tumors of the lung.2

The findings incited a flurry of research and speculation about how gut microbes contribute to cancer cell death, even in tumors far from the gastrointestinal tract. The most logical link between the microbiome and cancer is the immune system. Resident microbes can either dial up inflammation or tamp it down, and can modulate immune cells’ vigilance for invaders. Not only does the immune system appear to be at the root of how the microbiome interacts with cancer therapies, it also appears to mediate how our bacteria, fungi, and viruses influence cancer development in the first place.

“We clearly see shifts in the [microbial] community that precede development of tumors,” says microbiologist and immunologist Patrick Schloss, who studies the influence of the microbiome on colon cancer at the University of Michigan.

But the relationship between the microbiome and cancer is complex: while some microbes promote cell proliferation, others appear to protect us against cancerous growth. And in some cases, the conditions that spur one cancer may have the opposite effect in another. “It’s become pretty obvious that the commensal microbiota affect inflammation and, through that or through other mechanisms, affect carcinogenesis,” says Trinchieri. “What we really need is to have a much better understanding of which species, which type of bug, is doing what and try to change the balance.”

Gut feeling

In the late 1970s, pathologist J. Robin Warren of Royal Perth Hospital in Western Australia began to notice that curved bacteria often appeared in stomach tissue biopsies taken from patients with chronic gastritis, an inflammation of the stomach lining that often precedes the development of stomach cancer. He and Barry J. Marshall, a trainee in internal medicine at the hospital, speculated that the bacterium, now called Helicobacter pylori, was somehow causing the gastritis.3 So committed was Marshall to demonstrating the microbe’s causal relationship to the inflammatory condition that he had his own stomach biopsied to show that it contained no H. pylori, then infected himself with the bacterium and documented his subsequent experience of gastritis.4 Scientists now accept that H. pylori, a common gut microbe that is present in about 50 percent of the world’s population, is responsible for many cases of gastritis and most stomach ulcers, and is a strong risk factor for stomach cancer.5 Marshall and Warren earned the 2005 Nobel Prize in Physiology or Medicine for their work.

H. pylori may be the most clear-cut example of a gut bacterium that influences cancer development, but it is likely not the only one. Researchers who study cancer in mice have long had anecdotal evidence that shifts in the microbiome influence the development of diverse tumor types. “You have a mouse model of carcinogenesis. It works beautifully,” says Trinchieri. “You move to another institution. It works completely differently,” likely because the animals’ microbiomes vary with environment.

IMMUNE INFLUENCE: In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment. Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame.
See full infographic: WEB | PDF
© AL GRANBERG

Around the turn of the 21st century, cancer researchers began to systematically experiment with the rodent microbiome, and soon had several lines of evidence linking certain gut microbes with a mouse’s risk of colon cancer. In 2001, for example, Shoichi Kado of the Yakult Central Institute for Microbiological Research in Japan and colleagues found that a strain of immunocompromised mice rapidly developed colon tumors, but that germ-free versions of these mice did not.6 That same year, an MIT-based group led by the late David Schauer demonstrated that infecting mice with the bacterium Citrobacter rodentium spurred colon tumor development.7 And in 2003, MIT’s Susan Erdman and her colleagues found that they could induce colon cancer in immunocompromised mice by infecting them with Helicobacter hepaticus, a relative of? H. pylori that commonly exists within the murine gut microbiome.8

More recent work has documented a similar link between colon cancer and the gut microbiome in humans. In 2014, a team led by Schloss sequenced 16S rRNA genes isolated from the stool of 90 people, some with colon cancer, some with precancerous adenomas, and still others with no disease.9 The researchers found that the feces of people with cancer tended to have an altered composition of bacteria, with an excess of the common mouth microbes Fusobacterium or Porphyromonas. A few months later, Peer Bork of the European Molecular Biology Laboratory performed metagenomic sequencing of stool samples from 156 people with or without colorectal cancer. Bork and his colleagues found they could predict the presence or absence of cancer using the relative abundance of 22 bacterial species, including Porphyromonas andFusobacterium.10 They could also use the method to predict colorectal cancer with about the same accuracy as a blood test, correctly identifying about 50 percent of cancers while yielding false positives less than 10 percent of the time. When the two tests were combined, they caught more than 70 percent of cancers.

Whether changes in the microbiota in colon cancer patients are harbingers of the disease or a consequence of tumor development remained unclear. “What comes first, the change in the microbiome or tumor development?” asks Schloss. To investigate this question, he and his colleagues treated mice with microbiome-altering antibiotics before administering a carcinogen and an inflammatory agent, then compared the outcomes in those animals and in mice that had received only the carcinogenic and inflammatory treatments, no antibiotics. The antibiotic-treated animals had significantly fewer and smaller colon tumors than the animals with an undisturbed microbiome, suggesting that resident bacteria were in some way promoting cancer development. And when the researchers transferred microbiota from healthy mice to antibiotic-treated or germ-free mice, the animals developed more tumors following carcinogen exposure. Sterile mice that received microbiota from mice already bearing malignancies developed the most tumors of all.11

Most recently, Schloss and his colleagues showed that treating mice with seven unique combinations of antibiotics prior to exposing them to carcinogens yielded variable but predictable levels of tumor formation. The researchers determined that the number of tumors corresponded to the unique ways that each antibiotic cocktail modulated the microbiome.12

“We’ve kind of proven to ourselves, at least, that the microbiome is involved in colon cancer,” says Schloss, who hypothesizes that gut bacteria–driven inflammation is to blame for creating an environment that is hospitable to tumor development and growth. Gain or loss of certain components of the resident bacterial community could lead to the release of reactive oxygen species, damaging cells and their genetic material. Inflammation also involves increased release of growth factors and blood vessel proliferation, potentially supporting the growth of tumors. (See illustration above.)

Recent research has also yielded evidence that the gut microbiota impact the development of cancer in sites far removed from the intestinal tract, likely through similar immune-modulating mechanisms.

