https://orcid.org/0000-0003-4565-094X
Figures
Abstract
Introduction
Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune mechanisms in vitro.
Methods
Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10−7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs.
Results
Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1.
Citation: Stapleton EM, Keck K, Windisch R, Stroik MR, Thurman AL, Zabner J, et al. (2022) Vitamin D-mediated effects on airway innate immunity in vitro. PLoS ONE 17(6): e0269647. https://doi.org/10.1371/journal.pone.0269647
Editor: Y. Peter Di, University of Pittsburgh, UNITED STATES
Received: March 3, 2022; Accepted: May 24, 2022; Published: June 6, 2022
Copyright: © 2022 Stapleton et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This study was carried out with funding support from 5P01HL091842-13 (JZ): https://grantome.com/grant/NIH/P01-HL091842-13-5864. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Vitamin D is a well-established immunomodulator and potentiator of airway innate defense. Two observations support that vitamin D is associated with a reduction in respiratory infection risk. First, those with greater serum vitamin D (25(OH)D3) concentrations have a lower risk of respiratory tract infections and demonstrate higher lung function [1, 2]. Second, respiratory infection incidence increases during winter, when serum vitamin D levels are at their nadir [3, 4]. Vitamin D deficiency is also considered to be a risk factor for pulmonary inflammatory diseases [5], and respiratory exacerbations (asthma, chronic obstructive pulmonary disease, chronic rhinosinusitis with nasal polyps) [6–8]. Lastly, a recent meta-analysis of randomized controlled clinical vitamin D supplementation trials demonstrated that daily or weekly vitamin D supplementation provided a significant protective effect against acute respiratory tract infection, with stronger protective effects in participants with lower baseline serum vitamin D levels [9].
Calcitriol is a synthetic vitamin D analog that binds to the vitamin D receptor to increase serum blood calcium [10]. It can transport calcium and phosphate ions across epithelial cells [11]. Human airway epithelial cells (hAECs) constitutively generate calcitriol [12], and vitamin D increases antimicrobial and anti-inflammatory airway epithelial cell response in vitro [13, 14].
Airway innate immune defense consists of a variety of independent and coordinated mechanisms. First, antimicrobial peptides (AMPS), whose potency is affected by pH regulation, are embedded within airway surface liquid (ASL) and can immediately kill incoming viral and bacterial pathogens [15], After this first line of ASL defense, surviving pathogens can be cleared by mucociliary transport, which propels pathogens toward the larynx via ciliary beating [16]. Lastly, neutrophils phagocytose residual debris [17, 18]; Neutrophils express Triggering Receptor Expressed on Myeloid Cells 1 (TREM1) which can augment the immune response to infection and trigger an inflammatory cascade [18]. The role of calcitriol supplementation on ciliary beat frequency (CBF) or neutrophil migration in hAECs remains undetermined.
We hypothesized that calcitriol supplementation of hAEC media would improve ASL antiviral and antibacterial properties. We tested this in vitro by viral (229E-CoV) and bacterial (Staphylococcus aureus) challenge. Secondarily, we hypothesized that calcitriol supplementation would affect requisite airway host defense mechanisms, including ASL pH regulation, ASL viscosity, hAEC CBF and genetic expression. Lastly, we assessed whether increased TREM1 expression after calcitriol supplementation would lead to increased neutrophil migration across hAECs.
Methods
Previous viral and immunological research in human airway cells employed a calcitriol supplementation dose of 10−7 M in airway media [19–22]. Therefore, we applied a 10−7 M calcitriol (Tocris Bioscience, Bristol, UK) dose to basolateral media of primary bronchial/tracheal hAECs cultured at the air liquid interface (ALI) [23, 24] maintained with 5% CO2 at 37°C. Briefly, human donor lungs obtained following informed written consent were provided by the Iowa Donor Network, then dissected and seeded onto collagen coated filters at the ALI by the University of Iowa Cell Culture Core Repository. The study is approved by the University of Iowa Institutional Review Board (IRB #199507432). hAEC basolateral media consisted of DMEM/F-12 with 1% penicillin-streptomycin, 50 mg/ml gentamicin, and 2% Ultroser G (USG, Pall BioSepra, Cergy, France). The exact composition of USG is confidential, but correspondence with the manufacturer indicates it does not contain calcitriol. After 22-24h (unless otherwise noted), the following experiments and analyses were performed on donor-paired samples. GraphPad Prism Software (version 9.0.2) was used for statistical analysis, and data are presented as individual data points or mean ± SEM. Significance was determined at p<0.05.
