key: cord-1011487-nz36i2oa authors: Andonegui-Elguera, Sergio; Taniguchi-Ponciano, Keiko; Gonzales-Bonilla, Cesar Raul; Torres, Javier; Mayani, Hector; Herrera, Luis Alonso; Peña-Martínez, Eduardo; Silva-Román, Gloria; Vela-Patiño, Sandra; Ferreira-Hermosillo, Aldo; Ramirez-Renteria, Claudia; Carvente-Garcia, Roberto; Mata-Lozano, Carlos; Marrero-Rodríguez, Daniel; Mercado, Moises title: Molecular Alterations Prompted by SARS-CoV-2 Infection: Induction of Hyaluronan, Glycosaminoglycan and Mucopolysaccharide Metabolism date: 2020-06-18 journal: Arch Med Res DOI: 10.1016/j.arcmed.2020.06.011 sha: 12e35dfea3c015c958c21efea520dce7d766f8a3 doc_id: 1011487 cord_uid: nz36i2oa Abstract Background The SARS-CoV-2 is the etiological agent causing COVID-19 which has infected more than 2 million people with more than 200000 deaths since its emergence in December 2019. In the majority of cases patients are either asymptomatic or show mild to moderate symptoms and signs of a common cold. A subset of patients, however, develop a severe atypical pneumonia, with the characteristic ground-glass appearance on chest x-ray and computerized tomography, which evolves into an acute respiratory distress syndrome, that requires mechanical ventilation and eventually results in multiple organ failure and death. The Molecular pathogenesis of COVID-19 is still unknown. Aim of the study In the present work we performed a stringent metanalysis from the publicly available RNAseq data from bronchoalveolar cells and peripheral blood mononuclear cells to elucidate molecular alterations and cellular deconvolution to identify immune cell profiles. Results Alterations in genes involved in hyaluronan, glycosaminoglycan and mucopolysaccharides metabolism were over-represented in bronchoalveolar cells infected by SARS-CoV-2, as well as potential lung infiltration with neutrophils, NK cells, T CD4+ cell and macrophages. The blood mononuclear cells presented a proliferative state. Dramatic reduction of neutrophils, NK and T lymphocytes, whereas an exacerbated increase in monocytes. Conclusions In summary our results revealed molecular pathogenesis of the SARS-CoV-2 infection to bronchoalveolar cells inducing the hyaluronan and glycosaminoglycan metabolism that could shape partially the components of the ground-glass opacities observed in CT. And the potential immune response profile in COVID-19. Since its emerging in Wuhan, China in December 2019 the coronavirus disease epidemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has progressed rapid to pandemic (1) . As for April 27, 2020 more than 2 million people worldwide have been diagnosed with COVID-19 (2) . It is associated with significant mortality due to the development of acute respiratory distress syndrome (ARDS) and eventually in multiple organ failure, particularly in certain high-risk populations such as the elderly, as well as patients with obesity, diabetes and hypertension. This complicated course is thought to be due to a so-called "cytokine storm" characterized by over production of TNF, IL6 and IL1ß, which together lead to vascular hyperpermeability, multiorgan failure and eventually death (3) . The SARS-CoV-2 viral entry depends upon the binding of viral spike (S) protein to the host cell surface protein angiotensin-converting enzyme 2 (ACE2). Once internalized, the S protein undergoes proteolytic processing by transmembrane serine protease 2 (TMPRSS2), as well as by cathepsin L and B (CTSL and CTSB, respectively), resulting in cleavage at the S1/S2 site, and the ensuing S2dependent fusion of viral and cellular membranes (4) . Coronaviruses such as SARS-CoV and MERS-CoV have evolved strategies to dampen or delay IFN production, which usually trigger exacerbated inflammatory host response leading to severe lung pathology (5) . Among these mechanisms is the disruption of the pathogen associated molecular patterns recognition by the viral nonstructural protein 15 (nsp15) (6) . Despite the global treats of COVID-19, the host immune response against virus infection remains poorly understood (5) . Therefore, in the present work we carried out comprehensive and stringent transcriptomic metanalysis of SARS-CoV-2 infected bronchoalveolar cells and peripheral blood mononuclear cells to unveil the molecular alterations caused by viral infection as well as deconvolution analysis to identify the immune cell profiles in COVID-19 patients. Paired end fastq files were downloaded from BIGD-GSA and SRI from NCBI. Accession numbers are CRA002390 and SRR10571724, SRR10571730, and SRR10571732. Data files consisted in 3 COVID-19 patients bronchoalveolar fluid lavage (BALF) and 3 bronchoalveolar fluid lavage controls, and also 3 COVID-19 peripheral blood mononuclear cells (PBMC) and 3 control peripheral blood mononuclear cells. Fastq files quality control of was performed using FastQC v. 0.11.9. Samples adapter were trimmed with the Cutadapt v tool. The BALF and PBMC counts were subjected to quality control using the NOISeq package, biases associated with length, GC content, RNA composition and batch effect, were identified and removed. BALF and PBMC samples were processed using full-quantile normalization for GC content and length, finally, the ARSyN function was used to eliminate batch effect. The normalization process was carried out using the NOISeq and EDASeq packages in R. Differentially expressed genes were calculated on the normalized counts using the noiseqbio function of the NOISeq package, using the predefined parameters and filtered by a probability greater than 0.99. WebGestalt (http://www.webgestalt.org) was used for understanding the biological meaning behind the resulting list of genes, to obtain gene ontology and pathway information for significantly deregulated genes in COVID-19 patients. In order to identify potential cellular and molecular alterations in lung tissue we analyzed top 