key: cord-1030929-rirbffi6
authors: Shuai, Huiping; Chu, Hin; Hou, Yuxin; Yang, Dong; Wang, Yixin; Hu, Bingjie; Huang, Xiner; Zhang, Xi; Chai, Yue; Cai, Jian-Piao; Chan, Jasper Fuk-Woo; Yuen, Kwok-Yung
title: Differential immune activation profile of SARS-CoV-2 and SARS-CoV infection in human lung and intestinal cells: implications for treatment with IFN-β and IFN inducer
date: 2020-07-21
journal: J Infect
DOI: 10.1016/j.jinf.2020.07.016
sha: 4d42c280310dcbe03d8c4059ba31a6d0461233e1
doc_id: 1030929
cord_uid: rirbffi6

OBJECTIVES: Respiratory and intestinal tract were two primary target organs of SARS-CoV-2 infection. However, detailed characterization of the host-virus interplay in infected human lung and intestinal epithelial cells is lacking. METHODS: We utilized immunofluorescence assays, flow cytometry, and RT-qPCR to delineate the virological features and the innate immune response of the host cells against SARS-CoV-2 infection in two prototype human cell lines representing the human pulmonary (Calu3) and intestinal (Caco2) epithelium when compared with SARS-CoV. RESULTS: Lung epithelial cells were significantly more susceptible to SARS-CoV-2 compared to SARS-CoV. However, SARS-CoV-2 infection induced an attenuated pro-inflammatory cytokines/chemokines induction and type I and type II IFN responses. A single dose of 10 U/mL IFN-β (IFNβ) pretreatment potently protected both Calu3 and Caco2 against SARS-CoV-2 infection. Interestingly, SARS-CoV-2 was more sensitive to the pretreatment with IFNβ and IFN inducer than SARS-CoV in Calu3. CONCLUSIONS: Despite robust infection efficiency in both the human lung and intestinal epithelial cells, SARS-CoV-2 could attenuate the virus-induced pro-inflammatory response and IFN response. Pre-activation of the type I IFN signaling pathway primed a highly efficient antiviral response in the host against SARS-CoV-2 infection, which could serve as a potential therapeutic and prophylactic maneuver to COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported from Wuhan in December 2019 (1, 2) . It spread rapidly and has caused over 3.5 million laboratory-confirmed cases with over 0.25 million deaths globally in less than 6 months. SARS-CoV-2 is the seventh human coronaviruses and belongs to Betacoronavirus, the same genus as the other two highly pathogenic human coronaviruses, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) (3, 4) . A prominent feature of SARS-CoV-2 is its unusually high transmissibility among close contacts in family, nursing facility, cruise ship, and also the hospital setting (2, (5) (6) (7) (8) . The estimated basic reproductive number (R 0 ) of SARS-CoV-2 ranges from 1.4 to 6.49 (median 2.79) (9) , which is higher than that of SARS-CoV (10) .

Clinical symptoms of COVID-19 generally resemble those observed in SARS, ranging from relatively mild common cold-like illness such as cough, fever, myalgia and fatigue, to severe dyspnea, chest pain, multifocal peripheral ground-glass pneumonia, acute respiratory distress syndrome (ARDS), and multiorgan dysfunction syndrome (11, 12) . Unlike SARS-CoV, a significant proportion of COVID-19 cases were mildly symptomatic or asymptomatic (5, 13) .

Moreover, prolonged viral shedding in high titer was detected in the nasopharyngeal and oropharyngeal swabs from the asymptomatic individuals (6) , which might have facilitated the efficient transmission of SARS-CoV-2 in immune naïve population worldwide.

Apart from infection in the airway mucosa and the pulmonary alveoli, the gastrointestinal tract is also susceptible to highly pathogenic human coronaviruses, including SARS-CoV and MERS-CoV (14, 15) . Besides, viral RNA could also be detected in the intestinal tissues of SARS-CoV-2-infected hamsters (16) . Interestingly, SARS-CoV-2 appeared to replicate more efficiently in the human bronchus (17) and lung (18) than that of SARS-CoV. In contrast, while diarrhoea was the most common extrapulmonary clinical manifestation of SARS and was reported in up to 130 (20%) of 647 SARS patients (19) , it was uncommon in COVID-19 (42/1099, 3.8%) (12) , suggesting less enteric involvement of SARS-CoV-2 than SARS-CoV.

