Vitamin D increases the antiviral activity of bronchial epithelial cells in vitro

Aurica G. Telciana, Mihnea T. Zdrengheab,*, Michael R. Edwardsa, Vasile-Laza Stancaa, Patrick Malliaa,c, Sebastian L. Johnstona,c,1, Luminita A. Stanciua,b,1

aAirways Disease Infection Section, National Heart and Lung Institute, Imperial College London; Medical Research Council; Asthma UK Centre in Allergic Mechanisms of Asthma; Centre for Respiratory Infections, London, UK; b Department of Hematology, Iuliu Hatieganu University of Medicine and Pharmacy Cluj-Napoca and Ion Chiricuta Oncology Institute, Cluj-Napoca, Romania; cImperial College Healthcare NHS Trust, London, UK

1S.L.J. and L.A.S. contributed equally to this work.

Short title: Antivirus activity of calcitriol

* Correspondence: Mihnea T Zdrenghea, e-mail: mzdrenghea@umfcluj.ro

Abbreviations: 1α(OH)ase = 1-alpha-hydroxylase, 24(OH)ase = 24-hydroxylase, HPBECs = Human Primary Bronchial Epithelial Cells, IFN-β / IFN-λ = Interferon-β / -λ, ISGs = Interferon Stimulated Genes, IL-6 / IL-8 = Interleukin-6 / -8, mRNA = messenger RNA, MxA = myxovirus resistance A gene, RSV = Respiratory Syncytial Virus, RV-1B / RV-16 = Rhinovirus-1B / -16, 18S rRNA = 18S ribosomal RNA, TLR3 = Toll Like Receptor 3, VDR = Vitamin D Receptor

ABSTRACT

Background. By modulating the antiviral immune response via vitamin D receptor, the active form of vitamin D (1,25-dihydroxyvitamin D, calcitriol) could play a central role in protection against respiratory virus infections. This in vitro study tested the hypothesis that respiratory viruses modulate vitamin D receptor expression in human bronchial epithelial cells and this modulation affects the antiviral response to exogenous vitamin D.

Methods. Human primary bronchial epithelial cells were infected with rhinoviruses and respiratory syncytial virus in the presence or absence of vitamin D. Expression of vitamin D receptor, 1α-hydroxylase (1α(OH)ase), 24-hydroxylase (24(OH)ase), innate interferons, interferon stimulated genes and cathelicidin were measured by quantitative polymerase chain reaction. The antiviral effect of vitamin D on rhinovirus replication was determined by measurement of virus load. A direct inactivation assay was used to determine the antiviral activity of cathelicidin.

Results. Both RV and RSV decreased vitamin D receptor and 24(OH)ase and, in addition, RSV increased 1α(OH)ase expression in epithelial cells. Vitamin D decreased rhinovirus replication and release, and increased rhinovirus-induced interferon stimulated genes and cathelicidin. Furthermore, cathelicidin had direct anti-rhinovirus activity.

Conclusions. Despite lower vitamin D receptor levels in rhinovirus-infected epithelial cells, exogenous vitamin D increased antiviral defences most likely via cathelicidin and innate interferon pathways.

Keywords: cathelicidin; interferons; respiratory viruses.

1. Introduction

Vitamin D deficiency is linked to increased frequency of respiratory viral infections and supplementation with vitamin D may reduce the risk of respiratory tract infections (Ginde et al., 2009),(Sabetta et al., 2010),(Urashima et al., 2010),(Camargo et al., 2012),(Goodall et al., 2014). The biologically active metabolite 1,25-hydroxivitamin D (1,25(OH)D or calcitriol) is produced intracellularly when the major circulating metabolite, 25-hydroxyvitamin D (25(OH)D, calcidiol) is hydroxylated by 1α-hydroxylase (1α(OH)ase, CYP27B1). Human tracheobronchial epithelial cells are equipped to convert locally the inactive 25(OH)D to its active form 1,25(OH)D (Hansdottir et al., 2008). Calcitriol signals through the vitamin D receptor (VDR), a member of the nuclear receptor superfamily of transcription factors, decreasing pro-inflammatory cytokines and increasing peptides such as the innate antimicrobial peptide cathelicidin (hCAP-18/LL-37) (Liu et al., 2006),(Dhawan et al., 2015). Cathelicidin has direct antiviral activity against enveloped respiratory viruses such as influenza and respiratory syncytial virus (RSV) (Tripathi et al., 2013),(Currie et al., 2013).

