key: cord-0835502-zkt5nbro authors: Devadoss, D.; Acharya, A.; Manevski, M.; Pandey, K.; Borchert, G. M.; Nair, M.; Mirsaeidi, M. S.; Byrareddy, S. N.; Chand, H. S. title: Distinct Mucoinflammatory Phenotype and the Immunomodulatory Long Noncoding Transcripts Associated with SARS-CoV-2 Airway Infection date: 2021-05-18 journal: medRxiv : the preprint server for health sciences DOI: 10.1101/2021.05.13.21257152 sha: cfc3c35090e8ac306d786798fcc0854fc916a471 doc_id: 835502 cord_uid: zkt5nbro Respiratory epithelial cells are the primary target for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We investigated the 3D human airway tissue model to evaluate innate epithelial cell responses to SARS-CoV-2 infection. A SARS-CoV-2 clinical isolate productively infected the 3D-airway model with a time-dependent increase in viral load (VL) and concurrent upregulation of airway immunomodulatory factors (IL-6, ICAM-1, and SCGB1A1) and respiratory mucins (MUC5AC, MUC5B, MUC2, and MUC4), and differential modulation of select long noncoding RNAs (lncRNAs i.e., LASI, TOSL, NEAT1, and MALAT1). Next, we examined these immunomodulators in the COVID-19 patient nasopharyngeal swab samples collected from subjects with high- or low-VLs (~100-fold difference). As compared to low-VL, high-VL patients had prominent mucoinflammatory signature with elevated expression of IL-6, ICAM-1, SCGB1A1, SPDEF, MUC5AC, MUC5B, and MUC4. Interestingly, LASI, TOSL, and NEAT1 lncRNA expressions were also markedly elevated in high-VL patients with no change in MALAT1 expression. In addition, dual-staining of LASI and SARS-CoV-2 nucleocapsid N1 RNA showed predominantly nuclear/perinuclear localization at 24 hpi in 3D-airway model as well as in high-VL COVID-19 patient nasopharyngeal cells, which exhibited high MUC5AC immunopositivity. Collectively, these findings suggest SARS-CoV-2 induced lncRNAs may play a role in acute mucoinflammatory response observed in symptomatic COVID-19 patients. Initial host-viral interactions in the upper respiratory tract critically affect subsequent respiratory and systemic immune responses to other co-infections or environmental exposures. SARS-CoV-2 gains entry via the respiratory mucosa and any host factor dysregulations occurring during this interaction can result in pulmonary and/or extrapulmonary complications (1, 2) . Notably, the proteins necessary for viral entry are highly abundant in respiratory epithelial cells (RECs) (1, 3, 4) , rendering the respiratory tract highly susceptible to SARS-CoV-2 infection. SARS-CoV-2 is an enveloped β-coronavirus with a heavily glycosylated trimer spike (S) protein which facilitates viral attachment to ACE2 (angiotensin-converting enzyme 2) or CD147 (Basigin or BSG) (5, 6) . Following attachment, trimming of S protein by host cellular proteases such as TMPRSS2 (transmembrane serine protease 2) and furin mediates viral entry (7) (8) (9) . That said, the expression of these viral receptors is sensitive to various immunomodulators. For example, in addition to being the protective barrier and a competent airway lumen clearance mechanism, airway mucins normally moderate the mucosal immune responses (10) . During SARS-CoV-2 infection, however, induced inflammatory factors drive airway tissue remodeling and severe inflammation can cause mucus hyperexpression potentially leading to acute respiratory distress syndrome (ARDS) (11) . Managing this immune response constitutes of the major challenges faced in effectively treating COVID-19 for patients presenting with severe manifestations. Analyzing host-viral interactions is vital for understanding viral pathogenesis, and while several notable protein interactions necessary for SARS-CoV-2 infection and/or progression are now well documented, documented roles for specific host long noncoding RNAs (lncRNAs) during SARS-CoV-2 infection remain elusive. LncRNAs regulate virtually every cellular function including moderating the immune response of infected host cells and regulating the viral genome packing and replication (12, 13) . Although a few studies have reported on the expression profiling of lncRNAs in stratifying the COVID-19 disease severity and associated To model the acute respiratory infection conditions, a 3D airway tissue culture model of primary human respiratory tract epithelial cells (RTECs) was infected with SARS-CoV-2 primary isolate (USA-WI1/2020, BEI Cat # NR-52384) at 1 multiplicity of infection (MOI) (see Supplemental Methods for details). Viral RNA was assessed in apical washes, in the basal culture supernatant, and in cells; from RNA samples extracted at 0, 1, 4, 24, and 48 h post-infection (hpi). Viral RNA increased by 3.5-fold from 8.9x10 5 viral genomic