Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells

Christoph Muus; Malte D Luecken; Gokcen Eraslan; Avinash Waghray; Graham Heimberg; Lisa Sikkema; Yoshihiko Kobayashi; Eeshit Dhaval Vaishnav; Ayshwarya Subramanian; Christopher Smillie; Karthik Jagadeesh; Elizabeth Thu Duong; Evgenij Fiskin; Elena Torlai Triglia; Christophe Becavin; Meshal Ansari; Peiwen Cai; Brian Lin; Justin Buchanan; Sijia Chen; Jian Shu; Adam L Haber; Hattie Chung; Daniel T Montoro; Taylor Adams; Hananeh Aliee; Samuel J Allon; Zaneta Andrusivova; Ilias Angelidis; Orr Ashenberg; Kevin Bassler; Christophe Becavin; Inbal Benhar; Joseph Bergenstrahle; Ludvig Bergenstrahle; Liam Bolt; Emelie Braun; Linh T Bui; Mark Chaffin; Evgeny Chichelnitskiy; Joshua Chiou; Thomas M Conlon; Michael S Cuoco; Marie Deprez; David S Fischer; Astrid Gillich; Joshua Gould; Minzhe Guo; Austin J Gutierrez; Arun C Habermann; Tyler Harvey; Peng He; Xiaomeng Hou; Lijuan Hu; Alok Jaiswal; Peiyong Jiang; Theodoros Kapellos; Christin S Kuo; Ludvig Larsson; Michael A Leney-Greene; Kyungtae Lim; Monika Litvinukova; Ji Lu; Leif S Ludwig; Wendy Luo; Henrike Maatz; Elo Maddissoon; Lira Mamanova; Kasidet Manakongtreecheep; Charles-Hugo Marquette; Ian Mbano; Alexi M McAdams; Ross J Metzger; Ahmad N Nabhan; Sarah K Nyquist; Jose Ordovas-Montanes; Lolita Penland; Olivier B Poirion; Segio Poli; CanCan Qi; Daniel Reichart; Ivan Rosas; Jonas Schupp; Rahul Sinha; Rene V Sit; Kamil Slowikowski; Michal Slyper; Neal Smith; Alex Sountoulidis; Maximilian Strunz; Dawei Sun; Carlos Talavera-Lopez; Peng Tan; Jessica Tantivit; Kyle J Travaglini; Nathan R Tucker; Katherine Vernon; Marc H Wadsworth; Julia Waldman; Xiuting Wang; Wenjun Yan; Ali Onder Yildirim; William Zhao; Carly G K Ziegler; Aviv Regev

. CC-BY-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.19.049254 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.19.049254 doi: bioRxiv preprint (orange), and CTSL (green) in epithelial cells. Effect size (x axis) of the association, in log fold change (sex, smoking status) or slope of log expression with age. Colored bars: associations with an FDR-corrected p-value<0.05, where pseudo-bulk analysis shows a consistent effect direction. Error bars: standard errors around coefficient estimates. (e) Distribution of ACE2 and TMPRSS2 expression across level 3 lung cell types. Red shading indicates the main cell types that express ACE2 and TMPRSS2. (f) Hold out analysis shows the robustness of associations to holding out a dataset. The values show the number of held-out datasets that result in loss of association between a given covariate (rows) and ACE2, TMPRSS2, or CTSL expression in a given cell type (columns). Robust trends are determined by significant effects that are robust to holding out any dataset (0 values). (g) Low expression in pediatric samples. Mean expression level (log CPM, y axis) of ACE2 (blue), TMPRSS2 (orange), and CTSL (green) across age bins (x axis) in AT2 (left) and ciliated (right) cells. Pediatric samples: 0-10 years. Samples from past or current smokers were removed from this plot to avoid smoking confounders. Error bars are omitted due to y-axis limitations. They are typically 10-fold the mean value (Supplementary Table 5 ). Multiciliated and AT2 cells are shown as these cell types are present in fetal data, and show significant age associations with ACE2 expression. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.19.049254 doi: bioRxiv preprint UMAP of scRNA-seq profiles (dots) colored by experimental group (c) or by Ace2 + cells and indicated double positive cells (d). Alveolar epithelial cells (AT1 and AT2) and airway epithelial secretory and ciliated cells are marked. (e) The relative frequency of Ace2 + cells is increased by smoking in airway secretory cells but not AT2 cells. Relative proportion (y axis) of Ace2 + (red) and Ace2 -(grey) cells in smoking and control mice of different cell types (x axis). (f, g) Expression of Ace2 is increased in airway secretory cells, but not in AT2 cells. Distribution of Ace2 expression (y axis) in secretory (f) and AT2 (g) cells from control and smoking mice (x axis). (h-j) Re-analysis of published bulk mRNA-Seq 101 of lungs exposed to different daily doses of cigarette smoke show increased expression of (h) Ace2, (i) Tmprss2, and (j) Ctsl after five months of chronic exposure. Extended Data Figure 6 . Age, sex, and smoking status associations with expression of ACE2, TMPRSS2, and CTSL across level 2 cell type annotations. Effect size (y axis) of association as log fold changes (sex, smoking status) and slope of log expression with age. Bars that are colored in indicate associations with an FDR-corrected p-value of < 0.05 where the pseudo-bulk analysis shows a consistent effect direction. Error bars represent model uncertainties. The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.19.049254 doi: bioRxiv preprint Extended Data Figure 7 . Age