Bronchial epithelial cells are the primary site of respiratory virus infection. RSV is the major cause of acute bronchiolitis in infants, a main cause of respiratory morbidity in children and the elderly and viral load correlated with disease severity in adults (Falsey et al., 2005),(Hall et al., 2009),(DeVincenzo et al., 2010). RSV is also an important cause of asthma exacerbations in adults, and especially in the elderly (Westerly and Peebles, 2010). Rhinoviruses (RVs) are the most frequent cause of common cold and are associated with majority of asthma exacerbations in children and in adults (Johnston et al., 1995). Vitamin D deficiency is associated with severe asthma exacerbation in children and adolescence (Brehm et al., 2010),(Gupta et al., 2011). Deficiency of vitamin D is also common in adult asthma and most pronounced in patients with severe or uncontrolled asthma (Korn et al., 2013).

We hypothesised that respiratory viruses, by modulating expression of the VDR in human primary bronchial epithelial cells, could perturb the antiviral response to exogenous calcitriol.

We report that RV and RSV decreased the expression of VDR in human primary bronchial epithelial cells. Calcitriol treatment decreased pro-inflammatory cytokines and increased antiviral interferon stimulated genes (ISGs) and cathelicidin in RV-infected human primary bronchial epithelial cells (HPBECs). Calcitriol decreased rhinovirus replication in infected HPBECs. Our in vitro data suggest a beneficial effect of calcitriol in respiratory viral infection.

2. Materials and Methods

2.1. Cell Culture and Virus Infection

BEAS-2B cells, a bronchial epithelial cell line (European Collection of Animal and Cell Cultures) were cultured as previously described (Papadopoulos et al., 2001),(Hewson et al., 2005). HPBECs (Clonetics, Lonza) were cultured in bronchial epithelial cell growth medium with RSV A2 (American Type Culture Collection, Rockville, MD, USA), minor group serotype rhinovirus RV1B (American Type Culture Collection) or major group serotype rhinovirus RV16 (obtained from the Health Protection Agency Culture Collections, Salisbury, United Kingdom) (all at MOI 1) as previously described (Stanciu et al., 2006),(Edwards et al., 2006). Virus load was quantified in samples either as mRNA by Taqman (quantitative real-time PCR) or as released virus in supernatants by titration on HeLa cells.

The stocks of calcitriol (1α,25-Dihydroxyvitamin D3, Sigma) were prepared in ethanol, kept at -200C, and used at the concentration of 100 nmol, as previously reported by other groups (Yim et al., 2007),(Hansdottir et al., 2008),(Brockman-Schneider et al., 2014),(Dhawan et al., 2015) unless specified in the dose-response experiments. The ethanol vehicle control did not have a meaningful effect on the viral or cellular activity in the experiments performed (Fig. 3). The mRNA and cell-free supernatants were harvested at 24 h after virus infection, or as indicated for the time-course experiments.

2.2. Direct Inactivation of RV1B by Cathelicidin

LL-37 peptide (cathelicidin), AnaSpec Inc, Cat 61302, was resuspended in cell culture media.

A direct inactivation assay was used to determine the antiviral activity of the antimicrobial peptide cathelicidin on RV1B (Daher et al., 1986). RV1B was incubated with cathelicidin (from 1.25 to100 µg/mL) for one hour at 370C, and then the virus’ ability to infect was assessed by two methods. First, the mixtures were titrated immediately on HeLa cells (according to our standard protocol for titration). Briefly, we performed serial dilutions of the mixtures virus-cathelicidin and added them to HeLa cells. The results were read 4 days later by assessing the cytophatic effect on the cells. The number of wells where the cytophatic effect is present correlates directly with the viral titre (few wells – lower titre, more wells – higher titre).

A second method was to infect BEAS-2B cells with cathelicidin-pre-treated virus, and then assess the virus released by cells. The BEAS-2B cells in this assay represent only the system used to test the infectivity of cathelicidin-pre-treated virus, therefore BEAS-2B cells were convenient to use as they grow fast and are easy to split.

