For more than two decades, Paul Kubes has been studying ways to directly visualize the role of immune cells during inflammation, infection and tissue injury, both in vitro and in vivo.

During the Tuesday morning J. Fraser Mustard Plenary Lecture at the ISTH 2015 Congress, he discussed several examples of how cutting-edge technologies, such as spinning-disk confocal and multi-photon microscopy, are helping researchers better understand how immune cells – such as neutrophils, monocytes, natural killer T-cells and Kupffer cells – function under physiological and pathological disease states.

“What we’re trying to do is understand the speed bumps that are put up by the immune system that prevent bacteria from disseminating throughout the vasculature,” explained Kubes, of the Snyder Institute for Chronic Diseases at the University of Calgary, Canada. Kubes and his associates built a multi-channel fluorescence spinning-disk confocal microscopy device that enables them to image innate immunity in the vasculature. They opted for the spinning-disk technology “because it actually captures images much faster than what you can do with standard confocal microscopy, and it’s faster than two-photon microscopy,” he said.

Use of this system to image the livers of mice in real time revealed Kupffer cells engaging with platelets. “It took us some time to figure out what this normal process was,” Kubes said. “If you perturb the system with E. coli, a TLR4 (toll-like receptor 4) agonist or viruses, the platelets very rapidly become engaged. You can see that [platelets] really love the neutrophils. They’re surrounding them and interacting with them.”

Not long ago, he continued, clinicians trained in medical school learned that platelets were for hemostasis and white cells were for immunity. Today, it’s known that platelets “have mediators that recruit immune cells, they recognize bacteria, [and] they have TLRs,” he said. “They have molecules that kill bacteria, so they have the capacity to function as immune cells.”

Kubes and other researchers have published important work about neutrophil extracellular traps (NETs), which are DNA-based structures that capture and eliminate microbes. “I’ve always wondered: How do NETs stay in the vasculature? Why don’t they just float away?” Kubes said. In the liver, for example, he and his associates “found incredible hot spots of von Willebrand factor. This is on Kupffer cells, and we saw some of it on the endothelium. The liver has a very high level of von Willebrand factor, which suggests that perhaps these NETs are binding to the vessel wall via a histone-von Willebrand factor interaction.”

NETs don’t always form during injury, though. In an experiment conducted about four years ago, Kubes and his associates touched the surface of the liver with a thermal probe and monitored the impact with imaging. “Immediately about 200 cells died,” Kubes recalled. “The question was: can neutrophils be recruited to this injury site? Over the 2-3 hours that we imaged this, we saw this spectacular neutrophil recruitment to the injury site, almost overkill. As they got close to the injury site, they elongated. The reason they were elongating is that around this injury site were sinusoids packed with platelets. The platelets went to this injury site within about 30 seconds to a minute. The neutrophils were crawling across the platelets. So instead of a platelet-neutrophil interaction, we had a neutrophil-platelet interaction.”

The researchers looked for the formation of NETs during this experiment, but none formed. This means “the neutrophils are going in there, but they know to distinguish between an infection and a tissue that’s dying and needing repair,” he said.

Kubes stated that he had no relevant financial disclosures.

By Doug Brunk |June 23, 2015