||The landscape of lung bronchoalveolar immune cells in COVID-19 revealed by single-cell RNA sequencing
||The novel coronavirus SARS-CoV-2, etiological agent of recently named Coronavirus infected disease (COVID-19) by WHO, has caused more than 2, 000 deaths worldwide since its emergency in Wuhan City, Hubei province, China, in December, 2019. The symptoms of COVID-19 varied from modest, mild to acute respiratory distress syndrome (ARDS), and the latter of which is generally associated with deregulated immune cytokine production; however, we currently know little as to the interplay between the extent of clinical symptoms and the compositions of lung immune microenvironment. Here, we comprehensively characterized the lung immune microenvironment with the bronchoalveolar lavage fluid (BALF) from 3 severe and 3 mild COVID-19 patients and 8 previously reported healthy lung controls through single-cell RNA sequence (scRNA-seq) combined with TCR-seq. Our data shows that monocyte-derived FCN1+ macrophages, whereas notFABP4+ alveolar macrophages that represent a predominant macrophage subset in BALF from patients with mild diseases, overwhelm in the severely damaged lungs from patients with ARDS. These cells are highly inflammatory and enormous chemokine producers implicated in cytokine storm. Furthermore, the formation of tissue resident, highly expanded clonal CD8+ T cells in the lung microenvironment of mild symptom patients suggests a robust adaptive immune response connected to a better control of COVID-19. This study first reported the cellular atlas of lung bronchoalveolar immune microenvironment in COVID-19 patients at the single-cell resolution, and unveiled the potential immune mechanisms underlying disease progression and protection in COVID-19.
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acute respiratory infections
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medRxiv preprint Table 1
monocyte-derived FCN1 + macrophages
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First 5000 Characters:Since December, 2019, a disease outbreak caused by a novel coronavirus (now given the name SARS-CoV-2) started in Wuhan, and has quickly spread in China and subsequently inmany other countries  . The WHO has declared the new coronavirus (CoV) infection as a global public health emergency and named the coronavirus infected disease-19 . The genome analysis showed that the SARS-CoV-2 is a SARS-CoV like β-lineage coronavirus  and likely originates from bats  . SARS-CoV-2 uses human ACE2 protein as their receptors  , which explains its similarly high transmissibility. The clinical data indicated that the COVID-19 varied from asymptomatic to acute respiratory distress syndrome (ARDS), similar as SARS-CoV infection    . Although researchers are racing against time to develop vaccines and test anti-viral drugs in clinical trials  , there is no effective prophylactic and clinical treatment for COVID-19 yet.
Generally, the COVID-19 is less severe and less fatal than the SARS, however, some patients, especially aged populations with co-morbidities are prone to develop more severe symptoms and require emergent medical interventions  . It is not completely understood why some patients develop severe but others have mild or even asymptomatic diseases by the same SARS-CoV-2
infections. The immunopathogenesis of hCoVs-induced respiratory distress syndrome may involve deranged interferon production, hyper-inflammatory response and cytokine storms, inefficient or delayed induction of neutralizing antibody and specific T cell responses [10, 11] .
However, due to biosafety and ethics issues and technical limitations, most of the current knowledge was generated from animal model studies, and not directly from human subjects.
More investigations using patient samples are needed to explore the relevant protective or pathogenic mechanisms in clinical settings.
Here, we applied the emerging single-cell RNA sequence (scRNA-seq) and single-cell TCR-seq to comprehensively characterize the lung bronchoalveolar lavage fluid (BALF) cells from 6 of COVID-19 patients, including 3 severe and 3 mild cases. Our study depicts a high-resolution transcriptome atlas of lung resident immune subsets in response to SARS-CoV-2 infections. It reveals that compared to the lung immune microenvironment of mild symptom patients, monocyte-derived FCN1 + macrophages replacing the FABP4 + alveolar macrophages predominate macrophage lineage compostions in the severely damaged lung, which are highly inflammatory and potent chemokine producers. Furthermore, the lung resident highly expanded clonal CD8 + T All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.02. 23.20026690 doi: medRxiv preprint cells formed in the mildly infected patients support the notion that a rapid and robust adaptive immune response is potentially critical for controlling COVID-19.
To characterize the immune microenvironment of the SARS-CoV-2-infected lung, we performed scRNA-seq analysis of single cells in the lung BALF (37, 820 cells) using the 10X Genomics platform, from 3 of recovered mild cases and 3 of severe cases ( Figure 1A , Table 1 ). We also collected public available scRNA-seq data (43, 627 cells) of 8 normal lungs as control  . This dataset passed stringent high-quality filtering ( Figure S1 ), yielding a mean of 188K reads/cell with median gene and unique molecular identifier (UMI) counts of 2, 070 and 6, 852, respectively (Table S1 ). Figure 1D and S2B). We were also able to detect low levels of SARS-CoV-2 transcripts in various cell populations from severe COVID-19 patients but not mild cases and controls ( Figure S2C ). We assumed that these viral transcripts were likely ambient contaminations of viral loads in the BALF.
We then compared the distribution of different cellular compartments among control, mild and severe group. There were higher proportions of T and NK cells in the COVID-19 patients than those in controls, while epithelial cells in patients are fewer. As compared to mild cases, severe patients contained more macrophages but less proportion of T and NK cells ( Figure 1E-1F ).
Together, our data showed an increased recruitment of immune cells to the lung in response to SARS-CoV-2 infection, and that the lung immune cell compartments differed between mild and severe COVID-19 patients.
All rights reserved. No reuse allowed without permission. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is . https://doi.org/10.1101/2020.02. 23.20026690 doi: medRxiv preprint
The increased lung macrophage population was present in severe COVID-19 patients. To further understand the macrophage heterogeneity, we re-clustered the macrophages
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