ACE2 Expression Is Lower in the Respiratory Tracts of Children

Both the first SARS virus, SARS-CoV-1 discovered in 2003, and the SARS-CoV-2 virus bind to angiotensin-converting enzyme 2 (ACE2) (11, 12). The renin angiotensin system, originally known for its critical role in blood pressure and hypertension, is also a critical trigger of inflammation in various organ systems. ACE converts the 10-amino acid angiotensin I peptide into the 8-amino acid peptide, known as angiotensin II. ACE2 then converts angiotensin II to the 7-amino acid peptide known as angiontensin_1–7. Angiotensin-II, when binding to the angiotensin type (AT) 1 receptor, is proinflammatory in multiple organ systems including the lungs, the heart, the kidneys (13, 14), and the brain (15). In contrast, angiotensin_1–7 is antiinflammatory when it binds to the Mas receptor (14).

Coronaviruses are enveloped single-stranded RNA viruses that cause enteric and respiratory tract infections in mammals and birds that can range from mild to severe (16). After the spike protein of SARS-CoV-2 binds to cells in the respiratory tract via ACE2 (11⇓–13, 17, 18), the virus enters the cell via proteolytic cleavage involving two proteases, the transmembrane protease serine 2 (TMPRSS2) and cathepsin L (CTSL). In recent studies (17, 18) researchers analyzed the levels of RNA in cells of the respiratory tract, including nasal, airway, and lung parenchyma. Both of these studies showed that expression of ACE2 increases with age in the respiratory tract.

Wang et al. (17) analyzed gene expression in healthy lung tissue from three different age groups: approximately age 30 gestational weeks, approximately age 3 y, and approximately age 30 y. They analyzed gene expression with two platforms at single nuclear (sn) resolution, using snRNA sequencing (snRNA-seq) and sn assay for transposase−accessible chromatin (snATAC-seq), enabling the researchers to analyze specific genes and neighboring (cis-) regions that control cell-type expression of gene modules involved in processes like inflammation.

The respiratory tract has diverse cell types including alveolar epithelial type 1 (AEI) cells, that are responsible for the air−blood barrier in lung, and alveolar epithelial type 2 (AEII) cells that are critical for secreting pulmonary surfactant (17, 18). The cellular distribution of ACE2 and TMPRSS2 was distinct from CTSL. While ACE2 and TMPRSS2 were primarily in AEI, AEII, and airway cells such as club, ciliated, and basal cells, CTSL expression was found in epithelial cells, mesenchymal cells, endothelial cells, and macrophages. It is the alveolar cells that are the anatomic site of acute respiratory distress syndrome.

Wang et al. (17) noted “an increase in the proportion of alveolar epithelial cells expressing ACE2 and TMPRSS2 in adult compared to young lungs.” The investigators observed marked differences, particularly in AE cells (17), with the percentage of ACE2⁺ AEII cells increasing with age from the samples from those age 3 y upward to the samples from those age 30 y. Similar findings were seen for the TMPRSS2⁺ cells.

Analysis of the regulatory elements related to TMPRSS2⁺ cells revealed modules related to activation of an immune response in the lungs with a correlation related to increasing age. Noteworthy immune factors included interferon (IFN) regulatory molecules for both type 1 and type 2 IFNs, and signal transducer and activator of transcription molecules involved in activating inflammatory cytokine molecular pathways. Expression of TMPRSS2 was highest in ciliated cells and AEI. Expression of TMPRSS2 increased with age in mice and humans (19). For SARS-CoV-1, TMPRSS2 was shown to promote viral spread, with its protease activity serving to reduce viral recognition by neutralizing antibodies. Whether TMPRSS2 plays a similar role in the spread of SARS-CoV-2 is a subject of current investigation (19).

Muus et al. (18) analyzed gene expression in more than 4 million cells derived from 107 single-cell RNA expression studies, including 22 studies focusing on datasets from the lung and airways in both adults and children. In this large metaanalysis of multiple RNA expression studies, the researchers compiled data on cells from “nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups.” They found ACE2, TMPRSS2, and CTSL coexpressed at low levels in alveolar cells of children compared to adults. The researchers were able to confirm these results directly with triple fluorescence in situ hybridization in specific cell types. They concluded that there was “particularly low” ACE2 in the pediatric samples (18).

Although the number of samples of young children was limited in this study (18), the researchers found that “strikingly, ACE2 expression is very low in normal lungs of newborns, the one ∼3 months’ lung sample, and the 3-year-old lungs.” These data were supported on a second platform known as scTHS-Seq, single-cell chromatin accessibility by transposome hypersensitive site sequencing, from human pediatric samples with no lung disease, collected at day 1 of life, at 14 mo, at 3 y, and at 9 y. They found “no signal was present at birth, it was low in the 9-year-old and 3-year-old sample, and higher in the 14 month-old.” The researchers concluded that “these patterns may be important in understanding why children are more resistant to COVID-19” (18).