ASL antimicrobial activity against S. aureus
Bioluminescent S. aureus (Xen29, Caliper Lifesciences Bioware®) sub-cultured to log-phase growth was suspended in minimal media (5% TSB) and challenged (1:1) by ASL from calcitriol supplemented hAECs and by un-supplemented hAEC ASL [25]. Briefly, ASL was collected by serially washing (20 μl/well DI H20) hAEC apical surfaces from paired donor cells (n = 4 per experiment), after which media was changed back to baseline media for a >72h washout period. Subsequent calcitriol treatments were always applied to previously supplemented cells. Relative light units (RLUs), previously correlated to colony forming units [26], were read and immediate killing quantified by comparing RLUs within 12 minutes to RLUs at 0 min. To assess whether ASL was bactericidal, summary data (% live bacteria) per condition were compared to a hypothetical mean of 100 using a one-sample t-test. To assess differences between conditions, significance was assessed by paired t-test. All studies were performed in triplicate or quadruplicate. Vehicle and bleach controls were used to confirm ASL bacterial killing (S1 Fig). We also ensured that S. aureus growth between untreated and calcitriol-treated media was not different (S2 Fig).
ASL antiviral activity against 229E-CoV
Donor-paired (n = 4/experiment) ASL from supplemented and un-supplemented hAECs or vehicle control was combined with human coronavirus 229E-CoV (ATCC® VR-740™) for 10 min at 33°C, then applied to MRC-5 cells (ATCC® CCL171™) for 1h (33°C + 5% CO2). After 4–7 days we assessed plaque formation, as previously described [27]. Statistical significance was assessed by paired t-test and all studies were performed in triplicate.
Effect of calcitriol supplementation on hAEC ASL pH
To assess the effect of calcitriol supplementation on ASL pH, apical hAEC surfaces were treated with 40 μl warm saline +1 mM HEPES solution (Research Products International, Mt Prospect, IL, USA) concurrently with basolateral media changes. After 22h, apical fluid was collected, and pH read by a pH microelectrode (Orion 9810BN, Thermo Scientific). Duplicate or triplicate paired samples from 9 donors were collected and significance determined by paired t-test.
Effect of calcitriol supplementation on ASL relative viscosity
FITC-conjugated to 70kDa dextran (D1822; Thermo Fisher Scientific, Waltham, MA, USA) was applied into ASL using a sterile 100 μm nylon strainer (352360; Falcon, Durham, NC, USA) and left to absorb for ≥12 hours. For photobleaching experiments, cells were placed in fresh cell culture media ± calcitriol within a 37°C chamber with 5% CO2. FITC was excited with a 488 nm laser then two baseline images were acquired (Zeiss LSM880 microscope). Then, a small circular region (radius 5.2 μm) was photobleached and fluorescent recovery monitored for >1 min. Fluorescent recovery within ASL was compared to experiments with the FITC-dextran in phospho-buffered saline. ASL viscosity is represented as the relative viscosity to the saline sample.
Effect of calcitriol supplementation on hAEC ciliary beat frequency
To test the effect of calcitriol on CBF, we applied supplemented media to the basolateral surface of hAECs (n = 4 and n = 8, depending on timepoint) after first measuring baseline CBF. CBF was acquired using brightfield imaging (Zeiss LSM880) across timepoints (45 min, 90 min, 24h). Line scanning mode was used to acquire images every 1.02 milliseconds (~980 Hz). A 40x LD C-Apochromat 40x/1.1 W Korr M27 objective (Carl Zeiss) was used to obtain images. hAECs were studied within a 37°C chamber containing 5% CO2. Within a single image, line scans often captured several ciliated cells. Each line scan was averaged into one value and five line-scans were performed per donor. We confirmed results by obtaining microscopic images of CBF using a Zeiss (Carl Zeiss AG, Oberkochen, Germany) Axio Observer microscope, studied under 37°C and ambient air.
Effect of calcitriol supplementation on hAEC gene expression
We tested the effect of calcitriol supplementation on hAEC gene expression using RNA sequencing. After 24h of treatment, mRNA was isolated from paired hAEC (n = 6) on polycarbonate filters, using the RNeasy Lipid Tissue Kit (Qiagen) according to the manufacturer’s instructions. Genomic DNA was digested using DNase I (Qiagen). RNA samples were then quantified using fluorimetry (Qubit 2.0 fluorometer; Life Technologies), and RNA quality was assessed using an Agilent BioAnalyzer 2100 (Agilent Technologies).
RNA library prep and sequencing
Library preparation and sequencing were conducted at the Iowa Institute of Human Genetics Genomics Sequencing Division core facility, University of Iowa. Briefly, an Illumina Stranded mRNA Prep kit was used to isolate oligo-dT purified polyadenylated RNA. The samples were then reverse transcribed to create cDNA. The cDNA was fragmented, blunt-ended, and ligated to indexed adaptors. Following quantification of the cDNA generated for the library, the samples were clustered and loaded equally over two lanes on an SP flow cell on an Illumina NovaSeq 6000 Sequencing system for 50bp paired end reads.
