Vitamin D has been linked to reduced risk of viral respiratory illness. We hypothesized that vitamin D could directly reduce rhinovirus (RV) replication in airway epithelium. Primary human bronchial epithelial cells (hBEC) were treated with vitamin D, and RV replication and gene expression were evaluated by quantitative PCR. Cytokine/chemokine secretion was measured by ELISA, and transepithelial resistance (TER) was determined using a voltohmmeter. Morphology was examined using immunohistochemistry. Vitamin D supplementation had no significant effects on RV replication, but potentiated secretion of CXCL8 and CXCL10 from infected or uninfected cells. Treatment with vitamin D in the form of 1,25(OH)2D caused significant changes in cell morphology, including thickening of the cell layers (median of 46.5 µm [35.0–69.0] vs. 30 µm [24.5–34.2], p<0.01) and proliferation of cytokeratin-5-expressing cells, as demonstrated by immunohistochemical analysis. Similar effects were seen for 25(OH)D. In addition to altering morphology, higher concentrations of vitamin D significantly upregulated small proline-rich protein (SPRR1β) expression (6.3 fold-induction, p<0.01), suggestive of squamous metaplasia. Vitamin D treatment of hBECs did not alter repair of mechanically induced wounds. Collectively, these findings indicate that vitamin D does not directly affect RV replication in airway epithelial cells, but can influence chemokine synthesis and alters the growth and differentiation of airway epithelial cells.
Citation: Brockman-Schneider RA, Pickles RJ, Gern JE (2014) Effects of Vitamin D on Airway Epithelial Cell Morphology and Rhinovirus Replication. PLoS ONE 9(1): e86755. doi:10.1371/journal.pone.0086755
Editor: Gernot GU. Rohde, Maastricht University Medical Center (MUMC), The Netherlands
Received: July 25, 2013; Accepted: December 16, 2013; Published: January 24, 2014
Copyright: © 2014 Brockman-Schneider 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.
Funding: This work was supported by NIH grant #P01 HL070831-06A1 (http://www.nih.gov/). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have read the journal′s policy and have the following conflicts: Dr. Gern is a consultant for GlaxoSmithKline, Merck Inc., AstraZeneca, Gilead, and Boehringer Ingelheim, and has had investigator initiated research grants from Merck Inc, GlaxoSmithKline, and AstraZeneca. Co-author Raymond J.Pickles is a PLOS ONE Editorial Board member. This does not alter the authors′ adherence to all the PLOS ONE policies on sharing data and materials.
Vitamin D3 is a fat soluble hormone obtained primarily through sun exposure, and to a lesser extent from the diet. Activation of vitamin D3 requires two sequential hydroxylation steps. The first takes place in the liver, where the enzyme 25-hydroxylase converts vitamin D3 to 25-hydroxyvitamin D3 (25(OH)D): the circulating, pre-hormone form of the vitamin. Subsequently, 25-hydroxyvitamin D3 is converted to 1,25-dihydroxyvitamin D3 (1,25(OH)2D) through the action of the enzyme 1α-hydroxylase. This enzyme has traditionally been associated with the kidney, but has since been found elsewhere in the body, including in bronchial epithelial cells . This latter compound, 1,25(OH)2D, represents the active, hormonal form of vitamin D3. Recent evidence indicates that vitamin D3 is involved in regulating genes that play a role in immunity , . In addition, vitamin D acts on epithelial cells to stimulate the secretion of cathelicidin and other peptides that protect against infections with bacteria and enveloped viruses , .
There is clinical evidence that vitamin D levels are inversely related to respiratory illnesses, as well as exacerbations of asthma, which are often provoked by viruses such as rhinoviruses (RV) , . The respiratory epithelium plays a critical role in defending against RVs through the activation of antiviral pathways, and the secretion of chemokines that recruit effector cells to the site of infection. In addition, the barrier function of airway epithelium also protects against RV infection; disruption of an intact epithelial layer in vitro significantly enhances RV replication . Collectively, these findings suggest that vitamin D could inhibit the growth of RVs, either directly or indirectly by influencing the growth and/or differentiation of the airway epithelium.