2.3. RNA Isolation, cDNA Synthesis and Real-Time Quantitative Polymerase Chain Reaction

RNA was extracted (RNeasy Mini Kit, Qiagen), and 1 µg used for cDNA synthesis (Omniscript RT Kit, Qiagen). Quantitative real-time polymerase chain reaction (PCR) was performed using primers and probes for 1α(OH)ase, 24α(OH)ase, VDR, IL-6, IL-8, IFN-β, interferon-lambda (IFN-λ1/IL-29, IFN-λ2/IL-28A, and IFN-λ3/IL-28B), Viperin, MxA, cathelicidin, RVs and 18S rRNA. The sequences for primers and probes were as follows: 1α(OH)ase (forward, 5’-TTG GCA AGC GCA GCT GTA T-3’; reverse, 5’-TGT GTT AGG ATC TGG GCC AAA-3’, probe, FAM 5’-TTG CAA TTC AAG CTC TGC CAG GCG-3’ TAMRA), 24α(OH)ase (forward, 5’-CAA ACC GTG GAA GGC CTA TC-3’; reverse, 5’-AGT CTT CCC CTT CCA GCA TCA-3’, probe, FAM 5’-ACT ACC GCA AAG AAG GCT ACG GGC TG-3’ TAMRA), VDR (forward, 5’-CTT CAG GCG AAG CAT GAA GC-3’; reverse, 5’-CCT TCA TCA TGC CGA TGT CC-3’, probe, FAM 5’-AAG GCA CTA TTC ACC TGC CCC TTC AA-3’ TAMRA) and cathelicidin (forward, 5’-TCA CCA GAG GAT TGT GAC TTC AA-3’; reverse, 5’-TGA GGG TCA CTG TCC CCA TAC-3’, probe, FAM 5’-AAG GAC GGG CTG GTG AAG CGG-3’ TAMRA). Rest of the sequences were published elsewhere (Gielen et al., 2010). Data were analysed using version 1.4 ABI Prism 7500 SDS software (ABI), normalized to 18S rRNA and 45-CT (for 1α(OH)ase, 2(OH)ase, VDR and cathelicidin) or copy number (for all the other genes, using standard curves for plasmids of known concentration containing the amplified region of the specific genome) were calculated. The results are presented as relative expression to media or virus control.

2.4. ELISA for IL-6, IL-8 and IFN-λ1/3

Supernatants were used to measure IL-6, IL-8 and IFN-λ1/3 levels using paired antibodies and standards (R&D Systems for IL-6/IL-8 and Duoset kit from R&D Systems for IFN-λ1/3). Assay sensitivities were 3.9 pg/mL (IL-6 and IL-8) and 15.6 pg/mL (IFN-λ1/3).

2.5. Statistical Analysis

Results were analyzed using GraphPad Prism version 4.00 (GraphPad Software, California). Results were expressed as means ± standard deviation (SD). When analyzing multiple groups, a one-way ANOVA and Bonferroni’s multiple comparison test were used. When analyzing groups of two (Fig 1 C-H and Fig 5 A-B), a paired t-test for paired comparisons was used. P values less than < 0.05 were considered significant.

3. Results

3.1. Respiratory viruses decrease VDR and 24(OH)ase gene expression in HPBECs

Human tracheobronchial epithelial cells, isolated from tracheal and bronchial mucosa by enzymatic dissociation, express high baseline levels of activating vitamin D 1α-hydroxylase and low levels of inactivating 24-hydroxylase (Hansdottir et al., 2008) and human bronchial epithelial cells express VDR (Yim et al., 2007).

We confirmed that HPBECs constitutively expressed the 1(OH)ase, 24(OH)ase and VDR and that treatment with calcitriol for 24 h decreased VDR and 1α(OH)ase expression (Fig. 1A), and increased expression of 24(OH)ase (Fig. 1B) in a dose-response manner.

RSV infection of HPBECs significantly decreased VDR mRNA expression, starting at 24 h, up to 48 h (Fig. 1C and 1D), increased 1α(OH)ase mRNA expression significantly at 24 h (Fig. 1E and 1F) and decreased 24(OH)ase gene expression at 24 h and 6 h (Fig. 1G and 1H). RV infection also decreased VDR and 24(OH)ase expression in HPBECs (Fig. 1D and 1H) but did not increase statistically significant 1α(OH)ase expression (Fig. 1F).

3.2. Calcitriol decreases rhinovirus-induced pro-inflammatory cytokines in HPBECs

Calcitriol decreased RSV-induced pro-inflammatory cytokines in tracheobronchial epithelial cells (Hansdottir et al., 2008),(Hansdottir et al., 2010). We therefore examined if exogenous calcitriol has similar effects in RV-infected HPBECs. Calcitriol (at increasing doses) was added to the cell culture, and IL-6 and IL-8 mRNA and protein levels determined at different time points.

Calcitriol alone at the highest concentration (1000 nmol) significantly decreased IL-6 and IL-8 protein levels at 24 h culture in HPBECs (Fig. 2B and 2D).