RNA sequencing analysis
Raw sequencing reads were pseudoaligned to human reference transcriptome GRCh38.p13 using Kallisto [28] version 0.45.0, and a gene-by-sample count matrix was generated for differential gene expression analysis with iDEP v.0.92 [29]. Next, up- and downregulated genes (p≤0.10) from our calcitriol supplemented hAECs were compared with human AMP-related genes putatively involved in a variety of innate immune response, and human genes involved in motile cilium using QuickGO annotations [30].
Neutrophil migration across hAECs
Gene expression data revealed TREM1 to be highly upregulated in hAECs. TREM1 is abundantly expressed on human myeloid cells, and facilitates neutrophil migration across hAECs [18], therefore, we evaluated the role of calcitriol on neutrophil migration across hAECs in vitro.
Bronchial hAECs were obtained from human donor lungs, n = 4 (donors were on average 40 years old (SD = 18), and 50% female) and seeded at the ALI onto 3 μm-pore-size collagen-coated transwell filters (Corning Life Sciences, Durham, NC, USA) in an inverted fashion with basolateral support provided by growth-factor reduced Matrigel® Matrix basement membrane (Corning Life Sciences, Durham, NC, USA). Upon hAEC polarization (2 weeks), calcitriol (100 nM) was added to the basolateral media in half of the wells.
The following day (24h), fresh blood was obtained from one healthy volunteer donor, and neutrophils were isolated and suspended in HBSS-/- (Gibco), as previously described (with modifications) [31]. Next, hAEC Matrigel was removed and transwells were inverted into 400 μL USG, then neutrophils were applied to the basolateral membrane (200,000/well) to assess their migration to the apical membrane. Half of the transwells were suspended in 30% zymosan activated serum, ZAS, a chemoattractant for neutrophils [18]. After three hours, migrated neutrophils were collected from the plate using PBS -/-, 2mM EDTA, and TrypLE (Gibco/Invitrogen, Waltham, MA, USA). Migrated cells were stained with fixable aqua dead cell stain kit (Thermo Fisher Scientific, Waltham, MA, USA), Human BD Fc Block™ (BD Biosciences, San Jose, CA, USA), and APC anti-human CD66b antibody (Biolegend, San Diego, CA, USA). CD66b+ cells assessed by flow cytometry using an Attune™ NxT Flow Cytometer Autosampler (Invitrogen™/Thermo Fisher Scientific, Waltham, MA, USA). CD66b+ cells were subsequently analyzed using FloJo™ software (BD Biosciences, San Jose, CA, USA). Each condition was performed in triplicate and significance determined by one-sample t-test (theoretical mean = 100) of baseline-transformed replicate data by experiment.
Results
ASL antimicrobial and antiviral activity
Compared to control, untreated ASL killed S. aureus and ASL from hAECs treated with 100 nM calcitriol (for 24h) increased S. aureus killing (mean live S. aureus, untreated ASL = 40%, mean live S. aureus, treated ASL = 27%, one-sample t-test with hypothetical mean = 100, p<0.001 both conditions). ASL from supplemented hAECs was significantly more bactericidal than ASL from unsupplemented hAECs (paired t-test, p = 0.02), Fig 1A.
A. ASL from untreated (blue dots) and calcitriol-treated (orange dots) hAECs or vehicle control was challenged with bioluminescent S. aureus and RLUs were read within 12 minutes; individual points represent mean data from six individual experiments, ASL derived from 4–6 individual hAEC donors, * indicates p<0.05 B. ASL from untreated (blue dots) and calcitriol-treated (orange dots) hAECs compared to vehicle control. Conditions were challenged with 229E-CoV in MRC-5 human fibroblasts, and viral plaques quantified. Individual points represent mean data from seven individual experiments, ASL was derived from 4–6 individual hAEC donors; raw plaque values for control, untreated hAEC ASL, and supplemented hAEC ASL were compared by a paired Friedman test, ASL vs. vehicle control p<0.01, ASL from hAECs supplemented with calcitriol vs. vehicle control p = 0.049, treated vs. untreated ASL p>0.99. All studies were performed in triplicate and error bars represent mean± SEM.
ASL from both conditions significantly reduced 229E-CoV infectivity compared to vehicle (viral plaques with untreated ASL p<0.01 and with supplementation p<0.05). Calcitriol supplementation did not affect ASL-mediated 229E-CoV inactivation (paired t-test, p = 0.39), Fig 1B.