To test this hypothesis, we added vitamin D to primary cultures of human bronchial epithelial cells (hBEC), and measured effects of vitamin D on RV replication, hBEC morphology and growth, epithelium integrity by monitoring transepithelial resistance (TER), and alterations in select gene expression levels. Two different models, involving addition of vitamin D to cells either during or following differentiation, were enlisted to investigate effects of vitamin D on airway epithelial cells.
Here we report that vitamin D does not directly affect RV replication in airway epithelial cells. Vitamin D does induce the synthesis of two chemokines, CXCL8 and CXCL10, showing an additive effect in conjunction with viral infection. In the course of conducting these experiments, it was incidentally noted that vitamin D has significant effects on the morphology of cultured cell layers, and higher concentrations of vitamin D produce changes similar to those of vitamin A deficiency.
Materials and Methods
Primary hBECs were derived from residual human surgical specimens from healthy lung donors. The protocol was approved by the University of Wisconsin’s Institutional Review Board, which waived the need for consent.
Differentiation of hBECs with Vitamin D Metabolites
Passage 1 primary hBECs were prepared in permeable membrane supports (12-well; 0.4 µm pore size, Corning Incorporated, Corning, NY) as previously described , . After 24 hrs, BEGM medium was removed from both the upper and lower chamber, and medium in the lower chamber only was replaced with 1 mL of air-liquid interface (ALI) medium, consisting of a 50∶50 mixture of BEGM and DMEM (Mediatech, Manassas, VA) that had been supplemented with all the BEGM additives (Lonza) except retinoic acid. To this mixture, fresh all-trans retinoic acid (50 nM, Sigma-Aldrich) was added, along with either 1,25(OH)2D or 25(OH)D (0.1, 1, 10, and 100 nM, Sigma-Aldrich). Since serum levels of 30–80 ng/mL (75–200 nM) 25(OH)D are considered optimal for health, with levels of 1,25(OH)2D being dependent upon the degree of localized synthesis, the range of concentrations used in our experiments did not exceed the physiologic range . Medium with retinoic acid was replaced daily during the first week, and then every 2–3 days. Transepithelial resistance measurements were made using a voltohmmeter.
Treatment of Fully Differentiated hBECs with 1,25(OH)2D
Cultures were prepared as above, with 1,25(OH)2D withheld until differentiated cultures were established (4 weeks), after which 10 nM 1,25(OH)2D was added to medium in lower compartment.
Differentiated cell layers were fixed with 10% phosphate-buffered formalin, and were stained with hemotoxylin and eosin, and alcian blue (VA Hospital, Madison, WI), as well as acetylated alpha tubulin (mouse mAb, Abcam, Cambridge, MA) and fibroblast specific protein (polyclonal rabbit anti-FSP1/S100A4, Millipore, Temecula, CA; UW-Madison’s Carbone Cancer Center Experimental Pathology Laboratory). Staining for MUC5AC, smooth muscle actin (SMA), and cytokeratin (CK 5/6) was performed by US Labs (Brentwood, TN). Average thicknesses of cell layers were determined as follows: sequential photos were taken along the length of each stained section, the area (µm2) and length (µm) of the section in each photo was measured using ImageJ software, and area/length (µm) calculations represented the average thickness of the layers.
Wounding of Cell Layers
Differentiated cell layers were wounded with a sterile P200 pipet tip . Cell layers were washed to remove loose cells, and wound areas in photos were measured using SigmaScan Pro 5 image measurement software.
Infection of Cells
Differentiated cell layers were incubated with purified RV-A16 (5×106 pfu/chamber, MOI = 10). Experiments that examined lower inoculating doses included 10-fold dilutions of the virus (5×105 pfu/chamber, MOI = 1; 5×104 pfu/chamber, MOI = 0.1). Cultures were incubated for 4 hrs at 34°C and then cell layers were washed 3 times. Cultures were then incubated for an additional 24 hrs (34°C, 5% CO2), after which medium in the lower chamber was cryopreserved for future analysis, and cell lysates were prepared.