Calcitriol treatment of RV-infected HPBECs decreased in a concentration-dependent manner RV1B-induced IL-6 and IL-8 mRNA (Fig. 2A and 2C) and protein levels (Fig. 2B and 2D) in HPBECs. In time-course experiments, IL-6 gene expression in RV-infected cells was downregulated by calcitriol from 12 h up until 24 h post-virus infection (data not shown) and IL-6 protein levels were decreased by calcitriol at 24 h for RV1B and at 18 h and 24 h for RV16 infection (data not shown).

3.3. Calcitriol suppresses RV1B replication and release in HPBECs

It was reported that vitamin D had no effects on the quantity or replication of RSV in tracheobronchial epithelial cells (Hansdottir et al., 2010). We assessed the effects of calcitriol on the antiviral capacity of HPBECs towards RV.

We have found that the treatment of HPBECs with calcitriol during RV1B infection decreased viral RNA and virus released in cell culture in a concentration dependent manner (Fig. 3A and 3B).

Pre-treatment of HPBECs with calcitriol prior virus infection (without using calcitriol in culture media after virus infection), also decreased RV1B replication (mRNA and virus release, Fig. 3C and 3D). The ethanol, by itself did not significantly alter virus levels.

3.4. Calcitriol increases RV1B-induced ISGs and cathelicidin gene expression in HPBECs

Having found that calcitriol increases the antiviral capacity of respiratory epithelial cells towards RV, we assessed whether this effect is due to stimulation of antiviral innate IFN-production by calcitriol. Calcitriol did not increase RV1B-induced IFN-β and IFN-λ1/IFN- λ2/IFN-λ3 mRNA expression (Fig. 4A and 4B and data not shown) or IFN-λ1/3 protein levels at 24 h post-infection in HPBECs (data not shown). However, calcitriol increased in a dose-dependent manner RV1B-induced mRNA expression of the antiviral ISGs MxA and Viperin in HPBECs, reaching significance at the highest dose of calcitriol (Fig. 4C and 4D).

In our experimental model, we found that HPBECs constitutively express the cathelicidin gene (240.9±64.1 copy number at baseline). Cathelicidin mRNA levels in HPBECs were increased by calcitriol treatment in a time-dependent manner (fold increase over media 7.3±1.09 at 12 h, 18.4±1.3 at 18 h, 28.8±6.5 at 24 h, and 30.4±6.4 at 48 h for calcitriol 100 nmol, Fig. 5A) and in a dose-dependent manner (fold increase over media 12.9±2.06 for calcitriol 10 nmol, 38.8±6.4 for calcitriol 100 nmol and 79.3±14.4 for calcitriol 1000 nmol at 24 h, Fig. 5C, left panel).

RV infection by itself did not significantly modify cathelicidin mRNA, while RSV infection significantly increased cathelicidin mRNA in HPBECs (Fig. 5B).

Cathelicidin gene expression was increased by calcitriol in RV1B-infected HPBECs in a dose-dependent manner (Fig. 5C), but the levels in RV1B-infected HPBECs treated with higher doses of calcitriol were significantly lower as compared to calcitriol alone (Fig. 5C). In contrast, calcitriol had a synergistic effect with RSV and the increase of cahelicidin gene expression in RSV-infected HPBECs was higher as compared to calcitriol alone (Fig. 5C).

3.5. Cathelicidin decreases RV1B ability to infect respiratory epithelial cells

Cathelicidin was reported to have direct antiviral activity against enveloped respiratory viruses such as influenza and respiratory syncytial virus (RSV) (Tripathi et al., 2013),(Currie et al., 2013). The difference in cathelicidin expression induced by calcitriol in cells infected with RV1B versus RSV prompted us to test the influence of cathelicidin on RV1B replication.

When RV1B was pre-incubated with increasing concentrations of cathelicidin (Daher et al., 1986), virus titer was decreased as determined by titration in HeLa cells (80% reduction in of virus release by HeLa cells infected with RV previously treated with cathelicidin 100 µg/mL, Fig. 6A).

We also used cathelicidin pretreated RV to infect BEAS-2B cells, and we assessed the level of RV released in supernatants. Pre-incubation with cathelicidin suppressed the capacity of RV1B to infect BEAS-2B cells and consequently less virus was detected in supernatants (93% reduction of virus release by BEAS-2B cells infected with virus previously treated with cathelicidin 100 µg/mL, Fig. 6B).