Effect of calcitriol supplementation on hAEC ASL pH, viscosity, and ciliary beat frequency
ASL pH from hAECs treated with basolateral calcitriol supplementation was not significantly different than the ASL pH from untreated control hAECs (p = 0.11), Fig 2A. Acute and 24-hour ASL viscosity did not change after calcitriol treatment (0 vs. 45 min p = 0.98, 0 vs. 90 min p = 0.20, 0 vs. 24h, p = 0.36), Fig 2B. Calcitriol supplementation did not affect CBF of hAECs acutely nor over 24h (0 vs. 45 min p = 0.98, 0 vs. 90 min p = 0.20, 0 vs. 24h, p = 0.36), Fig 2C.
A. Calcitriol free and supplemented basolateral hAEC media (n = 9 donors) was changed when apical saline+1mM HEPES solution was applied; after 22h ASL was collected, and pH read. Significance determined by paired t-test, p = 0.11. B. FITC-D within ASL (≥12h) was excited (488 nm), baseline images were acquired then a small circular region (radius 5.2 μm) was photobleached and fluorescent recovery monitored for >1 min; recovery was compared to FITC-D in PBS. Significance determined by one-way ANOVA with Šídák’s multiple comparisons test, 0 vs. 45 min p = 0.98, 0 vs. 90 min p = 0.20, 0 vs. 24h, p = 0.36. C. Time-course of paired (n = 4 or n = 8 per timepoint) hAEC CBF acquired using brightfield imaging before (0 min) and after 100 nM basolateral calcitriol supplementation, significance determined by one-way ANOVA with Šídák’s multiple comparisons test, 0 vs. 45 min p = 0.40, 0 vs. 90 min p = 0.20, 0 vs. 24h, p = 0.39.
Effect of calcitriol supplementation on hAEC gene expression
We assessed gene expression changes to hAECs after 24h calcitriol supplementation. The top ten significantly upregulated genes in calcitriol treated hAECs (compared to controls) were the following: CYP24A1, CAMP, PLG, HRCT1, SULT1C2, CA9, PRKG2, TREM1, IL1RL1, and PADI3 (all p<0.001). Compared to control epithelia, the following genes were the most downregulated in calcitriol treated hAECs: IL19, IGF2, PRRT1B, MUC13, SLC5A5, LTK, SLURP2, DEPP1, LAMB1, and SLC9A4 (all p<0.05).
Because AMP expression and composition affect ASL antiviral and antibacterial activity, we compared our RNA sequencing results with AMP-related genes involved in innate immune response (p≤0.10) from a previously published database [30]. Results from genes involved in a wide-range of host-defense are reported, including those involved in overall innate immune response, defense response to virus, type 1 interferon signaling, defense response to gram negative and positive bacterium, and antifungal response, as well as others. AMP-related genes most upregulated in our sample are shown in Table 1A, and those most downregulated can be seen in Table 1B.
Using the same protocol, we compared genes involved in human motile cilium against hAEC genes affected by supplementation. FSIP2, HIF1A and CABYR were upregulated (log2 fold-change = 0.53; p = 0.075, 0.36; p = 0.006, and 0.26; p = 0.064, respectively) and ATP1B1 was downregulated (log2 fold-change = 0.39; p = 0.009). These genes’ roles include motile cilium and sperm components.
Because calcitriol alters the expression of genes that regulate Ca2+, we compared genes associated with intracellular Ca2+ in our sample. TMEM37, TRPV6, and CHRNA7 were significantly upregulated (log2 fold-change = 1.43; p<0.001, 0.802; p<0.001, and 0.776; p<0.001, respectively), while CACNB1 was significantly downregulated (log2 fold-change = 0.567; p<0.001).
Neutrophil migration across hAECs
Because TREM1 was significantly upregulated in our calcitriol-treated sample, we assessed the effect of calcitriol supplementation on neutrophil migration across hAECs at the ALI. Overnight calcitriol treatment did not significantly affect neutrophil migration (p = 0.39), while ZAS increased neutrophil migration regardless of basolateral hAEC supplementation (p<0.05). Under ZAS supplementation, neutrophil migration tended to be higher in calcitriol-treated cells (paired t-test, p = 0.09) compared to untreated cells.
Discussion
Vitamin D supplementation may reduce the risk of respiratory infection and improve airway innate immunity, but mechanisms supporting its effect are not well understood. We systematically tested the effect of calcitriol supplementation on key innate immune mechanisms. We found that calcitriol supplementation of hAECs increased ASL antibacterial activity but did not increase its antiviral activity. Within our calcitriol-treated sample, cathelicidin (CAMP) was the second most upregulated gene, which likely explains the observed increase in antibacterial activity. Calcitriol treatment of hAECs did not impact ASL pH or viscosity but did acutely affect CBF. While TREM1 was overexpressed in our calcitriol-treated sample, we did not find an appreciable effect on neutrophil migration across hAECs in vitro.
