Profiling epithelial viral receptor expression in amniotic membrane and nasal epithelial cells at birth

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Sutanto, Courtney Kidd, James Lim, Minda Amin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4374264/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Children with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the in utero environment may contribute to the development of airway epithelial vulnerabilities. The aims of the study were to establish whether the receptors for rhinovirus (RV), respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are expressed in the amniotic membrane and whether the pattern of expression is similar to newborn nasal epithelium. Amniotic and newborn nasal samples expressed various receptors for RV, RSV and SARS-CoV-2 at the gene level, quantified by qPCR. In addition, protein expression of these receptors was confirmed in the amniotic samples by western blot, which were localised to the epithelial layer of the membrane using immunohistochemistry. This proof-of-concept study indicates the potential of amniotic samples to facilitate investigation into the interactions between the in utero environment and prenatal programming of epithelial innate immune responses to viruses. General Cell Biology & Physiology Virology epithelium amnion viral infection receptors Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Respiratory diseases are a considerable burden for the individual, community and health care system, and often have their origins in early life [ 1 – 3 ] . Viral infections are the predominant trigger of recurrent respiratory illnesses in childhood [ 4 , 5 ] , with RV and RSV the most common infections experienced in infancy [ 6 , 7 ] . Both RV and RSV cause severe infection and often hospitalisation and are associated with poor respiratory outcomes such as asthma in later life [ 8 – 10 ] . Certain individuals are more susceptible to recurrent viral infection in early life and are more likely to go on to develop asthma in later childhood [ 6 , 11 – 14 ] . However, a causal link between individual susceptibility, recurrent viral infections and later respiratory morbidity is yet to be established. Impairments to the innate immune and inflammatory response to viral infections have been identified in patients with asthma and their airway epithelial cells (AECs), such as lowered levels of interferon Type I and II, and increased pro-inflammatory cytokines [ 15 – 17 ] . Viruses utilise host cell surface receptor proteins to access host cells to support replication and expression levels of these surface receptor proteins, are associated with increased susceptibility to respiratory viral infections [ 18 – 20 ] . The upregulation of cell surface expression of receptors for RV, such as Intracellular Adhesion Molecule 1 (ICAM-1) (e.g., RV-A and -B) and Cadherin related family member 3 (CDHR3) (e.g., RV-C), on AECs has been associated with a higher risk of infection [ 18 – 20 ] . The COVID-19 pandemic has led to further insights into the role of epithelial cell surface receptors and susceptibility to SARS-CoV-2. For example, variability in expression levels of the key receptor for SARS-CoV-2, Angiotensin-converting enzyme 2 (ACE2), appears to explain some of the age-related differences in susceptibility [ 21 – 23 ] and could potentially explain the heterogeneity of responses to exposure to SARS-CoV-2 between individuals of all ages. These observations highlight the need to elucidate the developmental patterns of epithelial innate responses to viral infections, their relations to early life susceptibility to viral infections and how they may contribute to the development of chronic respiratory illnesses such as asthma. Exposures in pregnancy (e.g. infections, smoking) can negatively impact the development of fetal tissues, including the lungs [ 24 ] , which increase the likelihood of poor postnatal respiratory outcomes of the developing infant [ 25 ] . Investigating fetal-derived tissue could provide unique insights to further understand the role of the in utero environment on the epithelial innate response. The amniotic membrane presents an attractive surrogate option for fetal airway epithelial cells, which may be impractical to obtain [ 26 ] . The amniotic membrane of the human placenta contains an abundance of amniotic epithelial cells that would be exposed to the same in utero environment as the developing lungs and could provide a source of fetal-origin respiratory-like epithelial progenitor [ 27 ] . Here we present a proof-of-concept study to determine whether the amniotic membrane expressed cell surface receptors required for common viral infections (RV, RSV and SARS-CoV-2, refer to Supplementary Table S1). We hypothesised that they could serve as markers of in utero reprogramming that affects respiratory epithelial cell development and individual susceptibility to early life respiratory outcomes. Results Profiling viral receptor gene expression in amniotic samples All viral receptors were expressed in the amniotic samples, however the magnitude of expression differed between individual samples and receptors (median(interquartile range (IQR)); ICAM-1 : 0.69(2.21) arbitrary units (AU); LDLR : 0.39(1.38) AU; CDHR3 : 1.0 x 10 − 4 (3.0 x 10 − 4 ) AU; NCL : 1.03(0.55) AU; CX3CR1 : 0.12(0.24) AU; TMPRSS2 : 3.0 x 10 − 4 (16.0 x 10 − 4 ) AU; ACE2 : 0.01(0.02) AU; Fig. 1). ICAM-1 was the most abundantly expressed gene in amniotic samples, followed by LDLR , both receptors for RV. The receptor with the least abundant expression was CDHR3 , a receptor for RV-C. Profiling viral receptor gene expression in newborn nasal epithelium samples Gene expression of viral receptors of interest was determined in newborn nasal epithelial samples. All viral receptors were found to be expressed in the nasal samples, and the magnitude of expression differed between individual samples and receptors (median(IQR); ICAM-1 : 11.44(63.18) AU; LDLR : 4.00(7.32) AU; CDHR3 : 0.40(1.14) AU; NCL : 2.32(2.18) AU; CX3CR1 : 2.17(2.33) AU; TMPRSS2 : 1.99(4.85) AU; ACE2 : 0.36(0.52) AU; Fig. 2). Similar to the amniotic samples, ICAM-1 was the most abundantly expressed receptor in nasal epithelial samples, followed by LDLR . Unlike amniotic samples, ACE2 was the receptor with the least expression in the nasal epithelial samples of newborn infants. Quantification of protein expression in amniotic samples Western blot analysis was used to investigate protein expression of the different viral receptors, ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2, for amniotic samples only. These samples expressed the receptors at the protein level, with varied magnitude between biological samples