key: cord-293691-ewerquin
authors: Sauerhering, Lucie; Kupke, Alexandra; Meier, Lars; Dietzel, Erik; Hoppe, Judith; Gruber, Achim D.; Gattenloehner, Stefan; Witte, Biruta; Fink, Ludger; Hofmann, Nina; Zimmermann, Tobias; Goesmann, Alexander; Nist, Andrea; Stiewe, Thorsten; Becker, Stephan; Herold, Susanne; Peteranderl, Christin
title: Cyclophilin Inhibitors Restrict Middle East Respiratory Syndrome Coronavirus Via Interferon λ In Vitro And In Mice
date: 2020-07-02
journal: Eur Respir J
DOI: 10.1183/13993003.01826-2019
sha: 
doc_id: 293691
cord_uid: ewerquin

RATIONALE: While severe coronavirus infections, including Middle East respiratory syndrome coronavirus (MERS-CoV) cause lung injury with high mortality rates, protective treatment strategies are not approved for clinical use. OBJECTIVES: We elucidated the molecular mechanisms by which the cyclophilin inhibitors Cyclosporin A (CsA) and Alisporivir (ALV) restrict MERS-CoV to validate their suitability as readily-available therapy in MERS-CoV infection. METHODS: Calu-3 cells and primary human alveolar epithelial cells (hAEC) were infected with MERS-CoV and treated with CsA or ALV or inhibitors targeting cyclophilin inhibitor-regulated molecules including Calcineurin, NFAT, or MAP kinases. Novel CsA-induced pathways were identified by RNA sequencing and manipulated by gene knockdown or neutralising antibodies. Viral replication was quantified by qRT-PCR and TCID(50). Data were validated in a murine MERS-CoV infection model. RESULTS: CsA and ALV both reduced MERS-CoV titers and viral RNA replication in Calu-3 and hAEC improving epithelial integrity. While neither Calcineurin nor NFAT inhibition reduced MERS-CoV propagation, blockade of c-Jun N-terminal kinase diminished infectious viral particle release but not RNA accumulation. Importantly, CsA induced interferon regulatory factor 1 (IRF1), a pronounced type-III-interferon (IFNλ) response and expression of antiviral genes. Down-regulation of IRF1 or IFNλ increased MERS-CoV propagation in presence of CsA. Importantly, oral application of CsA reduced MERS-CoV replication in vivo, correlating with elevated lung IFNλ levels and improved outcome. CONCLUSIONS: We provide evidence that cyclophilin inhibitors efficiently decrease MERS-CoV replication in vitro and in vivo via upregulation of inflammatory, antiviral cell responses, in particular IFNλ. CsA might therefore represent a promising candidate to treat MERS-CoV infection.

Saudi Arabia [1] and led to recurring human infections with more than 2,500 laboratory-confirmed cases and high case fatality rates of about 35% [2] . In ex vivo infection of human lung tissue, MERS-CoV targets bronchial and alveolar epithelial cells (AEC) and leads to a detachment and apoptosis of AEC [3] . Recent reports analyzing autopsy material of deceased MERS-CoV-infected patients showed MERS-CoV antigen in AEC and epithelial multinucleated syncytial cell conglomerates in vivo [4, 5] . Accordingly, severe human infection presents as pneumonia with progression to acute respiratory distress syndrome [4, 5] .

To date, no vaccine or specific treatment for MERS-CoV -or the recently ongoing pandemic caused by the novel severe acute respiratory syndrome CoV 2 (SARS-CoV-2) -is approved and therapy relies on supportive measures only [2, 6] . While in vitro studies and experiments in non-human primates demonstrated benefits of a combination of type-I-interferon and antiviral compounds including ribavirin against MERS-CoV [7] [8] [9] , results from retrospective patient cohorts applying similar treatment regimens remain controversial [10] [11] [12] . Cyclosporin A (CsA) has been found to inhibit several human-pathogenic CoV in cell lines originating from kidney or liver epithelia [13] [14] [15] [16] . However, the molecular mechanisms by which CsA affects CoV, including MERS-CoV, particularly in its primary target cells, the pulmonary epithelium, remain elusive. Moreover, preclinical studies addressing the effect of CsA or related compounds on MERS-CoV replication in vivo have been lacking to date.

CsA is known to block the peptidyl-prolyl cis-trans isomerase (PPI) activity of cyclophilins that is involved in diverse cellular processes (e.g. protein folding, 17).

Additionally, CsA forms together with cyclophilin A (CypA) and calcineurin (CnA) a ternary complex which blocks the CnA-dependent activation of NFAT (nuclear factor of activated T-cells), a process which accounts for the immunosuppressive effect of CsA [18] . In addition, CsA has also been shown to inhibit the MAP kinases JNK (c-Jun N-terminal kinase) and p38 [19, 20] .