equivalents per ml at 1 hpi to 3.1x10 6 at 24 hpi in apical washes ( Figure 1A) , there was no change in VL in basal culture supernatant which remained at ~3.0x10 5 genomic equivalents per ml at all the time-points ( Figure 1B) , and the level of vRNA in total cell RNA increased by ~6 fold at 24 hpi ( Figure 1C ). Thus, 3D airway cells productively infected with an actively replicating SARS-CoV-2 shed the virions almost exclusively from the apical surface. In addition, we found the expression of the viral entry host factor, ACE2, significantly reduced at all time points following infection (see Supplemental Figure 1A ). In contrast, the expression of S protein processing enzyme TMPRSS2 remained constant for first 24 hpi but was markedly suppressed at 48 hpi (Supplemental Figure 1B) . Thus, SARS-CoV-2 infection directs ACE2 and TMRPSS2 suppression in a RTEC 3D model, as reported earlier (8, 20) . We also found altered expressions of several inflammatory factors charged with directing airway tissue remodeling and/or controlling host immune responses and airway mucin expression (21, 22) . Among the inflammatory factors, although CXCL-8 expression was unaffected following infection (Supplemental Figure 1C ), interleukin-6 (IL-6) was significantly elevated (9-fold) at 1 hpi ( Figure 1D ), whereas intercellular adhesion molecule-1 (ICAM-1) was elevated (>5-fold) at 24 hpi ( Figure 1E ) and airway secretoglobulin 1A1 (SCGB1A1), also known as club cell All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 18, 2021. ; https://doi.org/10.1101/2021.05.13.21257152 doi: medRxiv preprint secretory protein (CCSP) was induced by 5-fold from 1 to 24 hpi with then returned to baseline levels at 48 hpi ( Figure 1F ). Notably, we also observed robust mucin hyperexpression in our 3D airway tissue model. SARS-CoV-2 infection robustly upregulated secretory mucin MUC5AC (21, (23) (24) (25) (26) transcription by more than 50-fold by 1 hpi and maintained hyperexpression at all the time-points ( Figure 1G ). In addition, expression of the constitutive epithelial mucin, MUC5B, involved in the airway antimicrobial response (27) (28) (29) was increased by 48 hpi ( Figure 1H ), and similar to MUC5AC, cell surface-associated mucins, MUC2 ( Figure 1I ), and MUC4 ( Figure 1J ) levels were also increased by 1 hpi then remain elevated at all time-points. Furthermore, known transcriptional regulators of mucin expression (30, 31) were similarly upregulated following infection with >2fold induction of SPDEF levels ( Figure 1K ) and >20-fold induction of FOXA3 levels at 1 hpi ( Figure 1L ). Next, protein expressions were assessed in transwell cells by immunostaining. In agreement with our quantitative RT-PCR analyses, immunostaining confirmed robust immunopositivity for MUC5AC expression ( Figure 1M ) with ~40% MUCAC+ cells at 48 hpi compared to <5% cells at 0 hpi ( Figure 1N ). Levels of secreted inflammatory factors, IL-6 ( Figure 1O ) and ICAM-1 ( Figure 1P ) in basal media supernatants were also elevated at 24 and 48 hpi. Thus, the inflammatory and mucous responses of respiratory epithelial cells are acutely moderated by SARS-CoV-2 infection. Although roles for airway mucins in innate antiviral immunity are well documented, SARS-CoV-2 associated mucin signatures have not been thoroughly evaluated to date (32) . Specifically, these mucins are heavily glycosylated and could result in higher viral binding/retention due to interaction with sialic acids (33) and contribute to viral infection and/or transmission. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 18, 2021. ; In addition to mucoinflammatory factor encoding mRNAs, another type of RNA, lncRNAs, have now been found to play vital roles in regulating the innate immune responses (12, 13) and recent studies have shown that specific lncRNAs are differentially expressed during hostpathogen interactions following viral infection (18, 19, 34, 35) . That said, our group identified two novel epithelial lncRNAs, LASI (lncRNA on antisense strand to ICAM-1) and TOSL (TNFAIP3opposite strand lncRNA) that are differentially expressed during the mucus hypersecretory response (36) and blocking these lncRNA expression alleviates the mucin hyperexpression. Notably, we find LASI transcripts are highly elevated (10-fold) at 1 hpi (Figure 2A) , whereas TOSL expression is increased by 1 hpi and were >6-fold higher at 48 hpi ( Figure 2B ). However, in contrast to recent clinical reports (19, 37, 38) , we find NEAT1 (Nuclear Enriched Abundant Therefore, our data suggest that these lncRNAs may be serve as biomarkers for measuring SARS-CoV-2 disease progression. However, these need to be confirmed using large number of clinical samples. Large