The vast majority of people infected with the Omicron variant of SARS-CoV-2 experience mild cold-like symptoms, moderate flu-like symptoms, or no symptoms at all, but the virus is so transmissible that it still spread deep into lung tissue to cause severe disease and death in thousands of people in the United States in 2022 alone.
Researchers at the University of North Carolina at Chapel Hill have revealed biological reasons for how disease progression happens and why a certain population of asthma patients are less susceptible to severe COVID.
This research, published in the Proceedings of the National Academy of Sciences, illustrates the importance of a well-known cytokine called interleukin-13 (IL-13) in protecting cells against SARS-CoV-2, which helps explain the mystery of why people with allergic asthma fare better than the general population despite having a chronic lung condition.
“We knew there had to be a bio-mechanistic reason why people with allergic asthma seemed more protected from severe disease,” said senior author Camille Ehre, Ph.D., assistant professor of pediatrics at the UNC School of Medicine and member of the UNC Marsico Lung Institute.
“Our research team discovered a number of significant cellular changes, particularly due to IL-13, leading us to conclude that IL-13 plays a unique role in defense against SARS-CoV-2 infection in certain patient populations.”
Although cytokines like IL-13 cannot be used as therapies because they trigger inflammation, it is important to understand natural molecular pathways that cells use to protect themselves from pathogen invasion, as these studies have the potential to reveal new therapeutic targets.
There are many health factors that increase a person’s risk of severe COVID, including chronic lung diseases such as COPD, but during the pandemic, epidemiologists found that people with allergic asthma were less susceptible to severe disease.
“These are patients with asthma caused by allergens, such as mold, pollen, and dander,” said Ehre, who is also a member of the UNC Children’s Research Institute. “To find out why they are less susceptible, we investigated specific cellular mechanisms in primary human airway epithelial cell cultures.”
The experiments were led by co-first authors Cameron Morrison, a medical student in the Ehre lab, and Caitlin Edwards, a research assistant and MPH student in the lab of Ralph Baric, Ph.D., Kenan Distinguished Professor of Epidemiology at the UNC Gillings School of Global Public Health and professor in the UNC Department of Microbiology and Immunology at the UNC School of Medicine.
The researchers used genetic analysis of human airway cell cultures infected with SARS-CoV-2 to discover that the expression of the human protein ACE2 governed which cell types were infected and the amount of virus found in this cell population (also known as “viral load.”)
The scientists then used electron microscopy (EM) to identify an intense exodus of virus from infected ciliated cells, which are cells tasked with moving mucus along the airway surface. EM also revealed severe cytopathogenesis—changes inside human cells due to viral infection. And these changes culminating in ciliated cells (packed with virions) shedding away from the airway surface.
“This shedding is what provides a large viral reservoir for spread and transmission of SARS-CoV-2,” Ehre said. “It also seems to increase the potential for infected cells to relocate to deeper lung tissue.”
Further experiments on infected airway cells revealed that a major mucus protein called MUC5AC was depleted inside cells, likely because the proteins were secreted to try to trap invading viruses. But the virus load kept increasing because the cells tasked with producing MUC5AC were overwhelmed in the face of a rampant viral infection.
The researchers knew from epidemiological studies that allergic asthma patients—known to overproduce MUC5AC—were less susceptible to severe COVID. Ehre and colleagues also knew the cytokine IL-13 increased MUC5AC secretion in the lungs when asthma patients faced an allergen.
The scientists decided to mimic asthmatic airways by treating human airway cells with IL-13. They then measured viral titers, viral mRNA, the rate of infected cell shedding, and the overall number of infected cells. Each one was significantly decreased. They found this remained true even when mucus was removed from the cultures, suggesting other factors were involved in the protective effects of IL-13 against SARS-CoV-2.
Bulk RNA-sequencing analyses revealed that IL-13 upregulated genes that control glycoprotein synthesis, ion transport, and antiviral processes—all of which are important in airway immune defense. They also showed that IL-13 reduced the expression of the viral receptor, ACE2, as well as reducing the amount of virus inside cells and cell-to-cell viral transmission.
Taken together, these findings indicate that IL-13 significantly affected viral entry into cells, replication inside cells, and spread of virus, thus limiting the virus’s ability to find its way deeper into the airways to trigger severe disease.