Systemic effects

In the mid-2000s, MIT’s Erdman began infecting a strain of mice predisposed to intestinal tumors withH. hepaticus and observing the subsequent development of colon cancer in some of the animals. To her surprise, one of the mice developed a mammary tumor. Then, more of the mice went on to develop mammary tumors. “This told us that something really interesting was going on,” Erdman recalls. Sure enough, she and her colleagues found that mice infected with H. hepaticus were more likely to develop mammary tumors than mice not exposed to the bacterium.13The researchers showed that systemic immune activation and inflammation could contribute to mammary tumors in other, less cancer-prone mouse models, as well as to the development of prostate cancer.

MICROBIAL STOWAWAYS: Bacteria of the human gut microbiome are intimately involved in cancer development and progression, thanks to their interactions with the immune system. Some microbes, such as Helicobacter pylori, increase the risk of cancer in their immediate vicinity (stomach), while others, such as some Bacteroides species, help protect against tumors by boosting T-cell infiltration.© EYE OF SCIENCE/SCIENCE SOURCE
http://www.the-scientist.com/images/April2016/immune_2.jpg

 

 

© DR. GARY GAUGLER/SCIENCE SOURCE  http://www.the-scientist.com/images/April2016/immune3.jpg

At the University of Chicago, Thomas Gajewski and his colleagues have taken a slightly different approach to studying the role of the microbiome in cancer development. By comparing Black 6 mice coming from different vendors—Taconic Biosciences (formerly Taconic Farms) and the Jackson Laboratory—Gajewski takes advantage of the fact that the animals’ different origins result in different gut microbiomes. “We deliberately stayed away from antibiotics, because we had a desire to model how intersubject heterogeneity [in cancer development] might be impacted by the commensals they happen to be colonized with,” says Gajewski in an email to The Scientist.

Last year, the researchers published the results of a study comparing the progression of melanoma tumors implanted under the mice’s skin, finding that tumors in the Taconic mice grew more aggressively than those in the Jackson mice. When the researchers housed the different types of mice together before their tumors were implanted, however, these differences disappeared. And transferring fecal material from the Jackson mice into the Taconic mice altered the latter’s tumor progression.14

Instead of promoting cancer, in these experiments the gut microbiome appeared to slow tumor growth. Specifically, the reduced tumor growth in the Jackson mice correlated with the presence of Bifidobacterium, which led to the greater buildup of T?cells in the Jackson mice’s tumors. Bifidobacteriaactivate dendritic cells, which present antigens from bacteria or cancer cells to T?cells, training them to hunt down and kill these invaders. Feeding Taconic mice bifidobacteria improved their response to the implanted melanoma cells.

“One hypothesis going into the experiments was that we might identify immune-suppressive bacteria, or commensals that shift the immune response towards a character that was unfavorable for tumor control,” says Gajewski.  “But in fact, we found that even a single type of bacteria could boost the antitumor immune response.”

http://www.the-scientist.com/images/April2016/immune4.jpg

 

Drug interactions

Ideally, the immune system should recognize cancer as invasive and nip tumor growth in the bud. But cancer cells display “self” molecules that can inhibit immune attack. A new type of immunotherapy, dubbed checkpoint inhibition or blockade, spurs the immune system to attack cancer by blocking either the tumor cells’ surface molecules or the receptors on T?cells that bind to them.

CANCER THERAPY AND THE MICROBIOME

In addition to influencing the development and progression of cancer by regulating inflammation and other immune pathways, resident gut bacteria appear to influence the effectiveness of many cancer therapies that are intended to work in concert with host immunity to eliminate tumors.

  • Some cancer drugs, such as oxaliplatin chemotherapy and CpG-oligonucleotide immunotherapy, work by boosting inflammation. If the microbiome is altered in such a way that inflammation is reduced, these therapeutic agents are less effective.
  • Cancer-cell surface proteins bind to receptors on T cells to prevent them from killing cancer cells. Checkpoint inhibitors that block this binding of activated T cells to cancer cells are influenced by members of the microbiota that mediate these same cell interactions.
  • Cyclophosphamide chemotherapy disrupts the gut epithelial barrier, causing the gut to leak certain bacteria. Bacteria gather in lymphoid tissue just outside the gut and spur generation of T helper 1 and T helper 17 cells that migrate to the tumor and kill it.

As part of their comparison of Jackson and Taconic mice, Gajewski and his colleagues decided to test a type of investigational checkpoint inhibitor that targets PD-L1, a ligand found in high quantities on the surface of multiple types of cancer cells. Monoclonal antibodies that bind to PD-L1 block the PD-1 receptors on T?cells from doing so, allowing an immune response to proceed against the tumor cells. While treating Taconic mice with PD-L1–targeting antibodies did improve their tumor responses, they did even better when that treatment was combined with fecal transfers from Jackson mice, indicating that the microbiome and the immunotherapy can work together to take down cancer. And when the researchers combined the anti-PD-L1 therapy with a bifidobacteria-enriched diet, the mice’s tumors virtually disappeared.14

Gajewski’s group is now surveying the gut microbiota in humans undergoing therapy with checkpoint inhibitors to better understand which bacterial species are linked to positive outcomes. The researchers are also devising a clinical trial in which they will give Bifidobacterium supplements to cancer patients being treated with the approved anti-PD-1 therapy pembrolizumab (Keytruda), which targets the immune receptor PD-1 on T?cells, instead of the cancer-cell ligand PD-L1.

Meanwhile, Zitvogel’s group at INSERM is investigating interactions between the microbiome and another class of checkpoint inhibitors called CTLA-4 inhibitors, which includes the breakthrough melanoma treatment ipilimumab (Yervoy). The researchers found that tumors in antibiotic-treated and germ-free mice had poorer responses to a CTLA-4–targeting antibody compared with mice harboring unaltered microbiomes.15 Particular Bacteroides species were associated with T-cell infiltration of tumors, and feedingBacteroides fragilis to antibiotic-treated or germ-free mice improved the animals’ responses to the immunotherapy. As an added bonus, treatment with these “immunogenic” Bacteroides species decreased signs of colitis, an intestinal inflammatory condition that is a dangerous side effect in patients using checkpoint inhibitors. Moreover, Zitvogel and her colleagues showed that human metastatic melanoma patients treated with ipilimumab tended to have elevated levels of B. fragilis in their microbiomes. Mice transplanted with feces from patients who showed particularly strong B. fragilis gains did better on anti-CTLA-4 treatment than did mice transplanted with feces from patients with normal levels of B. fragilis.