Here, we aimed to elucidate the distinct signaling pathways by which CsA affects MERS-CoV in clinically relevant models such as primary human AEC and a murine MERS-CoV infection model [21, 22] . We demonstrate that CsA blocks MERS-CoV infectious particle egress, which is dependent on JNK. Moreover, we for the first time provide evidence that CsA triggered the activation of an antiviral defense state in lung epithelial cells. We show that CsA is a potent inducer of Interferon regulatory factor 1 (IRF1), type-III-IFN (IFNλ) and multiple interferon-stimulated genes (ISGs).

Additionally, we demonstrate that oral application of CsA induced a robust IFNλ response in vivo and, importantly, significantly reduced MERS-CoV replication and improved disease progression in infected mice.

Experiments with MERS-CoV were performed under biosafety level 4 conditions at the Institute of Virology, Philipps-University of Marburg, Germany. Human alveolar epithelial cells (hAEC) were isolated and cultured as previously described [23] .

Human lung tissue was obtained from patients who underwent lobectomy after informed written consent (Departments of Pathology and Surgery, University of Giessen, approved by the University of Giessen Ethics Committee; Az.58/15). Calu-3 or hAEC were infected at a multiplicity of infection (MOI) of 0.1 diluted in DMEM/F12 without FCS at 37°C for 1 h. Cells were washed with DMEM/F12 with 10% FCS and supplemented with stimulatory/ inhibitory reagents as indicated. 24 h after infection (pi) cells were processed for quantitative PCR (Maxima-SYBR/ROX qPCR-Mastermix, Thermo Fisher) and supernatant was harvested for virus titration as described previously [24] .

All animal experiments were performed in accordance with the German animal protection laws and were authorized by the regional authorities (G73/2017). C57BL/6 mice were purchased from Charles River Laboratories and housed under pathogenfree conditions. Mice were intratracheally inoculated with Adenovirus-hDPP4-mCherry (cloned at ViraQuest Inc.) as described [21, 25] . Five days post transduction, mice were infected intranasally with 1.5x10 5 TCID 50 /ml MERS-CoV (EMC/2012). 50 mg/kg/day CsA diluted in DMSO or DMSO alone were mixed with a nut/chocolate-creme, and offered to the mice for voluntary uptake. Uptake was controlled daily. CsA feeding started 2 days before MERS-CoV challenge. Mice were sacrificed 4 or 7 days post MERS-CoV infection. 

All data are given as mean ± SEM. Statistical significance was analyzed by unpaired two-tailed Student's t-test or by 1-way ANOVA and post-hoc multicomparison tests as indicated in the respective figures. A P value of less than 0.05 was considered significant. *P < 0.05; **P < 0.01; ***P < 0.005.

Further experimental details can be found in the Online Supplement.

To address the previously proposed antiviral activity of CsA in clinically relevant cells, we infected the human bronchial epithelial cell line Calu-3 and primary human alveolar epithelial cells (hAEC) with MERS-CoV and analyzed intracellular viral RNA and infectious particle release in presence of DMSO or CsA ( Figure 1 ). In both Calu-3 and hAEC, CsA treatment led to a >95% decrease of viral RNA ( Figure 1A ) and a reduction of viral titers in the supernatant by 2.6-2.8 log 10 , respectively ( Figure 1B) .

Interestingly, and in accordance with reports from autopsy material of MERS-CoV patients [4] , MERS-CoV-infected Calu-3 and primary hAEC both showed apoptotic cell loss and formation of multinucleated cell foci ( Figure 1C ). Addition of CsA reduced cell foci formation and significantly reduced apoptosis induction ( Figure 1C , D). In line, both CFTR (cystic fibrosis transmembrane conductance regulator; Figure   1E ) and ENaCβ (epithelial sodium channel beta; Supplement Fig.E1) protein expression was improved after addition of CsA to MERS-CoV-infected Calu-3.

Moreover, epithelial structural integrity and ability for vectorial water transport were reduced in MERS-CoV-infected control cells and significantly improved to normal levels in MERS-CoV-infected, CsA-treated cells ( Figure 1F , G).