Despite these differential virological and clinical observations between SARS and COVID-19, the underlying mechanisms of these differences remain largely unexplored. We previously reported that Calu3 and Caco2 both supported robust replication of SARS-CoV-2 among nine human cell lines tested (20) . In this study, we further investigated the efficacy of infection, innate immune, and inflammatory response induced by SARS-CoV-2 in Calu3 and Caco2 as prototype cell lines that mimic virus infection in the human lung and the intestinal tract, respectively.

Using SARS-CoV as control, our results revealed substantial differences in the infection and host innate immune response between these two highly pathogenic human coronaviruses, thus providing scientific basis for the therapeutic or prophylactic treatment for COVID-19 patients.

SARS-CoV-2 HKU-001a (GenBank accession number: MT230940) was isolated from the nasopharyngeal aspirate specimen of a clinical patient with laboratory-confirmed COVID-19 as we previously described (20) . SARS-CoV GZ50 (GenBank accession number: AY304495) was a 2003 isolate archived in the Department of Microbiology at the University of Hong Kong. Both strains were propagated in VeroE6 cells. Viral titers from supernatants were assessed in VeroE6 with plaque assays. Experiments involving live viruses were performed in accredited biosafety level-3 (BSL-3) laboratory. VeroE6 and and Caco2 (ATCC) were cultured with the minimal essential medium (MEM) supplemented with 1% P/S, and 10% or 20% fetal bovine serum (FBS), respectively. Calu3 was cultured with Dulbecco's minimal essential medium F12 (DMEM/F12) supplemented with 20% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S). At the point of virus inoculation, FBS in the culture medium was removed. All cell lines used in this study was tested negative for mycoplasma contamination.

Infected cells and supernatants were lysed at 2, 24, 48, 72 and 120 hours post-infection (hpi) and extracted with QIAamp viral RNA mini kit (QIAGEN) or QIARneasy mini kit (QIAGEN) according to manufacturer's instructions. For virus replication kinetics assays, the extracted RNA was quantified with the one-step QuantiNova Probe RT-PCR kit (QIAGEN). For profiling the host genes, the extracted RNA was reverse transcribed with Transcriptor First Strand cDNA Synthesis Kit (Roche). Gene expression was quantified by qPCR with LightCycler 480 SYBR Green I Master (Roche) and normalized to mock-infected controls or GAPDH gene expression as we previously described (21) (22) (23) . Primer sequences used in this study were listed in supplementary table 1.

To determine the infectivity of SARS-CoV-2 and SARS-CoV, Calu3 and Caco2 cells were infected with SARS-CoV-2 or SARS-CoV at 0.002, 0.02, 0.2, 2 and 20 MOI. Cells were fixed with 10% neutral-buffered formalin solution at 24 hpi. for immunofluorescence staining to visualize viral antigen expression.

Cells were fixed with 10% neutral-buffered formalin solution at the designated time points. Viral antigen expression was detected with an in-house polyclonal rabbit antiserum against the nucleocapsid protein of SARS-CoV but also cross-react with that of the SARS-CoV-2 (20) . The Alexa Fluor 488 goat anti-rabbit antibody was obtained from ThermoFisher Scientific. Nuclei were stained with Prolong antifade mountant with DAPI (ThermoFisher Scientific). Images were obtained with Olympus BX53 fluorescence microscope. Relative fluorescence units of the fluorescence images were quantified with ImageJ.

To quantitatively compare the amount of antigen expression in cells infected by SARS-CoV-2 and SARS-CoV, flow cytometry was performed with Calu3 and Caco2 infected at 0.1, 1 and 10 MOI as previously described with slight modifications (24, 25) . Briefly, cells were detached with EDTA-trypsin, fixed with neutral-buffered 10% formalin solution, permeabilized with 0.1%

Triton-X-100 in PBS, and nucleocapsid protein was immunolabeled for flow cytometry analysis with a LSR Fortessa analyzer (BD). Data obtained were analyzed with FlowJo 7.6 Alias software (FlowJo LLC.).

