key: cord-0986599-rya21i2b
authors: Jeon, Yung Jin; Gil, Chan Hee; Jo, Ara; Won, Jina; Kim, Sujin; Kim, Hyun Jik
title: The influence of interferon-lambda on restricting Middle East Respiratory Syndrome Coronavirus replication in the respiratory epithelium
date: 2020-06-19
journal: Antiviral Res
DOI: 10.1016/j.antiviral.2020.104860
sha: 5571abe1d0735ceae3edf074d056f62b84b31075
doc_id: 986599
cord_uid: rya21i2b

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes severe respiratory in human with high mortality and it has been a challenge to determine optimum treatment for MERS-CoV-induced respiratory infection. Here, we observed the distribution of MERS-CoV receptors using human respiratory mucosa and also evaluated the contribution of interferon-lambdas (IFN-λs) in response to MERS-CoV infection using in vitro normal human nasal epithelial (NHNE) and bronchial epithelial (NHBE) cells. We found that the gene and protein expression of DPPIV, MERS-CoV receptor, were more dominantly located in nasal and bronchial epithelium although human nasal mucosa exhibited relatively lower DPPIV expression than lung parenchymal tissues. The quantitative mRNA level of the MERS-CoV envelope (upE) gene was significantly induced in MERS-CoV-infected cultured NHNE and NHBE cells until 3 days after infection. The induction of IFNs was identified in in NHNE and NHBE cells after MERS-CoV infection and IFN-λs were predominantly increased in MERS-CoV-infected respiratory epithelial cells. Inoculation of IFN-λs to NHNE and NHBE cells suppressed MERS-CoV replication and in particular, IFN-λ(4) showed a strong therapeutic effect in reducing MERS-CoV infection with higher induction of IFN-stimulated genes. Thus, IFN-λ has a decisive function in the respiratory epithelium that greatly limits MERS-CoV replication, and may be a key cytokine for better therapeutic outcomes against MERS-CoV infection in respiratory tract.

has since attracted increasing international interest in its epidemiology, clinical features, and options for therapy (Zaki et al., 2012) . Epidemiologic studies have established that human zoonotic transmission is suspected, with evidence of spread from dromedary camels as a MESR-CoV residue (Haagmans et al., 2014; Sabir et al., 2016) . The virus has continued to cause severe zoonotic human disease in the Middle East, sometimes associated with outbreaks of human-to-human transmission.

Accordingly, there is an urgent need to advance the development of therapeutic agents or a vaccine to treat and prevent MERS-CoV infection (Hotez et al., 2014) .

It has been suggested that MERS-CoV replicates in the human upper and lower respiratory tract after invasion through its specific receptors. Dipeptidyl Peptidase IV (DPPIV), a type Ⅱ transmembrane ectopeptidase, is a well-known receptor of MERS-CoV that plays a critical role in entry and infection of target cells (Lu et al., 2013; Raj et al., 2013) . DPPIV is mainly expressed on the apical surfaces of epithelial and acinar cells, and on capillary endothelial cells of various organs. A positive correlation between higher susceptibility to MERS-CoV infection and prominent DPPIV expression has been demonstrated (van Doremalen et al., 2014) .

Therefore, the evaluation of DPPIV distribution in tissues would help identify target tissues for effective suppression of MERS-CoV infection in the respiratory tract. The innate immune system of the respiratory epithelium serves as the first line of defense against respiratory viruses by producing interferon (IFN), a group of key molecules in the antiviral response . Emerging evidence has indicated that, among the IFN family of cytokines, IFN-λs such as IFN-λ 1, -λ 2 , -λ 3 and -λ 4 are critical immune modulators against viral infection in the epithelial mucosa, and rapid immune response to respiratory viruses occurs through activation of IFN-λ (Galani et al., 2017; Kim et al., 2019) . Based on our previous data, IFN-λ is believed to be primarily responsible for protection against viral invaders in the respiratory tract and to play an important role in local antiviral innate immunity . However, our knowledge of the contribution of IFN-λs to coronavirus clearance from the respiratory tract is limited and our understanding of the modulators involved in IFN-λ production, especially within the context of MERS-CoV infections in the upper and lower airway epithelium, remains lacking. Here, we assessed the distribution of DPPIV in human nasal mucosa and lung tissue to predict the infection route of MERS-CoV in the respiratory tract. In addition, we found that induction of IFN-λ signaling might be more dominant in MERS-CoV-infected respiratory epithelial cells. Administration of recombinant IFN-λs, especially IFN-λ 4 , resulted in a more significant suppressive effect on MERS-CoV replication as well as the induction of IFN-stimulated genes (ISG).