4. Discussion

We report here that the respiratory viruses RV and RSV downregulate VDR mRNA expression in primary bronchial epithelial cells. By decreasing VDR, viruses could limit the functional activity of the vitamin D active metabolite, calcitriol, in respiratory epithelial cells. However exogenous calcitriol added to epithelial cells enhances intracellular components important in controlling virus replication by increasing RV1B-induced ISGs and cathelicidin gene expression. Consequently, RV replication was reduced in calcitriol-treated HPBECs. Furthermore, we found that cathelicidin pretreatment of RV1B decreases its capacity to infect epithelial cells suggesting that this might contribute to the antiviral activity of calcitriol.

It was reported that tracheal and bronchial cells express 1α(OH)ase mRNA and convert 25(OH)D to active 1,25(OH)D and that exogenous 1,25(OH)D increased 24(OH)ase mRNA expression (Hansdottir et al., 2008). We confirm now that HPBECs express 1α(OH)ase, 24(OH)ase and VDR mRNA. In addition we show that calcitriol decreases VDR and 1α(OH)ase and increases 24(OH)ase mRNA expression in HPBECs suggesting autocrine regulation of vitamin D metabolism in respiratory epithelial cells (Hansdottir et al., 2008),(Takeyama and Kato, 2011).

We found that calcitriol treatment of RV-infected HPBECs decreased in a concentration-dependent manner RV1B-induced IL-6 and IL-8 mRNA and protein levels in HPBECs. It has been previously reported that calcitriol decreases RSV-induced pro-inflammatory cytokines in tracheobronchial epithelial cells (Hansdottir et al., 2008),(Hansdottir et al., 2010). In another report, calcitriol (10 nmol) enhanced RV-induced IL-8 secretion, but did not alter IL-6 levels, in an air-liquid interface model of primary bronchial cells derived from residual human surgical specimens from healthy lung donors, differentiated in a special medium enriched in vitamin D for 4 weeks (Brockman-Schneider et al., 2014).

RSV increased 1α(OH)ase gene expression in HPBECs and RSV and RV decreased VDR and 24(OH)ase mRNA expression in HPBECs. The finding that viruses decrease expression of the VDR would suggest that treatment with calcitriol may not increase host antiviral immunity. It has been reported that calcitriol did not affect RSV replication in tracheal and bronchial cells (Hansdottir et al., 2010). However, we found that calcitriol decreased RV1B RNA and also RV1B release in HPBECs. RV release was reduced by approximately 50% with calcitriol treatment and there remains the need to confirm in vivo if this in vitro finding is significant. Recently in a study of the role of vitamin D in prevention of respiratory infection in young adults, RV load was lower in the vitamin D supplementation group compared to the placebo group (Goodall et al., 2014).

Having found that calcitriol has antiviral activity towards RV in HPBECs we went further to determine if cacitriol increased antiviral IFNs and cathelicidin in our model. Interestingly, it was reported that calcitriol significantly decreased RSV-induction of IFN-β and ISGs mRNA in tracheal and bronchial cells (Hansdottir et al., 2010). We found that in primary bronchial epithelial cells calcitriol increased RV1B-induced ISGs MxA and Viperin mRNA expression. IFN-α/β protein levels could not be detected in RV-infected cell culture at 24 h, but the increases in ISG levels would suggest an earlier transient increase in the production of innate IFNs. It was also reported that calcitriol inhibited hepatitis C virus production via increasing the expression of IFN-β and MxA in hepatoma cells (Gal-Tanamy et al., 2011).

Cathelicidin was previously shown to be expressed and secreted by airway epithelial cells and levels were increased by locally activated exogenous calcitriol and by RSV±25(OH)D (Bals et al., 1998),(Yim et al., 2007),(Hansdottir et al., 2008),(Brockman-Schneider et al., 2014),(Dhawan et al., 2015). We also found that RSV and exogenous calcitriol increased cathelicidin expression in HPBECs demonstrating that VDR is functional in HPBECs. It was previously reported that in primary bronchial cells derived from residual human surgical specimens from healthy lung donors, differentiated in a special medium enriched in vitamin D for 4 weeks in an air-liquid interface model, re-exposure to 10 nmol calcitriol post-differentiation increased cathelicidin expression (Brockman-Schneider et al., 2014).

RV infection did not increase cathelicidin mRNA expression. However, RV infection in the presence of calcitriol significantly increased cathelicidin mRNA levels (although levels were lower compared to the increase induced by calcitriol in uninfected cells) suggesting that exogenous calcitriol has therapeutic potential in RV infections.