CsA is known to act via multiple signaling pathways including cyclophilin PPIase activity, the CnA-NFAT axis as well as MAP kinase signaling [17] [18] [19] [20] . Using specific inhibitors, we aimed to interfere with CsA-affected pathways to identify relevant molecular signaling events involved in the CsA-mediated reduction of MERS-CoV infection. Inhibition of CnA by its specific inhibitor calcineurin inhibitory peptide (CIP), as well as inhibition of the downstream transcription factor NFAT resulted in minor, statistically non-significant changes in MERS-CoV viral titers in both Calu-3 and hAEC (Figure 2A , B). The non-immunosuppressive derivate of CsA, Alisporivir (ALV), that binds the PPIase but does not induce ternary complex formation of CypA with CnA, reduced viral titers to a similar extent as CsA, suggesting that the CypA-PPIase activity elicits the restrictive effect on MERS-CoV replication rather than ternary complex-mediated signaling events. Moreover, ALV reduced cell foci formation and loss of epithelial integrity to a similar extent as CsA (Supplement Fig. E2 ). Applying specific MAPK inhibitors against JNK and p38, we revealed that inhibition of the MAP kinase JNK, but not of p38 reduced MERS-CoV titers in both Calu-3 and hAEC ( Figure 2A 

Our data suggest that, as opposed to its well-known CnA/NFAT- Figure 4C ). These data indicate that CsA treatment mounts a distinct interferon-driven antiviral response in lung epithelial cells.

To better understand the transcriptional programs leading to IFNλ induction in CsA-treated cells, we analyzed the regulation of interferon regulatory factors (IRFs).

Our data reveal significant upregulation of IRF1 mRNA levels upon CsA treatment, but not of IRF3, IRF7 or IRF9 ( Figure 5A ). IRF1 is known to be a specific activator of IFNL gene expression [26] . Accordingly, we identified a significantly increased number of IRF1-expressing cells in CsA-stimulated Calu-3 cells by immunofluorescence ( Figure 5B ). In line, IRF1 siRNA knockdown significantly reduced IFNL mRNA levels in CsA treated Calu-3 cells ( Figure 5C ). Accordingly, IRF1 knockdown inhibited IFNλ release by more than 75 % as compared to control ( Figure 5D ).

To understand the extent to which the inhibition of MERS-CoV propagation in CsA treated cells was mediated by IRF1-mediated production of IFNλ, we performed knockdown of IRF1 or neutralized cell-free IFNλ, respectively. Our data demonstrated that silencing of IRF1 but not treatment by control siRNA lead to a significant increase in MERS-CoV released viral particles in CsA-treated cells ( Figure   6A , B). Moreover, neutralizing antibodies directed against IFNλ1, IFNλ2 and IFNλ3 or against the less induced IFNβ were applied ( Figure 6B ). Neutralization of IFNβ had no significant impact on MERS-CoV replication after CsA treatment, whereas application of anti-IFNλ1/2/3 treatment significantly increased MERS-CoV viral titers by 1.05 log 10 level ( Figure 6B ). These data indicate that the antiviral effects of CsA were at least partially mediated by an IRF1-IFNλ signaling axis, and independent of type-I-IFN. As no specific treatment is approved for MERS-CoV or SARS-CoV(-2), current treatment strategies are supportive [29, 30] . Treatments including recombinant type-I-IFN and antivirals (e.g. Lopinavir/Ritonavir) have been applied off-label to treat MERS-CoV and yielded only moderate efficacy with controversial results in retrospective studies, and data from prospective studies or randomized controlled trials are lacking [29, [31] [32] [33] . Due to its receptor specificity to the human DPP4, only few animal models to study MERS-CoV pathogenesis and MERS-CoV-directed antiviral compounds have been accessible to date. For this study, MERS-CoV infection in the mouse was facilitated via intratracheal delivery of a human DPP4encoding adenovirus, that might cause low-level inflammation itself and inhomogeneous receptor distribution within the lung, present for a limited time frame.

However, even if this model might not fully recapitulate the native cellular distribution or density of the receptor as seen in the human lung, high transduction efficiencies (≥ 95%, data not shown) allow efficient viral spread in the upper and lower respiratory airways with quick progression to severe lung injury [22] and with moderate changes in morbidity [34] . Thus, model-specific neurotropism as seen in some of the transgenic hDPP4 mice [35] or the necessity to adapt virus isolates via multiple passages, which might potentially affect its susceptibility to interventional strategies, are circumvented. While prior exposure to adenovirus evokes moderate histological changes including perivascular and bronchiolar lymphocytic infiltration (data not shown), MERS-CoV infection led to a clearly distinguishable granulocytic, necrotizing interstitial pneumonia with alveolar edema formation as described previously [22] . Moreover, we demonstrate that inhibition of CypA via CsA or ALV, which both potently block the CypA PPIase activity at the used concentrations [42] , results in a pronounced upregulation of type-III-IFN on both mRNA and protein level, which was mediated via IRF1 and was accompanied by expression of antiviral ISGs. Among those, especially IFIT1 (Interferon-induced protein with tetratricopeptide repeats 1), has been reported to influence the pathogenesis of MERS-CoV, highlighting the relevance of our findings [43] .