Consistent with previous literature, calcitriol supplementation of hAECs led to significantly increased cathelicidin (CAMP/LL-37) expression (log2 FC = 7.36, p<0.001), Table 1A [32–34]; cathelicidin is one of many AMPs within the ASL [12, 20, 35, 36], and its expression has been shown to increase antibacterial (Mycobacterium tuberculosis) activity after calcitriol supplementation [14]. Additionally, silencing CAMP gene expression, and blocking LL-37, inhibits the cathelicidin-induced increase in antimicrobial activity [14, 37]. Therefore, the increased ASL-mediated S. aureus killing in calcitriol treated hAECs observed in our study is likely due to increased cathelicidin expression. However, given the abundance of AMP-related gene expression alterations, we cannot solely ascribe this finding to cathelicidin.
Antimicrobial bacterial killing is affected by ASL pH [38]. We did not find a difference in ASL pH between calcitriol-treated and untreated hAECs. Additionally, increased ASL viscosity has been observed in certain lung disease (e.g. cystic fibrosis), and viscosity changes can occur independently of ASL pH [39]; however, we found hAEC ASL viscosity after treatment with calcitriol was not significantly different compared to pre-treatment viscosity (p>0.20), Fig 2B. Next, we tested whether ciliary movement was altered upon calcitriol supplementation. Epithelial cilia work in a coordinated fashion to move incoming pathogens toward the larynx, and increased CBF abets this process. Changes to CBF may be due to activation of cellular signaling pathways (acutely) and/or alterations to gene expression (hours-days). As seen in Fig 2C, activation of the vitamin D receptor did not significantly change CBF over time. While CBF varied stochastically, it typically remained higher than prior to supplementation for most donors. This variation in CBF may result from gene expression-dependent (e.g., upregulation of FSIP2, HIF1A and CABYR or downregulation of ATP1B1) or gene expression-independent changes in cilia regulation. Fluctuations in CBF were observed within each set of experiments.
It has been proposed that vitamin D deficiency increases the risk of COVID-19 incidence and severity [40]. We recapitulated our previous work demonstrating ASL to be virucidal [41], however, we found no difference in ASL-mediated 229E-CoV viral inactivation between calcitriol-treated and untreated hAEC ASL. This is consistent with a recent finding from the United Kingdom where blood 25(OH)D concentrations did not correlate with COVID-19 infection risk [42]. However, vitamin D supplementation as a means to reduce respiratory infection in clinical trials yields inconsistent results [43]. In one retrospective cohort study (n = 489), likely vitamin D deficiency was imputed based on recent 25(OH)D3 or calcitriol serum levels and treatment and likely deficient vitamin D levels were associated with an increased rate of COVID-19 positivity [44]. In vitro work revealed that calcitriol treatment of hAECs can reduce inflammatory signaling after exposure to viral influenza (H1N1) infection, reducing autophagy and returning apoptosis to constitutive levels [22]. While evidence supports a role for vitamin D supplementation and reduced risk of viral infection, we focused on strictly innate immune ASL-mediated viral inactivation and found this not to be influenced by supplementation.
RNA sequencing data revealed calcitriol supplementation led to significantly increased TREM1 expression. Previous work has demonstrated increased TREM1 expression in calcitriol-treated hAECs from distinct cell lines [45], however, to our knowledge, ours is the first study to identify TREM1 expression in endogenous primary hAECs. TREM1 is a membrane-bound surface receptor with an unknown ligand, while its soluble form may act as a counterregulatory decoy receptor [46]. Because TREM1 is required for neutrophil migration across hAECs, we expected increased neutrophil migration in calcitriol-treated hAECs. We did not observe an increase in neutrophil migration after hAECs were treated with calcitriol, however, Fig 3. Zymosan activated serum, a neutrophil chemokine was used as a positive control and significantly increased neutrophil migration. We observed a slightly increased migration upon supplementation with ZAS (paired t-test p = 0.09) but did not find significant differences between control and calcitriol treated cells. This may indicate calcitriol supplementation can enhance migration in the presence of yeast pathogens.
Percent of migrated neutrophils across hAECs with (orange and black circles) and without (white circles) overnight calcitriol treatment, n = 4 hAEC donors, each dot represents the average of triplicate data per experiment, neutrophils isolated from one healthy donor. Neutrophil migration was performed under USG and 30% zymosan activated serum (ZAS) conditions; significance determined by comparing baseline-corrected data of each condition to untreated hAECs using a one-sample t-test with hypothetical mean of 100, * indicates p<0.05, ** indicates p<0.01, error bars indicate mean ±SEM.
