key: cord-0723012-5slfth6n
authors: Zhang, Zhao; Guo, Liyan; Huang, Li; Zhang, Che; Luo, Ruibang; Zeng, Liang; Liang, Huiying; Li, Qiuhui; Lu, Xiaoxia; Wang, Xianfeng; Yan Ma, Chui; Shao, Jianbo; Luo, Weiren; Li, Le; Liu, Li; Li, Ziyue; Zhou, Xiaoya; Zhang, Xiaoxian; Liu, Jie; Yang, Jinjian; Kwan, Ka Yi; Liu, Wei; Xu, Yi; Jiang, Hua; Liu, Hongsheng; Du, Hui; Wu, Yanheng; Yu, Guangyin; Chen, Junhui; Wu, Jieying; Zhang, Jinqiu; Liao, Can; Chen, Huanhuan Joyce; Chen, Zhiwei; Tse, Hung-fat; Xia, Huimin; Lian, Qizhou
title: Distinct disease severity between children and older adults with COVID-19: Impacts of ACE2 expression, distribution, and lung progenitor cells
date: 2021-01-03
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1911
sha: 46de47b6c7a9e32dd9c692f392c6798591682c35
doc_id: 723012
cord_uid: 5slfth6n

BACKGROUND: Children and older adults with coronavirus disease 2019 (COVID-19) display a distinct spectrum of disease severity yet the risk factors aren’t well understood. We sought to examine the expression pattern of angiotensin-converting enzyme 2 (ACE2), the cell-entry receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the role of lung progenitor cells in children and older patients. METHODS: We retrospectively analysed clinical features in a cohort of 299 patients with COVID-19. The expression and distribution of ACE2 and lung progenitor cells were systematically examined using a combination of public single-cell RNA-seq datasets, lung biopsies, and ex vivo infection of lung tissues with SARS-CoV-2 pseudovirus in children and older adults. We also followed up patients who had recovered from COVID-19. RESULTS: Compared with children, older patients (> 50 yrs.) were more likely to develop into serious pneumonia with reduced lymphocytes and aberrant inflammatory response (p = 0.001). The expression level of ACE2 and lung progenitor cell markers were generally decreased in older patients. Notably, ACE2 positive cells were mainly distributed in the alveolar region, including SFTPC positive cells, but rarely in airway regions in the older adults (p < 0.01). The follow-up of discharged patients revealed a prolonged recovery from pneumonia in the older (p < 0.025). CONCLUSION: Compared to children, ACE2 positive cells are generally decreased in older adults and mainly presented in the lower pulmonary tract. The lung progenitor cells are also decreased. These risk factors may impact disease severity and recovery from pneumonia caused by SARS-Cov-2 infection in older patients.

The pandemic of Coronavirus Disease 2019 (COVID- 19) due to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has affected over 73 million people over 1,630,000 of whom have died in the last 10 months (https://coronavirus.jhu.edu). [1] Currently, many therapeutic strategies are being explored in ongoing clinical trials, including antiviral drugs, biological response modifiers, and RAAS inhibitors. Ultimately, the development of effective vaccines is key to combat COVID-19 and appropriate measures are being taken [2, 3] Unfortunately, the understanding of how SARS-CoV-2 infection-provoked lung injury is not clear. Epidemiological studies indicate a broad spectrum of disease severity in children and older adults. Compared with the older adults, children and young adults generally display much milder symptoms with a lower mortality (< 1%), whereas mortality can be as high as 15% in older adults (> 50 yrs.). [4] The underlying factors for this variance in disease severity remain elusive. [5, 6] Lung is a main target of SARS-CoV-2. Pathological changes after SARS-CoV-2 infection include extensive necrotizing bronchiolitis and severe alveolitis. [7] Lung repair is associated with lung progenitor cells-modulated regeneration, [8] and ACE2 is the cell-entry receptor of SARS-CoV-2. [9] Nonetheless, little is known about the differences in clinical features, laboratory parameters, profiles of ACE2, and lung progenitor cells between infected children and adults. Several reports suggest that ACE2 expression increases with age, particularly in adults who smoke or have lung cancer. [10, 11] The increased ACE2 expression in adult lungs is thought to be a risk factor for the consequences of COVID-19. [12] Nevertheless, there is no direct evidence to support this assumption. In this study, we retrospectively analysed the clinical 