RNA was extracted from cell lysates with Trizol (Invitrogen, Carlsbad, CA), treated with DNase (Promega, Madison, WI) for 15 minutes at 37°C, and cleaned up with a MinElute kit (Qiagen, Valencia, CA). cDNA was synthesized using a TaqMan RT kit (Applied Biosystems, Foster City, CA). RV RNA was measured using real-time PCR (Prism 7000, Applied Biosystems, Foster City, Calif) with primers and probes described previously . The standard curve was developed by extracting a known concentration of RV16 (1×103–1×108 PFU). SPRR1β and cathelicidin (CAMP) mRNA were quantitated using RT2 qPCR primer assays for SYBR® Green human SPRR1β and human CAMP from SABiosciences (Frederick, MD).
CXCL8 protein in medium from lower compartments of wells was measured using a sandwich ELISA . The samples were assayed in duplicate and the results expressed in pg/mL, with an assay sensitivity of 3–6 pg/mL. CXCL10, CCL5, IL-29 and IL-6 were measured by multiplex ELISA (Milliplex® MAP Kit, Millipore Corporation, Billerica, MA), with samples assayed in duplicate. Assay sensitivity for CXCL10, CCL5, and IL-6 was 3 pg/mL, and 195 pg/mL for IL-29. The type III interferon, IL-29, was selected for measurement because it is the predominant interferon detected in this system. In preliminary experiments, IFN-β was present only at the threshold of detection, and in a minority of samples.
Thicknesses of cell layers were compared using paired t-test and Wilcoxon Signed Rank test using software (SigmaStat, Systat Inc.). SPRR1β and CAMP gene expression was analyzed by ΔΔCT method, with gene expression normalized to β-actin, and statistical analyses were done using paired t-test (SigmaStat). Treatment groups in RV16 replication experiments were compared via t-test or Mann-Whitney Rank Sum, also using software (SigmaStat). Effects of different levels of 1,25(OH)2D on cytokine/chemokine production in RV-infected cells were analyzed using three way ANOVA (Sigmastat).
1,25(OH)2D and RV Replication and RV-induced Cytokine Secretion
To determine whether vitamin D inhibits RV replication in epithelial cells, hBECs differentiated in the presence of 0, 0.1, 1, and 10 nM 1,25(OH)2D were infected with RV16 (MOI = 10) for 24 hours. Treatment of hBECs with 1,25(OH)2D had no significant effect on the replication of RV16 (Fig. 1A). This was true even at lower inoculating doses of virus (MOI of 0.1 and 1, Fig. 1B). RV16 induced CXCL8 (p<0.01) and CXCL10 (p<0.001). In addition, 1,25(OH)2D treatment enhanced secretion of CXCL8 (p<0.03) and CXCL10 (p<0.03) production, either in the presence or absence of infection (Fig. 2A, 2B). While RV infection stimulated increased production of CCL5, IL-6, and IL-29 by differentiated epithelial cells, treatment with 1,25(OH)2D did not alter this effect (Fig. 2C, 2D, 2E).
(A) Replication of RV16 at 24 hours in differentiated hBEC cultured at air-liquid interface for 24 days in the presence of various concentrations of 1,25(OH)2D (n = 7). (B) Replication of RV16 (MOI = 0.1, 1, or 10) at 24 hours in differentiated hBEC cultured at air-liquid interface for 27 days in the presence of 10 nM of 1,25(OH)2D (n = 3).
(A,B) 1,25(OH)2D enhanced RV-induced CXCL8 (n = 6), and CXCL10 (n = 5) secretion. (C–E) Production of CCL5, IL-29, and IL-6 (n = 6) in these same cells, with “n.d.” indicating non-detectable levels. *p≤0.05 for cells treated with 1,25(OH)2D compared to untreated cells.
