key: cord-0818152-ri9ncm86 authors: Zhou, Zhuo; Ren, Lili; Zhang, Li; Zhong, Jiaxin; Xiao, Yan; Jia, Zhilong; Guo, Li; Yang, Jing; Wang, Chun; Jiang, Shuai; Yang, Donghong; Zhang, Guoliang; Li, Hongru; Chen, Fuhui; Xu, Yu; Chen, Mingwei; Gao, Zhancheng; Yang, Jian; Dong, Jie; Liu, Bo; Zhang, Xiannian; Wang, Weidong; He, Kunlun; Jin, Qi; Li, Mingkun; Wang, Jianwei title: Heightened innate immune responses in the respiratory tract of COVID-19 patients date: 2020-05-04 journal: Cell host & microbe DOI: 10.1016/j.chom.2020.04.017 sha: 0e10bd6e8479cc0809b6b38cc8961b160749246c doc_id: 818152 cord_uid: ri9ncm86 Summary The outbreaks of 2019 novel coronavirus disease (COVID-19) caused by SARS-CoV-2 infection has posed a severe threat to global public health. It is unclear how the human immune system responds to this infection. Here, we used metatranscriptomic sequencing to profile immune signatures in the bronchoalveolar lavage fluid of eight COVID-19 cases. The expression of proinflammatory genes, especially chemokines, was markedly elevated in COVID-19 cases compared to community-acquired pneumonia patients and healthy controls, suggesting that SARS-CoV-2 infection causes hypercytokinemia. Compared to SARS-CoV, which is thought to induce inadequate interferon (IFN) responses, SARS-CoV-2 robustly triggered expression of numerous IFN-inducible genes (ISGs). These ISGs exhibit immunopathogenic potential, with overrepresentation of genes involved in inflammation. The transcriptome data was also used to estimate immune cell populations, revealing increases in activated dendritic cells and neutrophils. Collectively, these host responses to SARS-CoV-2 infection could further our understanding of disease pathogenesis and point towards antiviral strategies. ). An adjusted p-value (q-value < 0.05) and fold change (FC) 112 ratio (|log2FC| ≥ 2) were used to determine the DEGs (Tables S3A-3C We next collated genes whose expression is mostly regulated (|log2FC| ≥ 5) in 120 SARS2-H ( Figure S1A ). Expression levels of proinflammatory cytokine and 121 chemokine genes (IL-1B, CXCL17, CXCL8, and CCL2), typical antiviral ISGs (IFIT 122 and IFITM family genes), and calgranulin genes that exert pleiotropic functions in 123 inflammatory disorders (S100A8, S100A9, and S100A12) were upregulated in SARS2 124 as compared to Healthy ( Figure S1A ). Meanwhile, we observed marked upregulation Signaling" (z-score = 1.6) in IPA ( Figure 1C ), along with "Chemokine signaling 141 pathway", "IL-17 signaling pathway", "TNF signaling pathway", and "NF-κB 142 signaling pathway" in KEGG (q < 0.05, Figure S1B ). Moreover, both IPA and KEGG 143 analyses showed that DEGs from SARS2-H were strongly and specifically enriched 144 in mRNA translation-related categories, with up-regulated "EIF2 Signaling" (z-score 145 = 3.3, Figure 1C ) in IPA and "Ribosome" (q < 0.05, Figure S1B ) in KEGG. Intriguingly, several neuron function-related pathways were specifically 147 downregulated in SARS2 by IPA analysis, including "CREB Signaling in Neurons" 148 (z-score = -1.1), "Synaptogenesis Signaling Pathway" (z-score = -1.4), and 149 "Endocannabinoid Neuronal Synapse Pathway" (z-score = -2.1, Figure 1C ). This Next, we build protein-protein interaction (PPI) networks with the upregulated DEGs 153 in SARS2 by STRING ( Figures 1D and S2) . A dominant network comprising several 154 subnetworks were generated using the confidence score of 0.9. Genes in the top two 155 enriched KEGG categories (Figure S1B), namely "Ribosome" and "Chemokine 156 signaling pathway", formed two most densely connected subnetworks, which are 7 categories of "Chemokine" and "Receptor", multiple genes were more remarkably 166 upregulated in SARS2 as compared to those in CAPs. Several genes were regulated in 167 the "Interleukin" and "Tumor necrosis factor" categories, and few were modulated in 168 other categories (Figure 2A ). To examine potential cytokine expression dynamics, we 169 aligned individual cases according to the increasing days between sampling and 170 symptom onset. Expression levels of cytokine-related genes seem to decrease over 171 time, with one patient (C4) who eventually