Construction of a Human Cell Landscape of COVID-19 Infection at Single-cell Level

COVID-19 is now causing a global pandemic, there is a demand to explain the different clinical patterns between children and adults. To clarify the organs/cell types vulnerable to COVID-19 infection and the potential age-depended expression patterns of five factors (ACE2, TMPRSS2, MTHFD1, CTSL, CTSB) associated with clinical symptoms. In this study, we analyzed expression levels of five COVID-19 host dependency factors in multiple adult and fetal human organs. The results allowed us to grade organs at risk and also pointed towards the target cell types in each organ mentioned above. Based on these results we constructed an organ- and cell type-specific vulnerability map of the expression levels of the five COVID-19 factors in the human body, providing insight into the mechanisms behind the symptoms, including the non-respiratory symptoms of COVID-19 infection and injury. Also, the different expression patterns of the COVID-19 factors well demonstrate an explanation that the different clinical patterns between adult and children/infants.

and CD71 + early erythroid, while MTHFD1 is highly expressed in thymus, liver of organs and some types of cells ( Supplementary Fig. 1D, E). Furthermore, we explored scRNA-seq data from different parts of the adult human digestive system (Supplementary Fig. 2A-H). We found an interesting phenomenon that paneth cells rather than enterocyte, took the largest proportion in adult ascending colon, duodenum, epityphlon and transversecolon. Enterocyte only dominates in jejunum, rectum and sigmoid-colon parts in adult digestive system (Supplementary Fig. 3A Supplementary  Fig. 5A, B). This made fetal intestine also a high-risk organ as the adult intestine, even a higher risk grade, but via different infection targets, rather than paneth cell and enterocyte (in adult). Secondly, we also found that these factors were enriched in antibody-related cells, such as B cells and antigen-presenting cells, which may be implying fetal demonstrate a different immune response to the infection ( Supplementary Fig. 3A Moreover, the expression of ACE2 level in AT1 cells, which has been described as the "putative mechanism" of the lung infection, is quite low in adult donors, and we neither found any AT1 or AT2 cells in fetal donors ( Supplementary Fig. 4C, H; Supplementary Table 2). In contrast, high TMPRSS2 expression was detected in AT1, AT2 and other cell types in adult lung (Supplementary  Table 2), while the TMPRSS2 expression could not be detected in most cell types in fetal lung ( Supplementary  Fig. 5E, F). Positive TMPRSS2 expression was found in all cell types except in megakaryocyte and myeloid cells, and positive MTHFD1 expression was found in all cell types except bronchial chondrocyte, ciliated cells, and stromal cells in adult lung. CTSL and CTSB are both expressed in all cell types in adult lung (Supplementary Fig. 3E; Supplementary Fig. 5E). There was no cell coexpressed ACE2 or any other factors, while distal progenitor cells co-expressed TMPRSS2 and other factors, such as MTHED1, CTSL and CTSB. Also, the coexpression of MTHFD1-CTSL and MTHFD1-CTSB was detected in many cell types in the adult lung ( Supplementary Fig. 6). All these indicate that ACE2 may not be the key factor (or the only factor) of the COVID-19 infection, the high expression of other factors (even some other cofactors), such as TMPRSS2 and MTHFD1 could be considered as the target of COVID-19 infection make the adult lung as the high-risk organ. Also, positive MTHFD1 expression was detected in various cell types both in adult and fetal lung tissue ( Supplementary Fig. 3E, F). Compare with the fetal kidney, the universal expression of TMPRSS2 and high expression/coexpression level of CTSL and CTSB in adult lung make the adult lung as the high-risk organ than the fetal. Secondly, the large proportion of lung mesenchyme cells (cardiopulmonary progenitor cells) in adult with positive expression of the factors make adult more vulnerable to virus infection than fetal or infants ( Supplementary Fig.  3E, F; Supplementary Fig. 4C, H). Lastly, the factors were co-expressed in the macrophage and megakaryocyte/ erythroid progenitor cell, which implies that immune cells also could be the targets of the virus infection or involved in the cytokine storm leading to the so-called macrophage activation syndrome (MAS) (Supplementary Fig. 5E, F; Supplementary Fig. 6E, F). MTHFD1 expressed in hepatocytes in three adult liver donors and positive expressed in many immune cell types including neutrophil, effector T cell, monocyte, T cell and so on ( Supplementary Fig. 2N, O; Supplementary  Fig 3D, I; Supplementary Fig 4D, I; Supplementary Fig.  5M, N), whereas besides in epithelial cells, neither ACE2 nor TMPRSS2 was expressed in adult and fetal liver, which implying that liver as a high-risk organ due to the highly expressed MTHFD1 rather than ACE2 and TMPRSS2. Same as intestine, kidney and liver, the expression of CTSL and CTSB with a broader distribution in all cell types in liver ( Supplementary Fig. 5G, H). The co-express analysis demonstrated that sinusoidal endothelial cells and epithelial cells are the targets leading to adult liver vulnerable to COVID-19 infection ( Supplementary Fig. 3G, H; Supplementary Fig. 6G, H).