Patients with COVID-19 were enrolled from four hospitals in China, including Taihe Hospital of Hubei University of Medicine, the Third People's Hospital of Shenzhen, Wuhan Children's Hospital and Guangzhou Women and Children's Medical Centre. Patients who fulfilled the following criteria were included: (1) diagnosed with laboratory-confirmed COVID-19 according to the WHO guideline [13] and the recommendation of the National Health Commission of the People's Republic of China (NHC). [14] ; (2) age ≤ 80 years; (3) written informed consent was obtained. Briefly, SARS-CoV-2 was detected by quantitative polymerase chain reaction (qPCR) with samples from nasopharyngeal swabs. Infection was defined as at least two positive test results. Disease severity was classified into five levels from asymptomatic to critically ill according to the recommendation of NHC and WHO guidelines. [14] The medical history of adult patients was summarized in Supplementary Table 1. Clinical data, laboratory parameters, radiological findings were acquired from electronic medical records. The data collection forms were reviewed independently by 3 researchers. This study was approved by the Institutional Review Board of the four hospitals respectively. Written informed consent was obtained from patients and/or guardians before data collection.

The coding-sequence of spike protein (SARS-CoV-2, QHR63250) was cloned into pVAX-1 plasmid upon verification of Sanger-sequencing, and transfected into 293T cells with human immunodeficiency virus type I pNL4-3GFP + Env -Vprbackbone to package GFP-expressed SARS-CoV-2 pseudovirus as previously described. [ 

Paraffin-embedded sections were deparaffinized and incubated with primary antibodies at 4°C overnight, and secondary antibodies at room temperature for 1h as described previously. [16] Pseudoviral-infected tissue was embedded for cryosection. After rehydration, tissue sections were incubated with primary antibodies (4°C overnight), secondary antibodies (1h, room temperature) and DAPI. To exclude non-specific fluorescent staining of ACE2, only second antibody IgG with fluorescent conjugation was simultaneously used in slides as negative control and the ACE2 immunostaining in kidney sections was set as a positive control. [17] Fifty lung tissue biopsy samples in which 26 are from children and 24 from adults, were used for immunostaining against ACE2. ACE2 + cells were captured at different angles to generate 20 random views in a single lung slide for each patient. All slides with positive immunostaining signals were determined by two independent reviewers. The distribution of ACE2 + cells was determined as the average cell number per 0.025 cm 2 lung tissue. [18] Antibodies are described in the supplementary materials.

Twelve single-cell RNA-sequencing datasets of healthy lung tissues from three studies were used to explore the co-expression of genes. Four samples, "LMEX0000001623" (Homo sapiens, 1-day old, 2,342 cells), "LMEX0000001624" (Homo sapiens, 1-day old, 2,000 cells), "LMEX0000001625" (Homo sapiens, 21-month old, 2,000 cells), and "LMEX0000001626" (Homo sapiens, 9- project. [21] The raw read counts of each gene in each cell were downloaded in either the "tabdelimited table", "Matrix Market" or "Seurat" format, and loaded and processed using R 3.6.1. We aggregated the reads from all cells in each sample and normalized the expression of each gene using TPM (Transcripts Per Million). The coefficients were calculated using the "GGally" and visualized using "ggplot2".

Kruskal-Wallis test was utilized to analyse the blood and laboratory biochemical measurements in COVID-19 patients. The distribution of age and clinical severity, follow-up data, and ex vivo infection data were examined by Chi-square test or Fisher's exact test. ACE2 expression and distribution were analysed by students' t test or ANOVA. qPCR results were analysed using unpaired t test. Data are presented as mean ± SD, and p < 0.05 was considered significant. Statistical analysis was performed using SPSS software (version 20.0, IBM, Armonk, NY, USA).

In this retrospective study, 299 patients with COVID-19 were enrolled from four different hospitals in Table 1 ). There was no remarkable difference between males and females on the distribution of ages (Chi-square test, p = 0.995; Figure 1B) , whereas a significant relationship was revealed between disease severity and age ( Figure 1B , Chi-square test, p = 0.001).