deceased being the outlier (Figure 2A ). (Table S2) 187 exhibited the most pronounced chemokine upregulation ( Figure 2B ), suggesting that 188 higher virus replication resulted in a more robust proinflammatory response. Corroborating this, in three cases (C1, C2, and C5) sampled at the same time after Figure 3A ). Eighty-three ISGs were significantly elevated in SARS2, suggesting the 208 robust IFN response (Table S4) . ISGs known to exert direct antiviral activity were 209 upregulated, including IFIT and IFITM genes that exert broad-spectrum antiviral (Table S4) . Surprisingly, ISGs are Our analysis also showed a significantly higher NLR in COVID-19 cases ( Figure 250 S3A). The highest NLR was observed in the case with ultra-high viral reads and 251 robust cytokine/ISG expression (Figures S3B and S3C) . Table S4 ). In (B) and (C), ISGs were 354 assigned into five biclusters. innate immunity-related cell subtypes. Asterisks represent significant differences 359 between groups (*q-value < 0.05, **q-value < 0.01, ***q-value < 0.001, 360 Mann-Whitney test). See also Figure S3 . (Table S3A) , Virus-like CAP to 469 Healthy (Table S3B) , and Non-viral CAP to Healthy (Table S3C) IL1RN IL1B IL4 CXCL17 CXCL8 CCL2 CXCL1 CCL4 CXCL10 CXCL2 CCL3 CCL20 CXCL6 CCL7 CXCL11 CCL8 CXCL16 TNFSF10 TNFSF13B TNFSF12 OSM CCR1 CXCR2 IL5RA CXCR1 IFNGR2 IL1R1 OSMR IFNAR1 IL1R2 CSF3R IL17RA IL4R IL10RA Inflammation EIF4E3 SELL NMI IRF2 PCGF5 NBN TMEM123 TMEM60 ANXA1 PHACTR2 AIDA CAPZA2 TMX1 C1GALT1 CIR1 SSB ARL5B ELF1 DENND1B NME7 CD2AP HBP1 SMCHD1 ARHGAP12 ASCC3 GPD2 RNF138 FOXN2 TANK FMR1 BET1 RSBN1L IFIT1 IFITM3 IFITM2 IFITM1 IFIT2 BAG1 ISG15 IFIH1 TNFSF10 RSAD2 CASP4 IFIT3 CHMP5 EPSTI1 IRF7 STAT1 LYSMD2 SAT1 PSMB8 ZFP36 PHACTR4 CSRNP1 MARCKS IL1RN CCL2 CCR1 NINJ1 KLF6 SAMSN1 CXCL10 CCRL2 PLSCR1 DAPP1 CCL3 SNX10 C3AR1 B4GALT5 NAMPT SLAMF7 TNFSF13B RGS1 FPR3 GADD45B RNF19B LYN FGL2 GCH1 OPTN ADM P2RY14 PMAIP1 C −6 0 6 Log2FC C 8 C 7 C 1 C 2 C 5 C 6 C 4 C 3 National Key Research and Development Program 19 Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins 545 and receptor binding Infectious Diseases Society of Thoracic Society consensus guidelines on the management of community-acquired 549 pneumonia in adults Animal models of acute lung injury An official American Thoracic Society clinical practice guideline: 554 the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease Elevated levels of NAP-1/interleukin-8 are 558 present in the airspaces of patients with the adult respiratory distress syndrome and are associated 559 with increased mortality Parsing the Interferon Transcriptional Network and Its Disease Associations Database Resources of the National 564 Robust enumeration of cell subsets from tissue expression profiles ISG15 in antiviral immunity and beyond Proinflammatory activity in 571 bronchoalveolar lavage fluids from patients with ARDS, a prominent role for interleukin-1 Genomic diversity of SARS-CoV-2 in Coronavirus Disease 2019 patients Monocyte recruitment during infection and inflammation Inhibition of cytokine gene expression and induction of chemokine 590 genes in non-lymphatic cells infected with SARS coronavirus STRING v11: protein-protein association networks with 593 increased coverage, supporting functional discovery in genome-wide experimental datasets H5N1 Virus Attachment to Lower Respiratory Tract Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia 599 in IFITM proteins inhibit entry driven by the 601 MERS-coronavirus spike protein: evidence for cholesterol-independent mechanisms Characteristics of and Important Lessons From the Coronavirus 604 Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese 605 Center for Disease Control and Prevention clusterProfiler: an R package for comparing biological 607 themes among gene clusters A pneumonia outbreak associated with a new coronavirus of probable bat origin A 611 Novel Coronavirus from Patients with Pneumonia in China Severe acute respiratory syndrome coronavirus fails to activate 614 cytokine-mediated innate immune responses in cultured human monocyte-derived dendritic cells SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients