When the COVID-19 pandemic began to threaten the operations of Angelo D’Alessandro’s lab at the University of Colorado School of Medicine in early 2020, he didn’t realize that it would soon hit even closer to home. He had just visited collaborators at Columbia University in New York City, and shortly after returning, he tested positive for SARS-CoV-2. But for D’Alessandro and other researchers, the growing pandemic turned into an opportunity to study how an unlikely candidate responds to the virus: red blood cells.

Nilam Mangalmurti revealed a way that red blood cells behave like immune cells.

credit: Nilam Mangalmurti

“For a very, very long time, we've known that red cells have interactions with the immune system, but there haven't been very many studies solidifying the role of that immune response,” said Nilam Mangalmurti, a physician-scientist at the University of Pennsylvania. “It's interesting to think about this in critical illness because as an ICU doctor, I think about different things that affect the host response.”

The red blood cell conundrum in COVID-19

Red blood cells are not often thought of as a line of defense against foreign invaders. That’s where white blood cell types such as T cells, B cells, and monocytes take the spotlight. These cells can do everything from recognizing pathogens to eating them up. 

Red blood cells, on the other hand, are best known for their more routine job of carrying oxygen throughout the body. Filled with hemoglobin, these pillowy cells float through capillaries in the lung where they pick up oxygen that binds their hemoglobin. They then proceed through the bloodstream to the rest of the body to deliver this oxygen, ensuring that cells and organs function.

While these oxygen-ferrying cells have piqued the interest of some COVID-19 researchers, studies remain limited. A puzzling early observation in patients with COVID-19 was that they could have low levels of blood oxygen without difficulty breathing, sparking the use of pulse oximeters to identify people in the early stages of the disease. To some scientists, this signaled that something was going haywire in red blood cells. As more data have emerged, doctors have also noticed that anemia — a reduction in numbers of healthy red blood cells — associates with worse COVID-19 outcomes, including long COVID (1).

Now, evidence indicates that lung dysfunction plays a large role in dropping oxygen levels in COVID-19 cases, but red blood cells aren’t off the hook (2). Some researchers hypothesize that the SARS-CoV-2 virus can infect red blood cells as other pathogens do. (Although that would leave the virus in a predicament; because red blood cells don’t have nuclei or many organelles, they don’t have the machinery to make the new proteins the virus needs to replicate.) Evidence on whether this affects cells’ ability to carry oxygen is mixed, however (3,4).

Accelerated aging

D’Alessandro’s team had been studying the way that red blood cells’ metabolic processes change as cells aged, both in the body and in places where blood was stored, such as blood banks. His previous work with Zika and Chikungunya viruses — put on hold during the pandemic — had begun to suggest that infections affect how old red blood cells appear. Aged cells were less metabolically competent, leaving them with unabated signs of oxidative stress. As COVID-19 cases rose sharply in New York, D’Alessandro’s collaborators at Columbia University quickly built up a biobank.

The COVID-19 pandemic sparked Angelo D’Alessandro’s interest in studying red blood cells during infection. But first, he had to fight the virus himself.

credit: Angelo D’Alessandro

“It was like a totally serendipitous overlap of what was going on,” D’Alessandro said. “When COVID hit, we were asking the question, ‘what happens to red blood cells as a function of disease severity?’” With samples from the biobank, he could answer that question.

In a study published in the Journal of Proteome Research  in 2020, D’Alessandro’s team measured hundreds of molecules in red blood cells from 29 SARS-CoV-2-positive patients and 23 patients who tested negative (5). They found that cells from COVID-19 patients showed altered energy metabolism and signs of stress. Much of the damage appeared in membrane proteins, which have important structural and metabolic roles. 

This rang a bell for D’Alessandro; it closely mirrored what he had seen in aging blood stored in blood banks. Without all the required protein-assembling machinery, the red blood cells can’t replace the damaged proteins, which could leave them with reduced structural flexibility and make them more vulnerable to sudden stress. A more recent study confirmed that red blood cells from some COVID-19 patients are less able to bend, and this may alter their ability to navigate blood vessels and be filtered out by the spleen (6).

Seeing these lingering, hamstrung red blood cells in COVID-19 patients reminded D’Alessandro of another puzzling feature of the disease: long COVID, a condition that had plagued D’Alessandro and his wife for months after they recovered from their illnesses. “The damage makes a blood cell look like an older red blood cell, and now you have a blood cell that is damaged by COVID-19 that circulates for up to 120 days,” he said, hypothesizing that the effects of the damaged cells could outlast the viral infection itself.

