High-Throughput Method Finds Migrating Gut Bugs That Activate Immune Cells

September 1, 2022

By Deborah Borfitz 

September 1, 2022 | Investigators at Cedars-Sinai and the National Institute of Allergy and Infectious Diseases have used high throughput sequencing and human serum to characterize the reactivities of immunoglobulin G (IgG) to microorganisms in the gastrointestinal tract that crossed the intestinal mucosal barrier. In doing so, they have “unlocked the ability to go back to studies that have already been done and measure the immune responses to gut microbes in an unbiased way,” according to Ivan Vujkovic-Cvijin, Ph.D., an assistant professor in the department of biomedical sciences and gastroenterology at Cedars-Sinai.  

Up to now, the ability to measure the antibody molecule required stool from individuals. But blood is the most common sample type collected from the human body, he says, noting that most freezers used for research samples around the world are primarily stocked with serum.  

The novel technique for identifying translocating gut bacteria, described in a study newly published in Science Translational Medicine (DOI: 10.1126/scitranslmed.abl3927), sheds light on the interactions between the host and its gut microbiota. It may also provide an additional method to identify microbes linked to inflammatory diseases—including obesity, liver disease, inflammatory bowel disease (IBD), cancer, and some neurological diseases—and enable more personalized therapeutic treatment. 

Vujkovic-Cvijin is among a growing number of researchers obsessed with the microbiome, in his case triggered by a series of studies about a decade ago showing that certain bacteria in the gut of mice are responsible for inducing and recruiting immune cells into the gut that are important in a variety of human diseases and could also migrate throughout the body. He was particularly intrigued by the observation that gut bacteria with no clear signs of pathology could be responsible for inducing an immune cell response.    

“I became convinced that the microbiome may be something we can target therapeutically to mitigate or ameliorate disease,” he says. It is known that gut microbes can activate the immune system, stimulating inflammation, by breaching the gut barrier. Although that process is thought to underlie many diseases, he adds, few strategies currently exist to reliably identify which bacteria have made the journey in people. 

‘Not A Species Alone’ 

Many questions remain unanswered, including which gut bacteria cross the mucosal barrier so they can be studied to see if they share any characteristics enabling the translocation process, says Vujkovic-Cvijin, highlighting the value of methodologies for identifying the immunity-activating microbes. The molecules and strategies may differ between pathogenic and non-pathogenic gut microbes, as might their reasons for crossing the barrier. 

Humans living in an industrialized world are exposed to chemicals through diet and environmental pollution, and this may impact the integrity of the gut barrier, he says. There may also be some host-intrinsic components, such as general health and genetic differences, contributing to impairment of the gut barrier. 

The current estimate is that each individual harbors a hundred trillion gut bacteria, says Vujkovic-Cvijin, roughly equivalent to the number of cells in the body. “We are certainly not a species alone. Every human individual is a community of organisms and that community in many ways is an organ very important for the health of its host.” 

Unlike the human genome, the microbial community is highly heterogeneous and underscores the possibility that it is linked to different disease susceptibilities, he notes. While the genome of any two people is 99.9% identical, they are thought to share only about 10% of the same microbes. 

“Intriguingly, having a disease itself may influence the microbes,” Vujkovic-Cvijin says. He was involved with an earlier study that found differences seen between healthy people and those with a disease were the effects of lifestyle variables influencing the microbiome, many of which happen because of disease.  

“Just looking at what’s there and in what proportion is susceptible to confounding effects because your diet [and lifestyle habits] and mine are very different,” he says. The IgG scoring approach is more likely to shed light on what is contributing to disease because it is directly measuring immune response to microbes irrespective of their abundance. 

Unique Bacterium/Host Relationships 

For the new study, the research team used high throughput sequencing to calculate an IgG score providing a measure of antibody level against each gut microbe, says Vujkovic-Cvijin. That is, translocating microbes were identified indirectly by how robustly the immune system was responding to their presence.  

The methodology was demonstrated both in mouse models as well as humans who were undergoing bowel surgery, he continues. Bacteria that had crossed the gut barrier of patients with IBD “conspicuously elicited a high IgG response”. 

These included several typically innocuous bacteria: Collinsella, Bifidobacterium, Lachnospiraceae, and Ruminococcaceae. “Regardless of what kind of bacterium it is, if it enters the spaces where it is not supposed to be [e.g., the gut lumen], it has the potential to be a threat, and the immune system typically responds robustly in order to eliminate that threat,” Vujkovic-Cvijin says. 

But whether all bacteria that are targeted by IgG are universally bad for the host is a “totally unanswered question,” he adds. “It is not the case that ever bacterium that we identify with this technique is deleterious, but it does indicate a unique relationship with the host.” 

Pathogenic Role 

Bifidobacteria have traditionally been considered not only non-pathogenic but potentially protective against disease because they are fermenters, but the latest study suggests they might be pro-inflammatory, says Vujkovic-Cvijin. Yogurt and other fermented food have so many heathy metabolites that the assumption has been that the fermenting bacteria themselves might contribute to health. 

A closer look in the literature hints at the potential pathogenic role for the identified bugs, Vujkovic-Cvijin says. Many studies in mice suggest Bifidobacteria bacteria elicit an immune response thought to contribute to IBD. And in one large study of stool samples from individuals at high risk for developing severe complications in early-onset IBD, Collinsella enrichment was the top hit in terms of association (The Lancet, DOI: 10.1016/S0140-6736(17)30317-3

“We think of our study as a starting point in many ways and one of them is following up on the unique immune relationship [of these bacteria] with the host,” he says. 

The research team plans to explore observations in the study, particularly the finding of heightened immune response by the subset of gut microbiota constituents that translocate across the gut barrier. They also intend to apply the IgG scoring tool to other inflammatory diseases beyond IBD, including rheumatoid arthritis and liver disease.  

One study already underway in collaboration with the Department of Defense and researchers at the University of Nevada and Mount Sinai is examining whether some of the immune responses detected in IBD subjects are an effect of disease, using serum samples from individuals before they developed IBD. That longitudinal framework is expected to provide further evidence of the causal role of anti-microbiota responses.