Our study has several limitations. All studies were performed in vitro and extrapolation to in vivo effects is therefore limited. Our in vitro design relied on airway epithelia from organ donors whose vitamin D status was unknown at the time of organ harvest. Therefore, we are unable to speculate whether the epithelial innate immune response to vitamin D supplementation observed in our study was from donors with healthy or deficient vitamin D levels. Further, our calcitriol supplementation dose of 100 nM may be significantly greater than that found in healthy serum (~200 picomolar) [47], but we are unable to measure endogenous calcitriol in human airway epithelia and therefore selected the dose previously assayed with human airway cells [20–22]. For any given experiment, at least four donors were studied, so it is unlikely, but still possible, that all were deficient. To reduce variability in response to the vitamin D receptor, our experiments utilized active vitamin D (1,25(OH)2D3), thus our results cannot be extrapolated to the stimulation of cells with inactive vitamin D (25(OH)2D3). Our experimental design relied on supplementation ≤24 hours, and therefore effects from longer stimulation are unknown. We did not evaluate synergistic and additive effects of AMPs. Our antimicrobial results are limited to ASL effects on one type of relevant bacteria and virus, and we cannot extrapolate these towards all pathogen species, including other bacteria, viruses, fungal and mycobacterial agents. For neutrophil migration assays, only hAECs were stimulated and not neutrophils, which may have led to effects on migration. Finally, we used primary hAECs and our results cannot be extrapolated to other cell types, including alveolar epithelial cells.
Despite these limitations, our findings demonstrate that calcitriol treatment increases ASL bactericidal properties (S. aureus) and significantly alters hAEC gene expression, especially AMP-related genes, while calcitriol does not affect ASL-antiviral (229E-CoV) properties, pH, viscosity or CBF. In addition, neutrophil migration may be increased if chemokines are present in the air space. Vitamin D supplementation to increase active vitamin D concentrations may provide a useful tool to resolve respiratory bacterial infections.
Supporting information
S1 Fig. Relative light units of control conditions from S. aureus bacterial killing assays.
Control (vehicle) media and S. aureus resulted in a slight decline of bacteria over 12 minutes, while bleach and S. aureus led to complete bacterial killing over the same time.
https://doi.org/10.1371/journal.pone.0269647.s001
(DOCX)
S2 Fig. S. aureus growth in media with and without calcitriol supplementation.
A clinically isolated strain of methicillin-resistant S. aureus was cultured overnight in tryptic soy broth (TSB), then sub-cultured (1:100) in TSB +/- 10−7 M calcitriol. Paired t-tests of % growth compared to time 0 (at hours 1–6) demonstrated no statistically significant differences between control and supplemented media (all p>0.05). This experiment was independent of airway mechanisms.
https://doi.org/10.1371/journal.pone.0269647.s002
(DOCX)
Acknowledgments
We would like to thank Sankar Baruah at the University of Iowa for his TREM1 assistance and trouble shooting. We would also like to thank Christian Zirbes for providing us with an S. aureus clinical isolate for our bacterial growth experiments, and for his prompt and helpful assistance. The University of Iowa Cell Culture Core provided invaluable assistance in isolating primary human donor cells and providing hAECs at the ALI.
References
- 1. Berry DJ, Hesketh K, Power C, Hyppönen E. Vitamin D status has a linear association with seasonal infections and lung function in British adults. British Journal of Nutrition. 2011;106(9):1433–40. pmid:21736791
- 2. Jartti T, Ruuskanen O, Mansbach JM, Vuorinen T, Camargo CA Jr. Low serum 25-hydroxyvitamin D levels are associated with increased risk of viral coinfections in wheezing children. The Journal of allergy and clinical immunology. 2010;126(5):1074. pmid:21050947
- 3. Hope-Simpson RE. The role of season in the epidemiology of influenza. Epidemiology & Infection. 1981;86(1):35–47. pmid:7462597
- 4. Cannell J, Vieth R, Umhau J, Holick M, Grant W, Madronich S, et al. Epidemic influenza and vitamin D. Epidemiology & Infection. 2006;134(6):1129–40. pmid:16959053
- 5. Lin J, Liu J, Davies ML, Chen W. Serum vitamin D level and rheumatoid arthritis disease activity: review and meta-analysis. PloS one. 2016;11(1):e0146351. pmid:26751969
- 6. Burkes RM, Ceppe AS, Doerschuk CM, Couper D, Hoffman EA, Comellas AP, et al. Associations Among 25-Hydroxyvitamin D Levels, Lung Function, and Exacerbation Outcomes in COPD. 2020. pmid:31958447
- 7. Brehm JM, Schuemann B, Fuhlbrigge AL, Hollis BW, Strunk RC, Zeiger RS, et al. Serum vitamin D levels and severe asthma exacerbations in the Childhood Asthma Management Program study. Journal of Allergy and Clinical Immunology. 2010;126(1):52–8. e5. pmid:20538327
- 8. Wynne M, Atkinson C, Schlosser RJ, Mulligan JK. Contribution of epithelial cell dysfunction to the pathogenesis of chronic rhinosinusitis with nasal polyps. American journal of rhinology & allergy. 2019;33(6):782–90.
