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

First, we performed triple fluorescence in situ hybridization to identify ACE2, CTSL and TMPRSS2 on alveolar sections. We observed co-expression, albeit at low levels, of all three genes in alveolar cells (Fig. 1e) . We then performed co-staining with cell type-specific markers. We observed ACE2 transcripts in a subset of type 2 (AT2) cells identified by canonical AT2 protein markers, HTII-280 and pro-SFTPC (Fig. 1f,g) . Similarly, we observed TMPRSS2 gene expression in HTII-280+ AT2 cells (Fig. 1f) . Immunostaining for TMPRSS2 protein further confirmed AT2 cell expression (Extended Data Fig. 5a) . We also observed TMPRSS2 protein expression at low levels in some AT1 cells identified by the canonical AT1 protein marker AGER (Extended Data Fig. 5a) . Of note, some non-epithelial cells also expressed these three genes. We further validated the expression of ACE2 by bulk mRNA-seq in sorted AT2 cells, including those from long-term cultured alveolar organoids (Extended Data Fig. 5b) . We then performed immunohistochemistry and deployed three different available putative ACE2 antibodies to establish ACE2 protein expression (Supplementary Table 4 ). One of these 3 antibodies, the one used previously to functionally block cellular viral entry, specifically labeled adult pro-SFTPC-positive AT2 cells (Extended Data Fig. 5c) . As a cautionary note, the lack of agreement between antibody staining patterns suggests that some of these antibodies may be non-specific.