To investigate the sensitivity of SARS-CoV-2 to IFN and IFN inducer, Calu3 and Caco2 cells were pretreated with 0, 10, 50, 100, 500, or 1000 U/mL IFN-α (R&D Systems), -β (Abcam) or -γ (Abcam) and polyinosinic-polycytidylic acid (poly(I:C)) (Invivogen) for 18 h prior to infection.

After pretreatment, cells were inoculated with SARS-CoV-2 at a MOI of 2 and were fixed with 10% neutral-buffered formalin solution at 24 hpi for immunofluorescence staining. Cell lysates and supernatants were harvested at 24 and 48 hpi for viral load quantification with one-step RT-qPCR. Mock-infected cell lysates were harvested for RT-qPCR analysis at the point of infection to determine the expression levels of ISGs and SARS-CoV-2 entry-related host factors.

All data obtained were analyzed with GraphPad Prism 7.0 software. Statistical analyses for two groups were determined using unpaired Student's t-test. Statistical analyses among three or more groups were computed with one-way or two-way ANOVA. Two tailored-p value < 0.05 was considered statistically significant.

To explore the differential tropism of SARS-CoV-2 and SARS-CoV, we compared the virus susceptibility in Calu3 and Caco2 using MOIs ranging from 0.002 to 20. The efficacy of infection was visualized by the detection of viral nucleocapsid (N) protein upon immunofluorescence staining. Supporting our findings on the replication kinetics profile (Fig.   S1 ), the infectivity of SARS-CoV-2 in Calu3 and Caco2 were similarly efficient, as indicated by the comparable expression level of viral nucleocapsid protein at 0.02 MOI or above (Fig. 1 , left panels). However, for SARS-CoV infection, drastic differences in infection efficiency were observed between the two cell lines. Particularly, SARS-CoV is substantially less capable of infecting Calu3, which was indicated by the low abundance of virus antigen detected at 2 MOI and was further reduced to almost non-detectable level at 0.2 MOIs or below (Fig. 1, right   panels) . Notably, Calu3 infected with 20 MOI of SARS-CoV has a similar nucleocapsid protein expression level as those infected with only 0.2 MOI of SARS-CoV-2, thus amounting to approximately 100-fold difference in protein expression between the two viruses in this cell type ( Fig. 1 and Fig. S2 ). Since ACE2 and TMPRSS2 were reported to be essential for SARS-CoV-2 entry (26), we profiled the expression of these two genes in Calu3 and Caco2. In general, the expression of ACE2 in Calu3 was around 1-to-4 folds higher than Caco2 while the expression of TMPRSS2 was approximately 4-to-64 folds more abundant in Caco2 than Calu3 (Fig. S3 ). Next, we further quantitatively investigated the differential infection of SARS-CoV-2 and SARS-CoV Overall, our data suggested that SARS-CoV-2 infected human lung epithelial cells more efficiently than SARS-CoV, while SARS-CoV infected human intestinal cells more efficiently than that of SARS-CoV-2.

Next, we asked whether SARS-CoV-2 and SARS-CoV would activate the IFN and proinflammatory response in Calu3 and Caco2 cells. To this end, we evaluated the expression of five pro-inflammatory cytokines/chemokines and a panel of eight genes associated with type I or type II IFN response. To our surprise, despite robust SARS-CoV-2 replication in Caco2, only IP-10 was significantly upregulated among the five cytokines/chemokines tested (Fig. 3B,   p<0 .01). Besides, SARS-CoV also upregulated only IP-10 to a similar magnitude as that induced by SARS-CoV-2 in Caco2 (Fig. 3B, p<0.01) and IFsITM3 (Fig. 4A) . On the contrary, both type I and type II IFN response were attenuated upon SARS-CoV-2 infection in Calu3 (Fig. 4A ). In summary, our results demonstrated that the virus-induced innate immune response was largely diminished in the intestinal epithelial cells upon the infection of both SARS-CoV-2 and SARS-CoV. However, substantial differences were observed between the two cell lines. Importantly, SARS-CoV-2 induced a significantly attenuated innate immune response in pulmonary epithelial cells compared to SARS-CoV despite more robust virus infection and propagation.