First, patients aged over 50 yrs.-old were more likely to develop into severe (35.1%) and critical A c c e p t e d M a n u s c r i p t 8 (2.7%) pneumonia, whereas only 0.6% in severe and no critical pneumonia observed in patients aged below 16 yrs. Second, the older patients had more severe symptoms comparing to the counterpart of children patients, including fever, cough, and sore throat (Chi-square or Fisher's exact test, p < 0.05). Third, ICU admission was more common in older patients (Chi-square test, p = 0.001). Our results suggested older patients (> 50 yrs.) were more vulnerable to COVID-19 than younger adults and children. [4] Compared with children, white blood cell, lymphocyte, and platelet count were significantly decreased in adult patients with COVID-19, while the number of neutrophils, neutrophil over lymphocyte ratio, high-sensitivity C-reactive protein (hs-CRP), prothrombin time (PT), fibrogen (FIB), and total bilirubin were increased ( Figure 2 , Kruskal-Wallis test, p < 0.0001). Moreover, compared with children, alanine aminotransferase, creatine, and creatine kinase significantly increased in adult patients ( Figure 2 ). These laboratory findings indicated that adult patients were more sensitive to SARS-CoV-2 infection with multiple organs affected, which agreed with the previous report. [22] The distinct expression and distribution of ACE2 in lungs of children and older patients ACE2 is a cell-entry receptor for SARS-CoV-2 to invade the human respiratory system. [23] Recent studies indicate the high vulnerability of older adults to COVID-19 are possibly associated with ACE2 expression in the respiratory system. [11, 12, 22] To assess the role of ACE2 in SARS-CoV-2 infection and the potential relation between ACE2 expression and disease severity, we examined the spectrum of ACE2 expression and distribution in the lung tissue of age-matched children and adults (Supplementary Table 2 and 3) . were presented in the bronchus of older adults, indicating bronchus degeneration with aging [24] .

Second, lung tissue sections were subjected to immunostaining for human ACE2 using kidney as a positive control, and isotype IgG was used to exclude non-specific signals ( Figure 3A iii-vii and x-xiv).

The expression and distribution of ACE2 were determined in 26 lung samples from children (aged 2 months to 12 yrs.) and 24 lung samples from adults (aged 16 yrs. to 80 yrs.). The results revealed that ACE2 positive (ACE2 + ) cells were sporadically distributed in all lung regions, including the bronchus (Br) and pulmonary alveolar (PA) areas ( Figure 3A iii-v and x-xii). Due to increasing mortality in COVID-19 patients aged over 10 years, [4] samples were grouped as 0-10Y, 10-50Y, 50-60Y, 60-70Y and > 70Y based on patient ages, at least 3 patients' lung samples in each grouped age were subjected to analysis. 20 images were randomly captured from different view-fields of the slide for each sample. [18] Unexpectedly, our results indicated that ACE2 + cells were highly enriched in lung tissues of children and gradually decreased with increased age. Of note, ACE2 + cells were significantly decreased in older adults (50-70 yrs.) compared with children ( Figure 3B Because SARS-CoV-2 could infect ACE2 + cells in the regions of bronchus and alveoli, we further examined the distribution of ACE2 + cells only expressed in these regions in the lungs of children and older adults. We observed a reduction of ACE2 + cells in the bronchial region with increased age but A c c e p t e d M a n u s c r i p t 10 no such reduction in the pulmonary alveolar region ( Figure 3C i, Kruskal-Wallis test, p = 0.0005; Figure 3C ii, Kruskal-Wallis test, p = 0.0986), indicating that the alveolar region in older adults is at high-risk of SARS-CoV-2 infection. When compared with children (< 16 yrs.), ACE2 + cell number was remarkably decreased in older adults (> 50 yrs.) in the bronchial region ( Figure 3C iii, One-way ANOVA, p < 0.0001), but not obviously so in the alveolar region ( Figure 3C iii). With degenerative changes of the bronchus in older individuals, ACE2 + cells were mainly located in the alveolar region that may facilitate easy infection at the lower respiratory tract by SARS-CoV-2 and develop into severe pneumonia.

Lung progenitor cells play a putative role in lung development, injury and repair following SARS-CoV-2 infection and influenza virus-induced respiratory failure. [25, 26] Recent studies indicate that ACE2 is expressed in certain lung cells including lung progenitor cells at different stages of lung development. [19] [20] [21] To evaluate the expression profile of ACE2 in lung progenitor cells that may contribute to the disease severity of COVID-19 in children and older adults, we analysed the co-expression of ACE2, TMPRSS2, and lung progenitor marker genes using three public single-cell RNA-seq datasets of human lungs, including 12 samples from 4 children, 3 young adults and 5 elder adults [19] [20] [21] . Pearson correlation coefficient (PCC) was used to predict the possibility of co-expressions among each pair of "SFTPC", "PDPN", "ACE2", "TMPRSS2", "KRT5", "TP63", "SOX2", "NKX2-1", "SOX9" and "SCGB1A1" based on their TPM in 12 samples of datasets in tissue level [27] . The results showed that most of lung progenitor genes were not strongly coupled with ACE2 in lung tissues except for SFTPC and PDPN (Figure 4A i, PPC value >0.5). Second, we counted the percentage of cells with at least two reads in "ACE2", "TMPRSS2", and another progenitor Figure 1A and 1B) . Importantly, our results indicated that TMPRSS2 was widely expressed in human lung tissue, including bronchus, submucosa, and alveoli, and no significant difference between children and older adults (Supplementary Figure 1C i-iii) . These data suggested TMPRSS2, different from ACE2 which was mainly expressed in AT II specific cells, was much more abundantly expressed in lung tissues.

We used a spike driven GFP-expression SARS-CoV-2 pseudovirus to determine the capacity of SARS- A c c e p t e d M a n u s c r i p t 13 25%) children were considered partially recover since the lesion in lung was not absorbed completely based on the radiological images. In contrast, only one (1/10; 10%) older patient recovered completely, while 9 (9/10; 90%) patients recovered partially with residual lung lesions.

Four (4/17, 23.5%) patients in the young adults recovered completely, while 13 (13/17, 76.5%) patients recovered partially ( Figure 5D, E) . Notably, of the 10 older patients, one had evidence of lung fibrosis detected on a CT scan ( Figure 5C ).

Among patients suffered from SARS-CoV-2 infection, the older are more likely to become severely and critically ill with higher mortality than children and young adults. [22] Older patients with comorbidities and acute respiratory distress syndrome (ARDS) are at risk of increased mortality. [36, 37] Nonetheless, little direct evidence was available about the spectrum of disease severity linked with the expression pattern of ACE2 and lung progenitor cells. ACE2 is the cell-entry receptor for SARS-CoV-2, and lung progenitor cells are critically involved in lung repair and regeneration after injury. We systematically assessed the profile of ACE2 and lung progenitor cells in children and older patients using a combination of clinical cohort analysis, public single-cell RNA-seq datasets, and lung biopsies. This study presents several key findings.

First, in patients who suffered from SARS-CoV-2, older patients (> 50 yrs.) were particularly vulnerable to COVID-19 ( Figure 1 ). In those aged above 50 yrs., 35.1% and 2.7% developed into severe and critical pneumonia respectively, compared with children (< 16 yrs.-old) of whom only 0.6% presented with severe pneumonia and the others suffered a mild or moderate illness. This distinct variance in disease severity between children and older patients was also reflected by the laboratory parameters of inflammation (Figure 2) . Table 2 and 3, Figure 3 ). Our finding suggests that the ACE2 expression level is not sufficient to determine the higher vulnerability of COVID-19 in older patients. Recent reports indicated that ACE2 was highly expressed in the sinonasal cavity and pulmonary alveoli, proposing sites of ACE2 distribution were more important in disease transmission and disease severities in patients with COVID-19. [38] We therefore examined sites of ACE2 distribution and found a remarkable difference in the distribution of ACE2 between children and older adults. ACE2 + cells were mainly located in the alveolar region and notably decreased at the bronchus in the older ( Figure 3B ). ex vivo infection of pseudoviral SARS-CoV-2 indicated the similar susceptibility of ACE2expressed cells in the alveolus between children and older adults ( Figure 5A-B) .

Several contrasting findings exist in the literature on ACE2 and ages as well as cigarette exposure. [39, 40] Some prior bioinformatic studies indicated that the ACE2 expression in the respiratory system was increased with ages, smoking or cancers, arising an assumption that the higher ACE2 expression contributes to the high-risk of COVID-19 in older individuals. [10, 11, 41] [42, 43] On the other hand, other reports indicated ACE2 expression levels were not changed with ages. [44] It is difficult to compare our results with above reports because most of these bioinformatic studies were mainly focused on adult population RNA-seq datasets without comparison to children's samples parallelly, and no clear information for disease conditions used for studies. We examined ACE2 expressions in 26 children and 24 adults' lung tissues at both transcription and protein levels with negative (IgG) and positive controls (Kidney). The smoking factor was also considered. Our findings indicated that compared to children, the older adults A c c e p t e d M a n u s c r i p t 15 present generally reduced ACE2 amount and expression levels, and these ACE2 positive cells mainly were detected in the lower pulmonary tract.

