Transcriptome Analaysis of humanized lungs in COVID-19 [Bulk_RNAseq]

Mice with a human immune system (humanized mice), generated by transplantation of human hematopoietic stem and progenitor cells (HSPCs), serve as invaluable tools to study the development and function of the human immune system in vivo. MISTRG6 mouse model, engineered by a human/mouse homolog gene-replacement strategy, provides physiological factors for essentially all classes of human HSPCs to develop in mice. MISTRG6 (acronym for genes replaced) mice encode humanized M-CSF (enabling monocytes and tissue macrophage development), GM-CSF/IL-3 (to provide lung alveolar macrophages), SIRPa (establish macrophage tolerance to human cells), ThPO (hematopoiesis and platelets), and IL-6 (better engraftment allowing study of adult human patients and improved antigen-specific antibody responses as well as human IL-6 per se), in a Rag2/Gamma common chain deleted background. By adapting recombinant adeno-associated virus (AAV)-driven gene therapy to deliver hACE2 to the lungs, which allows infection with SARS-CoV-2 of MISTRG6 mice engrafted with HPSCs, we created a humanized mouse model of COVID-19 that recapitulates the distribution and function of the human innate and adaptive immune system and is amenable to the mechanistic study of COVID-19 and its myriad of complications. We evaluated the lung transcriptional landscape and response to therapeutics in this model. First, we assessed the impact of anti-IFNAR2 and Remdesivir combined therapy and control dexamethasone therapy on the immunological transcriptome of SARS-CoV-2 infected MISTRG6-hACE2 mice by focusing on differentially regulated human genes in whole lung tissue. We treated infected MISTRG6-hACE2 mice with Remdesivir, anti-IFNAR2 antibody or a combination of the two starting 7dpi, to sequentially target viral replication and the IFN-dependent cascade downstream of infection. As a control, we also treated mice with dexamethasone, one of the few treatments that significantly reduced hospitalization and mortality in the clinic. Next, we compared the single cell transcriptomes of human immune cells from infected mice late in disease (28dpi) with their uninfected counterparts to gain a deeper understanding of transcriptional changes at the cellular level. Finally, we focused our efforts on deeper characterization of monocyte/macrophage clusters at early (4dpi) or late (14 and 28dpi) infection. Overall design: We successfully delivered AAV-hACE2 to lung tissues of immune-reconstituted MISTRG6 mice (MISTRG6-hACE2) that have been engrafted with human HSPCs. MISTRG6-hACE2 mice were then infected with SARS-CoV-2. Mice were treated with therapeutics starting at 7dpi. For whole tissue transcriptional analysis in response to therapeutics: whole lungs, collected at 14dpi and 28dpi, were homogenized. RNA was RNA was extracted with the RNeasy mini kit (Qiagen) per the manufacturer's protocol.For single cell transcriptional analysis: lungs from infected mice were collected and digested at 37°C for 20 min using digestion cocktail containing 1 mg/ml collagenase D (Sigma) and 30 µg/ml DNase I (Sigma-Aldrich) in RPMI. Single cell suspensions from digested lungs were stained with antibodies against human CD45 and mouse CD45. Human immune cells were sorted (humanCD45+mouseCD45-DAPI-) and stained with TotalSeq (TotalSeq™-B0251 anti-human Hashtag 1 Antibody: GTCAACTCTTTAGCG; TotalSeq™-B0252 anti-human Hashtag 2 Antibody: TGATGGCCTATTGGG) antibodies (Biolegend) prior to processing for droplet based scRNA-seq. Duplicates from each condition/time point were pooled in equal numbers to ensure 10000 cells were encapsulated into droplets using 10X Chromium GEM technology. Libraries were prepared in house using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics). scRNA-seq libraries were sequenced using Nova-Seq. Raw sequencing reads were processed with Cell Ranger 3.1.0 using a human-mouse combined reference to generate a gene cell count matrix.