Single-Cell RNA-seq Tracks Immune Response to Microbial Pathogens

November 21, 2016
Single-Cell RNA-seq Tracks Immune Response to Microbial Pathogens
This is a fluorescence microscopy showing on the left, a macrophage (nucleus in blue) infected with a non-replicating bacteria in yellow indicated by an arrow and on the right infected with bacteria that has replicated (red). [Antoine-Emmanuel Saliba]

A research team from the University of Würzburg, Germany, in collaboration with scientists at Imperial College, London has just developed a novel technique that allows them to investigate the interplay of individual host cells with infecting bacteria through the use of single-cell genomics. The findings from this new study were published recently in Nature Microbiology through an article entitled “Single-cell RNA-seq ties macrophage polarization to growth rate of intracellular Salmonella.”

The investigators used a technique called single-cell RNA-sequencing (RNA-seq) to study the infection of macrophages by Salmonella. Macrophages are immune cells that belong to the group of white blood cells. Salmonella, on the other hand, are pathogenic bacteria that may be taken up by the ingestion of contaminated water or food to cause local gastroenteritis and diarrhea. However, for immunocompromised patients, Salmonella may disseminate throughout the entire body and cause life-threatening illness.

Upon the invasion of macrophages, Salmonella pursues two strategies: The bacteria either replicate to high numbers inside the host cell or adopt a non-growing state that allows them to persist for years within the body of their host. "This disparate growth behavior impacts disease progression and plays a major role in the success of antibiotic treatment," remarked senior study investigator Jörg Vogel, Ph.D., professor and group leader in the Institute of Molecular Infection Biology (IMIB) at the University of Würzburg.

Since little is known if and how macrophages respond to these disparate lifestyles of intracellular Salmonella, the Würzburg scientists began their investigation by culturing macrophages in the laboratory and infected them with Salmonella.

The RNA from the infected cells was subsequently extracted and analyzed using deep-sequencing, leading to the detection of more than 5,000 different transcripts per macrophage—which was combined with information about the growth behavior of the intracellular pathogens.

Interestingly, the scientists found that macrophages containing non-growing bacteria adopted the hallmark signature typically associated with inflammation. They expressed signaling molecules to attract further immune cells to the site of infection. In this respect, the dominantly infected macrophages responded similarly to macrophages that have encountered Salmonella but have not been infected. "These macrophages cannot detect their intracellular bacteria—they are below their radar" explained lead study investigator Antoine- Emmanuel Saliba, Ph.D., postdoctoral researcher at IMIB.

Conversely, macrophages with fast-growing bacteria develop an anti-inflammatory response. While these results are as compelling as they are interesting, they do lead to a series of questions that should keep researchers busy for some time: Do Salmonella induce this different response? Do they manipulate the macrophages, so they do not raise the alarm to facilitate the bacteria to evade an immune response? Are there situations where Salmonella are unable to perform this trick? In these cases, will there still be an immune response forcing the bacteria to switch to their resting growth state?

“Currently we have just looked at a single time point after infection and thus cannot differentiate between cause and consequence" noted study co-author Alexander Westermann, Ph.D., postdoctoral researcher at IMIB. Follow-up studies are required. Nevertheless, the current findings already provide a new perspective on the host response to pathogenic microbes. And using the new technology, bacterial infections can be studied in unprecedented resolution—namely on the single-cell level.

The investigators were excited by their findings, and their new method should be of great interest for many further biomedical projects. "Among others, heterogeneity amongst tumor cells or the effect of drugs on single cells may be analyzed in unknown accuracy," concluded Dr. Vogel.