Using real-time PCR and Western blot analysis, mRNA and protein expression of DPPIV was studied in human palatine tonsillar tissues, nasal mucosal tissues (N=8) and lung parenchymal tissues (N=4) of subjects, respectively. mRNA expression of DPPIV in lung parenchymal tissue was significantly higher than DPPIV expression in nasal mucosa (Fig. 1a) . Moreover, protein expression of DPPIV was also relatively higher in lung parenchyma than nasal mucosa (Fig. 1b) . Immunohistochemistry to detect DPPIV was performed on human nasal mucosal tissues and lung parenchymal tissues. In the nasal mucosa, the positive staining of DPPIV protein was dominantly detected in human nasal epithelial cells, particularly ciliated cells, but DPPIV staining was limited to goblet cells of the nasal epithelium (Fig. 1c ). In addition, some staining of DPPIV protein was observed in submucosal glands in the nasal mucosa (Fig. 1c) . DPPIV immunostaining was detected in the multifocal cells lining the terminal bronchial epithelium in human lung parenchyma (Fig. 1d) . Overall, DPPIV expression was higher in human lung tissue than nasal mucosa and was more dominant in nasal or bronchial epithelial cells in the human respiratory tract. Therefore, respiratory epithelial cells might be the main target for MERS-CoV infection and transmission in the human respiratory tract.

Normal nasal mucosa was obtained from four healthy volunteers who underwent septal surgery, and human lung tissue was also extracted from subjects who underwent pneumonectomy for unilateral lung cancer. Normal human nasal epithelial The upE mRNA level was more completely reduced in MERS-CoV-infected NHNE cells treated with IFN-λ 4 (5.2×10 6 ) compared to cells treated with IFN-λ 1/2 (3.8×10 9 ).

In addition, mRNA of IFN-stimulated genes (ISGs) such as CXCL10, IFIT1, Mx1, and 

This study was approved by the institutional review board ( 

Formalin-fixed and paraffin-embedded tissues (FFPE) were sectioned at 4㎛thickness and immunohistochemical staining were performed. Briefly, after deparaffinization, the sections were incubated with 3% hydrogen peroxidase to inhibit endogenous peroxidases. Heat-induced epitope retrieval was then performed by microwaving samples in a 10 mmol/l citrate buffer (pH 6.0). nonspecific staining was prevented by treating tissue sections with rabbit serum (2.5% in PBS) for 30 min.

The sections were incubated overnight at 4℃ in a primary antibody. probe, 5'-CTCTTCACATAATCGCCCCGAGCTCG-3'). Primer for human DPPⅣ was purchased from Applied Biosystems (Foster City, CA, USA).

Real-time PCR was performed using the PE Biosystems ABI PRISM® 7700

Sequence Detection System. Thermocyling parameters were as follows: 50°C for 2 min, 95°C for 10 min, and then 40 cycles of 95°C for 15 s and 60°C for 1 min.

Target mRNA levels were quantified using target-specific primer and probe sets for MERS-CoV. All PCR assays were quantitative and utilized plasmids containing the target gene sequences as standards. All reactions were performed in triplicate, and all real-time PCR data were normalized to the level of the housekeeping gene glyceraldehyde phosphate dehydrogenase (1 × 10 6 copies) to correct for variation between samples.

Total protein lysates were harvested in RIPA lysis buffer (Thermo Fisher Scientific, Wilmington, DE, USA). The cell lysates (25μg per lane, as measured by a BCA protein assay purchased from Thermo Fisher Scientific) were electrophoresed in 10% SDS gels and transferred to polyvinylidene difluoride membranes in Trisbuffered saline (TBS; 50mM Tris-Cl, pH 7.5, and 150 mM NaCl) for one hour at room temperature. Each membrane was incubated overnight with a primary antibody with an anti-human DPPⅣ antibody (mouse monoclonal antibody, Clone OTI11D7, OriGene, USA) in Tween-Tris-buffered saline (TTBS; 0.5% Tween-20 in TBS) at 4°C.

After washing with TTBS, each blot was incubated for one hour at room temperature with a secondary anti-mouse antibody (Cell Signaling, Beverly, MA, USA) in TTBS.

Expression was detected using an enhanced chemiluminescence system (Amersham, Little Chalfont, UK).

Real-time PCR results are presented as median values (interquartile ranges for 25 and 75%). The statistical significance of differences between two groups was determined by the Mann-Whitney test. All statistical analysis was performed with GraphPad Prism software (version 5; GraphPad Software, La Jolla, CA, USA). Pvalues <0.05 were considered to be statistically significant.

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