100 Consistently with the observed in the up-regulated genes, the down-regulated genes showed involvement in negative regulation of cell proliferation (CDKN1C, SFN and RUNX3) and cell maturation (SOX8, RHEX and GATA2) at GO biological process. In the GO molecular function terms cytokine receptor binding (CXCL1, EFNA5 and RHEX) among others (Figure 3 ). Very interestingly the cellular deconvolution analysis showed dramatic reduction of neutrophils, NK and T cells, whereas an exacerbated increase in monocytes (Figure 4 ). The present study constitutes a comprehensive meta-analysis of the available RNA sequencing data from pulmonary and blood mononuclear cells from patients with COVID-19 infection. In order to be able to design effective therapeutic strategies against SARS-CoV-2 infection, it is crucial to understand the molecular basis of its interaction with host cells. The infection of SARS-CoV-2 can cause severe pulmonary disease and complications with severe mortalities, currently there is no effective drug (5). The acute respiratory distress syndrome that complicates a substantial number of patients with COVID-19 infection is characterized by a generalized endothelial dysfunction with a substantial fluid leak into the interstitial space thereby reducing lung compliance and thus, effective ventilation (7, 8) . This is the basis for the so-called ground-glass appearance on chest CT (9) and correlates with recent autopsy findings showing filling of the lungs with a clear fluid or gel-like material, much resembling what occur in wet drowning (10) . Although the fluid filling components of the lungs are not yet defined, hyaluronan accumulation has been documented in patients with ARDS, and the potential role of polysaccharides for the interstitial edema of the lung have not received much attention (11) . Hyaluronan is glycosaminoglycan component of the extracellular matrix found in lung, and can absorb water 1000 times its molecular weight, and the alteration in hyaluronan synthesis and accumulation in ARDS is established (12) . Our molecular findings in bronchoalveolar cells regarding the upregulation of genes encoding proteins involved in hyaluronan, glycosaminoglycan, aminoglycan and mucopolysaccharide metabolism support the notion that these complex polysaccharides play indeed a role in the genesis of this severe form of interstitial pulmonary edema. Thus, inhibition of hyaluronan synthesis could represent a useful measure in the management of these patients (13) . Several inflammatory cytokines produced in COVID-19 could be strong inducers of hyaluronan metabolism (10) therefore the relevance to identify immune cells infiltrating lung tissue. Using molecular deconvolution analysis, we identified the presence of neutrophils, NK cells, T CD4+ lymphocytes and macrophages infiltrating the lungs of COVID-19 patients, consistently our findings with the previously reported (14) . Increased infiltrating macrophages are identified in autopsies and are thought to be responsible for fueling and perpetuating the inflammatory process (14) . Neutrophils recruited to the site of infection can induce a prominent proinflammatory response directly by recognizing and clearing pathogens or by recruiting other immune cells (15) . In summary, potentially the ARDS could be prompted by SARS-CoV-2 and fueled by host immune response. Our in-silico findings revealed that peripheral mononuclear blood cells are in a proliferative state, perhaps, these proliferation events could represent clonal expansion of the remaining immune cells specific to viral infection. Interestingly, the molecular deconvolution analysis showed an evident shift of the immune profiles between COVID-19 and controls showing dramatic reduction of neutrophils, NK and T lymphocytes, whereas an exacerbated increase in monocytes. Changes in myeloid, NK and B cells in the COVID-19 patients has been reported (16) . Important lymphopenia is also observed, indicating an impairment of the immune system during the SARS-CoV-2 infection (17) . During viral infections it is generally accepted that host immune responses determine both protection against viral infections and the pathogenesis of respiratory injury (16) . Coordinated immune response, innate and adaptative, may lead to rapid control of virus, whereas failed immune response may lead to viral spreading, cytokine storm and eventually death (18) . Epidemiology and transmission of COVID-19 in 391 cases and 1286 of their close contacts in Shenzhen, China: a retrospective cohort study Atypical presentation of COVID-19 in young infants COVID-19 cytokine storm: the interplay between inflammation and coagulation SARS-CoV-2 cell entry depends on ACE2 TMPRSS2 and is blocked by a clinically proven protease inhibitor Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients Coronavirus endoribonuclease targets viral polyuridine sequences to evade activating host sensors Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China COVID-19 pneumonia: what has CT taught us? COVID-19 infection: the perspectives on immune responses Accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome Defective lung function following influenza virus is due to prolonged, reversible hyaluronan synthesis Inhibition of hyaluronan synthesis attenuates pulmonary hypertension associated with lung fibrosis The landscape of lung bronchoalveolar immune cells in COVID-19 revealed by single-cell RNA sequencing Neutrophils in viral infection Immune Cell Profiling of COVID-19 Patients in the Recovery Stage by Single-Cell Sequencing Characteristics of peripheral lymphocyte subset alteration in COVID-19 pneumonia Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8⁺ T cells Mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy COVID-19, pulmonary mast cells, cytokine storms, and beneficial actions of luteolin The authors declare no conflict of interest.