for each of the different proteins (Fig. 3 a). Band densitometry analysis showed varied levels of expression between amniotic samples (n = 16) for each protein (median(IQR); ICAM-1: 0.04(0.05) AU; LDLR: 0.08(0.1) AU; CDHR3: 0.27(0.16) AU; NCL: 1.13(1.11) AU; CX3CR1: 0.15(0.13) AU; TMPRSS2: 0.08(0.07) AU; ACE2: 0.35(0.96); Fig. 3 b). The most abundant signal across the samples were ACE2 and NCL, whilst ICAM-1 produced the lowest signal. Localisation of protein expression in amniotic samples Immunohistochemistry was used to investigate the localised expression of ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2 within the amniotic membrane. Positive staining was observed for all receptors in the amniotic epithelium as indicated by the arrows marked with an ‘E’ (Fig. 4 ), and within the subepithelial layer as indicated by the arrows marked with an ‘S’ for LDLR, NCL, CX3CR1, TMPRSS2 and ACE2 (Fig. 4 C, E, F, G and H). In addition, positive staining patterns varied across the proteins of interest, such as ACE2 presenting strong cytoplasmic staining (Fig. 4 H), NCL showing strong nuclear staining (Fig. 4 E) and ICAM-1 with weak cytoplasmic and nuclear staining (Fig. 4 B). Discussion 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. Methods Study participants and sample collection Samples were obtained from participants from two sub-studies nested within the ORIGINS Project, a unique long-term study and a collaboration between Telethon Kids Institute and Joondalup Health Campus. It is one of the most comprehensive studies of pregnant women and their families in Australia to date, recruiting 10,000 families over a decade from the Joondalup and Wanneroo communities of Western Australia [ 45 ] . The Airway Epithelium Respiratory Illnesses and ALlergy (AERIAL) [ 37 ] and the NewbOrn nasal Sampling Evaluation (NOSE) studies are being conducted in accordance with the Helsinki Declaration and was approved by the Ramsey Health Care HREC WA-SA (# 1746 and 1908 respectively). Written informed consent was obtained from each participant’s legal guardian after being fully informed about the nature and purpose of the study prior to collection of samples. Two sample types were utilised for this study: amniotic membrane samples from 33 participants (20 male, AERIAL Study), and nasal brushings from 20 newborn infants (14 male, NOSE Study), all births were greater than 38 weeks’ gestational age. Placental samples were collected and processed within 72-hours (19.3 ± 15.8 hours, mean ± standard deviation) post-birth, and the amniotic membrane manually separated from the chorion and sectioned into three strips, two of which were cryopreserved in either DNA/RNA Shield™ or cell extraction buffer (CEB) at -80°C for downstream gene and protein expression analyses respectively. The remaining amniotic membrane strip was fixed in 10% (v/v) neutral buffered formalin (NBF) at 4°C and paraffin embedded for histological assessments. Newborn nasal samples were collected within 72 hours (36.0 ± 16.5 hours) post birth, as previously described [ 30 , 37 ] and cryopreserved in DNA/RNA Shield™ at -80°C until extracted for RNA. RNA extraction and gene expression analysis The amniotic sample cryopreserved in 500µL of DNA/RNA Shield™ was transferred into 1 mL of QIAzol® in a Precellys® CK14 soft tissue ceramic bead homogenising tube (Bertin Instruments, Montigny-le-Bretonneux, France) and homogenised at 12,833x g for 30 seconds using the Precellys® 24 Tissue Homogeniser (Bertin Instruments). The homogenate was mixed with chloroform, centrifuged, the aqueous phase collected, and an equal volume of 70% (v/v) ethanol was added for total RNA extraction using the PureLink™ mini RNA extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer’s instructions. Newborn nasal RNA was extracted using Chemagic™ 360 RNA blood kits (PerkinElmer), as per manufacturer’s instructions. For all samples, total RNA purity was determined via NanoDrop™ 1000 spectrophotometer (A260/280 > 2.0), yield by Qubit fluorometer and quality determined using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyser, according to manufacturer's instructions (Agilent Technologies, Santa Clara, CA, USA). The gene expression of viral receptors of interest ( ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2 ) (refer to Supplementary Table S1) and housekeeping gene (Peptidylprolyl Isomerase A ( PPIA) ), was determined using two-step RT-qPCR reactions as previously described [ 46 ] . Reverse transcription was performed to synthesise cDNA and mRNA expression of genes of interest and housekeeping genes were assessed using validated TaqMan primer/probes (refer to Supplementary Table S2) and real-time qPCR as previously described [ 46 , 47 ] on a Quantstudio™ 7 Flex Real-Time PCR (Thermo Fisher Scientific). Gene expression of all samples was expressed as 2 −ΔΔCT values relative to the expression of the housekeeping gene, PPIA , and a positive control. All samples that had an undetectable cycle threshold value were presented as half of the lowest 2 −ΔΔCT value for that gene, to represent an undetectable level of expression. Total protein extraction and western blot The cryopreserved amniotic membrane samples stored in CEB was transferred to a soft tissue ceramic bead homogenisation tube (Bertin Instruments) and homogenised for two cycles of 60 seconds at 2,833x g with a 30-second break in between, using the Precellys® 24 Tissue Homogeniser (Bertin Instruments). The homogenate was centrifuged at 10,000x g for 15 minutes at 4°C and the supernatant total protein was quantified using the micro-BCA™ Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer’s instructions. The protein expression of receptors of interest (ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2, ACE2) was determined using western blot assay. A total protein of 40µg per sample, along with a Chameleon Duo pre-stained protein ladder were loaded on Bolt 4–12% Bis-Tris Plus Polyacrylamide Gels (Invitrogen, Carlsbad, CA, USA). Gel electrophoresis and wet transfer were performed as previously described [ 48 ] . Membranes were blocked with Odyssey Blocking Buffer (PBS) and blotted for each protein using primary and fluorophore-tagged secondary antibodies. The membranes were scanned using the LI-COR Odyssey Near-Infrared scanner and protein expression was semi-quantified using LI-COR Odyssey® v.5.2 software, where the integrated intensity of each protein band was normalised to the housekeeping protein, β-actin, in the corresponding lane. The list of primary and secondary antibodies used for protein expression analysis is shown in Supplementary Table S3 and S4. Immunohistochemistry To localise protein expression in the amniotic sample, formalin-fixed