Since type I IFN response, which was reported to be critical for host defense against various viral infections (27-29), were not significantly activated by SARS-CoV-2, we hypothesized that preactivation of IFN signaling pathway to prime the antiviral response of the host might be highly beneficial to restricting SARS-CoV-2 infection. We tested this hypothesis by pretreating Calu3 and Caco2 cell lines with IFNβ (type I IFN) at increasing concentrations of 0, 100, 500 and 1000 U/mL. We first verified the potency of IFNβ by examining the expression levels of five representative IFN-stimulated genes (ISGs). Expectedly, all five ISGs were efficiently upregulated by IFNβ pretreatment (Fig. S4) . Next, we investigated the antiviral potency of IFNβ and found that a single dose of 100 U/mL IFNβ pretreatment prior to SARS-CoV-2 infection reduced around 70% of virus production in Calu3 with the inhibitory effect lasted at least until 48 hpi ( Fig. 5A and 5B ). Under the same IFNβ pretreatment, we observed a less than 25% reduction in virus production in Calu3 infected with SARS-CoV ( Fig. 5A and 5B ). Consistently, IFNβ pretreatment was more potent against SARS-CoV-2 than SARS-CoV in Caco2 ( Fig. 5C and 5D). We further reduced the dosage of IFNβ pretreatment to more physiologically feasible levels and the results showed that the IFNβ pretreatment effectively suppressed the expression of viral antigen at a concentration as low as 10 U/mL (Fig. 5E) . ACE2 was recently identified as a potential ISG (30) , which might compromise the effectiveness of IFNβ treatment. By evaluating the expression of ACE2 upon IFNβ treatment, our data showed that IFNβ treatment did not upregulate ACE2 expression in Calu3 and Caco2 cells (Fig. 5F , 5G and S5). As an alternative strategy to IFNβ pretreatment, we pretreated cells with poly(I:C), a stimulator of the host innate immune response via activating the cellular pattern recognition receptors. In this setting, SARS-CoV-2 was similarly sensitive to the pre-induced antiviral immune response ( Figure 5H-5K) .

Interestingly, SARS-CoV-2 also demonstrated a higher sensitivity to poly(I:C) pretreatment than that of SARS-CoV in Calu3 cells ( Figure 5H and 5I) . These results highlighted the potential of activating the IFN signaling pathway as an antiviral strategy against SARS-CoV-2.

Despite the differential tissue-specific virological and clinical observations between SARS and COVID-19, the underlying mechanisms of the differences remain largely unexplored. Using Type I IFN response is known to be important for restricting virus replication before adequate adaptive immunity is mounted (36) . To facilitate efficient virus propagation, coronaviruses encode multiple structural and nonstructural viral proteins (37) (38) (39) to antagonize the IFN signaling pathway upon infection. Our data showed that SARS-CoV-2 launched an attenuated innate immune response despite efficient virus replication, suggesting the virus can also effectively modulate IFN signaling. Early administration of type I IFN was associated with efficient virus clearance and alleviated disease severity in SARS-CoV-infected mice and SARS patients (40) (41) (42) . Therefore, we investigated the therapeutic potential of type I IFN by priming the host with IFNβ pretreatment and poly(I:C) as an upstream stimulator. Our results demonstrated that compared to SARS-CoV, SARS-CoV-2 was more sensitive to IFNβ and poly(I:C) pretreatment, indicating treatment with IFNβ in COVID-19 patients could be more effective and beneficial than that in SARS patients. Importantly, several lines of evidence suggested the potential use of IFNβ as a treatment strategy for COVID-19 infection. First, treatment with IFNβ or its inducer may jump-start the host immune system as the innate immune response by IFN and proinflammatory cytokines are markedly suppressed by SARS-CoV-2 in both model cell lines and in ex vivo human lung tissue explants (18) . Second, IFNβ did not increase the expression of SARS-CoV-2 receptor ACE2 in our lung and intestinal model cell lines, which was recently suggested to be an IFN-stimulated gene (30) . Third, IFNβ was shown to decrease virus-induced lung fibrosis in a mouse model, which might improve outcomes of patients with COVID-19 complicated by acute respiratory distress syndrome (43) . Forth, synergistic effects were reported for leukocytic IFNα with ribavirin, and IFNβ with ribavirin (44) .

Finally, IFNβ exhibited potent in vitro and/or in vivo antiviral activity against SARS-CoV and MERS-CoV (45, 46) . These findings formed the basis of a recent randomized clinical treatment trial which showed that the triple combination of antiviral therapy with IFNβ-1b, lopinavirritonavir, and ribavirin is safe and highly effective in shortening the duration of virus shedding, decreasing cytokine responses, alleviating symptoms, and facilitating the discharge of patients with mild to moderate COVID-19 (47) . 