“There are bugs that allow the therapy to work, and at the same time, they protect against colitis,” says Trinchieri. “That is very exciting, because not only [can] we do something to improve the therapy, but we can also, at the same time, try to reduce the side effect.”

And these checkpoint inhibitors aren’t the only cancer therapies whose effects are modulated by the microbiome. Trinchieri has also found that an immunotherapy that combines antibodies against interleukin-10 receptors with CpG oligonucleotides is more effective in mice with unaltered microbiomes.1He and his NCI colleague Goldszmid further found that the platinum chemotherapy oxaliplatin (Eloxatin) was more effective in mice with intact microbiomes, and Zitvogel’s group has shown that the chemotherapeutic agent cyclophosphamide is dependent on the microbiota for its proper function.

Although the mechanisms by which the microbiome influences the effectiveness of such therapies remains incompletely understood, researchers once again speculate that the immune system is the key link. Cyclophosphamide, for example, spurs the body to generate two types of T?helper cells, T?helper 1 cells and a subtype of T?helper 17 cells referred to as “pathogenic,” both of which destroy tumor cells. Zitvogel and her colleagues found that, in mice with unaltered microbiomes, treatment with cyclophosphamide works by disrupting the intestinal mucosa, allowing bacteria to escape into the lymphoid tissues just outside the gut. There, the bacteria spur the body to generate T?helper 1 and T?helper 17 cells, which translocate to the tumor. When the researchers transferred the “pathogenic” T?helper 17 cells into antibiotic-treated mice, the mice’s response to chemotherapy was partly restored.

Microbiome modification

As the link between the microbiome and cancer becomes clearer, researchers are thinking about how they can manipulate a patient’s resident microbial communities to improve their prognosis and treatment outcomes. “Once you figure out exactly what is happening at the molecular level, if there is something promising there, I would be shocked if people don’t then go in and try to modulate the microbiome, either by using pharmaceuticals or using probiotics,” says Michael Burns, a postdoc in the lab of University of Minnesota genomicist Ran Blekhman.

Even if researchers succeed in identifying specific, beneficial alterations to the microbiome, however, molding the microbiome is not simple. “It’s a messy, complicated system that we don’t understand,” says Schloss.

So far, studies of the gut microbiome and colon cancer have turned up few consistent differences between cancer patients and healthy controls. And the few bacterial groups that have repeatedly shown up are not present in every cancer patient. “We should move away from saying, ‘This is a causal species of bacteria,’” says Blekhman. “It’s more the function of a community instead of just a single bacterium.”

But the study of the microbiome in cancer is young. If simply adding one type of microbe into a person’s gut is not enough, researchers may learn how to dose people with patient-specific combinations of microbes or antibiotics. In February 2016, a team based in Finland and China showed that a probiotic mixture dubbed Prohep could reduce liver tumor size by 40 percent in mice, likely by promoting an anti-inflammatory environment in the gut.16

“If it is true that, in humans, we can alter the course of the disease by modulating the composition of the microbiota,” says José Conejo-Garcia of the Wistar Institute in Philadelphia, “that’s going to be very impactful.”

Kate Yandell has been a freelance writer living Philadelphia, Pennsylvania. In February she became an associate editor at Cancer Today.

GENETIC CONNECTION

The microbiome doesn’t act in isolation; a patient’s genetic background can also greatly influence response to therapy. Last year, for example, the Wistar Institute’s José Garcia-Conejo and Melanie Rutkowski, now an assistant professor at the University of Virginia, showed that a dominant polymorphism of the gene for the innate immune protein toll-like receptor 5 (TLR5) influences clinical outcomes in cancer patients by changing how the patients’ immune cells interact with their gut microbes (Cancer Cell, 27:27-40, 2015).

More than 7 percent of people carry a specific mutation in TLR5 that prevents them from mounting a full immune response when exposed to bacterial flagellin. Analyzing both genetic and survival data from the Cancer Genome Atlas, Conejo-Garcia, Rutkowski, and their colleagues found that estrogen receptor–positive breast cancer patients who carry the TLR5 mutation, called the R392X polymorphism, have worse outcomes than patients without the mutation. Among patients with ovarian cancer, on the other hand, those with the TLR5 mutation were more likely to live at least six years after diagnosis than patients who don’t carry the mutation.

Investigating the mutation’s contradictory effects, the researchers found that mice with normal TLR5produce higher levels of the cytokine interleukin 6 (IL-6) than those carrying the mutant version, which have higher levels of a different cytokine called interleukin 17 (IL-17). But when the researchers knocked out the animals’ microbiomes, these differences in cytokine production disappeared, as did the differences in cancer progression between mutant and wild-type animals.

“The effectiveness of depleting specific populations or modulating the composition of the microbiome is going to affect very differently people who are TLR5-positive or TLR5-negative,” says Conejo-Garcia. And Rutkowski speculates that many more polymorphisms linked to cancer prognosis may act via microbiome–immune system interactions. “I think that our paper is just the tip of the iceberg.”

