key: cord-0756010-0ffshsat
authors: Yamaya, Mutsuo; Shimotai, Yoshitaka; Ohkawara, Ayako; Bazarragchaa, Enkhbold; Okamatsu, Masatoshi; Sakoda, Yoshihiro; Kida, Hiroshi; Nishimura, Hidekazu
title: The clinically used serine protease inhibitor nafamostat reduces influenza virus replication and cytokine production in human airway epithelial cells and viral replication in mice
date: 2020-12-17
journal: J Med Virol
DOI: 10.1002/jmv.26700
sha: e3245fc9b99178a66d9a949423dd00d433b951a6
doc_id: 756010
cord_uid: 0ffshsat

The effects of the clinically used protease inhibitor nafamostat on influenza virus replication have not been well studied. Primary human tracheal (HTE) and nasal (HNE) epithelial cells were pretreated with nafamostat and infected with the 2009 pandemic [A/Sendai‐H/108/2009/(H1N1) pdm09] or seasonal [A/New York/55/2004(H3N2)] influenza virus. Pretreatment with nafamostat reduced the titers of the pandemic and seasonal influenza viruses and the secretion of inflammatory cytokines, including interleukin‐6 and tumor necrosis factor‐α, in the supernatants of the cells infected with the pandemic influenza virus. HTE and HNE cells exhibited mRNA and/or protein expression of transmembrane protease serine 2 (TMPRSS2), TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced cleavage of the precursor protein HA0 of the pandemic influenza virus into subunit HA1 in HTE cells and reduced the number of acidic endosomes in HTE and HNE cells where influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus [A/Hyogo/YS/2011 (H1N1) pdm09] in mouse lung washes. These findings suggest that nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infection‐induced airway inflammation by modulating cytokine production.

camostat, suppress viral HA cleavage and reduce influenza virus replication. [12] [13] [14] [15] [16] The serine protease inhibitor nafamostat, which has been clinically used to treat patients with acute pancreatitis and disseminated intravascular coagulation, 17, 18 also reduces influenza virus replication in MDCK cells. 12 However, the effects of nafamostat have not been studied in human airway epithelial cells.

In this study, primary cultures of human tracheal epithelial (HTE) cells, which retain the functions of the original tissue, 19 and human nasal epithelial (HNE) cells 20 were infected with the 2009 pandemic or a seasonal influenza virus, and the effects of nafamostat on viral replication and release of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which are associated with disease severity, 21 were

examined. An animal study using mice was also utilized to examine the effects of nafamostat on viral replication, survival, and body weight loss after influenza virus infection.

2 | METHODS 2.1 | Human tracheal and nasal epithelial cell culture HTE and HNE cells were isolated as described previously 16, 20 and cells were cultured in 24-well plates in a mixture of Dulbecco's modified Eagle's medium (DMEM)-Ham's F-12 (DF-12) (Life Technologies) medium containing 2% Ultroser G (USG) serum substitute.

The tracheas used for cell cultures were obtained from 9 patients after death (age: 55 ± 6 years; mean ± SEM; 3 females, 6 males). HNE cells were obtained excised from the nasal polyps of subjects undergoing endoscopic surgery (n = 21; age: 60 ± 3 years; 7 females, 14 males). The cause of death or reason for surgery and statuses for allergic rhinitis and bronchial asthma are shown in Table 1 . None of the patients were being treated with nafamostat at the time of death or surgery. This study was approved by the Tohoku University Ethics Committee.

MDCK cells were cultured in T 25 flasks in Eagle's minimum essential medium (MEM) supplemented with 10% fetal calf serum. 16 The cells were then cultured in 96-well plates. 16 The cells were then cultured in a DF-12 medium containing 2% USG at 37°C in 5% 

The detection and titration of influenza viruses in culture supernatants were performed using the endpoint method involving infection of MDCK cell replicates in plastic 96-well plates with 10-fold dilutions of virus-containing supernatants as previously described. 16 After exposing MDCK cells to the virus-containing supernatants, the supernatants were aspirated, and the cells were rinsed with phosphate-buffered saline (PBS); fresh MEM containing trypsin was then added. The presence of the characteristic cytopathic effects of the influenza virus was then determined. The TCID 50 (TCID, tissue culture infective dose) was calculated, and viral titers in supernatants are expressed as TCID 50 /ml. 16 

Infection of cells with influenza virus was performed using previously described methods. 16 

The treatment of cells with 10 μg/ml (20 μM) nafamostat 12 was started 30 before infection and continued during infection and after infection until the end of the experimental period. 16 To examine the concentration-dependent effects of nafamostat, the cells were treated with nafamostat at concentrations ranging from 0.001 to 10 μg/ml using the same methods previously used for camostat studies. 16 

A portion of supernatant (300 μl) was collected 24 and 72 h after infection, and an equal volume (300 μl) of fresh medium supplemented with nafamostat or vehicle was added to the cell culture. 16 The entire supernatant volume (1 ml) was collected 120 h after infection.

Viral RNA in cells was measured to confirm the differences in the magnitude of viral replication. A two-step real-time quantitative reverse-transcription PCR (RT-PCR) assay was performed using TaqMan® Gene Expression Master Mix (Applied Biosystems) as described previously. 16 The primers and TaqMan probe used for each virus were designed as previously reported. 16, 23 The expression of viral RNA was normalized to the constitutive mRNA expression of β-actin.