A Novel Coronavirus from Patients with Pneumonia in China

A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease

Asymptomatic and Presymptomatic SARS-CoV-2 Infections in Residents of a Long-Term Care Skilled Nursing Facility -King County

Estimating the infection and case fatality ratio for coronavirus disease (COVID-19) using age-adjusted data from the outbreak on the Diamond Princess cruise ship

Epidemiology of Covid-19 in a Long-Term Care Facility

Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan

The reproductive number of COVID-19 is higher compared to SARS coronavirus

Model parameters and outbreak control for SARS

Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Clinical Characteristics of Coronavirus Disease 2019 in China

First Mildly Ill, Non-Hospitalized Case of Coronavirus Disease 2019 (COVID-19) Without Viral Transmission in the United States

Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways

Human intestinal tract serves as an alternative infection route for Middle East respiratory syndrome coronavirus

Simulation of the clinical and pathological manifestations of Coronavirus Disease 2019 (COVID-19) in golden Syrian hamster model: implications for disease pathogenesis and transmissibility

Tropism of the Novel Coronavirus SARS-CoV-2 in Human Respiratory Tract: An Analysis in Ex Vivo and In Vitro Cultures. The Lancet Respiratory Medicine

Comparative replication and immune activation profiles of SARS-CoV-2 and SARS-CoV in human lungs: an ex vivo study with implications for the pathogenesis of COVID-19

Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection

Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 with impliccations for clinical manifestaations, transmimssibility, and laborattory studies of COVID-19: an observational study. The Lancet Microbe

Targeting the inositol-requiring enzyme-1 pathway efficiently reverts Zika virus-induced neurogenesis and spermatogenesis marker perturbations

Middle East respiratory syndrome coronavirus and bat coronavirus HKU9 both can utilize GRP78 for attachment onto host cells

Improved Molecular Diagnosis of COVID-19 by the Novel, Highly Sensitive and Specific COVID-19-RdRp/Hel Real-Time Reverse Transcription-PCR Assay Validated In Vitro and with Clinical Specimens

Middle East Respiratory Syndrome Coronavirus Efficiently Infects Human Primary T Lymphocytes and Activates the Extrinsic and Intrinsic Apoptosis Pathways

Productive replication of Middle East respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response

Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The host type I IFN response to viral and bacterial infections

Decoding type I and III IFN signalling during viral infection

Type I IFNs in viral control and immune regulation

SARS-CoV-2 receptor ACE2 is an IFN-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues

Virological assessment of hospitalized patients with COVID-2019

Severe acute respiratory syndrome coronavirus evades antiviral signaling: role of nsp1 and rational design of an attenuated strain

PARP1 Enhances Influenza A Virus Propagation by Facilitating Degradation of Host Type I IFN Receptor

HIV-1 evades innate immune recognition through specific cofactor recruitment

Links between innate and adaptive immunity via type I IFN

The Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Inhibits Type I IFN Production by Interfering with TRIM25-Mediated RIG-I Ubiquitination

Middle East respiratory syndrome coronavirus M protein suppresses type I IFN expression through the inhibition of TBK1-dependent phosphorylation of IRF3

Severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type I IFN, in infected cells

Dysregulated Type I IFN and Inflammatory Monocyte-Macrophage Responses Cause Lethal Pneumonia in SARS-CoV-Infected Mice

IFN alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study

Coronaviruses -drug discovery and therapeutic options

TLR9-induced IFN beta is associated with protection from gammaherpesvirus-induced exacerbation of lung fibrosis

SARS: systematic review of treatment effects

Broad-spectrum antivirals for the emerging Middle East respiratory syndrome coronavirus

Treatment With Lopinavir/Ritonavir or IFN-beta1b Improves Outcome of MERS-CoV Infection in a Nonhuman Primate Model of Common Marmoset

Triple combination of IFN beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. The Lancet

We thank the staff at the Imaging and Flow Cytometry Core of CPOS of the Li Ka Shing Faculty of Medicine, The University of Hong Kong, for facilitating this study.

This study was partly supported by the donations of May Tam