paraffin-embedded amniotic membrane samples were sectioned and rehydrated for immunohistochemical analysis as previously described [ 49 ] . Briefly, the tissue sections underwent heat-induced antigen retrieval in citrate buffer, followed by hydrogen peroxide treatment and blocking buffer incubation. The tissue was incubated with primary antibody overnight at 4°C. List of primary antibodies used for protein expression analysis is shown in Supplementary Table S3. A secondary antibody from the VECTASTAIN® Elite ABC-HRP Kit (Vector Laboratories, Newark, CA, USA) was incubated with the tissue section for 1 hour at room temperature (RT) followed by a 30-minute RT incubation using the Elite ABC solution from the same kit. NovaRED™ solution was added to the tissue for approximately 2 minutes before being removed and tissues rehydrated. The slides were imaged using the ScanScope Digital Slide Scanner with the 50x objective (Leica Microsystems, Mt Waverly, VIC, Australia). Statistics Analysis and presentation of the data were completed using GraphPad Prism 9®. Data were assumed to be nonparametric and presented as median with interquartile range (IQR, 3rd -1st quartile), unless otherwise stated in the Figure legends. No tests to determine statistical differences for two-group comparisons or multiple comparisons were completed in this study, as they were unnecessary to address the study aims and hypothesis. Declarations Funding Statement This work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC115648) and Western Australian Department of Health (Near Miss Merit Awards). SMS is supported by an NHMRC Investigator Grant (NHMRC2007725). TI is supported by a Future Health Research Innovation Fund (FHIRF) Innovation Fellowship. Acknowledgments We would like to thank the contribution of the AERIAL and NOSE families and the dedicated research team, for the recruitment, liaising and sample collection over the duration of the studies. We are grateful to all the ORIGINS families who support the project. We would also like to acknowledge and thank the following teams and individuals who have made The ORIGINS Project possible: The ORIGINS Project team; Joondalup Health Campus (JHC); members of ORIGINS Community Reference and Participant Reference Groups; Research Interest Groups and the ORIGINS Scientific Committee; Telethon Kids Institute; City of Wanneroo; City of Joondalup; and Professor Fiona Stanley. The ORIGINS Project has received core funding support from the Telethon Perth Children’s Hospital Research Fund, Joondalup Health Campus, the Paul Ramsay Foundation and the Commonwealth Government of Australia through the Channel 7 Telethon Trust. Substantial in-kind support has been provided by Telethon Kids Institute and Joondalup Health Campus. Author contributions Conception of study: S.S., and T.I. Design of study: B.R., E.N.S, L.K.S., S.S., and T.I. Management of study: L.K.S. Sample acquisition and processing: B.R., C.K., J.L., M.A., N.D.V, L.K.S., and T.I. Acquisition, analysis and interpretation of data: B.R., E.N.S., L.B., G.F.H. and T.I. Drafting or revising, critically reviewing and approving the final manuscript: B.R., E.N.S, C.K., J.L., M.A., L.B., G.F.H., N.D.V, L.K.S., S.S., and T.I. Competing interest statement The authors declare that they have no competing interests. Data availability statement The data presented in this study are available from the corresponding author on reasonable request. References Australian Bureau of Statistics. Asthma. (2022). https://www.abs.gov.au/statistics/health/health-conditions-and-risks/asthma/latest-release; accessed 22/01/2024. Australian Institute of Health and Welfare. 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A prenatally disrupted airway epithelium orchestrates the fetal origin of asthma in mice. J Allergy Clin Immunol 145 , 1641-1654 (2020). https://doi.org:10.1016/j.jaci.2020.01.050 Kicic-Starcevich, E. et al. Airway Epithelium Respiratory Illnesses and Allergy (AERIAL) birth cohort: study protocol. medRxiv (2023). https://doi.org:10.1101/2023.04.29.23289314 D'Vaz, N. et al. The ORIGINS Project Biobank: A Collaborative Bio Resource for Investigating the Developmental Origins of Health and Disease. Int J Environ Res Public Health 20 (2023). https://doi.org:10.3390/ijerph20136297 Looi, K. et al. Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability. Exp Lung Res 42 , 380-395 (2016). https://doi.org:10.1080/01902148.2016.1235237 Kast, J. I. et al. Respiratory syncytial virus infection influences tight junction integrity. Clin Exp Immunol 190 , 351-359 (2017). https://doi.org:10.1111/cei.13042 Khoo, S. K. et al. Upper Airway Cell Transcriptomics Identify a Major New Immunological Phenotype with Strong Clinical Correlates in Young Children with Acute Wheezing. J Immunol 202 , 1845-1858 (2019). https://doi.org:10.4049/jimmunol.1800178 Coenen, I. et al. Impaired interferon response in plasmacytoid dendritic cells from children with persistent wheeze. J Allergy Clin Immunol (2023). https://doi.org:10.1016/j.jaci.2023.11.920 Kim, J. et al. Transcriptome landscape of the human placenta. BMC Genomics 13 , 115 (2012). https://doi.org:10.1186/1471-2164-13-115 Gong, S. et al. The RNA landscape of the human placenta in health and disease. Nat Commun 12 , 2639 (2021). https://doi.org:10.1038/s41467-021-22695-y Silva, D. T. et al. Introducing the ORIGINS project: a community-based interventional birth cohort. Rev Environ Health 35 , 281-293 (2020). https://doi.org:10.1515/reveh-2020-0057 He, J. Q. et al. Selection of housekeeping genes for real-time PCR in atopic human bronchial epithelial cells. Eur Respir J 32 , 755-762 (2008). https://doi.org:10.1183/09031936.00129107 Iosifidis, T. et al. Aberrant cell migration contributes to defective airway epithelial repair in childhood wheeze. JCI Insight 5 (2020). https://doi.org:10.1172/jci.insight.133125 Prele, C. M. et al. Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155. Biomedicines 9 (2021). https://doi.org:10.3390/biomedicines9050498 Buck, J. et al. Veliparib Is an Effective Radiosensitizing Agent in a Preclinical Model of Medulloblastoma. Front Mol Biosci 8 , 633344 (2021). https://doi.org:10.3389/fmolb.2021.633344 Additional Declarations The authors declare no competing interests. Supplementary Files RenoufB2024SciReportsSupplementaryMaterial.docx Profiling epithelial viral receptor expression in amniotic membrane and nasal epithelial cells at birth_Supplementary-Material_BRenouf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4374264","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":299067635,"identity":"288b292c-a51c-484c-bce8-8b4d1ecd2c17","order_by":0,"name":"Bailee Renouf","email":"","orcid":"","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Bailee","middleName":"","lastName":"Renouf","suffix":""},{"id":299067636,"identity":"c92cefd0-597c-495e-b10c-f8f75bb62901","order_by":1,"name":"Erika N. Sutanto","email":"","orcid":"https://orcid.org/0000-0002-4224-2839","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Erika","middleName":"N.","lastName":"Sutanto","suffix":""},{"id":299067637,"identity":"67e26fc2-0ffe-4e07-b476-041d2b35d38c","order_by":2,"name":"Courtney Kidd","email":"","orcid":"https://orcid.org/0000-0003-1330-4073","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Courtney","middleName":"","lastName":"Kidd","suffix":""},{"id":299067638,"identity":"a5d691e7-b6bc-4d2f-8a14-4427262e89a6","order_by":3,"name":"James Lim","email":"","orcid":"","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"","lastName":"Lim","suffix":""},{"id":299067639,"identity":"025900b3-d196-4be7-9788-538ab3190128","order_by":4,"name":"Minda Amin","email":"","orcid":"","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Minda","middleName":"","lastName":"Amin","suffix":""},{"id":299067640,"identity":"55113f76-8e61-488d-af80-9ddc08221e91","order_by":5,"name":"Luke Berry","email":"","orcid":"","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Luke","middleName":"","lastName":"Berry","suffix":""},{"id":299067641,"identity":"2d7f6477-6519-47ef-a85f-6121016d1e97","order_by":6,"name":"Gerard Hoyne","email":"","orcid":"https://orcid.org/0000-0002-7370-9139","institution":"University of Notre Dame Australia: Fremantle, Western Australia, AU","correspondingAuthor":false,"prefix":"","firstName":"Gerard","middleName":"","lastName":"Hoyne","suffix":""},{"id":299067642,"identity":"82f90b43-c131-40c5-8db5-9b170f858199","order_by":7,"name":"Nina D'Vaz","email":"","orcid":"","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Nina","middleName":"","lastName":"D'Vaz","suffix":""},{"id":299067643,"identity":"6e0b7d32-2ecf-44e1-9c30-ce0b704b8d7f","order_by":8,"name":"Elizabeth Starcevich-Kicic","email":"","orcid":"https://orcid.org/0009-0002-9113-8939","institution":"Telethon Kids Institute","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"","lastName":"Starcevich-Kicic","suffix":""},{"id":299067644,"identity":"e384c96d-46cc-436b-ac4c-b518d92ab174","order_by":9,"name":"Stephen M. Stick","email":"","orcid":"https://orcid.org/0000-0002-5386-8482","institution":"Perth Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Stephen","middleName":"M.","lastName":"Stick","suffix":""},{"id":299067645,"identity":"021c9451-a651-4f24-846c-aa1eb947c598","order_by":10,"name":"Thomas Iosifidis","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0001-8462-5865","institution":"Telethon Kids Institute","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"","lastName":"Iosifidis","suffix":""}],"badges":[],"createdAt":"2024-05-06 05:41:50","currentVersionCode":1,"declarations":{"humanSubjects":true,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":true,"humanSubjectConsent":true,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4374264/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4374264/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56282204,"identity":"fc1115a7-a479-4f2f-8a3e-2106ae96dd7e","added_by":"auto","created_at":"2024-05-10 21:27:09","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61269,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression profiles of respiratory virus receptors in amniotic samples.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression of viral receptors for RV, RSV, and SARS-CoV-2 were analysed using qPCR in amniotic samples from 33 participants. Gene expression was profiled for \u003cem\u003eICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 \u003c/em\u003eand\u003cem\u003e ACE2\u003c/em\u003e. The data were normalised to the house-keeping gene and positive control, and results presented as 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e in AU.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/855944d1f5ad3ba86dade983.png"},{"id":56282108,"identity":"3f50688e-55db-4e6b-846e-ec3b69b7cb78","added_by":"auto","created_at":"2024-05-10 21:25:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65751,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene expression profiles of respiratory virus receptors in nasal epithelial samples from newborn infants.\u003c/strong\u003e Gene expression of viral receptors for RV, RSV, and SARS-CoV-2 was analysed using qPCR in nasal epithelial samples from 20 newborn infants. Gene expression was profiled for \u003cem\u003eICAM-1\u003c/em\u003e, \u003cem\u003eLDLR\u003c/em\u003e, \u003cem\u003eCDHR3\u003c/em\u003e, \u003cem\u003eNCL\u003c/em\u003e, \u003cem\u003eCX3CR1\u003c/em\u003e, \u003cem\u003eTMPRSS2 \u003c/em\u003eand\u003cem\u003e ACE2\u003c/em\u003e. The data were normalised to the house-keeping gene and positive control, and results presented as 2\u003csup\u003e-ΔΔCt\u003c/sup\u003e in AU.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/e0b13e57ddf9de6b9b57f9b9.png"},{"id":56282020,"identity":"5e2df348-8740-46b3-bc5b-b5c119e7777f","added_by":"auto","created_at":"2024-05-10 21:23:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292006,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProtein expression profiles in amniotic samples.\u003c/strong\u003e \u003cstrong\u003ea)\u003c/strong\u003e Representative western blot showing the band size (according to the Chameleon Duo Stain ladder) and fluorescent signal for protein expression of viral receptors LDLR, ACE2, NCL, CX3CR1, TMPRSS2, CDHR3, ICAM-1 and loading control protein (β-actin) in amniotic samples. \u003cstrong\u003eb) \u003c/strong\u003eSemi-quantitative analysis of the densitometry signal produced using a western blot of protein expression of viral receptors from 16 amniotic samples for ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2 (• = individual amniotic sample). The data were normalised relative to expression of house-keeping protein, β-actin and results presented as AU.