Sounding the alarm

Other researchers have also found a key role for red blood cells in the heat of the moment during an infection. In a study recently published in Science Translational Medicine, researchers from the University of Pennsylvania reported that red blood cells can behave a little bit like pathogen-fighting white blood cells by expressing a toll-like receptor (TLR) on their surface (7). These signaling molecules usually bind molecules characteristic of invading pathogens, including proteins, sugars, and nucleic acids such as DNA or RNA. 

Red blood cells expressed a variety called TLR9, which bound certain DNA sequences. When this happened, the cells were eaten up by other immune cells, not only reducing the number of red blood cells, but also activating an inflammatory response. This clearance process is a normal step for aging red blood cells, but binding DNA can accelerate it. And in COVID-19, there’s plenty of mitochondrial DNA floating around that has been released by damaged cells. Red blood cell TLR9 receptors from 100 patients with COVID-19 were more likely to bind mitochondrial DNA than those from healthy patients, and this was especially true in patients with anemia or more severe COVID-19.

This might answer an important question about the anemia that arises in ICU patients and heralds worse outcomes in diseases like COVID-19. “Almost all of our patients become anemic during their time in the intensive care unit, and we don't quite know why,” said Mangalmurti, senior author of the study. “Years after we looked at this in the non-COVID sepsis patients, it was validating to find that the same process was occurring during COVID-19.” 

Lessons for RBC diseases

COVID-19 isn’t the only disease where red blood cells start to appear metabolically haggard. D’Alessandro has seen the same phenomenon in sickle cell disease, a blood disorder where a mutation in the hemoglobin protein causes red blood cells to become deformed and leads to anemia and excruciating pain. D’Alessandro was also intrigued to learn that some of the metabolic pathways hampered in COVID-19 patients’ red blood cells — such as carnitines involved in lipid metabolism — are also dysregulated in sickle cell disease.

Angela Brandow organized a registry of patients with sickle cell disease and COVID-19.

credit: Angela Brandow 

It’s not surprising to clinicians that COVID-19 and sickle cell disease could have intersecting effects on patients. “Really early on, the sickle cell disease community collectively had increased concern about how it would affect our patients,” said Amanda Brandow, a hematologist at the Medical College of Wisconsin. “Some of this stemmed from the fact that we know influenza in our patients can trigger pulmonary complications of sickle cell disease.” 

Brandow co-leads SECURE-SCD, a registry of patients with sickle cell disease and COVID-19, and has shown that Black people with both conditions are at a higher risk of hospitalization or severe disease. She hasn’t looked at red blood cells specifically, but she thinks that inflammation and other organ system damage might leave people with sickle cell disease more susceptible to severe COVID-19 (8). “When you live with a disease that is already a chronic inflammatory state, you've already primed your immune system to be active,” Brandow said. “Then you put a virus on top of that.”

Inflammation might also explain the high levels of mitochondrial DNA that Mangalmurti has noticed in both sickle cell disease and COVID-19. She hopes that what they’ve learned about DNA-bearing red blood cells can lead to better ways to detect and eliminate these potential instigators of further inflammation. “I'd be very interested to know what happens when we take cells from a patient with sickle cell disease and incubate them with excess amounts of DNA,” Mangalmurti said. “I’d be curious to see if we get increased sickling.”

For Mangalmurti and D’Alessandro, red blood cells are the future of research to better understand the immune system.

“I would love for the whole community to start looking at red blood cells for their role in responses to infection and inducing immune activation,” D’Alessandro said.

References

1. Lenehan, P. J. et al. Anemia during SARS-CoV-2 infection is associated with rehospitalization after viral clearance. iScience  24(7), 102780 (2021).
2. Dhont, S. et al. The pathophysiology of 'happy' hypoxemia in COVID-19. Respir Res  21(1), 198 (2020).
3. Park, K. C. et al. Single-cell oxygen saturation imaging shows that gas exchange by red blood cells is not impaired in COVID-19 patients. Br J Haematol  190(4), e229-e232 (2020).
4. Vogel, D. J. et al. A left shift in the oxyhaemoglobin dissociation curve in patients with severe coronavirus disease 2019 (COVID-19). Br J Haematol  191(3), 390-393 (2020).
5. Thomas, T. et al. Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients. J Proteome Res  19(11), 4455-4469 (2020).
6. Kubánková, M. et al. Physical phenotype of blood cells is altered in COVID-19. Biophys J  120(14), 2838-2847 (2021).
7. Lam, L. K. L. et al. DNA binding to TLR9 expressed by red blood cells promotes innate immune activation and anemia. Sci Trans Med  13(616), eabj1008 (2021).
8. Singh, A. et al. COVID-19 in individuals with sickle cell disease/trait compared with other Black individuals. Blood Adv  5 (7), 1915-1921 (2021).