key: cord-0898034-xpia3xpm authors: Dobosh, Brian; Zandi, Keivan; Giraldo, Diego Moncada; Goh, Shu Ling; Musall, Kathryn; Aldeco, Milagros; LeCher, Julia; Giacalone, Vincent D.; Yang, Junkai; Eddins, Devon; Bhasin, Manoj; Ghosn, Eliver; Sukhatme, Vikas; Schinazi, Raymond F.; Tirouvanziam, Rabindra title: BARICITINIB ATTENUATES THE PROINFLAMMATORY PHASE OF COVID-19 DRIVEN BY LUNG-INFILTRATING MONOCYTES date: 2022-05-25 journal: Cell Rep DOI: 10.1016/j.celrep.2022.110945 sha: 3f039eb72c7c25bfa0fce763d92e8e54c27f97dd doc_id: 898034 cord_uid: xpia3xpm SARS-CoV-2-infected subjects are generally asymptomatic during initial viral replication, but may suffer severe immunopathology after the virus has receded and monocytes have infiltrated the airways. In bronchoalveolar lavage fluid from severe COVID-19 patients, monocytes express mRNA encoding inflammatory mediators and contain SARS-CoV-2 transcripts. We leverage a human small airway model of infection and inflammation whereby primary blood monocytes transmigrate across SARS-CoV-2-infected lung epithelium to characterize viral burden, gene expression and inflammatory mediator secretion by epithelial cells and monocytes. In this model, lung-infiltrating monocytes acquire SARS-CoV-2 from the epithelium and upregulate expression and secretion of inflammatory mediators, mirroring in vivo data. Combined use of baricitinib (Janus kinase inhibitor) and remdesivir (nucleoside analog) enhances antiviral signaling and viral clearance by SARS-CoV-2-positive monocytes while decreasing secretion of pro-neutrophilic mediators associated with acute respiratory distress syndrome. These findings highlight the role of lung-infiltrating monocytes in COVID-19 pathogenesis and their importance as a therapeutic target. mechanistic effects of antiviral and immunomodulatory drugs during the course of COVID-19 airway model of infection and inflammation (Dobosh et al., 2021) previously developed and 71 validated by our group to study lung-infiltrating leukocytes in cystic fibrosis (CF) and ARDS 72 (Forrest et al., 2018; Grunwell et al., 2019) . This model features a human lung epithelium (HLE) 73 monolayer differentiated at air-liquid interface (ALI) that enables luminal infection with SARS- CoV-2 and subsequent infiltration by primary human leukocytes. Mimicking the early, epithelium-restricted, stage of SARS-CoV-2 infection in humans 76 (Ravindra et al., 2021) , HLE-ALI cells infected with SARS-CoV-2 in vitro did not upregulate 77 canonical antiviral pathways nor express inflammatory cytokines. Next, we observed by The chemoattractants, CCL2 (250 pg/mL) and LTB4 (100 nM), were used in all conditions. To understand the impact of monocyte transmigration on the epithelium, we identified 180 induce proinflammatory signaling, which is attenuated by baricitinib treatment. To investigate the expression profile of lung-infiltrating monocytes during SARS-CoV-2 182 infection (compared to uninfected conditions), a set of 67 genes was measured by multiplexed 183 qRT-PCR and grouped by unsupervised clustering ( Figure 3B ). Nucleic acid signaling sensor 184 (CGAS and STING1, DDX58, OAS1/2/3), kinase (TBK1), effector (RNAse L), and transcription 185 factor (IRF3, IRF7) genes as well as critical neutrophil chemoattractant genes such as CXCL8 186 and IL-1 were upregulated in monocytes recruited across SARS-CoV-2-infected HLE-ALI cells. The same set of genes were extracted from the RNA-seq dataset of the HLE-ALI cells pre-and 188 post-transmigration. Although the post-transmigration HLE-ALI cells did not show global 189 enrichment of antiviral pathways by analysis of GO terms compared to the pre-transmigration 190 control, specific antiviral genes such as DDX58 and OAS1/2/3 and the pro-neutrophilic from the bronchoalveolar lavage fluid (BALF) of COVID-19 patients hospitalized with COVID-19 with severe rather than mild disease ( Figure 4A, Supplementary Figure 6 ). To our knowledge 198 this is the only dataset that has obtained scRNA-seq from BALF of hospitalized patients 199 spanning mild and severe COVID-19, pre-remdesivir. Another study, which analyzed BALF from 200 patients with severe COVID-19 found that there was expression of numerous inflammatory 201 cytokines in BALF cells from a variety of cell types, particularly neutrophils (Bost et al., 2021) . However, that study was conducted later during the course of the COVID-19 pandemic and did 203 not report on BALF from relatively milder COVID-19. Epithelial cells recovered in BALF of severe COVID-19 patients upregulated cell-death-205 associated pathways