Effects of Vitamin D on Cell Morphology and Transepithelial Resistance
While preparing differentiated cells for the RV inoculation experiments, we noted that adding 1,25(OH)2D to the cultures led to thickened cell layers by day 25 (Fig. 3A). In addition, vitamin D also altered cell morphology, and some flattened cells at the base of the cell layer were observed. These sections stained negative for smooth muscle actin, vimentin and fibroblast specific protein, and apical cells stained positive for acetylated alpha tubulin; a robust ciliated cell marker (data not shown). Enhanced expression of cytokeratin 5/6, a basal cell marker, was seen in the subapical cells (Fig. 3B). Alcian blue staining revealed the presence of increasing numbers of mucin-filled cysts with higher levels of 1,25(OH)2D (Fig. 3C). These changes became so exaggerated in cell layers exposed to the upper limit of the concentration range (100 nM) as to create a dysmorphic appearance. Treatment with 10 nM 1,25(OH)2D caused significant thickening whether it was added during differentiation (Fig. 3D) or after differentiation (Fig. 3E).
(A) Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)2D. (B) Staining of paraffin cross-sections from 1,25(OH)2D-treated primary hBEC cell layers with antibody against cytokeratin 5/6 (CK5/6). (C) Alcian blue stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 24 days in the presence of 10 nM 1,25(OH)2D. (D) Average thicknesses of primary hBEC layers differentiated in the presence of 10 nM 1,25(OH)2D for 4 to 6 weeks (n = 8). (E) Average thicknesses of fully differentiated primary hBEC layers after treatment with 10 nM 1,25(OH)2D for 6 weeks (n = 5).
Respiratory epithelial cells can express 1α-hydroxylase , suggesting that the vitamin D precursor 25(OH)D and the active hormone should have similar effects on cell morphology. To test this theory, we treated differentiating hBEC cultures with 0, 0.1, 1, and 10 nM of either 1,25(OH)2D or 25(OH)D over the course of 21 days. Both forms of vitamin D caused similar thickening of the hBEC layers (Fig. 4).
Hematoxylin and eosin stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of various concentrations of 1,25(OH)2D or 25(OH)D.
Notably, culturing hBEC in the presence of 0, 0.1, 1 or 10 nM 1,25(OH)2D had no effect on transepithelial resistance (data not shown), an indicator of tight junction formation. Transepithelial resistance was somewhat lower for cell layers treated with 100 nM of 1,25(OH)2D, likely a function of the dysmorphic changes noted previously. Time until cilia development also was not affected by 1,25(OH)2D treatment, with cilia detected between day 17 and 21 as is typical in this culture system (data not shown). Finally, vitamin D did not affect healing of differentiated cells after mechanical wounding (data not shown).
Expression of SPRR1β and Cathelicidin
To further characterize the nature of the cells in the thickened cell layers of calcitriol-treated cultures, expression of small proline-rich protein 1β (SPRR1β; also “cornifin”), a marker of squamous metaplasia –, was examined via quantitative PCR. Treatment of fully differentiated cultures with 10 nM 1,25(OH)2D for 6 weeks caused a 14-fold upregulation of SPRR1β expression (p<0.05), suggesting that the cells making up the bulk of the thickened layers were squamous in nature. As a positive control, we also measured expression of cathelicidin (CAMP/hCAP18/LL-37), a vitamin D-induced antimicrobial peptide . Treatment of epithelial cells with 10 nM 1,25(OH)2D either during or after differentiation significantly induced SPRR1β (Table I), and similar trends were noted for cathelicidin.