References

  1. N. Iida et al., “Commensal bacteria control cancer response to therapy by modulating the tumor microenvironment,” Science, 342:967-70, 2013.
  2. S. Viaud et al., “The intestinal microbiota modulates the anticancer immune effects of cyclophosphamide,” Science, 342:971-76, 2013.
  3. J.R. Warren, B. Marshall, “Unidentified curved bacilli on gastric epithelium in active chronic gastritis,”Lancet, 321:1273-75, 1983.
  4. B.J. Marshall et al., “Attempt to fulfil Koch’s postulates for pyloric Campylobacter,” Med J Aust, 142:436-39, 1985.
  5. J. Parsonnet et al., “Helicobacter pylori infection and the risk of gastric carcinoma,” N Engl J Med, 325:1127-31, 1991.
  6. S. Kado et al., “Intestinal microflora are necessary for development of spontaneous adenocarcinoma of the large intestine in T-cell receptor β chain and p53 double-knockout mice,” Cancer Res, 61:2395-98, 2001.
  7. J.V. Newman et al., “Bacterial infection promotes colon tumorigenesis in ApcMin/+ mice,” J Infect Dis, 184:227-30, 2001.
  8. S.E. Erdman et al., “CD4+ CD25+ regulatory T lymphocytes inhibit microbially induced colon cancer in Rag2-deficient mice,” Am J Pathol, 162:691-702, 2003.
  9. J.P. Zackular et al., “The human gut microbiome as a screening tool for colorectal cancer,” Cancer Prev Res, 7:1112-21, 2014.
  10. G. Zeller et al., “Potential of fecal microbiota for early-stage detection of colorectal cancer,” Mol Syst Biol, 10:766, 2014.
  11. J.P. Zackular et al., “The gut microbiome modulates colon tumorigenesis,” mBio, 4:e00692-13, 2013.
  12. J.P. Zackular et al., “Manipulation of the gut microbiota reveals role in colon tumorigenesis,”mSphere, doi:10.1128/mSphere.00001-15, 2015.
  13. V.P. Rao et al., “Innate immune inflammatory response against enteric bacteria Helicobacter hepaticus induces mammary adenocarcinoma in mice,” Cancer Res, 66:7395, 2006.
  14. A. Sivan et al., “Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-L1 efficacy,” Science, 350:1084-89, 2015.
  15. M. Vétizou et al., “Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota,”Science, 350:1079-84, 2015.

……..

 

Microbially Driven TLR5-Dependent Signaling Governs Distal Malignant Progression through Tumor-Promoting Inflammation

Melanie R. Rutkowski, Tom L. Stephen, Nikolaos Svoronos, …., Julia Tchou,  Gabriel A. Rabinovich, Jose R. Conejo-Garcia
Cancer cell    12 Jan 2015; Volume 27, Issue 1, p27–40  http://dx.doi.org/10.1016/j.ccell.2014.11.009
Figure thumbnail fx1
  • TLR5-dependent IL-6 mobilizes MDSCs that drive galectin-1 production by γδ T cells
  • IL-17 drives malignant progression in IL-6-unresponsive tumors
  • TLR5-dependent differences in tumor growth are abrogated upon microbiota depletion
  • A common dominant TLR5 polymorphism influences the outcome of human cancers

The dominant TLR5R392X polymorphism abrogates flagellin responses in >7% of humans. We report that TLR5-dependent commensal bacteria drive malignant progression at extramucosal locations by increasing systemic IL-6, which drives mobilization of myeloid-derived suppressor cells (MDSCs). Mechanistically, expanded granulocytic MDSCs cause γδ lymphocytes in TLR5-responsive tumors to secrete galectin-1, dampening antitumor immunity and accelerating malignant progression. In contrast, IL-17 is consistently upregulated in TLR5-unresponsive tumor-bearing mice but only accelerates malignant progression in IL-6-unresponsive tumors. Importantly, depletion of commensal bacteria abrogates TLR5-dependent differences in tumor growth. Contrasting differences in inflammatory cytokines and malignant evolution are recapitulated in TLR5-responsive/unresponsive ovarian and breast cancer patients. Therefore, inflammation, antitumor immunity, and the clinical outcome of cancer patients are influenced by a common TLR5 polymorphism.

see also… Immune Influence

In recent years, research has demonstrated that microbes living in and on the mammalian body can affect cancer risk, as well as responses to cancer treatment.

By Kate Yandell | April 1, 2016

http://www.the-scientist.com/?articles.view/articleNo/45644/title/Immune-Influence

Although the details of this microbe-cancer link remain unclear, investigators suspect that the microbiome’s ability to modulate inflammation and train immune cells to react to tumors is to blame. Here are some of the hypotheses that have come out of recent research in rodents for how gut bacteria shape immunity and influence cancer.

HOW THE MICROBIOME PROMOTES CANCER

Gut bacteria can dial up inflammation locally in the colon, as well as in other parts of the body, leading to the release of reactive oxygen species, which damage cells and DNA, and of growth factors that spur tumor growth and blood vessel formation.

http://www.the-scientist.com/images/April2016/ImmuneInfluence1_640px.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence2_310px1.jpg

Helicobacter pylori can cause inflammation and high cell turnover in the stomach wall, which may lead to cancerous growth.

HOW THE MICROBIOME STEMS CANCER

Gut bacteria can also produce factors that lower inflammation and slow tumor growth. Some gut bacteria (e.g., Bifidobacterium)
appear to activate dendritic cells,
which present cancer-cell antigens to T cells that in turn kill the cancer cells.

http://www.the-scientist.com/images/April2016/ImmuneInfluence3_310px1.jpg

http://www.the-scientist.com/images/April2016/ImmuneInfluence4_310px1.jpg

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Intestinal inflammatory pharmaceutics

Curator: Larry H. Bernstein, MD, FCAP

 

AbbVie Invests in Synthetic Microbes for Treatment of Intestinal Disorders

Aaron Krol    http://www.bio-itworld.com/2016/2/10/abbvie-invests-synthetic-microbes-treatment-intestinal-disorders.html

 

February 10, 2016 | This morning, AbbVie announced a partnership with Synlogic of Cambridge, Mass., to create microbiome-based therapies for the treatment of inflammatory bowel disease (IBD). The two companies have sketched out a suggested three-year timeline for preclinical research and development, after which AbbVie will take over advancing any drug candidates into clinical trials.