cohort studies revealed there was no difference in SARS-CoV-2 viral load between symptomatic and asymptomatic patients, whereas disease severity and mortality in symptomatic All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 18, 2021. ; https://doi.org/10.1101/2021.05.13.21257152 doi: medRxiv preprint patients is directly correlated with viral load (38, 41, 42) . The mucosal immune response of upper respiratory tract plays a critical role in viral shedding and replication. To explore this correlation, in our study, we divided subjects into 2 groups based on the qRT-PCR cycle threshold (CT) of SARS-CoV-2 N1 nucleocapsid, with a CT>30 considered a low viral load (Lo-VL) and a CT<30 high viral load (Hi-VL). As detailed in Supplemental Table 1 , nasopharyngeal swabs collected from a total of 20 subjects (10 Hi-VL and 10 Lo-VL) were examined. Twelve symptomatic subjects were hospitalized and had at least one and/or combination of comorbidities viz., obesity, chronic disorders, hypertension, and diabetes. Hi-VL subjects were found to contain >100-fold higher vRNA levels than Lo-VL subjects ( Figure 3A) . Interestingly, no significant differences were observed in ACE2 ( Figure 3B ) and TMPRSS2 ( Figure 3C ) mRNA levels between groups. In contrast, Hi-VL samples exhibited ~1.5-fold higher IL-6 ( Figure 3D) and ICAM-1 ( Figure 3E ) inflammatory factors mRNA expressions, as well as 4-fold higher SCGB1A1 expression ( Figure 3F ). Hi-VL patient samples also exhibited robust secretory mucin levels, with ~10-fold higher MUC5AC ( Figure 3G ) and 4-fold higher MUC5B ( Figure 3H) expressions. Notably, there was a trend towards reduced MUC2 expression ( Figure 3I ) in Hi-VL subjects with ~10-fold higher MUC4 ( Figure 3J ) expression compared to Lo-VL subjects. Expression of SPDEF was also increased by ~5-fold in Hi-VL subjects ( Figure 3K ), but in contrast to our 3D models, we failed to detect any FOXA3 transcripts in patient nasopharyngeal swab samples. These data clearly suggests that SARS-CoV-2 can regulate the expression of several cellular receptors by inducing inflammatory responses. While we found no difference in MALAT1 lncRNA levels between Hi-VL and Lo-VL subjects (Supplemental Figure 2A (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. showed enriched SARS-CoV-2 nucleocapsid vRNA and LASI transcripts in nuclear and cytosolic regions ( Figure 4D ). The H-score quantitation confirmed vRNA and LASI transcripts were 1.5-fold higher in Hi-VL samples compared to Lo-VL samples ( Figure 4F ), corroborating our qRT-PCR data. Nasal epithelial cell mucin protein expression was also analyzed for the mucin protein expression by immunostaining for MUC5AC ( Figure 4E ) and MUC5B (Supplemental Figure 2B) Figure 3B ). In terms of lncRNAs, MALAT1 trended lower in COVID-19 patients, LASI lncRNA expression trended higher and was significantly elevated in at least one COVID-19 patient, and there was no observed difference in NEAT1 lncRNA levels between COVID-19 patients and healthy controls (Supplemental Figure 3C ). That said, these data do further corroborate the association of mucoinflammatory response and lncRNAs with COVID-19 pathophysiology observed in our 3D models and clearly warrant further analyses of larger data sets and the underlying molecular mechanisms. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In conclusion, the present study employing both a 3D airway tissue model of SARS-CoV-2 infection and COVID-19 patient samples provides the first experimental association of SARS-CoV-2 viral load with mucoinflammatory phenotype and specific airway mucosa lncRNAs despite limited cohort sample size, possible variation in swab sampling, and/or the cell numbers per sample potentially contributing to data incongruity. Importantly, we have recently shown that blocking lncRNA LASI can help suppress mucus hyperexpression (34) and hence, could be a feasible strategy to regulate COVID-19 associated mucoinflammatory response. As initial hostviral interactions in the upper respiratory tract are crucial for modulating subsequent respiratory and systemic immune responses to SARS-CoV-2 and other co-infections and environmental exposures; targeting these lncRNAs could potentially rescue the debilitating outcomes of the COVID-19 sequelae and help improve current disease management strategies. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. Respiratory epithelial cells, SARS-CoV-2, COVID-19, long noncoding RNAs, mucoinflammatory response All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 18, 2021. ; LASI transcripts per cell at 4 and 24 hpi (n=9-10 cells/gp; *p<0.05; **p<0.01; by Student's ttest). 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