It was reported that compared with the adult population, children with COVID-19 generally display milder symptoms and lower mortality. [4, 45] The discrepancy between the abundant expression ACE2 in paediatric patients that generally display milder symptoms and lower mortality as compared to aged adults has not been fully understood. One possibility is that the milder disease in paediatric patients with SARS-CoV-2 infection might be associated with the different patterns of immune responses. [5] Our clinical anlaysis also indicated that high sensitivity C-reactive protein (hs-CRP) level was significantly lower in paediatric patients with COVID-19 than older adult counterparts ( Figure 1B ). In addition, although ACE2 is expressed in various human tissues and organs, ACE2expressing organs do not equally participate in COVID-19 pathophysiology, indicating that other mechanisms are also involved in orchestrating cellular infection resulting in tissue damage.

Third, lung progenitor cells were reduced in the older compared with children. It is essential to maintain sufficient lung progenitor cells for lung development and repair. [46] [47] [48] ACE2 marker was in SFTPC + AT II cells, but not in lung progenitor cells including TP63, KRT5, SOX2, PDPN, NKX2.1, or SCGB1A1 positive cells (Figure 4 ). SFTPC presented in AT II cells is also considered as bipotent progenitor cells capable of differentiating into AT I cells during the process of lung injury-repair. [28] [29] [30] Although ACE2/SFTPC AT II cells are targeted by SARS-CoV-2 infection during the subsequent inflammatory response, [49] other putative lung progenitor cells without expression of ACE2 should not be attacked by SARS-CoV-2. However, decreased lung progenitor cells in the older limit their capacity for lung repair and regeneration. Our follow-up study also revealed that infected older patients displayed a poorer recovery from pneumonia ( Figure 5C -E).

There are several limitations in this study. First, a histological study in normal areas of lung tissue that was adjacent to diseased tissue. Whether diseases such as lung abscess or lung A c c e p t e d M a n u s c r i p t 16 cancer, affect ACE2 expression in normal areas of lung tissue requires further investigation. Second, the relationship between age and ACE2 expression remains controversial. It could not exclude the possibility that such a difference could be affected by selected tissues from older or younger individuals. Additionally, it is possible that ACE2 expression within singlecell types correlates with age, but such differences are not reflected in protein level analysis.

Third, at the current stage, only 12 children and 27 adult patients were in the followed-up study. Long term follow-up of more patients will provide more putative information.

Nevertheless, this study is the first time to characterize ACE2 expression and distribution as well as the profile of lung progenitor cells in children and older adults.

In summary, compared to children, older patients are more vulnerable to develop severe pneumonia with poor recovery potential from COVID-19. Older patients present generally reduced ACE2expressing cells and were mainly distributed in the lower pulmonary tract. The lung progenitor cells were also reduced in older adults. These risk factors may be in part contribute to distinct disease severity between children and older patients following SARS-CoV-2 infection.

A c c e p t e d M a n u s c r i p t M a n u s c r i p t year-old patient with moderate pneumonia. Lung lesions were detected in the child (i) and the older patient (iv) on admission. Alleviation of lesion was observed at discharge for the child (ii). A new lesion was detected for the older patient (v) who had met the criteria for discharge with improvement in symptoms and negative result of SRAS-CoV-2 test. Based on the radiological assessment in follow-up visit, the child completely recovered without abnormal finding in CT images (iii), while stripe-like high density shadows were detected for the older patient (vi).

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We thank Clinical Biological Resource Bank of Guangzhou Women and Children's Medical Center for providing clinical samples. We thank Sarah Aglionby for revising the manuscript. 

Qizhou Lian supervised the whole project. Qizhou Lian and Zhao Zhang conceptualized and designed the study, collected data, wrote, and revised the manuscript; Che Zhang, Li Huang and Huimin Xia 

The authors declare no competing interests.

Data are available upon reasonable request. Deidentified participant data.A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 21