The inhibitory effects of nafamostat on HA cleavage by serine proteases in HTE cells were examined as previously described. 16 2.10 | Expression of transmembrane protease serine 2, transmembrane protease serine 4 and transmembrane protease serine 11D

The mRNA expression of TMPRSS2, TMPRSS4, and TMPRSS11D was measured using the RT-PCR methods described above (Quantification of influenza virus RNA levels) by utilizing primers that were designed previously. 8, 16, 25 The protein concentrations of TMPRSS2 in supernatants were measured using a human transmembrane protease 2 TMPRSS2

ELISA kit (MYBioSourse).

An indirect immunofluorescence assay was performed as reported previously. 16 Cells were fixed with 4% paraformaldehyde in PBS for 

The Probes) as previously described. 20 The fluorescence intensity was calculated using a fluorescence image analyzer system (Lumina Vision®; Mitani).

IL-6 and TNF-α levels in supernatants were measured using a solidphase chemiluminescent enzyme-linked immunosorbent assay For the cytotoxicity assay, adhered cells were collected by treating cell sheets with trypsin, and the live cells were quantified by the exclusion of trypan blue using a hemocytometer. 16 

The effects of the serine protease inhibitor camostat (10 μg/ml) 16 were also examined and compared with those of nafamostat (10 μg/ml Figure 1H ,I). The potency of the inhibitory effect of camostat on the viral titer was significantly lower than that of nafamostat ( Figure 1H ,I).

Significant amounts of TMPRSS2, TMPRSS4, and TMPRSS11D mRNA were detected in HTE and HNE cells (Figure 2A,B ).

An indirect immunofluorescence assay confirmed the expression of the TMPRSS2 proteins in HTE and HNE cells ( Figure 2C,D) .

In addition, significant amounts of the TMPRSS2 protein were detected in the supernatants of HTE and HNE cells ( Figure 2E ,F).

The mRNA expression levels of TMPRSS2, TMPRSS4, and TMPRSS11D in HTE and HNE cells and the TMPRSS2 protein concentration in supernatants did not differ between nafamostat-and vehicle-treated cells (Figure 2A,B,E,F) . 

In the absence of nafamostat, the cleaved HA1 subunit predominated in the supernatants of HTE cells ( Figure 2G ). In contrast, the amount of cleaved HA1 subunit decreased as the nafamostat concentration increased, while the amount of uncleaved HA0 correspondingly increased ( Figure 2G ). In contrast, treatment with nafamostat (10 μ/ml) reduced the number of acidic endosomes in HTE and HNE cells ( Figure 3B,E) . Moreover, treatment with nafamostat reduced the fluorescence intensity compared to that in cells treated with vehicle and cells before treatment ( Figure 3G ,H). Treatment with camostat (10 μ/ml) also reduced the fluorescence intensity of the cells (Figure 3G,H) . The potency of the inhibitory effects of nafamostat did not differ from that of camostat. Treatment with a decreased amount of nafamostat (2 mg/kg/ day) 27 did not reduce the viral titers in lung samples ( Figure 5D ) or improve the survival rate after infection ( Figure 5E ).

In the present study, the serine protease inhibitor nafamostat, which has been used to treat patients with acute pancreatitis and disseminated intravascular coagulation, 17 Treatment of cells with nafamostat reduced the production of IL-6 and TNF-α, which are associated with disease symptoms and severity in influenza-infected patients 21, 33 and with cell damage. 34 41 is stored in secretory granules in the Caco-2 cells. 42 They also suggested that the secretory granules move to the cell surface, fuse with the plasma membrane and secrete matriptase. Therefore, this pathway may be associated with the shedding of TMPRSS2 from HTE and HNE cells.

In an in vivo study using mice, treatment with intraperitoneal injection of 30 mg/kg/day nafamostat or camostat reduced lung viral titers. These findings are consistent with those of previous reports using mice treated with camostat. 13 Furthermore, the viral titers in mice treated with nafamostat were lower than those in mice treated with oseltamivir.

Thus, treatment with nafamostat reduced mouse lung viral titers but did not improve the survival rate or bodyweight reduction after infection. The reasons for the discrepancy are uncertain; however, metabolic and hematological adverse effects reported in patients treated with nafamostat, including hyperkalemia and eosinophilia, 43, 44 might be associated with the mechanisms of the discrepant effects on mice treated with 30 mg/kg/day nafamostat in the present study. Further studies are required to confirm the most efficient doses and administration routes of nafamostat, including the nasal and intravenous routes, as shown in studies using zanamivir, peramivir or laninamivir. 26, [45] [46] [47] Nafamostat has been suggested to inhibit SARS-CoV-2 replication by inhibiting TMPRSS2-mediated viral entry and to be a candidate drug to treat COVID-19 patients. 11, 48 Therefore, nafamostat may become a candidate drug to treat patients infected with SARS-CoV-2 and/or influenza viruses in the winter season.

In conclusion, nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infectioninduced airway inflammation by modulating cytokine production. 

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