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Summary
Full article available at https://www.frontiersin.org/articles/10.3389/fimmu.2020.607314/full
Acute lung injury (ALI) is an important cause of morbidity and mortality after viral infections, including influenza A virus H1N1, SARS-CoV,MERS-CoV, and SARS-CoV-2. The angiotensin I converting enzyme 2 (ACE2) is a key host membrane-bound protein that modulates ALI induced by viral infection, pulmonary acid aspiration, and sepsis. However, the contributions of ACE2 sequence variants to individual differences in disease risk and severity after viral infection are not understood. In this study, we quantified H1N1 influenza-infected lung transcriptomes across a family of 41 BXD recombinant inbred strains of mice and both parents—C57BL/6J and DBA/2J. In response to infection Ace2 mRNA levels decreased significantly for both parental strains and the expression levels was associated with disease severity (body weight loss) and viral load (expression levels of viral NA segment) across the BXD family members. Pulmonary RNA-seq for 43 lines was analyzed using weighted gene co-expression network analysis (WGCNA) and Bayesian network approaches. Ace2 not only participated in virusinduced ALI by interacting with TNF, MAPK, and NOTCH signaling pathways, but was also linked with high confidence to gene products that have important functions in the pulmonary epithelium, including Rnf128, Muc5b, and Tmprss2. Comparable sets of transcripts were also highlighted in parallel studies of human SARS-CoV-infected primary human airway epithelial cells. Using conventional mapping methods, we determined that weight loss at two and three days after viral infection maps to chromosome X—the location of Ace2. This finding motivated the hierarchical Bayesian network analysis, which defined molecular endophenotypes of lung infection linked to Ace2 expression and to a key disease outcome. Core members of this Bayesian network include Ace2, Atf4, Csf2, Cxcl2, Lif, Maml3, Muc5b, Reg3g, Ripk3, and Traf3. Collectively, these findings define a causally-rooted Ace2 modulatory network relevant to host response to viral infection and identify potential therapeutic targets for virus-induced respiratory diseases, including those caused by influenza and coronaviruses.
About cases
Mice We used females from 41 BXD RI strains and both parental strains—B6 and D2. Mice were between 8 and 12 weeks of age when infected. They were housed and maintained on a 12:12 light/dark cycle, with ad libitum access to food and water. Virus Original stocks of mouse-adapted A/Puerto Rico/8/34 (H1N1, PR8M) virus were obtained from Stefan Ludwig, University of Münster (28). Virus stocks were propagated in the chorioallantoic cavity of 10-day-old pathogen-free embryonated chicken eggs for 48 h at 37°C as described previously (29). Viral titer was determined using a focus-forming unit (FFU) assay as described previously (29). Infection of Mice Animals were anesthetized by intraperitoneal injection of ketamine/xylazine (10 % (v/v) of 100 mg/ml ketamine and 5 % (v/v) of 20 mg/ml xylazine in 0.9 % (w/v) NaCl with a dose adjusted to body weight (200 μl/20 g body weight). Infection was performed by intranasal application of virus solution in 20 μl sterile phosphate-buffered saline (PBS), with a PR8M dosage of 2×103 FFU. Mice were bred and infected at the animal facilities at UTHSC
About tissue
RNA Isolation and Sequencing Mice were sacrificed 3 days post-infection (dpi) and both lungs were extracted and transferred immediately to RNAlater (Qiagen), stored at 4°C for one day, and then stored at −20°C. RNA was isolated using Qiagen Midi kit (30). RNA quality was evaluated on a 2100 Bioanalyzer (Agilent). Five-hundred nanograms of total RNA was used to prepare libraries for sequencing using the Lexogen SENSE RNA-seq library kit for Ion Torrent. Libraries were amplified for 11 cycles as the final step of library preparation. Before sequencing, 1-μl aliquots were pooled and sequenced on an Ion Torrent PGM 314 chip. Barcoded data from the PGM was used to balance the final pool before sequencing. Library pools were sized to ~260 bp on a Pippin Prep instrument using 2% Pippin agarose gel. The sized libraries were evaluated on an Agilent High Sensitivity chip, quantified using real-time PCR, and used to prepare beads using a One-Touch 2 device. Beads were sequenced on an Ion Torrent Proton P1 chip. On average, 67 million reads were obtained per strain.
About data processing
Read Mapping and Gene Expression Quantification RNA-seq reads were quality-trimmed using Trim Galore (31) and mapped to the mm10 reference genome or to the IAV PR8M genome using STAR (32). Counts were summarized at the gene level using the R-package Rsubread (33), normalized and log transformed using the R-package DESeq2 (34), and batchcorrected using the ComBat function of the R-package sva (35, 36). For annotations of genes, ENTREZID from Rsubread were matched to RefSeq annotations using R-package biomart (37).
Contributors
LL, KS, RW, and RB conceived the study. SB and AS conducted the experiments. FX, KS, YC, and RW performed data analysis. LL, FX, KS, RW, and JG wrote the manuscript. FX, KS, and JG prepared the figures and tables. RW, KL, DA, and CJ edited the manuscript. All authors contributed to the article and approved the submitted version.
Citation
Xu F, Gao J, Bergmann S, Sims AC, Ashbrook DG, Baric RS, Cui Y, Jonsson CB, Li K, Williams RW, Schughart K and Lu L (2021) Genetic Dissection of the Regulatory Mechanisms of Ace2 in the Infected Mouse Lung. Front. Immunol. 11:607314. doi: 10.3389/fimmu.2020.607314
Acknowledgment
Primary human bronchial epithelial cells were provided by Scott H. Randell (Marsico Lung Institute, Tissue Procurement and Cell Culture Core, The University of North Carolina at Chapel Hill, USA). The cells were obtained under protocol 03-1396 approved by the University of North Carolina at Chapel Hill Biomedical Institutional Review Board. The RNA-seq was carried out by the Molecular Resource Center at UTHSC.
Notes
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.07314/full#supplementary-material