How the Nose Responds to SARS-CoV-2, Cell by Cell

When people get infected with the SARS-CoV-2 virus, which causes COVID-19, why do some people get mildly sick while others get very sick? Although we know that SARS-CoV-2 enters the body via the nose and mouth, we do not fully understand how nasal cells respond to this infection or why some people get sicker than others. To answer these questions, we studied the immune response—the way the body fights infectious agents like bacteria and viruses—of cells from nasal swabs of healthy people and people with COVID-19. We found that nasal cells of very sick COVID-19 patients had a weaker immune response against the virus compared to that of cells from mildly sick patients, despite their virus levels being similar. Understanding how nasal cells interact with viruses and how their responses to infection are linked to how sick people get can help us better identify and manage the effects of viral infection.

are linked to how sick people get can help us better identify and manage the e ects of viral infection.
Have you ever gotten sick at the same time as a friend, with a runny nose, cough, or chills, maybe by catching the same cold at school? A cold is actually a viral infection that can be caused by hundreds of di erent kinds of viruses, including coronaviruses. Even though you both caught the same cold, you might be sick for a week while your friend feels better in a few days. Why did you get sicker than your friend? Did your friend's cells respond di erently to infection than yours did? This di erence in responses between individuals has also been seen with other respiratory viruses, including SARS-CoV-and the disease it causes, COVID- [ , ]. We studied cells from nasal swabs of people without COVID-as well as people with mild or severe COVID-, to better understand people's varying responses to the virus and how these responses influence how sick people might get after infection.

LOOKING AT THE NOSE CELL BY CELL
To study the immune response of nasal cells infected with SARS-CoV-, we used a technology called single-cell RNA sequencing (scRNA-seq) (Figure ). While this may sound complicated, this SINGLE-CELL RNA SEQUENCING (scRNA-seq) A technology used to study cells' behavior on a cell-by-cell basis, by studying their gene expression.
technology simply lets us take a bunch of cells from nasal swabs and analyze them, cell by cell [ ]. To illustrate, imagine a fruit salad with apples, pomegranates, and grapes. We can count how many of each type of fruit we have, determine which fruits have seeds, or compare how each fruit tastes. scRNA-seq lets us do something similar with mixtures of cells, allowing us to describe their similarities and di erences by measuring what each cell is doing at a certain time. We do this by counting the mRNA molecules in individual cells, which tell us about that cell's activity.
To make this clear, let us take a step back. Every human cell with a nucleus contains DNA-the genetic material passed down by our they should make a person's eyes blue versus brown, or whether a cell should become a heart cell or a lung cell. The instructions in a cell's genes are conveyed to the cell in the form of a message molecule called messenger RNA (mRNA). When a cell makes mRNA from a gene,

MESSENGER RNA (mRNA)
A molecule found in all living cells that is produced during gene expression.
it is said to be "expressing" that gene. The "single" part of "single-cell RNA sequencing" means we can study each cell one by one, while the "sequencing" part means we can read each cell's mRNA to figure out what the cells are doing. Studying gene expression in this way

GENE EXPRESSION
The process by which a gene's information is expressed by a cell, where it is used to make RNA or protein that help that cell fulfill certain functions. We collected nasal swabs from over fifty people who visited the hospital ( Figure ). Swabs were classified based on whether the person had COVID-or not, and if they did, how bad they had it. We isolated cells from the swabs and used scRNA-seq to count how many specific RNA molecules each cell contained, as well as to read those messages to give us a look at their gene expression. This helped us determine how the nasal cells were responding to infection, known as their immune response. But what is an immune response, exactly?

IMMUNE RESPONSE
The reaction the body launches to recognize and defend itself against viruses, bacteria, and foreign substances.

Figure
To study the immune responses of nasal cells from people with or without COVID-, we collected nasal swabs from these individuals and processed the swabs to isolate the cells from them.

WHAT IS AN IMMUNE RESPONSE?
The immune response is the reaction the immune system mounts to protect a person exposed to potentially harmful organisms, like bacteria and viruses. The immune system recognizes certain "danger" proteins on the surfaces of these organisms, which signal that these organisms must be removed. You are probably familiar with the immune response that occurs when you get vaccinated against flu, for example. Vaccination trains the immune system to recognize and attack the virus before you actually catch it.
One of the first steps infected cells take is to release a substance called interferon, which tells other cells to fight back and stop the