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/01036c9553d26072c9c6e258.png"},{"id":56282162,"identity":"9dc5b728-8cc3-4f77-9405-a76b53b5c17c","added_by":"auto","created_at":"2024-05-10 21:26:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1073617,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemical staining of viral receptors in the amniotic membrane. \u003c/strong\u003eImmunohistochemical staining of A) negative control, B) ICAM-1, C) LDLR, D) CDHR3, E) NCL, F) CX3CR1, G) TMPRSS2 and H) ACE2 in the amniotic membrane. Images are representative of 8 biological replicates. Arrows show positive ‘E’ epithelial cell staining and ‘S’ subepithelial staining. Scale bar is 50µm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/a216314ef05b66d291d7f635.png"},{"id":56282084,"identity":"403cd848-aea3-4b05-9d47-51f1b41ddb9f","added_by":"auto","created_at":"2024-05-10 21:24:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":903739,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/00a55b70-77d6-4c76-b20e-5e709950102a.pdf"},{"id":56282161,"identity":"194d7a8b-76d6-4813-ac3c-0c244dae7102","added_by":"auto","created_at":"2024-05-10 21:26:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":19006,"visible":true,"origin":"","legend":"\u003cp\u003eProfiling epithelial viral receptor expression in amniotic membrane and nasal epithelial cells at birth_Supplementary-Material_BRenouf\u003c/p\u003e","description":"","filename":"RenoufB2024SciReportsSupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4374264/v1/e317855140adb907bb2154bf.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003eProfiling epithelial viral receptor expression in amniotic membrane and nasal epithelial cells at birth\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRespiratory diseases are a considerable burden for the individual, community and health care system, and often have their origins in early life\u003csup\u003e[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Viral infections are the predominant trigger of recurrent respiratory illnesses in childhood\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, with RV and RSV the most common infections experienced in infancy\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Both RV and RSV cause severe infection and often hospitalisation and are associated with poor respiratory outcomes such as asthma in later life\u003csup\u003e[\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Certain individuals are more susceptible to recurrent viral infection in early life and are more likely to go on to develop asthma in later childhood\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12 CR13\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. However, a causal link between individual susceptibility, recurrent viral infections and later respiratory morbidity is yet to be established.\u003c/p\u003e \u003cp\u003eImpairments to the innate immune and inflammatory response to viral infections have been identified in patients with asthma and their airway epithelial cells (AECs), such as lowered levels of interferon Type I and II, and increased pro-inflammatory cytokines\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Viruses utilise host cell surface receptor proteins to access host cells to support replication and expression levels of these surface receptor proteins, are associated with increased susceptibility to respiratory viral infections\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe upregulation of cell surface expression of receptors for RV, such as Intracellular Adhesion Molecule 1 (ICAM-1) (e.g., RV-A and -B) and Cadherin related family member 3 (CDHR3) (e.g., RV-C), on AECs has been associated with a higher risk of infection\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. The COVID-19 pandemic has led to further insights into the role of epithelial cell surface receptors and susceptibility to SARS-CoV-2. For example, variability in expression levels of the key receptor for SARS-CoV-2, Angiotensin-converting enzyme 2 (ACE2), appears to explain some of the age-related differences in susceptibility\u003csup\u003e[\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e and could potentially explain the heterogeneity of responses to exposure to SARS-CoV-2 between individuals of all ages. These observations highlight the need to elucidate the developmental patterns of epithelial innate responses to viral infections, their relations to early life susceptibility to viral infections and how they may contribute to the development of chronic respiratory illnesses such as asthma.\u003c/p\u003e \u003cp\u003eExposures in pregnancy (e.g. infections, smoking) can negatively impact the development of fetal tissues, including the lungs\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, which increase the likelihood of poor postnatal respiratory outcomes of the developing infant\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Investigating fetal-derived tissue could provide unique insights to further understand the role of the \u003cem\u003ein utero\u003c/em\u003e environment on the epithelial innate response. The amniotic membrane presents an attractive surrogate option for fetal airway epithelial cells, which may be impractical to obtain\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. The amniotic membrane of the human placenta contains an abundance of amniotic epithelial cells that would be exposed to the same \u003cem\u003ein utero\u003c/em\u003e environment as the developing lungs and could provide a source of fetal-origin respiratory-like epithelial progenitor\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere we present a proof-of-concept study to determine whether the amniotic membrane expressed cell surface receptors required for common viral infections (RV, RSV and SARS-CoV-2, refer to Supplementary Table S1). We hypothesised that they could serve as markers of \u003cem\u003ein utero\u003c/em\u003e reprogramming that affects respiratory epithelial cell development and individual susceptibility to early life respiratory outcomes.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eProfiling viral receptor gene expression in amniotic samples\u003c/h2\u003e\n\u003cp\u003eAll viral receptors were expressed in the amniotic samples, however the magnitude of expression differed between individual samples and receptors (median(interquartile range (IQR)); \u003cem\u003eICAM-1\u003c/em\u003e: 0.69(2.21) arbitrary units (AU); \u003cem\u003eLDLR\u003c/em\u003e: 0.39(1.38) AU; \u003cem\u003eCDHR3\u003c/em\u003e: 1.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e(3.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) AU; \u003cem\u003eNCL\u003c/em\u003e: 1.03(0.55) AU; \u003cem\u003eCX3CR1\u003c/em\u003e: 0.12(0.24) AU; \u003cem\u003eTMPRSS2\u003c/em\u003e: 3.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e (16.0 x 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) AU; \u003cem\u003eACE2\u003c/em\u003e: 0.01(0.02) AU; Fig.\u0026nbsp;1). \u003cem\u003eICAM-1\u003c/em\u003e was the most abundantly expressed gene in amniotic samples, followed by \u003cem\u003eLDLR\u003c/em\u003e, both receptors for RV. The receptor with the least abundant expression was \u003cem\u003eCDHR3\u003c/em\u003e, a receptor for RV-C.