Drugs inspired by the microbes that live in the human gut are a hot topic in biotech. Companies like Seres Health and Vedanta Biosciences are pursuing the idea from a variety of angles, from making traditional small molecule drugs that interact with the microbiome, to creating probiotics or microbial cocktails that restore a healthy balance to the gut ecosystem. IBD, including Crohn’s disease and ulcerative colitis, is an especially popular target for these companies, thanks to strong suggestions that bacterial populations can affect the course of the disease. Already, Second Genome and Coronado Biosciences have taken prospective treatments into the clinic (though the latter has been dealt serious setbacks in Phase II trials).

But even among this peculiar batch of startups, Synlogic’s approach to drug design is exquisitely odd. The company calls its products “synthetic biotics”―in fact, they’re genetically engineered bacteria whose DNA contains intricately designed “gene circuits,” built to start producing therapeutic molecules when and only when the patient needs them.

“We are not looking at correcting the dysregulation of microbes in the gut, like other microbiome companies,” CEO José-Carlos Gutiérrez-Ramos tells Bio-IT World. “We have one bacterium, and it’s engineered to do different functions.”

Synlogic was founded in 2013 by two synthetic biologists at MIT, Timothy Lu and Jim Collins. (Bio-IT World has previously spoken with Lu about his academic work on bacterial gene circuits.) Gutiérrez-Ramos joined almost two years later, leaving a position as the head of Pfizer’s BioTherapeutics R&D group, where he had plenty of opportunity to turn emerging biotechnology ideas into drug candidates ready for submission to the FDA.

Still, synthetic biotics are a good deal more unusual than the biologic drugs he worked on at Pfizer.

His new company doesn’t quite spin functions for its microbes out of whole cloth. All the genes the company uses are copied either from the human genome, or from the bacteria living inside us. But by recombining those genes into circuits, Gutiérrez-Ramos believes Synlogic can finely control whether and when genes are expressed, giving its synthetic biotics the same dosage control as a traditional drug. Meanwhile, choosing the right bacterium to engineer―the current favorite is a strain called E. coli Nissle―ensures the biotics do not form stable colonies in the gut, but can be cleared out as soon as a patient stops treatment.

“We’re pharma guys,” he says. “What we want is to have pharmacologically well-defined products.”

The Molecular Circuit Board

Even before the partnership with AbbVie, Synlogic had a pipeline of drug candidates in development, all meant to treat rare genetic disorders caused by single mutations that shut down the activity of a crucial gene. In principle, there seems to be no reason that bacteria carrying the right genes couldn’t pick up the slack. “We know the patient is missing a function that is typically performed by the liver, or the kidney, or the pancreas,” says Gutiérrez-Ramos. “What we do is shift that function from an organ to a stable fraction of the microbiome.”

The approach is in some ways analogous to gene therapy, where a corrected version of a broken gene is inserted into a patient’s own DNA. “We don’t use that word, but the fact is it’s a non-somatic gene therapy,” Gutiérrez-Ramos says. “And if something goes wrong, you can control it just by stopping treatment.” The most advanced synthetic biotic in Synlogic’s pipeline targets urea cycle disorder, exactly the sort of disease that might otherwise be addressed by gene therapy: patients are missing a single enzyme that helps remove nitrogen from the body and prevent it from forming ammonia in the bloodstream. Synlogic will meet with the FDA this March to discuss whether and how this first product can be tested in humans.

Gutierrez Ramos

The new IBD program with AbbVie, however, adds a whole new level of complexity. Executives from the two companies have been in discussions for around six months, and both agree that no single mechanism will be enough to provide significant relief for patients. Crohn’s and ulcerative colitis are painful autoimmune diseases that involve both a weakening of the epithelial lining in the stomach, and a buildup of inflammatory molecules. The development plan that AbbVie and Synlogic have agreed on includes three separate methods of attack to relieve these symptoms.

“One approach AbbVie is very interested in is for our synthetic biotics to produce substances that could tighten the epithelial barrier,” says Gutiérrez-Ramos. “Another approach is to degrade pro-inflammatory molecules”―the same tack taken by AbbVie’s current leading IBD drug, Humira, which targets the inflammatory protein TNFα. “Finally, we can produce anti-inflammatory molecules.”

Uniquely, synthetic biotics can perform all three functions at once; it’s just a matter of inserting the right genes. But that alone might not be a decisive advantage over some sort of combination therapy. The biggest selling point of Synlogic’s microbes is not the genes they can be engineered to express―what you might call the “output” of their gene circuits―but the input, the DNA elements called “inducible promoters” that decide when those genes should be activated.

The core idea is that patients will have a constant population of synthetic biotics in their bodies, taken daily―but those microbes will only generate their therapeutic payloads when needed. In IBD, Gutiérrez-Ramos explains, “it’s not that the patient is always inflamed, but they have flares. Our vision, and AbbVie’s vision, is that the bacteria that you take every day sense when the flare is coming, and then trigger the genetic output.”

This would be a major improvement over a drug like Humira, which after all is constantly inhibiting a part of the immune system. Patients taking Humira, or one of the many other immunosuppressant drugs for IBD, are at a constantly heightened risk of infection; tuberculosis is a particular specter for these patients. If Synlogic can find a genetic “on-switch” that responds to a reliable indicator of IBD flares, it could potentially create a much more precisely administered treatment, while still giving patients the simple dosing schedule of one pill every day.

The company has leads on two inducible promoters that might do the trick: one that reacts to nitric oxide, and another tied to reactive oxygen species. Of course, there’s no guarantee that either will respond sensitively to IBD flares in a real clinical setting. “This is an early time for the technology,” says Gutiérrez-Ramos. “We have demonstrated this in animals, but we have to demonstrate it in humans.”

Although it’s far too early to say if synthetic biotics will become an ordinary part of the pharma toolkit, AbbVie’s decision to invest in the technology offers the means to test this approach on a large scale. Synlogic expects to raise its own funding for trials of its rare disease products, which the FDA does not expect to enroll huge numbers of patients, but IBD is a problem of a very different order.