Cathelicidin had direct antiviral activity in vitro particularly against enveloped respiratory viruses such as influenza and RSV (Tripathi et al., 2013),(Currie et al., 2013),(Barlow et al., 2014). Cathelicidin enhanced viral double strand (ds)RNA TLR3-signalling and augmented IFN‐β expression and antiviral activity induced by dsRNA in keratinocytes and cathelicidin increased RV-induced cytokine production in BEAS-2B cells via activating TLR3 (Lai et al., 2011),(Singh et al., 2013),(Takiguchi et al., 2014),(Hewson et al., 2005). We found that cathelicidin has a direct antiviral activity and suppresses RV1B infectivity. Our data that pre-treatment of rhinovirus with LL-37 attenuated rhinovirus replication in bronchial epithelial cells suggest that LL-37 is a major antiviral component. However, we were unable to determine the anti-rhinovirus mechanism of LL-37 at present.

Unlike enveloped viruses, direct virus death through damage of the viral envelope cannot be the operative mechanism for rhinoviruses. Further studies are needed to determine how exogenous and endogenous LL-37 decreased the virus load in respiratory epithelial cells: blocking viral entry into cells by alteration of virus binding or uptake, protective effect on epithelial cells, inhibiting virus internalization? We cannot exclude that LL-37 and RV administered together do not increase antiviral proteins such innate IFNs via TLR3 (Lai et al., 2011).

In this study, we found some similarities but also notable differences in the effects of RSV and RV infection on the vitamin D pathways in HPBECs. Calcitriol had no antiviral effects on RSV (Hansdottir et al., 2010) whereas it suppressed RV1B replication and release. Another group recently reported that vitamin D does not directly affect RV replication in airway epithelial cells (Brockman-Schneider et al., 2014) but they used a different experimental set-up with primary bronchial cells derived from residual human surgical specimens from healthy lung donors, differentiated in a special medium enriched in vitamin D for 4 weeks and an air-liquid interface model. Similarly to the reported cathelicidin anti-RSV effect (Currie et al., 2013), we found that cathelicidin has antiviral activity against RV1B. It has been reported that calcitriol decreased RSV-induced IFN-β and ISGs in tracheal and bronchial cells (Hansdottir et al., 2010), while we found that IFN levels were not affected and the ISGs MxA and Viperin were increased in RV1B-infected HPBECs.

The strengths of our study include the use of primary bronchial epithelial cells, different respiratory viruses, and different doses of calcitriol. There are several limitations of our study including the assessment of the results mostly at a single time point, due to the difficulty to grow and expand HPBECs in culture. Furthermore, we did not have the possibility to verify if the anti-viral effect of calcitriol against RV1B is relevant in vivo and to identify mechanisms by which vitamin D induces antiviral activity towards non-enveloped viruses such as rhinoviruses in respiratory epithelium.

In conclusion, although rhinoviruses modify the vitamin D machinery in respiratory epithelial cells in vitro, antiviral properties of exogenous calcitriol are not decreased, suggesting a net beneficial effect of calcitriol in respiratory virus infections.

Potential conflict of interest

Professor Johnston consulted to Boehringer Ingelheim, Centocor, Chiesi, GlaxoSmithKline, Grunenthal, Novartis, Sanofi Pasteur and Synairgen, and received grant from Centocor Inc.

All other authors have no potential conflicts.

Authorship

S.L.J. and L.A.S. designed the research. A.G.T., M.R.E., M.T.Z. and V.L.S. performed analyses, A.G.T., P.M., S.L.J and L.A.S. wrote the manuscript. All authors approved the final version to be published. S.L.J and L.A.S. are guarantors of the paper.

Corresponding author for the Editorial Office: Luminita A Stanciu, l.stanciu@imperial.ac.uk

Acknowledgments

This work was supported by the European Respiratory Society (Fellowships to AGT, MTZ); the British Medical Association (AGT); Romanian Ministry of National Education, CNCS – UEFISCDI (project number PN-II-ID-PCE-2012-4-0417 to LAS and MTZ); the Wellcome Trust (Grant numbers 063717 and 083567/Z/07/Z for the Centre for Respiratory Infection and travel grant 063967 to MTZ); the University of Medicine and Pharmacy Cluj, Romania (Grant number 1494 /2014 to MTZ); ERC FP7 Advanced Grant number 233015 (SLJ); Asthma UK (grant numbers 02/027 and 05/067); the British Lung Foundation (Grants numbers P04/13 and P06/3 and Programme Grant number 00/02); MRC Centre Grant number G1000758 (SLJ) and UCB Institute of Allergy Fellowships (MTZ and VLZ). The sponsors had no involvement in the study design, data collection, analysis and interpretation, or the decision to submit the work for publication.

