“Two proteins, named CXCL10 and IFN-gamma, popped up. We think these two are the major drivers of myocarditis,” Wu said. They operate like a tag team.

CXCL10 and IFN-gamma both belong to a class of proteins called cytokines: signaling substances that immune cells secrete to carry on chemical conversations with one another.

Hoping to listen in on these communications, the scientists generated human immune cells called macrophages — fierce first-responder cells of the immune system — in a dish and incubated them with mRNA vaccines.

The macrophages responded by pumping out various cytokines but, most notably, pronounced amounts of CXCL10. They also otherwise generally mimicked the vaccine responses of macrophages reported in humans, as shown by comparison with published data from vaccinated individuals.

When the scientists further supplied the dish with an additional kind of immune cell — T cells, roving sentinels that can recognize and mount immune attacks on specific pathogens but can also incite general arousal of the immune system — or even when they merely steeped T cells in the solution in which vaccine-administered macrophages had bathed, they saw a marked uptick in the T cells’ output of IFN-gamma. But T cells incubated with mRNA vaccine in the absence of macrophages or their bathwater produced only standard amounts of IFN-gamma. These results showed that macrophages are the chief source of CXCL10 and that T cells are the chief source of IFN-gamma in response to mRNA vaccination.

Tag-teaming

But did the two cytokines, together, contribute directly to cardiac injury? The researchers vaccinated young male mice, then found heightened levels of cardiac troponin, the widely used clinical marker of heart muscle damage.

The investigators also noticed infiltration of macrophages and another frontline take-no-prisoners immune-cell type, neutrophils — short-lived first responders that live to die in glorious battle (typically with bacterial or fungal pathogens) and are the main component of pus — into the mice’s cardiac tissue. This also occurs in post-vaccination myocarditis patients.

This macrophage and neutrophil infiltration into the heart — which comes at a cost, as these shoot-first-and-ask-questions-later warrior immune cells often unload friendly fire, causing collateral damage to healthy tissue, including heart muscle — could be minimized by blocking CXCL10 and IFN-gamma activity.

Also seen in the mice’s hearts were increased populations of cell-surface molecules that snag macrophages, neutrophils and other white-blood-cell types, causing them to adhere to endothelial cells, which line all blood vessels including those in the heart.

So, yes, CXCL10 and IFN-gamma did contribute directly to cardiac injury in these mice. And blocking them largely preserved the immune response to the vaccination while lowering levels of cardiac troponin induced by vaccination.

Wu’s lab excels at a technology involving the transformation of human skin cells or blood cells into blank cells that can then be guided to differentiate into cardiomyocytes, macrophages and endothelial cells and to coalesce into spherical structures that mimic the heart’s rhythmic contractions.

The researchers treated these “cardiac spheroids” with CXCL10- and IFN-gamma-enriched bathwater from vaccine-stimulated macrophages and T cells, respectively. They witnessed a significant increase in markers of cardiac stress, rescued by inhibitors of the two cytokines.

The cardiac spheroids’ squeezing capacity, beating rate and other measures of healthy heart function were all impaired but, again, partially restored by the cytokine inhibitors.

Saved by a soybean

Wu had a hunch that a common dietary supplement could help prevent such damage. Given higher myocarditis rates among males and estrogen’s known anti-inflammatory properties, he revisited a compound he’d studied a few years earlier.

In a 2022 paper published in Cell, Wu’s team had identified genistein, a mild estrogen-like substance derived from soybeans, as having anti-inflammatory activity and the ability to counter marijuana-induced damage to blood vessels and heart tissue.

“Genistein is only weakly absorbed when taken orally,” Wu said. “Nobody ever overdosed on tofu.”

Wu and his colleagues conducted a series of experiments closely paralleling those described above, pre-treating cells, cardiac spheres and mice (the latter by oral administration of large quantities) with genistein. Doing this prevented much of the deleterious effects of mRNA vaccines or the CXCL10/IFN-gamma combo to heart cells and tissue.

The genistein Wu and his associates used was purer and more concentrated than the dietary supplement found in health food stores.

“It’s reasonable to believe that the mRNA-vaccine-induced inflammatory response may extend to other organs,” Wu said. “We and others have seen some evidence of this in lung, liver and kidney. It’s possible that genistein may also reverse these changes.”

Elevated inflammatory cytokine signaling could be a class effect of mRNA vaccines. Notably, IFN-gamma signaling is a fundamental defense mechanism against foreign DNA and RNA molecules, including viral nucleic acids, Wu said.

“Your body needs these cytokines to ward off viruses. It’s essential to immune response but can become toxic in large amounts,” he said. IFN-gamma secreted in large amounts, however lofty its purpose, can trigger myocarditis-like symptoms and degradation of structural heart muscle proteins.

That risk probably extends beyond mRNA-based COVID-19 vaccines.

“Other vaccines can cause myocarditis and inflammatory problems, but the symptoms tend to be more diffuse,” Wu said. “Plus, mRNA-based COVID-19 vaccines’ risks have received intense public scrutiny and media coverage. If you get chest pains from a COVID vaccine you go to the hospital to get checked out, and if the serum troponin is positive, then you get diagnosed with myocarditis. If you get achy muscles or joints from a flu vaccine, you just blow it off.”

The study was funded by the National Institutes of Health (grants R01 HL113006, R01 HL141371, R01 HL141851, R01 HL163680 and R01 HL176822) and the Gootter-Jensen Foundation.