This study presents new insights into the epithelium at birth, being the first to investigate viral receptor expression for RV, RSV and SARS-CoV2 in both amniotic and newborn nasal epithelial samples. Our data show that amniotic samples expressed ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2 receptors at the gene level, and were similarly expressed in the nasal airway epithelium of newborns. The magnitude of expression varied across samples and receptors, with the most abundantly expressed receptor being ICAM-1 in both sample types, and CDHR3 the least abundantly expressed in amniotic and ACE2 in nasal epithelial cells. Furthermore, protein expression of all receptors was confirmed in amniotic membrane samples, although the degree of staining varied between samples and receptors. The expression of proteins was localised to the amniotic epithelial cell layer with differing subcellular locations. Collectively, these novel findings support the use of amniotic membrane and nasal epithelial samples for the study of viral receptor expression at birth and potential susceptibility to repeated respiratory viral infection in early life.
Lower airway epithelial samples in newborn infants are largely inaccessible for both ethical and practical reasons, with surrogacy between lower airway epithelial and nasal epithelial samples previously established in children[26, 28]. Furthermore, previous studies have successfully sampled nasal brushings from infants, three days to 12 months of life[29–31]. However, the low epithelial sample yields that can be collected from newborn nasal brushings limits the potential to complete extensive downstream analysis on these samples, for example from this study, the inability to complete both gene and protein expression analysis on nasal epithelial samples. These limitations prompted the investigation into validating an alternative, more-accessible fetal-origin epithelial source that is exposed to the same in utero environment during pregnancy and may be a suitable surrogate for airway epithelial cells from infants at birth. The amniotic membrane was identified as a suitable candidate as it is comprised of predominantly fetal-origin epithelial progenitors and is easily accessible at birth[27].
This study established that the amniotic membrane expressed a full range of respiratory viral receptors, suggesting that there is potential to further explore the use of this sample type as a surrogate tissue for newborn airway epithelial samples. Previous studies have identified respiratory-like features of amniotic epithelial cells, showing that surfactant proteins that are typically expressed in surfactant producing alveolar epithelial cells of the lungs are also present in samples from the amniotic membrane[32]. However, there is still a paucity of data assessing the surrogacy between the amniotic and newborn nasal samples, and specifically in assessing the respiratory viral expression profiles in newborns to understand the role of in utero exposures on susceptibility to respiratory viral infection in early life. This study has shown that the same respiratory viral receptors for RV, RSV and SARS-CoV-2 are expressed in both amniotic and newborn nasal cells, however, the levels of expression for some receptors varied between sample types. For example, this study identified a discrepancy in CDHR3 gene expression levels between newborn nasal (high-expressing) and amniotic (low-expressing) samples. This could be explained by the different cellular composition in the two epithelial tissues; the progenitor characteristics of amniotic epithelial cells may not display identical morphological and physiological characteristic to nasal epithelial cells[33], where CDHR3 is identified to be expressed in terminally differentiated ciliated cells in the airway epithelium[34]. A limitation of this study is the non-matched amniotic and nasal epithelial samples, which impacts any direct associations in receptor expression across tissue types. Nevertheless, this pilot study provides proof-of-concept for the expression of receptor expression in the amniotic membrane and the mature airway epithelium of infants. Further studies are required to evaluate the broader surrogacy of matched amniotic and nasal epithelial tissues.
Data from this study highlight significant biological variation between participants and receptors for both amniotic and nasal epithelial samples. For example, a 10,000-fold difference in gene expression was observed for the most abundantly expressed receptor, ICAM-1, and least abundantly expressed receptor, CDHR3, in the amniotic samples. Similar differences in the magnitude of expression were also observed in the nasal epithelial samples where receptor expression for RV and RSV was higher than SARS-CoV2. These viral receptor expression profiles could align with the lower frequency and burden of disease for SARS-CoV-2 compared to other respiratory viral infections reported in young infants[5]. Furthermore, it highlights the potential role of in utero exposures in influencing the varied expression levels and opportunity to intervene in early life to modify infection responses. Exposure to viruses during the in utero period would occur during a critical period of lung development and could have a significant influence on future respiratory outcomes of infants and susceptibility to lung disease development[24, 25]. Thus, exploring the potential mechanisms underlying the imprinting of in utero exposures on the fetus could prove significant on predicting and treating adverse respiratory disease in early life. Poor maternal asthma control in pregnancy increases risk of asthma development in offspring [35]. Furthermore, respiratory viral infections experienced by a mother with asthma during pregnancy increase risk and severity of respiratory infections and wheezing in the infant within the first year of life[25]. The mechanisms of these susceptibilities are poorly understood but may be due to the induction of an abnormal fetal innate immune response, or abnormal development of the lungs and respiratory system[25]. Furthermore, a recent study utilising a mouse model of maternal asthma emphasised that a disrupted airway epithelium underlies the fetal origins of asthma[36]. The lack of longitudinal respiratory health outcomes of the infants at the time of this study limits our ability to determine susceptibility to viral infections and adverse respiratory outcomes. Future research to assess in utero exposures, viral receptor expression levels in fetal tissues at birth and tracking of postnatal respiratory health outcomes to understand how these exposures modify the airway epithelium and future respiratory outcomes in infants should be undertaken. Both the AERIAL Study and ORIGINS Project with bio banked samples[37, 38] and data would be able to facilitate such research.
In addition to viral receptor expression, other factors could contribute to susceptibility to viral infection and recurrent viral-induced wheezing, including impaired antiviral defence mechanisms, defective barrier structure and aberrant pro-inflammatory response to viral infections[39–42]. Using high-throughput genetic technologies, such as single-cell and bulk RNA-sequencing, on primary tissues would further our understanding of the physiological role of cell and tissue types beyond the profiling of a limited panel of genes. For example, previously published bulk RNA-sequencing datasets of placental samples, including the amnion, chorion and decidua tissue components, identified the global gene expression profiles within the membrane components[43, 44]. These studies demonstrated the feasibility to isolate RNA from the amniotic membrane suitable for RNA-sequencing. Future studies could utilise these technologies to further elucidate the surrogacy of the amniotic membrane to nasal epithelial samples, mechanisms of susceptibility to infection and wheeze, and contributions of maternal exposures to the development of epithelial vulnerabilities that may exist at birth.
In summary, this study is the first to assess and demonstrate the expression of respiratory viral receptors for RV, RSV and SARS-CoV-2 in both amniotic membrane and newborn nasal epithelial samples. We have provided new insights regarding the expression of respiratory viral receptors at birth and further rationale for understanding the impact of the in utero environment on the fetal epithelium. Further studies are needed to elucidate if expression of viral receptors in amniotic samples could suggest a vulnerability that exists in the epithelial cells at birth, which then associates with increased susceptibility to recurrent viral infections in infants.