Disclosures: The authors declare no conflict of interest.

Figure Legends

Fig. 1. Calcitriol, RSV and RVs modulate the VDR and the enzymes involved in vitamin D metabolism in HPBECs. HPBECs cultured in medium with different doses of calcitriol, or infected with RSV or RV were harvested at 24 h (A, B, D, F, H) or at 8-48 h (C, E, G) for mRNA expression. Calcitriol decreased VDR and 1(OH)ase and markedly increased 24(OH)ase (A, B). RSV decreased VDR (C, D), increases 1(OH)ase (E, F) and decreased 24(OH)ase (G, H) mRNA expression in HPBECs. Similar trends in regulating VDR (D) and 24(OH)ase (H) mRNA expression in HPBECs were seen for RV1B and RV-16 infection.

Data are presented as % expression relative to non-infected control (i.e. cells treated with media), *: P<0.05; **: P<0.01; ***: P<0.001 compared to media control, as determined by One-way ANOVA with Bonferroni comparison (A, B) or as determined by paired t-test (C, D, E, F, G, H); : P<0.05, : P<0.01, : P<0.001 compared between different doses of calcitriol in HPBECs, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: VDR n=5 100%=14.04±0.1 45-δCT, 1α(OH)ase n=5 100%=19.2±0.32 45-δCT; Graph B: 24(OH)ase n=5, 100%=12.88±0.85 45-δCT; Graph C: n=4, 100% for 8/24/48h: 8.1±0.37/7.19±0.44/6.8±0.77 45-δCT; Graph D: RSV n=10, 100%=12.37±0.89 45-δCT, RV1B n=25, 100%=12.21±0.31 45-δCT, RV16 n=10, 100%=12.15±0.35 45-δCT; Graph E: n=5, 100% for 8/24/48h: 13.6±0.26/12.28±0.32/11.65±0.72 45-δCT; Graph F: RSV n=16, 100%=17.92±1.04 45-δCT, RV1B n=30, 100%=19.07±0.31 45-δCT, RV16 n=11, 100%=19.2±0.62 45-δCT; Graph G: n=4, 100% for 8/24/48h: 13.58±1.16/13.63±0.45/12.86±0.91 45-δCT; Graph H: RSV n=10, 100%=15.22±0.59 45-δCT, RV1B n=25, 100%=13.21±0.68 45-δCT, RV16 n=10, 100%=15.27±0.78 45-δCT.

Fig. 2. Calcitriol down-regulates virus-induced pro-inflammatory cytokines. HPBECs were pre-treated with calcitriol for 16 h before infection with RV1B or RV-16, then cells were washed and fresh media ± calcitriol was added; supernatants for ELISA and cell lysates for mRNA isolation were harvested at 24 h post virus infection. Calcitriol treatment of RV-infected HPBECs decreased in a concentration-dependent manner RV-1B-induced IL-6 (A) and IL-8 (C) mRNA expression and protein levels (B and D) in HPBECs.

Data are presented as % expression relative to infected control (i.e. RV1B infected cells), *: P<0.05; ***: P<0.001, compared to media, as determined by one-way ANOVA with Bonferroni comparison; &&: P<0.01, &&&: P<0.001 compared to RV1B-infected HPBECs, as determined by One-way ANOVA with Bonferroni comparison; : P<0.05, : P<0.01, : P<0.001 compared between different doses of calcitriol in RV1B-infected HPBECs, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: n=9, 100%=22177±7363 copy numbers; Graph B: n=12, 100%=1158±476.5 pg/mL; Graph C: n=8, 100%=1399000±569351 copy numbers; Graph D: n=7, 100%=566.5±93.66 pg/mL.

Fig. 3. Calcitriol decreases the replication and release of RV1B. HPBECs were pre-treated with calcitriol for 16 h before infection with RV1B, then cells were washed and fresh media ± calcitriol added (except where stated in the graph), supernatants for virus titration and cell lysates for mRNA isolation were harvested at 24 h post virus infection. Ethanol (eth) was used in a concentration similar to what was used to prepare calcitriol 1000 nmol concentration, as the stocks of calcitriol were prepared in ethanol. Calcitriol decreased RV1B mRNA expression (A, C) and RV1B release in HPBECs (B, D) in a dose-response manner.