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003eProfiling viral receptor gene expression in newborn nasal epithelium samples\u003c/h2\u003e\n\u003cp\u003eGene expression of viral receptors of interest was determined in newborn nasal epithelial samples. All viral receptors were found to be expressed in the nasal samples, and the magnitude of expression differed between individual samples and receptors (median(IQR); \u003cem\u003eICAM-1\u003c/em\u003e: 11.44(63.18) AU; \u003cem\u003eLDLR\u003c/em\u003e: 4.00(7.32) AU; \u003cem\u003eCDHR3\u003c/em\u003e: 0.40(1.14) AU; \u003cem\u003eNCL\u003c/em\u003e: 2.32(2.18) AU; \u003cem\u003eCX3CR1\u003c/em\u003e: 2.17(2.33) AU; \u003cem\u003eTMPRSS2\u003c/em\u003e: 1.99(4.85) AU; \u003cem\u003eACE2\u003c/em\u003e: 0.36(0.52) AU; Fig.\u0026nbsp;2). Similar to the amniotic samples, \u003cem\u003eICAM-1\u003c/em\u003e was the most abundantly expressed receptor in nasal epithelial samples, followed by \u003cem\u003eLDLR\u003c/em\u003e. Unlike amniotic samples, \u003cem\u003eACE2\u003c/em\u003e was the receptor with the least expression in the nasal epithelial samples of newborn infants.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eQuantification of protein expression in amniotic samples\u003c/h2\u003e\n\u003cp\u003eWestern blot analysis was used to investigate protein expression of the different viral receptors, ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2, for amniotic samples only. These samples expressed the receptors at the protein level, with varied magnitude between biological samples for each of the different proteins (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\n\u003cp\u003eBand densitometry analysis showed varied levels of expression between amniotic samples (n\u0026thinsp;=\u0026thinsp;16) for each protein (median(IQR); ICAM-1: 0.04(0.05) AU; LDLR: 0.08(0.1) AU; CDHR3: 0.27(0.16) AU; NCL: 1.13(1.11) AU; CX3CR1: 0.15(0.13) AU; TMPRSS2: 0.08(0.07) AU; ACE2: 0.35(0.96); Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). The most abundant signal across the samples were ACE2 and NCL, whilst ICAM-1 produced the lowest signal.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eLocalisation of protein expression in amniotic samples\u003c/h2\u003e\n\u003cp\u003eImmunohistochemistry was used to investigate the localised expression of ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2 within the amniotic membrane. Positive staining was observed for all receptors in the amniotic epithelium as indicated by the arrows marked with an \u0026lsquo;E\u0026rsquo; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), and within the subepithelial layer as indicated by the arrows marked with an \u0026lsquo;S\u0026rsquo; for LDLR, NCL, CX3CR1, TMPRSS2 and ACE2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC, E, F, G and H). In addition, positive staining patterns varied across the proteins of interest, such as ACE2 presenting strong cytoplasmic staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH), NCL showing strong nuclear staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE) and ICAM-1 with weak cytoplasmic and nuclear staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis 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 \u003cem\u003eICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2\u003c/em\u003e and \u003cem\u003eACE2\u003c/em\u003e 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 \u003cem\u003eICAM-1\u003c/em\u003e in both sample types, and \u003cem\u003eCDHR3\u003c/em\u003e the least abundantly expressed in amniotic and \u003cem\u003eACE2\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eLower 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\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Furthermore, previous studies have successfully sampled nasal brushings from infants, three days to 12 months of life\u003csup\u003e[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. 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 \u003cem\u003ein utero\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThis 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\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. 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 \u003cem\u003ein utero\u003c/em\u003e 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 \u003cem\u003eCDHR3\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e, where CDHR3 is identified to be expressed in terminally differentiated ciliated cells in the airway epithelium\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eData 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, \u003cem\u003eICAM-1\u003c/em\u003e, and least abundantly expressed receptor, \u003cem\u003eCDHR3\u003c/em\u003e, 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\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Furthermore, it highlights the potential role of \u003cem\u003ein utero\u003c/em\u003e exposures in influencing the varied expression levels and opportunity to intervene in early life to modify infection responses. Exposure to viruses during the \u003cem\u003ein utero\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Thus, exploring the potential mechanisms underlying the imprinting of \u003cem\u003ein utero\u003c/em\u003e 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 \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Furthermore, a recent study utilising a mouse model of maternal asthma emphasised that a disrupted airway epithelium underlies the fetal origins of asthma\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. 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 \u003cem\u003ein utero\u003c/em\u003e 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\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e and data would be able to facilitate such research.\u003c/p\u003e \u003cp\u003eIn 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\u003csup\u003e[\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]\u003c/sup\u003e. 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\u003csup\u003e[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. 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.\u003c/p\u003e \u003cp\u003eIn 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 \u003cem\u003ein utero\u003c/em\u003e 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.