There was an association of cells expressing both ACE2 and TMPRSS2 that were more frequent with increasing age (18). Cells expressing both TMPRSS2 and ACE2 in children are quite rare (18). These double-positive cells were particularly notable for expression of associated modules mediating viral and immune responses. These double-positive cells were the likely locus where an exuberant inflammatory response emerges. In these double-positive cells in lung epithelium, expression of interleukin 6 (IL-6) and IL-6R was increased. In so-called cytokine storms, where there is overactivation of the immune system in the alveoli, activation of these associated gene modules may underlie the pathophysiologic response (18). Monoclonal antibodies targeting inflammatory cytokines including IL-6 and its receptor, as well as tumor necrosis factor (TNF), are in therapeutic trials for cytokine storm in COVID-19.

A reasonable conjecture might be that, if ACE2 and/or TMPRSS2 expression is diminished in children, then viral infection of respiratory cells by SARS-CoV-2 might be less likely at any given viral load, and, additionally, there might be reduced expression of associated inflammatory modules. It is known that, in healthy children, there is an age-related increase in the production of IFN-γ and TNF-α. For IL-6, there was a trend for lower IL-6 concentrations in children below 2 y of age compared to older children (20). Thus, children have a lowered propensity to transcribe inflammatory cytokines in lung cells, and this may reduce the chance for an explosive immune reaction within the lungs resulting in the so-called “cytokine storm.”

Impact of Frequent Respiratory Infections in Childhood and Virologic and Immunologic Interference

Numerous studies (21⇓–23) show that children younger than 2 y of age have five or more respiratory infections per year, and spend a median of 44 d with mild upper respiratory illnesses (21). The activation of adaptive immunity to common coronaviruses as well as the activation of the innate immune system in the respiratory tract may indeed provide some protection from microbial infection, including SARS-CoV-2. The frequent respiratory infections in children may provide further clues to why children are resistant. One line of reasoning is based on the phenomenon of viral interference. A second line of reasoning is based on the concept of immune interference.

A Surprising Potential Protective Benefit of Th2 Immunity in Children

There are three major arms of the immune response characteristics of human T helper cells, named Th1, Th2, and Th17 (33). The Th1 arm described above is mediated by gamma IFN. The Th2 arm is associated with allergic disease, and is mediated by IL-4, IL-5, and IL-13 (44). Sajuthi et al. (33) reported on gene expression studies on 695 children with asthma and healthy controls from the Genes-Environment & Admixture in Latino Americans study, an ongoing case-control study of asthma in Latino children and adolescents. They found that TMPRSS2 is part of a mucus secretory network, driven by Th2 inflammation via the actions of IL-13 (33). They found that Th2 responses driven by IL-4, IL-5, and IL-13 “dramatically” reduced ACE2 in the respiratory tract and are associated with better clinical outcomes with COVID-19, while the type 1 IFN response to respiratory viruses increased ACE2 expression (33).

Th2 cytokines drive an increase of a cell type called the eosinophil in the blood and tissues. Eosinophilia is a hallmark of Th2 inflammation in the airways, most notably in asthma (33, 45). Sajuthi et al. (33) concluded that, at least “provisionally… T[h]2 inflammation may predispose individuals to experience better COVID-19 outcomes through a decrease in airway levels of ACE2 that override any countervailing effect from increased expression of TMPRSS2.” It is indeed surprising that the Th2 immune type associated with allergic diseases including asthma, and with eosinophilia, provides some protection to COVID-19 in children. These findings from Sajuthi et al. (33) on 695 children and adolescents may help explain why low levels of eosinophilia were seen in fatalities in the elderly. Du et al. (45) reported that, in a study of 85 fatal COVID-19 adult subjects, 81.2% exhibited very low levels of blood eosinophils. A connection between knowledge gained in studying children (33) versus studying older adults, may help explain surprising outcomes in the elderly (45). This is one of the unexpected benefits of investigations on the “extremes of outcome” based on comparing children with the elderly populations at highest risk.

Another independent verification of the possible protective role of Th2 immunity was seen in a study on MIS-C. The very low IgE levels seen in individuals with MIS-C indicate that they may have lacked an adequate Th2 response to attenuate the increased inflammation associated with this hyperinflammatory complication in children (46).