and downregulated genes associated with antigen presentation, both of 206 which may promote inflammation by innate immune cells (Figure 4B, Supplementary Table 7) . In addition to CXCL8 mentioned above, infiltrated monocytes present in the BALF of severe Next, we sought to confirm that changes in the expression of immune mediator genes 216 was reflected in protein levels present in the apical aspect of our small airway model. Monocyte Our re-analysis of publicly available scRNA-seq data from lung-infiltrating monocytes in 237 the BALF of patients with severe COVID-19 revealed that some of them harbored SARS-CoV-2 238 transcripts ( Figure 4A ). In severe COVID-19 patients included in another BALF study, infiltrating 239 monocytes also contained SARS-CoV-2, although the vast majority of viral transcripts were 240 found in the neutrophil compartment (Bost et al., 2021) . Similarly, lung-infiltrating monocytes in 241 our model, which were not directly infected but rather were made to cross an infected In order to gain a better understanding of how SARS-CoV-2 enters monocytes we 263 analyzed the expression of surface ACE2 and also included various treatments during 264 transmigration known to affect SARS-CoV-2 entry into cells. Blood monocytes were found to 265 express surface ACE2 as measured by flow cytometry, and ACE2 appeared to be slightly Since SARS-CoV-2 genome copies were detectable in lung-infiltrating monocytes, we 284 examined whether these genomes were replication-competent. To this end, we isolated and Finally, to better understand the inflammatory state of lung-infiltrating monocytes 308 harboring viral genome copies, we performed a multiplexed electrochemiluminescent assay to contribute not only to fueling the CRS, but also pave the way for neutrophil-driven ARDS. Furthermore, it is possible that replicative SARS-CoV-2 in airway monocytes serves as a 356 continual source of dsRNA-stimulation resulting in inflammatory cytokine release contributing to 357 symptomatology seen in so-called "long-haulers", i.e., patients who have relatively mild disease 358 weeks or even months after the initial infection. Indeed, it was previously observed that CD16 + 359 blood monocytes expressed SARS-CoV-2 S1 spike protein up to 15 months after initial SARS- The finding that monocytes harbor cell-associated SARS-CoV-2 copies has been observed by 362 others and poses a key therapeutic opportunity for early host-directed disease intervention. In 363 one study, blood monocytes were able to be infected by SARS-CoV-2 in an antibody-dependent . A third study described that blood monocytes and differentiated macrophages could be 368 infected by SARS-CoV-2, resulting in abortive viral products (Boumaza et al., 2021) . However, 369 these prior instances were in blood monocytes, not lung-infiltrating cells as used in our study, 370 which we previously showed to acquire a distinct transcriptional poise (Ford et al., 2021) . Recently, another group showed that tissue-resident alveolar macrophages take up and In conclusion, we showed that human lung epithelial cells differentiated at ALI propagate Beads and cells were incubated at room temperature for 15 min and the supernatant was chloroform (Sigma) was added to the TriPure and RNA was extracted using the standard 577 purification procedure in the manufacturer's protocol followed by a sodium acetate precipitation 578 to further clean the RNA. For isolation of RNA (and DNA) from monocytes, the tubes were 579 thawed and placed on a magnet to remove the beads from solution. The TriPure supernatant 580 was transferred to a clean tube and chloroform was added and spun following the 581 manufacturer's protocol. Due to the small amount of expected RNA yield, the aqueous phase 582 (containing the RNA) was mixed 1:1 with 100% ethanol (Sigma) and loaded onto an RNA clean 583 and concentrator-5 column (Zymo). RNA was isolated following the manufacturer's protocol. DNA was isolated from the organic phase following the manufacturer's protocol. Deciphering the state of immune silence in fatal 742 COVID-19 patients Monocytes and Macrophages Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 746 Dysregulation of Immune Response in Patients With Coronavirus COVID-19: Loss of bridging between innate and adaptive 859 immunity? Med Hypotheses 144 Single-cell longitudinal analysis 862 of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in 863 gene expression, and cell state changes Severe COVID-19 Is Marked 867 by a Dysregulated Myeloid Cell Compartment Inflammasome activation in infected macrophages