“We are very honored to work with truly the leader in treatment of inflammatory bowel disease,” says Gutiérrez-Ramos. With the backing of big pharma, it will be possible to trial microbiome-based therapies for the kinds of common, chronic diseases that are the biggest drain on our healthcare system. What’s more, the AbbVie partnership is an important signal of the industry’s faith in synthetic biology as an approach to treating disease.

 

 

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Contribution to Inflammatory Bowel Disease (IBD) of bacterial overgrowth in gut on a chip

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a 

human gut-on-a-chip 
Gut-On-a-Chip Holds Clues for Treating Inflammatory Bowel Diseases
Greg Watry
Human intestinal epithelial cells cultured in the Wyss Institute's human gut-on-a-chip form differentiated intestinal villi when cultured in the presence of lifelike fluid flow and rhythmic, peristalsis-like motions. Here the villi are visible using a traditional microscope (left) or a confocal microscope (right); when the same villi are stained with fluorescent antibodies, it clearly reveals the nuclei in the intestinal cells (blue) and their specialized apical membranes when they contact the intestinal lumen (green). Credit: Wyss Institute at Harvard University
Human intestinal epithelial cells cultured in the Wyss Institute’s human gut-on-a-chip form differentiated intestinal villi when cultured in the presence of lifelike fluid flow and rhythmic, peristalsis-like motions. Here the villi are visible using a traditional microscope (left) or a confocal microscope (right); when the same villi are stained with fluorescent antibodies, it clearly reveals the nuclei in the intestinal cells (blue) and their specialized apical membranes when they contact the intestinal lumen (green). Credit: Wyss Institute at Harvard University

Roughly the size of a computer memory stick and made of clear flexible polymer, the human gut-on-a-chip was created by Harvard Univ.’s Wyss Institute in 2012. Three years later, researchers are utilizing the technology in hopes of creating new therapies for inflammatory bowel diseases (IBD).

The Centers for Disease Control and Prevention estimates that between 1 and 1.3 million people suffer from IBD, including such diseases as ulcerative colitis and Crohn’s disease. With origins still mysterious, IBD is currently incurable.

“It has not been possible to study…human intestinal inflammatory diseases, because it is not possible to independently control these parameters in animal studies or in vitro models,” wrote the researchers in Proceedings of the National Academy of the Sciences. “In particular, given the recent recognition of the central role of the intestinal microbiome in human health and disease, including intestinal disorders, it is critical to incorporate commensal microbes into experimental models, however, this has not been possible using conventional culture systems.”

Additionally, static in vitro methods fail to replicate the pathophysiology of human IBD.

But the hollow-channeled microfluidic gut-on-a-chip successfully simulates the human intestine’s physical structure, microenvironment, peristalsis-like motion, and fluid flow.

“With our human gut-on-a-chip, we can not only culture the normal gut microbiome for extended times, but we can also analyze contributions of pathogens, immune cells, and vascular and lymphatic endothelium, as well as model specific diseases to understand the complex pathophysiological responses of the intestinal tract,” said Donald Ingber, founding director of the Wyss Institute.

The device was “used to co-culture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation,” the researchers wrote.

Thus far, use of the device has yielded two interesting observations.

Four proteins—called cytokines—work together to trigger an inflammatory responses that exacerbate the bowel, the researchers found. Potentially, this new discovery could lead to the development of treatments that block the cytokine interaction.

Another observation, the researchers noted, is that “by ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease,” according to the researchers.

The researchers believe the micro-device may one day be applicable to precision medicine. Eventually, a custom treatment may arise from scientists using a patient’s gut microbiota and cells on a human gut-on-a-chip.

 

 

Contributions of microbiome and mechanical deformation to intestinal bacterial overgrowth and inflammation in a human gut-on-a-chip
Hyun Jung Kima,1, Hu Lia,2, James J. Collinsa,b,c,d,e,f,3, and Donald E. Ingbera,g,h,
http://www.pnas.org/content/early/2015/12/09/1522193112.full.pdf

A human gut-on-a-chip microdevice was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells for more than a week in vitro and to analyze how gut microbiome, inflammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inflammation. This in vitro model replicated results from past animal and human studies, including demonstration that probiotic and antibiotic therapies can suppress villus injury induced by pathogenic bacteria. By ceasing peristalsis-like motions while maintaining luminal flow, lack of epithelial deformation was shown to trigger bacterial overgrowth similar to that observed in patients with ileus and inflammatory bowel disease. Analysis of intestinal inflammation on-chip revealed that immune cells and lipopolysaccharide endotoxin together stimulate epithelial cells to produce four proinflammatory cytokines (IL-8, IL-6, IL-1β, and TNF-α) that are necessary and sufficient to induce villus injury and compromise intestinal barrier function. Thus, this human gut-on-a-chip can be used to analyze contributions of microbiome to intestinal pathophysiology and dissect disease mechanisms in a controlled manner that is not possible using existing in vitro systems or animal models.

 

Significance The main advance of this study is the development of a microengineered model of human intestinal inflammation and bacterial overgrowth that permits analysis of individual contributors to the pathophysiology of intestinal diseases, such as ileus and inflammatory bowel disease, over a period of weeks in vitro. By studying living human intestinal epithelium, with or without vascular and lymphatic endothelium, immune cells, and mechanical deformation, as well as living microbiome and pathogenic microbes, we identified previously unknown contributions of specific cytokines, mechanical motions, and microbiome to intestinal inflammation, bacterial overgrowth, and control of barrier function. We provide proof-of-principle to show that the microfluidic gut-on-a-chip device can be used to create human intestinal disease models and gain new insights into gut pathophysiology.

 

Various types of inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis, involve chronic inflammation of human intestine with mucosal injury and villus destruction (1), which is believed to be caused by complex interactions between gut microbiome (including commensal and pathogenic microbes) (2), intestinal mucosa, and immune components (3). Suppression of peristalsis also has been strongly associated with intestinal pathology, inflammation (4, 5), and small intestinal bacterial overgrowth (5, 6) in patients with Crohn’s disease (7) and ileus (8). However, it has not been possible to study the relative contributions of these different potential contributing factors to human intestinal inflammatory diseases, because it is not possible to independently control these parameters in animal studies or in vitro models. In particular, given the recent recognition of the central role of the intestinal microbiome in human health and disease, including intestinal disorders (2), it is critical to incorporate commensal microbes into experimental models; however, this has not been possible using conventional culture systems. Most models of human intestinal inflammatory diseases rely either on culturing an intestinal epithelial cell monolayer in static Transwell culture (9) or maintaining intact explanted human intestinal mucosa ex vivo (10) and then adding live microbes and immune cells to the apical (luminal) or basolateral (mucosal) sides of the cultures, respectively. These static in vitro methods, however, do not effectively recapitulate the pathophysiology of human IBD. For example, intestinal epithelial cells cultured in Transwell plates completely fail to undergo villus differentiation, produce mucus, or form the various specialized cell types of normal intestine. Although higher levels of intestinal differentiation can be obtained using recently developed 3D organoid cultures (11), it is not possible to expose these cells to physiological peristalsis-like motions or living microbiome in long-term culture, because bacterial overgrowth occurs rapidly (within ∼1 d) compromising the epithelium (12). This is a major limitation because establishment of stable symbiosis between the epithelium and resident gut microbiome as observed in the normal intestine is crucial for studying inflammatory disease initiation and progression (13), and rhythmical mechanical deformations driven by peristalsis are required to both maintain normal epithelial differentiation (14) and restrain microbial overgrowth in the intestine in vivo (15).

Thus, we set out to develop an experimental model that would overcome these limitations. To do this, we adapted a recently described human gut-on-a-chip microfluidic device that enables human intestinal epithelial cells (Caco-2) to be cultured in the presence of physiologically relevant luminal flow and peristalsislike mechanical deformations, which promotes formation of intestinal villi lined by all four epithelial cell lineages of the small intestine (absorptive, goblet, enteroendocrine, and Paneth) (12, 16). These villi also have enhanced barrier function, drug-metabolizing cytochrome P450 activity, and apical mucus secretion compared with the same cells grown in conventional Transwell cultures, which made it possible to coculture a probiotic gut microbe (Lactobacillus rhamnosus GG) in direct contact with the intestinal epithelium for more than 2 wk (12), in contrast to static Transwell cultures (17) or organoid cultures (11) that lose viability within hours under similar conditions. In the present study, we leveraged this human gut-on-a-chip to develop a disease model of small intestinal bacterial overgrowth (SIBO) and inflammation. We analyzed how probiotic and pathogenic bacteria, lipopolysaccharide (LPS), immune cells, inflammatory cytokines, vascular endothelial cells and mechanical forces contribute individually, and in combination, to intestinal inflammation, villus injury, and compromise of epithelial barrier function. We also explored whether we could replicate the protective effects of clinical probiotic and antibiotic therapies on-chip to demonstrate its potential use as an in vitro tool for drug development, as well as for dissecting fundamental disease mechanisms.

 

Fig. 1. The human gut-on-a-chip microfluidic device and changes in phenotype resulting from different culture conditions on-chip, as measured using genome-wide gene profiling. (A) A photograph of the device. Blue and red dyes fill the upper and lower microchannels, respectively. (B) A schematic of a 3D cross-section of the device showing how repeated suction to side channels (gray arrows) exerts peristalsis-like cyclic mechanical strain and fluid flow (white arrows) generates a shear stress in the perpendicular direction. (C) A DIC micrograph showing intestinal basal crypt (red arrow) and villi (white arrow) formed by human Caco-2 intestinal epithelial cells grown for ∼100 h in the gut-on-achip under medium flow (30 μL/h) and cyclic mechanical stretching (10%, 0.15 Hz). (Scale bar, 50 μm.) (D) A confocal immunofluorescence image showing a horizontal cross-section of intestinal villi similar to those shown in Fig. 1C, stained for F-actin (green) that labels the apical brush border of these polarized intestinal epithelial cells (nuclei in blue). (Scale bar, 50 μm.) (E) Hierarchical clustering analysis of genome-wide transcriptome profiles (Top) of Caco-2 cells cultured in the static Transwell, the gut-on-a-chip (with fluid flow at 30 μL/h and mechanical deformations at 10%, 0.15 Hz) (Gut Chip), or the mechanically active gut-on-a-chip cocultured with the VSL#3 formulation containing eight probiotic gut microbes (Gut Chip + VSL#3) for 72 h compared with normal human small intestinal tissues (Duodenum, Jejunum, and Ileum; microarray data from the published GEO database). The dendrogram was generated based on the averages calculated across all replicates, and all branches in the cluster have the approximately unbiased (AU) P value equal to 100. The y axis next to the dendrogram represents the metric for Euclidean distance between samples. Corresponding pseudocolored GEDI maps analyzing profiles of 650 metagenes between samples described above (Bottom).

 

Fig. 2. Reconstitution of pathological intestinal injury induced by interplay between nonpathogenic or pathogenic enteroinvasive E. coli bacteria or LPS endotoxin with immune cells. (A) DIC images showing that the normal villus morphology of the intestinal epithelium cultured on-chip (Control) is lost within 24 h after EIEC (serotype O124:NM) are added to the apical channel of the chip (+EIEC; red arrows indicate bacterial colonies). (B) Effects of GFP-EC, LPS (15 μg/mL), EIEC, or no addition (Control) on intestinal barrier function (Left). Right shows the TEER profiles in the presence of human PBMCs (+PBMC). GFP-EC, LPS, and EIEC were added to the apical channel (intestinal lumen) at 4, 12, and 35 h, respectively, and PBMCs were subsequently introduced through the lower capillary channel at 44 h after the onset of experiment (0 h) (n = 4). (C) Morphological analysis of intestinal villus damage in response to addition of GFP-EC, LPS, and EIEC in the absence (−PBMC) or the presence of immune components (+PBMC). Schematics (experimental setup), phase contrast images (horizontal view, taken at 57 h after onset), and fluorescence confocal micrographs (vertical cross-sectional views at 83 h after onset) were sequentially displayed. F-actin and nuclei were coded with magenta and blue, respectively. (D) Quantification of intestinal injury evaluated by measuring changes in lesion area (Top; n = 30) and the height of the villi (Bottom; n = 50) in the absence (white) or the presence (gray) of PBMCs. Intestinal villi were grown in the gut-on-a-chip under trickling flow (30 μL/h) with cyclic deformations (10%, 0.15 Hz) during the preculture period for ∼100 h before stimulation (0 h, onset). Asterisks indicate statistical significance compared with the control at the same time point (*P < 0.001, **P < 0.05). (Scale bars, 50 μm.)

 

Recapitulating Organ-Level Intestinal Inflammatory Responses. During inflammation in the intestine, pathophysiological recruitment of circulating immune cells is regulated via activation of the underlying vascular endothelium. To analyze this organ-level inflammatory response in our in vitro model, a monolayer of human microvascular endothelial cells (Fig. 3 C and D and Fig. S6 A and C) or lymphatic endothelial cells (Fig. S6 B and C) was cultured on the opposite (abluminal) side of the porous ECM-coated membrane in the lower microchannel of the device to effectively create a vascular channel (Fig. 3C). To induce intestinal inflammatory responses, LPS (Fig. 3 C and D) or TNF-α (Fig. S6) was flowed through the upper epithelial channel for 24 h, and then PBMCs were added to the vascular channel for 1 h without flow (Fig. 3 C and D). Treatment with both LPS (or TNF-α) and PBMCs resulted in the activation of intercellular adhesion molecule-1 (ICAM-1) expression on the surface of the endothelium (Fig. 3 C and D, Left, and Fig. S6) and a significant increase (P < 0.001) in the number of PBMCs that adhered to the surface of the capillary endothelium compared with controls (Fig. 3D). These results are consistent with our qPCR results, which also showed up-regulation of genes involved in immune cell trafficking (Fig. S5). Neither addition of LPS nor PBMCs alone was sufficient to induce ICAM-1 expression in these cells (Fig. 3D), which parallels the effects of LPS and PBMCs on epithelial production of inflammatory cytokines (Fig. 3A) as well as on villus injury (Fig. 2 B and D).

Evaluating Antiinflammatory Probiotic and Antibiotic Therapeutics On-Chip. To investigate how the gut microbiome modulates these inflammatory reactions, we cocultured the human intestinal villi with the eight strains of probiotic bacteria in the VSL#3 formulation that significantly enhanced intestinal differentiation (Fig. 1E and Fig. S1B). To mimic the in vivo situation, we colonized our microengineered gut on a chip with the commensal microbes (VSL#3) first and then subsequently added immune cells (PBMCs), pathogenic bacteria (EIEC), or both in combination. The VSL#3 microbial cells inoculated into the germ-free lumen of the epithelial channel primarily grew as discrete microcolonies in the spaces between adjacent villi (Fig. 4A and Movie S3) for more than a week in culture (Fig. S7A), and no planktonic growth was detected. These microbes did not overgrow like the EIEC (Fig. 2A and Movie S2), although occasional microcolonies also appeared at different spatial locations in association with the tips of the villi (Fig. S7 B and C). The presence of these living components of the normal gut microbiome significantly enhanced (P < 0.001) intestinal barrier function, producing more than a 50% increase in TEER relative to control cultures (Fig. 4B) without altering villus morphology (Fig. 4C). This result is consistent with clinical studies suggesting that probiotics, including VSL#3, can significantly enhance intestinal barrier function in vivo (18).

To mimic the effects of antibiotic therapies that are sometimes used clinically in patients with intestinal inflammatory disease (29), we identified a dose and combination of antibiotics (100 units per mL penicillin and 100 μg/mL streptomycin) that produced effective killing of both EIEC and VSL#3 microbes in liquid cultures (Fig. S9) and then injected this drug mixture into the epithelial channel of guton-a-chip devices infected with EIEC. When we added PBMCs to these devices 1 h later, intestinal barrier function (Fig. 4B) and villus morphology (Fig. 4C) were largely protected from injury, and there was a significant reduction in lesion area (Fig. 4D). Thus, the gut-on-a-chip was able to mimic suppression of injury responses previously observed clinically using other antibiotics that produce similar bactericidal effects.

Analyzing Mechanical Contributions to Bacterial Overgrowth. Finally, we used the gut-on-a-chip to analyze whether physical changes in peristalsis or villus motility contribute to intestinal pathologies, such as the small intestinal bacterial overgrowth (SIBO) (5, 6) observed in patients with ileus (8) and IBD (7). When the GFPEC bacteria were cultured on the villus epithelium under normal flow (30 μL/h), but in the absence of the physiological cyclic mechanical deformations, the number of colonized bacteria was significantly higher (P < 0.001) compared with gut chips that experienced mechanical deformations (Fig. 5A). Bacterial cell densities more than doubled within 21 h when cultured under conditions without cyclic stretching compared with gut chips that experienced physiological peristalsis-like mechanical motions, even though luminal flow was maintained constant (Fig. 5B). Thus, cessation of epithelial distortion appears to be sufficient to trigger bacterial overgrowth, and motility-induced luminal fluid flow is not the causative factor as assumed previously (7).

 

Discussion One of the critical prerequisites for mimicking the living human intestine in vitro is to establish a stable ecosystem containing physiologically differentiated intestinal epithelium, gut bacteria, and immune cells that can be cultured for many days to weeks. Here we leveraged a mechanically active gut-on-a-chip microfluidic device to develop an in vitro model of human intestinal inflammation that permits stable long-term coculture of commensal microbes of the gut microbiome with intestinal epithelial cells. The synthetic model of the human living intestine we built recapitulated the minimal set of structures and functions necessary to mimic key features of human intestinal pathophysiology during chronic inflammation and bacterial overgrowth including epithelial and vascular inflammatory processes and destruction of intestinal villi.

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