- 9. Martineau AR, Jolliffe DA, Hooper RL, Greenberg L, Aloia JF, Bergman P, et al. Vitamin D supplementation to prevent acute respiratory tract infections: systematic review and meta-analysis of individual participant data. bmj. 2017;356. pmid:28202713
- 10. Lung BE, Mowery ML, Komatsu DEE. Calcitriol. 2018.
- 11. Barminko J, Reinholt BM, Emmanuelli A, Lejeune AN, Baron MH. Activation of the vitamin D receptor transcription factor stimulates the growth of definitive erythroid progenitors. Blood advances. 2018;2(11):1207–19. pmid:29844206
- 12. Hansdottir S, Monick MM. Vitamin D effects on lung immunity and respiratory diseases. Vitamins & hormones. 2011;86:217–37. pmid:21419273
- 13. Zdrenghea MT, Makrinioti H, Bagacean C, Bush A, Johnston SL, Stanciu LA. Vitamin D modulation of innate immune responses to respiratory viral infections. Reviews in medical virology. 2017;27(1):e1909. pmid:27714929
- 14. Liu PT, Stenger S, Tang DH, Modlin RL. Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin. The journal of immunology. 2007;179(4):2060–3. pmid:17675463
- 15. Pezzulo AA, Tang XX, Hoegger MJ, Alaiwa MHA, Ramachandran S, Moninger TO, et al. Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature. 2012;487(7405):109–13. pmid:22763554
- 16. Delmotte P, Sanderson MJ. Ciliary beat frequency is maintained at a maximal rate in the small airways of mouse lung slices. Am J Respir Cell Mol. 2006;35(1):110–7.
- 17. Segal AW. How neutrophils kill microbes. Annu Rev Immunol. 2005;23:197–223. pmid:15771570
- 18. Klesney-Tait J, Keck K, Li X, Gilfillan S, Otero K, Baruah S, et al. Transepithelial migration of neutrophils into the lung requires TREM-1. The Journal of clinical investigation. 2012;123(1). pmid:23241959
- 19. Gui B, Chen Q, Hu C, Zhu C, He G. Effects of calcitriol (1, 25-dihydroxy-vitamin D3) on the inflammatory response induced by H9N2 influenza virus infection in human lung A549 epithelial cells and in mice. Virology journal. 2017;14(1):1–11.
- 20. Hansdottir S, Monick MM, Lovan N, Powers L, Gerke A, Hunninghake GW. Vitamin D decreases respiratory syncytial virus induction of NF-κB–linked chemokines and cytokines in airway epithelium while maintaining the antiviral state. The journal of immunology. 2010;184(2):965–74. pmid:20008294
- 21. Banerjee A, Damera G, Bhandare R, Gu S, Lopez‐Boado Y, Panettieri R Jr, et al. Vitamin D and glucocorticoids differentially modulate chemokine expression in human airway smooth muscle cells. British journal of pharmacology. 2008;155(1):84–92. pmid:18552877
- 22. Khare D, Godbole NM, Pawar SD, Mohan V, Pandey G, Gupta S, et al. Calcitriol [1, 25 [OH] 2 D3] pre-and post-treatment suppresses inflammatory response to influenza A (H1N1) infection in human lung A549 epithelial cells. European journal of nutrition. 2013;52(4):1405–15. pmid:23015061
- 23. Pezzulo AA, Starner TD, Scheetz TE, Traver GL, Tilley AE, Harvey BG, et al. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol. 2011;300(1):L25–31. pmid:20971803
- 24. Karp PH, Moninger TO, Weber SP, Nesselhauf TS, Launspach JL, Zabner J, et al. An in vitro model of differentiated human airway epithelia. Epithelial cell culture protocols: Springer; 2002. p. 115–37.
- 25. Stapleton EM, Manges R, Parker G, Stone EA, Peters TM, Blount RJ, et al. Indoor particulate matter from smoker homes induces bacterial growth, biofilm formation and impairs airway antimicrobial activity. A pilot study. Public Health Front. 2019;7:418.
- 26. Buonfiglio LGV, Mudunkotuwa IA, Alaiwa MHA, Calderón OGV, Borcherding JA, Gerke AK, et al. Effects of Coal Fly Ash Particulate Matter on the Antimicrobial Activity of Airway Surface Liquid. Environ Health. 2017;125(7).
- 27. Payne S. Methods to study viruses. Viruses. 2017:37.