Data are presented as % expression relative to infected control (i.e. RV1B infected cells), *: P<0.05; **: P<0.01; ***: P<0.001 compared to RV1B-infected HPBECs, as determined by one-way ANOVA with Bonferroni comparison; : P<0.05, : P<0.01, : P<0.001 compared between different doses of calcitriol used in RV1B-infected HPBECs, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: n=11, 100%= 3,521e+008± 1,328e+008 copy numbers; Graph B: n=13, 100%= 311278± 95695 TCID50; Graph C: n=4, 100%= 1,978e+007± 5,591e+006 copy numbers; Graph D: n=4, 100%= 413737± 73656 TCID50.

Fig. 4. Calcitriol increases virus-induced ISGs. HPBECs were pre-treated with calcitriol for 16 h before infection with RV1B, then cells were washed, fresh media ± calcitriol was added and cell lysates for mRNA isolation were harvested at 24 h post virus infection. Calcitriol increases RV1B-induced ISGs MxA and Viperin mRNA expression in HPBECs (C, D).

Data are presented as % expression relative to infected control (i.e. RV1B infected cells), **: P<0.01, compared to media, as determined by one-way ANOVA with Bonferroni comparison; &: P<0.05 compared to RV1B, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: n=5, 100%=348198±213290 copy numbers; Graph B: n=9, 100%=117657±68386 copy numbers; Graph C: n=9, 100%=219223±139922 copy numbers; Graph D: n=9, 100%=616280±375292 copy numbers.

Fig. 5. Calcitriol increases virus-induced cathelicidin mRNA expression. HPBECs were pre-treated with calcitriol for 16 h before infection with RV1B, cells were washed, fresh media ± calcitriol was added and cell lysates for mRNA isolation harvested at 24 h post virus infection (B and C) or indicated time points (A). HPBECs were not pre-treated with calcitriol in the time-course experiments (A). Calcitriol increases cathelicidin mRNA expression in a time-course and dose-dependent manner in HPBECs (A, C left pannel). RSV increase cathelicidin mRNA expression in HPBECs (B, C first column right pannel). Cathelicidin mRNA expression was increased by calcitriol in a dose-response manner in RV1B- and in RSV-infected HPBECs (C middle and right pannels).

Data are presented as % expression relative to non-infected control (i.e. cells treated with media), *: P<0.05; **: P<0.01; ***: P<0.001, compared to media, as determined by paired t-test (A, B) or as determined by one-way ANOVA with Bonferroni comparison (C); &&&: P<0.001 compared to RV1B, as determined by one-way ANOVA with Bonferroni comparison; #: P<0.05; ##: P<0.01, compared to RSV, as determined by one-way ANOVA with Bonferroni comparison; : P<0.001 compared between each dose of calcitriol alone and same dose of calcitriol added to RV1B-infected HPBECs, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: n=4, 100% for 2/4/8/12/18/24/48h: 14,81±1,03/14.16±0.96/13.5±1.19/13.39±0.91/13.24±0.51/13.22±1.07/11.96±0.47 45-δCT; Graph B: RV1B n=25, 100%=9.92±0.4 45-δCT, RV16 n=11, 100%=10.4±0.73 45-δCT, RSV n=11, 100%=10.33±0.45 45-δCT; Graph C: RV1B n=12,100%=7.81±1.04 45-δCT; RSV n=5, 100%=10.58±0.57 45-δCT.

Fig. 6. Cathelicidin decreases RV1B infection in human bronchial epithelial cells. RV1B was preincubated for 1 hour at 370C with cathelicidin in different concentrations and then titrated immediately on HeLa cells (A). Alternatively, cathelicidin-pretreated RV1B was used to infect BEAS-2B cells and the virus released in the supernatants was measured by titration on HeLa cells at 24 h postinfection (B). Cathelicidin decreases RV1B-infectivity in BEAS-2B cells in a dose-response manner.

Data are presented as % expression relative to control (i.e. cells infected with RV1B without addition of cathelicidin), *: P<0.05; **: P<0.01; ***: P<0.001 compared to HPBECs infected with RV1B without addition of cathelicidin, as determined by one-way ANOVA with Bonferroni comparison; : P<0.05, : P<0.01, : P<0.001 compared between different doses of cathelicidin from virus-cathelicidin mixture used in experiments, as determined by one-way ANOVA with Bonferroni comparison.

Graph A: n=8, 100%=1,778e+006±415630 TCID50; Graph B: n=4, 100%=20538±9446 TCID50.

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