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStudy participants and sample collection\u003c/h2\u003e \u003cp\u003eSamples were obtained from participants from two sub-studies nested within the ORIGINS Project, a unique long-term study and a collaboration between Telethon Kids Institute and Joondalup Health Campus. It is one of the most comprehensive studies of pregnant women and their families in Australia to date, recruiting 10,000 families over a decade from the Joondalup and Wanneroo communities of Western Australia\u003csup\u003e[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe Airway Epithelium Respiratory Illnesses and ALlergy (AERIAL)\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e and the NewbOrn nasal Sampling Evaluation (NOSE) studies are being conducted in accordance with the Helsinki Declaration and was approved by the Ramsey Health Care HREC WA-SA (# 1746 and 1908 respectively). Written informed consent was obtained from each participant\u0026rsquo;s legal guardian after being fully informed about the nature and purpose of the study prior to collection of samples.\u003c/p\u003e \u003cp\u003eTwo sample types were utilised for this study: amniotic membrane samples from 33 participants (20 male, AERIAL Study), and nasal brushings from 20 newborn infants (14 male, NOSE Study), all births were greater than 38 weeks\u0026rsquo; gestational age. Placental samples were collected and processed within 72-hours (19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;15.8 hours, mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation) post-birth, and the amniotic membrane manually separated from the chorion and sectioned into three strips, two of which were cryopreserved in either DNA/RNA Shield\u0026trade; or cell extraction buffer (CEB) at -80\u0026deg;C for downstream gene and protein expression analyses respectively. The remaining amniotic membrane strip was fixed in 10% (v/v) neutral buffered formalin (NBF) at 4\u0026deg;C and paraffin embedded for histological assessments. Newborn nasal samples were collected within 72 hours (36.0\u0026thinsp;\u0026plusmn;\u0026thinsp;16.5 hours) post birth, as previously described\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e and cryopreserved in DNA/RNA Shield\u0026trade; at -80\u0026deg;C until extracted for RNA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and gene expression analysis\u003c/h2\u003e \u003cp\u003eThe amniotic sample cryopreserved in 500\u0026micro;L of DNA/RNA Shield\u0026trade; was transferred into 1 mL of QIAzol\u0026reg; in a Precellys\u0026reg; CK14 soft tissue ceramic bead homogenising tube (Bertin Instruments, Montigny-le-Bretonneux, France) and homogenised at 12,833x\u003cem\u003eg\u003c/em\u003e for 30 seconds using the Precellys\u0026reg; 24 Tissue Homogeniser (Bertin Instruments). The homogenate was mixed with chloroform, centrifuged, the aqueous phase collected, and an equal volume of 70% (v/v) ethanol was added for total RNA extraction using the PureLink\u0026trade; mini RNA extraction kit (Thermo Fisher Scientific, Waltham, MA, USA) as per manufacturer\u0026rsquo;s instructions. Newborn nasal RNA was extracted using Chemagic\u0026trade; 360 RNA blood kits (PerkinElmer), as per manufacturer\u0026rsquo;s instructions. For all samples, total RNA purity was determined via NanoDrop\u0026trade; 1000 spectrophotometer (A260/280\u0026thinsp;\u0026gt;\u0026thinsp;2.0), yield by Qubit fluorometer and quality determined using the Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyser, according to manufacturer's instructions (Agilent Technologies, Santa Clara, CA, USA).\u003c/p\u003e \u003cp\u003eThe gene expression of viral receptors of interest (\u003cem\u003eICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2 and ACE2\u003c/em\u003e) (refer to Supplementary Table S1) and housekeeping gene (Peptidylprolyl Isomerase A (\u003cem\u003ePPIA)\u003c/em\u003e), was determined using two-step RT-qPCR reactions as previously described \u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e. Reverse transcription was performed to synthesise cDNA and mRNA expression of genes of interest and housekeeping genes were assessed using validated TaqMan primer/probes (refer to Supplementary Table S2) and real-time qPCR as previously described\u003csup\u003e[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e on a Quantstudio\u0026trade; 7 Flex Real-Time PCR (Thermo Fisher Scientific). Gene expression of all samples was expressed as 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e values relative to the expression of the housekeeping gene, \u003cem\u003ePPIA\u003c/em\u003e, and a positive control. All samples that had an undetectable cycle threshold value were presented as half of the lowest 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e value for that gene, to represent an undetectable level of expression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTotal protein extraction and western blot\u003c/h2\u003e \u003cp\u003eThe cryopreserved amniotic membrane samples stored in CEB was transferred to a soft tissue ceramic bead homogenisation tube (Bertin Instruments) and homogenised for two cycles of 60 seconds at 2,833x\u003cem\u003eg\u003c/em\u003e with a 30-second break in between, using the Precellys\u0026reg; 24 Tissue Homogeniser (Bertin Instruments). The homogenate was centrifuged at 10,000x\u003cem\u003eg\u003c/em\u003e for 15 minutes at 4\u0026deg;C and the supernatant total protein was quantified using the micro-BCA\u0026trade; Protein Assay Kit (Thermo Fisher Scientific) as per manufacturer\u0026rsquo;s instructions.\u003c/p\u003e \u003cp\u003eThe protein expression of receptors of interest (ICAM-1, LDLR, CDHR3, NCL, CX3CR1, TMPRSS2, ACE2) was determined using western blot assay. A total protein of 40\u0026micro;g per sample, along with a Chameleon Duo pre-stained protein ladder were loaded on Bolt 4\u0026ndash;12% Bis-Tris Plus Polyacrylamide Gels (Invitrogen, Carlsbad, CA, USA). Gel electrophoresis and wet transfer were performed as previously described \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Membranes were blocked with Odyssey Blocking Buffer (PBS) and blotted for each protein using primary and fluorophore-tagged secondary antibodies. The membranes were scanned using the LI-COR Odyssey Near-Infrared scanner and protein expression was semi-quantified using LI-COR Odyssey\u0026reg; v.5.2 software, where the integrated intensity of each protein band was normalised to the housekeeping protein, β-actin, in the corresponding lane. The list of primary and secondary antibodies used for protein expression analysis is shown in Supplementary Table S3 and S4.