- 28. Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nature biotechnology. 2016;34(5):525–7. pmid:27043002
- 29. Ge SX, Son EW, Yao R. iDEP: an integrated web application for differential expression and pathway analysis of RNA-Seq data. BMC bioinformatics. 2018;19(1):1–24. pmid:30567491
- 30. Binns D, Dimmer E, Huntley R, Barrell D, O’donovan C, Apweiler R. QuickGO: a web-based tool for Gene Ontology searching. Bioinformatics. 2009;25(22):3045–6. pmid:19744993
- 31. Oh H, Siano B, Diamond S. Neutrophil isolation protocol. J Vis Exp JOVE-J VIS EXP. 2008(17). pmid:19066523
- 32. White JH. Vitamin D as an inducer of cathelicidin antimicrobial peptide expression: past, present and future. The Journal of steroid biochemistry and molecular biology. 2010;121(1–2):234–8. pmid:20302931
- 33. Vukić M, Neme A, Seuter S, Saksa N, de Mello VD, Nurmi T, et al. Relevance of vitamin D receptor target genes for monitoring the vitamin D responsiveness of primary human cells. PloS one. 2015;10(4):e0124339. pmid:25875760
- 34. Gombart AF, Borregaard N, Koeffler HP. Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up‐regulated in myeloid cells by 1, 25‐dihydroxyvitamin D3. The FASEB journal. 2005;19(9):1067–77. pmid:15985530
- 35. Liu PT, Stenger S, Li H, Wenzel L, Tan BH, Krutzik SR, et al. Toll-like receptor triggering of a vitamin D-mediated human antimicrobial response. Science. 2006;311(5768):1770–3. pmid:16497887
- 36. Hansdottir S, Monick MM, Hinde SL, Lovan N, Look DC, Hunninghake GW. Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense. The Journal of Immunology. 2008;181(10):7090–9. pmid:18981129
- 37. Buonfiglio LGV, Cano M, Pezzulo AA, Calderon OGV, Zabner J, Gerke AK, et al. Effect of vitamin D 3 on the antimicrobial activity of human airway surface liquid: preliminary results of a randomised placebo-controlled double-blind trial. BMJ open respiratory research. 2017;4(1):e000211. pmid:28883932
- 38. Alaiwa MHA, Reznikov LR, Gansemer ND, Sheets KA, Horswill AR, Stoltz DA, et al. pH modulates the activity and synergism of the airway surface liquid antimicrobials β-defensin-3 and LL-37. Proceedings of the National Academy of Sciences. 2014;111(52):18703–8. pmid:25512526
- 39. Tang XX, Ostedgaard LS, Hoegger MJ, Moninger TO, Karp PH, McMenimen JD, et al. Acidic pH increases airway surface liquid viscosity in cystic fibrosis. The Journal of clinical investigation. 2016;126(3):879–91. pmid:26808501
- 40. Grant WB, Lahore H, McDonnell SL, Baggerly CA, French CB, Aliano JL, et al. Evidence that vitamin D supplementation could reduce risk of influenza and COVID-19 infections and deaths. Nutrients. 2020;12(4):988.
- 41. Stapleton EM, Welch JL, Ubeda EA, Xiang J, Zabner J, Thornell IM, et al. Urban particulate matter impairs airway-surface-liquid-mediated coronavirus inactivation. The Journal of infectious diseases. 2021.
- 42. Hastie CE, Mackay DF, Ho F, Celis-Morales CA, Katikireddi SV, Niedzwiedz CL, et al. Vitamin D concentrations and COVID-19 infection in UK Biobank. Diabetes & Metabolic Syndrome: Clinical Research & Reviews. 2020;14(4):561–5.
- 43. Jolliffe DA, Griffiths CJ, Martineau AR. Vitamin D in the prevention of acute respiratory infection: systematic review of clinical studies. The Journal of steroid biochemistry and molecular biology. 2013;136:321–9. pmid:23220552
- 44. Meltzer DO, Best TJ, Zhang H, Vokes T, Arora V, Solway J. Association of vitamin D status and other clinical characteristics with COVID-19 test results. JAMA network open. 2020;3(9):e2019722–e. pmid:32880651
- 45. Rigo I, McMahon L, Dhawan P, Christakos S, Yim S, Ryan LK, et al. Induction of triggering receptor expressed on myeloid cells (TREM-1) in airway epithelial cells by 1, 25 (OH) 2 vitamin D3. Innate immunity. 2012;18(2):250–7. pmid:21690199
- 46. Baruah S, Keck K, Vrenios M, Pope MR, Pearl M, Doerschug K, et al. Identification of a novel splice variant isoform of TREM-1 in human neutrophil granules. The Journal of Immunology. 2015;195(12):5725–31. pmid:26561551
- 47. Souberbielle J-C, Cavalier E, Delanaye P, Massart C, Brailly-Tabard S, Cormier C, et al. Serum calcitriol concentrations measured with a new direct automated assay in a large population of adult healthy subjects and in various clinical situations. Clinica Chimica Acta. 2015;451:149–53.