“We think this research further shows how important it is to treat SARS-CoV-2 infection as early as possible,” Ehre said. “And it shows just how important specific mechanisms involving ACE2 and IL-13 are, as we try our best to protect patients from developing severe infections.”
Other authors of the PNAS paper are Kendall Shaffer, Kenza Araba, Jason Wykoff, Danielle Williams, Takanori Asakura, Hong Dang, Lisa Morton, Rodney Gilmore, Wanda O’Neal, and Ric Boucher, all at UNC-Chapel Hill.
Asthma is associated with changes in the structure and function of the airway epithelium, a critical site for SARS-CoV-2 infection (16, 17). Airway epithelial gene expression changes attributable to the type 2 cytokine IL-13 are seen in approximately half of individuals with asthma (18, 19).
IL-13 stimulation of airway epithelial cells decreases expression of ACE2, which encodes the SARS-CoV-2 receptor, and increases expression of TMPRSS2, which encodes a transmembrane protease that primes the viral spike protein (20–22). Similar changes are seen in airways of individuals with type 2 high asthma (21, 22).
IL-13 has also been reported to protect against other RNA viruses, including respiratory syncytial virus (RSV) (23) and rhinovirus (24), that do not rely on ACE2 and TMPRSS2 for entry, indicating that other IL-13-regulated genes can also protect against viral infection.
We therefore hypothesized that IL-13 induces changes in expression of airway epithelial cell genes important in SARS-CoV-2 infection in a large subset of individuals with asthma and that IL-13 reduces susceptibility of these cells to SARS-CoV-2 infection.
Our studies reveal that IL-13 stimulation of HBECs affects expression of many SARS-CoV-2-associated genes and substantially inhibits SARS-CoV-2 infection of these cells. Genes encoding the large majority of SARS-CoV-2-interacting proteins identified in a previous study of HEK293T cells were expressed in HBECs. Expression of many SARS-CoV-2-associated genes differed between basal, ciliated, and secretory cells, potentially affecting how these cell types respond to SARS-CoV-2 infection.
Many IL-13-induced SARS-CoV-2-associated gene expression changes we detected in culture were also seen in bronchial epithelium obtained directly from individuals with type 2 high asthma. This provides a plausible mechanism for protection against COVID-19, although the impact of asthma on COVID-19 risk is still incompletely understood and other factors may also influence COVID-19 risk in individuals with asthma (13–16).
We also found significant associations of many IL-13-induced SARS-CoV-2-associated genes with type 2 inflammation in a large group of smokers with and without COPD, suggesting that the effects of IL-13 on SARS-CoV-2 risk may also be relevant in some individuals without asthma.
The effects of IL-13 on SARS-CoV-2-associated genes were clearly different than the effects of IFN-α, suggesting that these two cytokines induce different antiviral mechanisms. In experiments that established the inhibitory effect of IL-13 on SARS-CoV-2 infection of epithelial cells, we found evidence that another barrier component, the mucus gel, also provides protection against infection. Taken together, these studies provide insights into airway epithelial responses that can protect against SARS-CoV-2 and might influence COVID-19 susceptibility and severity in individuals with asthma or other airway diseases.
IL-13 had a substantial effect on SARS-CoV-2 infection of HBECs as demonstrated by measurements of viral RNA and dsRNA following viral inoculation. Prior studies report a variety of effects of asthma and IL-13 on development of illnesses caused by other viruses. IL-13 can increase susceptibility of HBECs to rhinovirus infection by suppressing induction of interferons (40–42), although another study reported that prolonged pre-treatment with IL-13 of HBECs reduced rhinovirus infection (24).
Mice with acute allergic airway inflammation (43) and people with pre-existing asthma (44) are reportedly protected from H1N1 influenza. Studies in IL-13-overexpressing transgenic mice and IL-13-deficient mice showed that IL-13 reduced respiratory syncytial virus replication and severity of illness (23). While effects of IL-13 on the airway epithelium are an important contributor to asthma pathogenesis, it is intriguing to speculate that IL-13 responses may have evolved at least in part to provide protection against viral infections. The finding that levels of IL-13 and the related type 2 cytokine IL-4 were higher in patients with moderate COVID-19 compared with severe COVID-19 or healthy controls is also consistent with an antiviral role for these cytokines (45).