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eTo localise protein expression in the amniotic sample, formalin-fixed paraffin-embedded amniotic membrane samples were sectioned and rehydrated for immunohistochemical analysis as previously described\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. Briefly, the tissue sections underwent heat-induced antigen retrieval in citrate buffer, followed by hydrogen peroxide treatment and blocking buffer incubation. The tissue was incubated with primary antibody overnight at 4\u0026deg;C. List of primary antibodies used for protein expression analysis is shown in Supplementary Table S3. A secondary antibody from the VECTASTAIN\u0026reg; Elite ABC-HRP Kit (Vector Laboratories, Newark, CA, USA) was incubated with the tissue section for 1 hour at room temperature (RT) followed by a 30-minute RT incubation using the Elite ABC solution from the same kit. NovaRED\u0026trade; solution was added to the tissue for approximately 2 minutes before being removed and tissues rehydrated. The slides were imaged using the ScanScope Digital Slide Scanner with the 50x objective (Leica Microsystems, Mt Waverly, VIC, Australia).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAnalysis and presentation of the data were completed using GraphPad Prism 9\u0026reg;. Data were assumed to be nonparametric and presented as median with interquartile range (IQR, 3rd -1st quartile), unless otherwise stated in the Figure legends. No tests to determine statistical differences for two-group comparisons or multiple comparisons were completed in this study, as they were unnecessary to address the study aims and hypothesis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding Statement\u003c/h2\u003e\n\u003cp\u003eThis work was supported by grants from the National Health and Medical Research Council of Australia (NHMRC115648) and Western Australian Department of Health (Near Miss Merit Awards). SMS is supported by an NHMRC Investigator Grant (NHMRC2007725). TI is supported by a Future Health Research Innovation Fund (FHIRF) Innovation Fellowship.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments\u003c/h2\u003e\n\u003cp\u003eWe would like to thank the contribution of the AERIAL and NOSE families and the dedicated research team, for the recruitment, liaising and sample collection over the duration of the studies. We are grateful to all the ORIGINS families who support the project. We would also like to acknowledge and thank the following teams and individuals who have made The ORIGINS Project possible: The ORIGINS Project team; Joondalup Health Campus (JHC); members of ORIGINS Community Reference and Participant Reference Groups; Research Interest Groups and the ORIGINS Scientific Committee; Telethon Kids Institute; City of Wanneroo; City of Joondalup; and Professor Fiona Stanley. The ORIGINS Project has received core funding support from the Telethon Perth Children\u0026rsquo;s Hospital Research Fund, Joondalup Health Campus, the Paul Ramsay Foundation and the Commonwealth Government of Australia through the Channel 7 Telethon Trust. Substantial in-kind support has been provided by Telethon Kids Institute and Joondalup Health Campus.\u003c/p\u003e\n\u003ch2\u003eAuthor contributions\u003c/h2\u003e\n\u003cp\u003eConception of study: S.S., and T.I. Design of study: B.R., E.N.S, L.K.S., S.S., and T.I. Management of study: L.K.S. Sample acquisition and processing: B.R., C.K., J.L., M.A., N.D.V, L.K.S., and T.I. Acquisition, analysis and interpretation of data: B.R., E.N.S., L.B., G.F.H. and T.I. Drafting or revising, critically reviewing and approving the final manuscript: B.R., E.N.S, C.K., J.L., M.A., L.B., G.F.H., N.D.V, L.K.S., S.S., and T.I.\u003c/p\u003e\n\u003ch2\u003eCompeting interest statement\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eData availability statement\u003c/h2\u003e\n\u003cp\u003eThe data presented in this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAustralian Bureau of Statistics. Asthma. (2022). https://www.abs.gov.au/statistics/health/health-conditions-and-risks/asthma/latest-release; accessed 22/01/2024.\u003c/li\u003e\n\u003cli\u003eAustralian Institute of Health and Welfare. 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M.\u003cem\u003e et al.\u003c/em\u003e Reduced SOCS1 Expression in Lung Fibroblasts from Patients with IPF Is Not Mediated by Promoter Methylation or Mir155. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e (2021). https://doi.org:10.3390/biomedicines9050498\u003c/li\u003e\n\u003cli\u003eBuck, J.\u003cem\u003e et al.\u003c/em\u003e Veliparib Is an Effective Radiosensitizing Agent in a Preclinical Model of Medulloblastoma. \u003cem\u003eFront Mol Biosci\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 633344 (2021). https://doi.org:10.3389/fmolb.2021.633344\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[{"identity":"3fee53a0-bd1e-4928-bacc-6171348bd551","identifier":"10.13039/501100000925","name":"National Health and Medical Research Council","awardNumber":"115648","order_by":0}],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Telethon Kids Institute","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"epithelium, amnion, viral infection, receptors","lastPublishedDoi":"10.21203/rs.3.rs-4374264/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4374264/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChildren with wheeze and asthma present with airway epithelial vulnerabilities, such as impaired responses to viral infection. It is postulated that the \u003cem\u003ein utero\u003c/em\u003e environment may contribute to the development of airway epithelial vulnerabilities. The aims of the study were to establish whether the receptors for rhinovirus (RV), respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are expressed in the amniotic membrane and whether the pattern of expression is similar to newborn nasal epithelium. Amniotic and newborn nasal samples expressed various receptors for RV, RSV and SARS-CoV-2 at the gene level, quantified by qPCR. In addition, protein expression of these receptors was confirmed in the amniotic samples by western blot, which were localised to the epithelial layer of the membrane using immunohistochemistry. This proof-of-concept study indicates the potential of amniotic samples to facilitate investigation into the interactions between the \u003cem\u003ein utero\u003c/em\u003e environment and prenatal programming of epithelial innate immune responses to viruses.\u003c/p\u003e","manuscriptTitle":"Profiling epithelial viral receptor expression in amniotic membrane and nasal epithelial cells at birth","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-10 21:05:42","doi":"10.21203/rs.3.rs-4374264/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d20e1956-eb57-4f69-8800-7f7588962951","owner":[],"postedDate":"May 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31549520,"name":"General Cell Biology \u0026 Physiology"},{"id":31549521,"name":"Virology"}],"tags":[],"updatedAt":"2024-05-10T21:05:42+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-10 21:05:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4374264","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4374264","identity":"rs-4374264","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}