Vitamin D vs. Vitamin A Effects on Differentiation of hBECs
The vitamin D receptor (VDR) shares a common subunit (retinoid X receptor, “RXR”) with the vitamin A receptor (RAR), suggesting that vitamin D could interfere with vitamin A-induced cell differentiation , . To examine possible interaction between the two compounds on epithelial cell differentiation, hBECs were differentiated in the presence of increasing levels of 1,25(OH)2D and all-trans retinoic acid (vitamin A). Retinoic acid is required for epithelial cell differentiation , and the layers failed to successfully differentiate with 0 nM and 1 nM retinoic acid, regardless of the level of 1,25(OH)2D that was present. Interestingly, cells that were differentiated in the presence of a suboptimal level (10 nM) of retinoic acid (and without vitamin D) exhibited some of the same morphological features (thickened layers, mucous cysts) as those differentiated in the presence of increased levels of 1,25(OH)2D (Fig. 5). For example, treatment with 10 nM vs. 50 nM retinoic acid caused increased epithelial thickness (median of 35.5 µm [26.7–40.7] vs. 27 µm [24.5–31.7], p<0.05). In addition, RA-deficiency induced SPRR1β mRNA (7-fold increase, p<0.01) and a there was a nonsignificant trend for increased cathelicidin (Table 1).
Hematoxylin and eosin-stained paraffin cross-sections of primary hBEC layers cultured at an air-liquid interface for 28 days in the presence of 10 nM (deficient) or 50 nM (normal) all-trans retinoic acid, with 0 nM or 10 nM 1,25(OH)2D.
Vitamin D status has been inversely associated with the prevalence of common colds , , suggesting the possibility that it inhibits viral replication or the induction of pro-inflammatory cytokine response. Here, we have demonstrated that treatment of differentiating primary hBECs with vitamin D in the form of either 25(OH)D (pre-hormone) or 1,25(OH)2D (active hormone) had no direct effects on RV replication, even after low-dose inoculation. Vitamin D had small but significant effects on epithelial cell chemokine production, but did not affect RV-induced secretion of the type III interferon IL-29. These findings suggest that associations between vitamin D levels and the frequency or severity of cold infections are not due to a direct antiviral effect related to RVs.
It is of note that Hansdottir et al conducted experiments in airway epithelial cell monolayers to determine whether vitamin D inhibited replication of respiratory syncytial virus (RSV), an enveloped RNA virus . Similar to our results, vitamin D did not affect RSV replication. In contrast to our results, the authors found that vitamin D inhibited induction of certain pro-inflammatory cytokines and chemokines, including IL-29. The discrepant results could be due to the use of different viral pathogens, or use of a monolayer cell culture system as opposed to a multi-layered, differentiated system.
We noted that vitamin D had obvious effects on epithelial cell growth and differentiation. Vitamin D produced marked changes in cellular morphology and increased expression of markers of basal cells (cytokeratin 5) and squamous metaplasia (SPRR1β). Notably, effects on growth and differentiation were similar when the cells were treated with 25(OH)D or 1,25(OH)2D. This finding is consistent with recent reports that respiratory epithelial cells convert inactive vitamin D to its active form . This finding has important implications since circulating levels of 25(OH)D are approximately 100-fold higher than the active form of the hormone. We used 0.1–100 nM vitamin D in our experiments, which is in the same range as optimal circulating 25(OH)D serum levels (30–80 ng/mL; 75–200 nM). Levels of vitamin D in airway fluids are unknown. In healthy airways, vitamin D levels may be considerably lower than in the bloodstream. Airway inflammation is associated with an influx of serum proteins , and under these conditions vitamin D levels could be considerably higher. This prompted us to evaluate potential effects of vitamin D on wound repair, however, the findings showed no significant effects on this process.
While the vitamin D-treated airway epithelial cell layers in our system became noticeably thicker, data from previous studies demonstrated that vitamin D can inhibit smooth muscle cell proliferation . Incidentally, we found in preliminary experiments that both 25(OH)D and 1,25(OH)2D had a mild inhibitory effect on growth of undifferentiated airway epithelial cell monolayers (data not shown). Collectively, these findings suggest that effects of vitamin D on cell growth may depend on the type of cell and differentiation state.