One important caveat about the potential protective effect of eosinophilia comes from studies done 50 y ago in making a vaccine against RSV. “In 1967, infants and toddlers immunized with a formalin-inactivated vaccine against RSV experienced an enhanced form of RSV disease characterized by high fever, bronchopneumonia, and wheezing when they became infected with wild-type virus in the community. Hospitalizations were frequent, and two immunized toddlers died upon infection with wild-type RSV. The enhanced disease was initially characterized as a ‘peribronchiolar monocytic infiltration with some excess in eosinophils’” (47). As new SARS-CoV2 vaccines are tested, the potential appearance of Th2 immunity and eosinophilia must be scrutinized through a lens of caution. Although Th2 responses appear to be associated with some degree of protection to COVID-19, based on the experience with development of a novel vaccine to RSV 50 y ago, the dreaded development of immune enhancement, rather than immunization, must be assessed.

Unanswered Questions and Lessons to Learn

Although children have milder outcomes, thus far in the first few months of the COVID-19 pandemic, much remains unknown (1⇓⇓⇓⇓⇓⇓⇓⇓–10). The list of questions is considerable. We do not know, at this point, whether children who are asymptomatic and who are major carriers of virus can spread disease. When children leave a “shelter-in-place” environment and return to school, how will this impact more-vulnerable populations? Recent reports of outbreaks at summer camps and schools raise serious concerns (48).

The emergence of MIS-C is particularly worrisome, and it is unclear whether there will be long-term sequelae of SARS-CoV-2 infection in patients who were asymptomatic or mildly symptomatic, in those who developed severe COVID-19, or in those who subsequently developed MIS-C (5⇓⇓⇓–9, 49). Other pandemics have been associated with postinfectious phenomena including postencephalitic Parkinson’s in the 1918 influenza pandemic (50). However, the simultaneous emergence of MIS-C in this current pandemic may allow elucidation of the pathophysiological underpinnings of this COVID-19 syndrome with systemic manifestations. Studies on MIS-C might provide insightful comparisons with the Kawasaki syndrome that has some clinical similarities. It is important to remember that intense inflammation activates the coagulation cascade, driving it in a procoagulant direction. Coagulation disorders and thrombosis are seen in MIS-C (51, 52). Autopsy studies on adults have revealed intense thrombotic microangiopathy with accompanying abnormalities in the coagulation cascade (52). The nexus between the coagulation cascade and the inflammatory response perhaps deserves increased attention for the possible therapeutic modalities that emerge from such a perspective (53, 54). Increased research emphasis on the connection between coagulation and inflammation is recommended.

The types of comorbidity underlying those at risk for COVID-19 accentuate some major differences between adults and children. Although children, like adults, with COVID-19 had a high incidence of comorbid conditions, there were significant differences in the types of comorbidity. The most common comorbidity in children, 40%, was those who were dependent on technological support. These children on technological support had congenital conditions, including developmental delay and genetic anomalies (4). In contrast, the comorbid conditions in adults are often considered acquired, including hypertension, pulmonary disease from smoking, and obesity. One comorbidity common to adults and children is obesity. Obesity was considered a risk factor in 48% of adults, while a lower percentage, 20.5%, of children with COVID-19 were obese (6, 55).

We should remember that, although there is widespread relief that children are hospitalized far less frequently than adults due to COVID-19 infection, we do not know how the carrier status of children affects their caregivers. We again have some relief that studies on pregnant women who test positive for the SARS-CoV-2 virus show that they and their offspring have, so far, done well. There appears to be negligible intrauterine transmission of disease, although larger studies are necessary to exclude this dreaded possibility (56).

While it seems that children are spared the most severe disease manifestations of COVID-19, they will undoubtedly be seriously impacted in many domains, beyond the direct effects of the pathogen. These potential negative impacts include how the pandemic and social distancing from other children affect psychological health, how it impacts education, and how it affects body weight and childhood obesity when getting sufficient exercise is more difficult during shelter-in-place and safe-distancing restrictions. One wonders how closure of playgrounds may further impact the obesity epidemic in children. Active programs to reduce childhood obesity have been designed so that this comorbidity does not further exacerbate the risk of COVID-19 in children (57). How the pandemic has changed routine medical and dental care, including immunizations to other preventable illnesses, needs to be examined.

The economic crisis associated with the pandemic has its own toll on children, as it is well known that deterioration in social determinants of health has significant adverse effects on children (https://www.cdc.gov/socialdeterminants/). Finally, sheltering in place and social distancing may even impact the frequency of infections typically seen in children (21⇓–23). We shall learn, for example, whether these frequent respiratory infections had provided some benefit to children as they form their immune repertoires and gain immune memory.

Dedicating adequate resources to understand the molecular and immune underpinnings of COVID-19 in children may answer many of these questions and potentially allow development of novel therapeutic strategies for enhancement of host resistance to viral infections. We might consider that we may learn as much or more about this virus, from studying the relative resistance to COVID-19 in children, as we will learn from studying the vastly increased level of susceptibility in adults. There is still much to learn.