Faster diagnosis? Chlorinated lipids in sepsis

In a study published last year, Dr. Ford and coauthors described data that suggested a “novel role for 2-CLFA as a lipid mediator” of the formation of neutrophil extracellular traps in neutrophil death, or netosis (Palladino END, et al. Arch Biochem Biophys. 2018;641:​31–38).

Dr. Ford also shared his laboratory’s study of endothelial cells, which used click chemistry with a synthesized analog of 2-chlorohexadecanoic acid (2-CLHA) to examine the subcellular location of the 2-CLFA in coronary artery endothelial cells (Hartman CL, et al. J Lipid Res. 2018;59[1]:113–122).

“We synthesized a molecule that had an alkyne at the methyl terminal of the chlorofatty acid. This allows us to perform click chemistry. We incubated this molecule with cells and then clicked the alkyne to an azide, and this azide had a reporter on it,” Dr. Ford said. The azide reporter was TAMRA (tetramethylrhodamine), and using fluorescence microscopy they could then see where the chlorofatty acid goes in the cell.

“We found that the chlorolipids co-localize with P-selectin, von Willebrand factor, and Cox IV. These co-localizations indicate that the chlorolipid is in the Weibel-Palade bodies, because that’s what stores P-selectin and von Willebrand factor in the endothelium, as well as the mitochondria (Cox IV) of the endothelial cells,” he said.

Weibel-Palade bodies contain P-selectin, VWF, and angiopoietin-2, and they change from a rod shape to a circular shape under activation. “Whenever we treated cells with chlorolipid, they went to this round shape to indicate that the Weibel-Palade bodies got activated,” Dr. Ford said.

The molecules inside the Weibel-Palade bodies perform significant roles during sepsis: The selectins increase neutrophil adherence to the endothelium, VWF causes platelets to adhere, and angiopoietin-2 increases permeability of the endothelial barrier, allowing for the formation of an edema.

Other experiments also showed that 2-CLFA increases the surface expression of P-selectin on endothelial cells. “We treated endothelial cells either with palmitic acid, chloropalmitic acid, or the click analog of 2-chloropalmitic acid,” Dr. Ford said. Under those conditions, P-selectin surface-expressed on those cells. Similar experiments showed that “along with the P-selectin surface expression, we see neutrophils adhering to the endothelium. We also see von Willebrand factor release in a similar sort of graph, and platelets adhering.” The laboratories of Dr. Ford and Dr. McHowat measured leakiness of the endothelium using an electric cell-substrate impedance sensing-resistance measure across the monolayer of endothelial cells. “The chlorolipids caused the endothelium to get leaky.”

Dr. Ford and colleagues also looked at the impact of chlorolipids on netosis. “Phorbol ester is a well-known stimulant to cause netosis,” he said. Myeloperoxidase is released and sticks on the neutrophil DNA that is extruded during netosis. “DNA is released during netosis and it binds to the heme protein, which is myeloperoxidase,” he said.

“After 90 minutes of stimulation, phorbol ester causes very little netosis. But the chloropalmitic acid elicits netosis at 90 minutes and also at 180 minutes. The latter—180 minutes—is when we saw the netosis with the phorbol esters.”

In summary, Dr. Ford said his laboratory’s targeted approach for chlorinated lipids showed that with leukocyte activation there is a respiratory burst, and plasmalogens get targeted by the bleach made by myeloperoxidase, liberating chlorinated fatty acids and chlorinated fatty aldehydes.

“We’ve shown they cause endothelial surface adhesion molecule expression that leads neutrophils and platelets to adhere to the endothelium,” Dr. Ford said. “We’ve seen this in both isolated cells as well as in intravital microscopy work” performed by Dr. Korthuis.

“That ultimately is going to lead to organ failure.”

Their studies have shown that netosis occurs when neutrophils are treated with chlorinated lipids, he said. “Many think of netosis as being an event to capture microbes, and that can be a great thing to happen during sepsis. But we envision with netosis that microbial killing is insignificant compared to neutrophil phagocytosis of microbes. When you have netosis, you’re going to plug the microcirculation, which will lead to further organ failure during sepsis.”

“Additionally, we have shown that these chlorinated lipids are predictive biomarkers of sepsis outcomes with death and acute respiratory distress syndrome,” and the studies are ongoing, Dr. Ford said.

Dr. Ford addressed a concern about the stability of 2-chlorofatty acid for clinical laboratory testing purposes.

“Fortunately, the chlorinated fatty acid is pretty stable. We are doing studies where we’ve spiked a chlorofatty acid in the plasma and measured it after six months, 12 months. We’re up to five years now, and we don’t see a drop with storage at -80°C for frozen plasma.”

A more pressing concern is the cleanliness of the preparation of the plasma, he said. “Often, someone will send us plasma, and we can tell without extracting it that something is wrong because everything is red. Hemolysis is a huge issue that we run into. To me, that is a bigger issue compared to 2-chlorofatty acid stability in our analyses.”

Amy Carpenter Aquino is CAP TODAY senior editor. Dr. Ford presented in the same AACC session as Dr. Isbell; see story.