Many mechanisms might account for IL-13-driven inhibition of SARS-CoV-2 infection. A recent study identified 65 interferon-stimulated genes that mediate restriction of SARS-CoV-2 infection (46), illustrating how a single cytokine can activate a large set of antiviral pathways. We found that gene expression changes induced by IL-13 were quite distinct from those induced by IFN-α, suggesting that these cytokines activate different antiviral pathways.
We confirmed prior studies (20, 21) showing that IL-13 induced a decrease in expression of the SARS-CoV-2 receptor ACE2, which could contribute to decreased infection. However, the reduction in ACE2 expression was modest compared with the effects of IL-13 on infection, suggesting that other IL-13 effects should also be considered.
As in the previous reports, we found that IL-13 increased expression of TMPRSS2, a protease that is important for viral entry. However, we found that the IL-13 effects were cell type-dependent: TMPRSS2 expression was increased in secretory cells but decreased in ciliated cells. Since ciliated cells were the primary cell type infected in our experiments, it is possible that decreased TMPRSS2 in ciliated cells contributed to an overall reduction in infection. Many other host cell factors influence viral entry, RNA synthesis and translation, and egress, and further studies will be required to determine which of these contribute to the antiviral effects of IL-13.
Our studies provided clear evidence that the mucus barrier produced by HBECs in cell culture inhibits SARS-CoV-2 infection. Airway mucus is a complex hydrogel that derives its characteristic viscoelastic properties from the mucin glycoproteins MUC5B and MUC5AC (47). Prior studies establish that mucins play important roles as restriction factors for other viruses, including influenza (48, 49).
We found that SARS-CoV-2 infection was decreased when mucus gels were left in place at the time of viral inoculation. We studied two subjects with distinct patterns of mucin expression and found somewhat different levels of protection from the gel. While further studies are clearly required to investigate this further, this result suggests that differences in mucus gels are also likely to be important in SARS-CoV-2 infection.
Changes in airway mucus volume, composition, and organization are prominent features of many airway diseases, including asthma (47). IL-13 is an important regulator of mucins, and we speculate that IL-13-driven increases in MUC5AC, which results in tethering of the mucus gel to the epithelium (25), might contribute to IL-13-induced inhibition of SARS-CoV-2 infection. However, this is unlikely to completely account for the antiviral effect since IL-13 inhibited viral infection even when the mucus gel was removed immediately prior to inoculation.
Our study has some important limitations. While we focused on a set of SARS-CoV-2-associated genes that have been defined in previous studies, other IL-13-regulated genes are also likely to be important for anti-viral effects. Some IL-13-regulated genes we identified in cell culture were not associated with a type 2 signature in cells from individuals with asthma or COPD, reflecting the influence of other factors, including other asthma mediators, or differences in IL-13 responses in cell culture versus in vivo.
As individual genes that contribute to inhibition of viral infection in HBECs are identified, it will be important to specifically examine the expression of those genes in asthma and COPD. Our HBEC infection studies used only one strain of SARS-CoV-2 and cells from only two donors, and further experiments with additional strains and more donors (including donors with asthma), will be required to better understand the interactions between virus, epithelial cells, and IL-13. Finally, our infection model focuses solely on the role of epithelial cells, but the effects of IL-13 on other cell types found in the lung are also deserving of further study.
In conclusion, we found that the central asthma mediator IL-13 has a strong inhibitory effect on SARS-CoV-2 infection of HBECs. The mechanisms that account for this effect are unknown, but widespread effects of IL-13 on expression of SARS-CoV-2 associated genes that are distinct from those induced by interferons suggest that some of these mechanisms may be novel. While the use of IL-13 itself as a therapeutic may well be prevented by the pro-asthmatic effects of this cytokine, identification of IL-13-induced antiviral pathways could help address the urgent need for development of novel targeted treatments for COVID-19.
reference link :https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7924269/
More information: Cameron B. Morrison et al, SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13, Proceedings of the National Academy of Sciences (2022). DOI: 10.1073/pnas.2119680119