INVESTIGATING CELLULAR RESPONSES TO SARS-CoV-INFECTION
We used scRNA-seq to analyze the immune response of nasal cells from swabs taken from healthy people or people with mild or severe COVID-, to try to understand why some people got very sick and others did not. In comparing gene expression, we found that cells from people who were mildly ill and those who were severely ill both showed higher levels of interferon-related antiviral genes compared to cells from healthy people. This illustrates that the infected cells were trying to defeat the virus, which is what we expect to happen. Cells from people with mild COVID-showed high levels of antiviral genes. However, cells from people with severe COVID-showed lower levels of these antiviral genes, even early on during the infection. In some cases, these cells even behaved like those from healthy patients-as if they were not fighting a virus at all! Because these cells were having di culty responding to the virus, the immune response in these people was not very strong, which seemed to result in these individuals getting very sick. On the other hand, when the cells could fight back by initiating a strong immune response, individuals seemed to get only mildly sick.
You might be wondering, "What if the really sick people just had to fight o more virus than the slightly sick people?" Surprisingly, we found that even when the levels of virus were the same, individuals with severe COVID-still had a much weaker immune response overall (Figure ). This might be because the nasal cells from individuals with severe COVID-are impaired in some way,  We also wondered what types of nasal cells were getting infected with SARS-CoV-. As we mentioned earlier, scRNA-seq let us determine which types of cells were present in the nasal swabs, based on their gene expression. We found that a large portion of the infected nasal cells were ciliated cells, goblet cells, and secretory cells. Ciliated cells are like cellular brooms that help sweep things out of the respiratory system, using tiny hair-like structures called cilia. Secretory and goblet cells secrete mucus, which helps trap viruses, bacteria, or allergens and move them out of the body. The fact that these cells were the ones infected most often might indicate that these cell types are especially good at helping the virus spread.
Although we are now aware of the di erences in immune responses between healthy people and those experiencing varying severities of COVID-, there are still unanswered questions waiting for curious minds like yours to help answer them. For instance, we still do not know why this di erence in immune response is happening in the first place, whether this di erence depends on other factors (like age), or why the interferon response varies so significantly between individuals. We also do not know how COVID-vaccines might influence this initial immune response, or how these responses might di er in other cell types or parts of the body besides the nose. More work on this immune response and the reasons why it might vary between individuals will need to be done by future scientists like you. SARS-CoV-and the Nose cells can launch an immune response. In response to SARS-CoV-, some individuals' nasal cells cannot express enough antiviral and interferon-related genes, thus preventing them from ringing the alarm to alert other cells to fight. This results in a weakened immune response, which causes these people to get severely sick. However, some people's nasal cells can launch a robust immune response, so they only get mildly sick.

CONCLUSION
As we learn more about viruses such as SARS-CoV-or influenza, it is important that we work to decipher the immune response on a cellular level, to better understand why and how people get sick. Studying how cells respond to viruses will not only help us learn more about the viruses themselves but might also teach us how to help people a ected by them. Going forward, we hope that this work will lead to treatments that improve the lives of people dealing with viral infections.  . doi: . /frym. .

CONFLICT OF INTEREST:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
COPYRIGHT © Taliaferro, Long and Ordovás-Montañés. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

CAROLINE, AGE:
Hello! I am Caroline and I am years old. I really enjoy biology, chemistry, and math. I am interested in studying medicine, specifically immunology. Aside from school, I play violin and like to read. Something else I like to do in my free time is creative writing. I am excited about the opportunity to serve as a young reviewer.

KRITIKA, AGE:
I am a -year-old going to th grade. Science is my favorite subject. I love doing experiments, we grow chicken eggs in the incubator and look at the embryos under a light to see when they will hatch. I want to become a veterinary doctor because I want to take care of animals, and I want to have a farm day with horses. My hobbies are taking my dog for a walk, feeding our chickens, parakeets, and Japanese quails. I love reading books and my favorite books are Harry Potter and Keeper of The Lost Cities.

MADELINE, AGE:
Hi my name is Madeline and I am going into the th grade at the Boston Latin School. I enjoy reading, drawing, and playing sports. I am interested in a career in biology and science. My love for science first began last school year when I did my science fair project about mushrooms. I think mushrooms are fascinating!

JACLYN M. LONG
Jaclyn Long is a Ph.D. candidate in the immunology program at Harvard Medical School. She grew up in Massachusetts and attended Northeastern University for her undergraduate studies, where she completed degrees in bioengineering and biology and did research on the immune system. As a graduate student, she now works in the labs of José Ordovás-Montañés and Arlene Sharpe. Her research is focused on how we develop immune memory of viral infections in the nasal mucosa.

JOSÉ ORDOVÁS-MONTAÑÉS
Dr. José Ordovás-Montañés, Ph.D. is a principal investigator at Boston Children's Hospital. He received his B.A. in biology from Tufts University, and Ph.D. from Harvard University, where he studied how the nervous system and the immune system function together as the principal sensory interfaces between the internal and external environments. As a Damon Runyon Postdoctoral Fellow at MIT, the Broad Institute, and the Ragon Institute, he discovered how human stem cells can be shaped by, and remember, inflammation. Jose started his group in , and they seek to understand the principles of how inflammation drives immunological memory in human barrier tissues like the nasal mucosa. *jose.ordovas-montanes@childrens.harvard.edu