Of note, type-III-IFNs have recently emerged as key antiviral players in the innate immune response to viral infections at mucosal and epithelial surfaces [44] [45] [46] [47] . They efficiently restrict different respiratory viruses, and act e.g. by limiting spread from the upper to the lower airways [44, [46] [47] [48] . As opposed to type-I-IFN, type-III-IFN do not trigger detrimental immune responses that contribute to immunopathology in influenza infection [23, 25, 44, 49] . This might prove to be pivotal in the context of CsA-dependent stimulation of IFNλ during CoV, as severe human CoV infections, like MERS-CoV and-while data are still limitedalso SARS-CoV-2, are characterized by an immunopathology with a strong cytokine induction [5, 50, 51] .

In addition to defining a novel pro-inflammatory, antiviral expression profile induced by CsA on lung epithelial cells, this study also demonstrated for the first time Gen. Virol. 2017. Statistical significance was calculated using unpaired two-way student's t-test (A, B, C, D, G) with *P < 0.05. OneStep RT-PCR System (Invitrogen Life Technologies) as described previously [2, 3] . 

Intracellular localization of endogenous IRF1 protein was analyzed 3 and 4 hour post CsA stimulation. DMSO-treated cells were used as negative control. Stimulated cells were fixed with 4% PFA and permeabilized with methanol/acetone for 10 min.

Cells were incubated with a rabbit monoclonal-anti-IRF1 (1:100; Cell Signaling) and

an AlexaFluor 594-conjugated secondary antibody (1:400; Dianova). Cell nuclei were counterstained with DAPI. The samples were mounted in Fluoprep (Biomérieux) and

images were recorded with a confocal laser scanning microscope (Leica SP5).

Calu3 cells were seeded in 0.4µm pore size transwell cell culture dishes (Corning) and cultured until achieving electrochemical resistances (TER) of ≥800Ω /cm 2 as measured by Millicell-ERS2 device. Cells were infected apically with MERS-CoV at ; as reported previously [4] .

For quantification of MERS-CoV induced apoptosis a Caspase 3/7 Gloassay® 

SDS-Page and western blot was analyzed as described previously [5] . Calu-3 cells were infected with MERS-CoV using a MOI of 0.1 and stimulated with CsA 1 hour after virus adsorption. 24 h pi cells were scratched off with 500 µl PBS supplemented with protease-inhibitor mix (Calbiochem) and centrifuged for 5 min at 5,000 rpm. Cell pellets were resuspended in sample buffer [6] containing 4% SDS and boiled at 100°C for 10 min. After discharge of the probes out of the BSL4 laboratory another 10 min boiling step was performed before the samples were separated using an 7,5 % SDS-Gel. After blotting on a nitrocellulose membrane and blocking using PBSdef with 5% milk powder first antibodies (Anti-CFTR Antibody, clone MM13-4 and mouse monoclonal Anti-Vinculin antibody both Sigma-Aldrich;

ENaCβ antibody (E-10), sc-48428; Santa Cruz Biotechnology) diluted in PBSdef with 1% milk powder were incubated overnight followed by secondary antibody-incubation for 1 h (Goat Anti-Mouse/HRP and Swine Anti-Rabbit/HRP; both Dako). For visualization of the signals Image Lab software was used.

All animal experiments were performed in accordance with the regulations of German animal protection laws and as authorized by the regional authorities 

For histopathological analyses of formalin-fixed, paraffin-embedded murine lung tissues, sections of 2 µm thickness were cut from four to six evenly distributed planes throughout the entire lungs and mounted on adhesive glass slides. The slides were stained with hematoxylin and eosin and coverslipped. Histopathological evaluation was performed using an established four grade scoring scheme [7] including the following parameters: affected area, severity and distribution of interstitial inflammation, infiltration of macrophages, lymphocytes and granulocytes, necrosis, alveolar hemorrhage and edema as well as formation of Bronchus-associated lymphoid tissue (BALT) and perivascular, lymphocytic cuffing. Figure OneStep RT-PCR kit as described previously [2, 3] . Quantification was carried out using a standard curve based on 10-fold serial dilutions of appropriately cloned RNA ranging from 10 2 to 10 5 copies. Bar graphs in represent mean ± SEM of n = 4 - 

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Work with live MERS-CoV was performed in the BSL-4 facility of the Philipps University, Marburg, Germany. We thank Julia Spengler, Larissa Hamann, Stefanie Jarmer, Dirk Becker and Marc Ringel for excellent technical and experimental assistance. We thank Ralf Bartenschlager for providing Alisporivir.