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells representing the interface between blood cells on the one side and hepatocytes and hepatic stellate cells on the other side. LSECs represent a permeable barrier. This would be a well explanation for the liver failure in adult COVID-19 patients rather than in children/infant patients. Finally, we found that most factors enrichment in endothelial cells and epithelial cells (specific in adult with large proportion) rather than in hepatocyte in adult, which may be indicating that endothelial cells and epithelial cells may be the reason that adult are more vulnerable to COVID-19 infection than infants (Supplementary Fig. 3D, I; Supplementary Fig.  5G, H; Supplementary Fig. 6G, H).
None of the cells expressed TMPRSS2, and only a few types of cells, such as fibroblast, cardiomyocyte, neutrophil with the positive expression of ACE2 in two adult and two fetal heart donors ( Supplementary Fig. 2P, Q). Besides immune cells, CTSL and CTSB also both enriched in ventricle cardiomyocytes, vascular endothelial cells, cardiomyocyte and apoptotic cells ( Supplementary Fig. 3I, J). We found that most factors enrichment in ventricle cardiomyocyte (specific in fetal with the largest proportion) and in proliferating cells in fetus, which may be indicating that children/infants could also be victim to COVID-19. And fibroblast is enriched most factors in adult heart, so fibroblast might be the target of the virus leading to adult heart vulnerable to COVID-19 infection ( Supplementary Fig. 6I, J).
In summary, the results above allowed us to point the target cell types in each organ, also graded organs at risk (Fig. 1). Overall ranking is the average of the individual ranking. From the overall ranking, we found that the liver and kidney of adult and fetal share same risk value, and kidney is the highest risk to the COVID-19 infection (liver rank value 3 and kidney rank value 1, rank 1 is the highest risk value and rank 7 is the lowest value). Fetal intestine and heart are more likely infected by COVID-19 than that of adult, and fetal intestine like fetal kidney, both of them are top risk to be infected. Adult lung is more vulnerable to infection due to the much higher expression of CTSL and CTSB and slightly higher expression of ACE2. Also, the different expression patterns of the COVID-19 factors (TMPRSS2, ACE2, MTHFD1, CTSL and CTSB) well demonstrates an explanation that the different clinical patterns between adult and children/infants.
The results allowed us to grade organs at risk and pointed towards the target cell types in each organ mentioned above. A comparison of fetal and adult organs and cell types suggest that low ACE2 and TMPRSS2 expression level in various subpopulation of enterocyte cells in fetal intestine, lower ACE2 expression in various subpopulations of intercalated cells in fetal kidney and lacking ACE2 expression in most subtypes cells in fetal lung could be major factors determining the welldocumented reduced risk of infants.
Based on these results we constructed an organ-and cell type-specific vulnerability map of the expression levels of the five COVID-19 factors (TMPRSS2, ACE2, MTHFD1, CTSL and CTSB) in the human body, providing insight into the mechanisms hidden behind the symptoms, including the non-respiratory symptoms of COVID-19 infection and injury. Also, the different expression patterns of the COVID-19 factors well demonstrate an explanation that the different clinical patterns between adult and children/infants.
In summary, our study provides an overview of COVID-19 infection-related human vulnerable organs based on single-cell analysis. We first time managed to elucidate vulnerable organs and stratify organs of fetal and adult into high and low risk according to the expression level of COVID-19 receptors, including ACE2, TMPRSS2 and the key enzyme for viral replication, MTHFD1 in certain cell types. Also, we study the different receptors expression level between adult and fetal. This finding may explain why adults are more likely to suffer COVID-19 infection and the non-respiratory symptoms observed in COVID-19 pneumonia patients, such as diarrhea and multiple organ failure.