A recent study found that vitamin D deficiency in mice inhibited lung growth and caused deficits in lung function . It joins a prior body of work that indicates vitamin D deficiency can affect lung development via multiple mechanisms: structural, functional, and immunological, both in utero and postnatally . When considered together with these findings, our results support the concept that vitamin D could affect epithelial growth and differentiation during periods of human lung growth and development.
Vitamin D is part of a complex system of interacting elements, and other agents, such as vitamin A, can modulate its effects . These two vitamins, when bound to their respective receptors, form heterodimers with a shared partner protein (retinoic X receptor), and our experiments suggest that vitamins A and D may have opposing effects on epithelial cell differentiation. Similarly, vitamins A and D can have opposing effects on the skeletal system, with high levels of retinoic acid interfering with the vitamin D-regulated maintenance of normal serum calcium levels . As a result, high vitamin A intake is associated with an increased incidence of osteoporosis and hip fractures , . Our findings suggest that excessive amounts of vitamin D in the airway could have detrimental effects on epithelial cell differentiation, especially if vitamin A status is marginal. In this situation, cell layers exhibit dysmorphic changes, including reduced transepithelial resistance and squamous metaplasia, a mechanism that has been associated with airway pathology in smoking .
Vitamin D levels have been inversely related to the frequency and severity of respiratory illnesses including influenza and the common cold , , , . Protective effects of vitamin D against bacteria and certain enveloped viruses such as influenza virus are presumed to occur through increased expression of antimicrobial peptides such as cathelicidin, which is secreted by epithelial cells into the airways fluid , . This protein, upon binding to microbial membranes, changes membrane permeability through a pore-forming mechanism, thus disrupting metabolism and destroying the microbe , . In contrast to enveloped viruses and bacteria, RVs have a protein capsid, and we found no evidence that vitamin D treatment inhibited RV replication. Our findings suggest that the inverse association between vitamin D status and cold frequency could be due to indirect mechanisms , . For example, vitamin D could reduce colonization with bacterial pathogens (e.g. S. pneumoniae) that might synergize with viruses to produce more severe respiratory illnesses .
Strengths of this study include the use of primary cultures of airway epithelial cells, with consideration of vitamin D effects on cells grown at air-liquid interface. Effects on growth and morphology were reproducible using the cells from multiple primary cell donors. It is also important to consider that our model lacks immunologically important accessory cells, such as neutrophils, eosinophils, and macrophages, all of which could contribute to antiviral defenses in vivo. Thus, while vitamin D had no direct antiviral effect on epithelial cells, there could be indirect antiviral effects mediated by other cells. Since epithelial cells can convert 25(OH)D into 1,25(OH)2D, they could serve as a source of the active hormone for other cells in the airway microenvironment.
Vitamin D has been linked to reduced respiratory illnesses in several studies, but the mechanisms for these effects are unclear. Our findings demonstrate that vitamin D did not have direct anti-RV effects in epithelial cells, but could affect the quality of the antiviral immune response by inducing CXCL8 and CXCL10. Incidentally, we found that vitamin D also affects epithelial cell growth and differentiation, especially if vitamin A status is marginal.
The authors thank Richard Strauss of US Labs and Joe Hardin with Experimental Pathology, University of Wisconsin Carbone Cancer Center for help with immunohistochemical staining.
Conceived and designed the experiments: RAB RJP JEG. Performed the experiments: RAB. Analyzed the data: RAB JEG. Contributed reagents/materials/analysis tools: RAB JEG. Wrote the paper: RAB JEG. Helped edit the manuscript: RAB RJP JEG.
- 1. Hansdottir S, Monick MM, Hinde SL, Lovan N, Look DC, et al. (2008) Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defense. J Immunol 181: 7090–7099. doi: 10.4049/jimmunol.181.10.7090
- 2. Schauber J, Gallo RL (2009) Antimicrobial peptides and the skin immune defense system. J Allergy Clin Immunol 124: R13–R18. doi: 10.1016/j.jaci.2009.07.014
- 3. Lin R, White JH (2004) The pleiotropic actions of vitamin D. Bioessays. 26: 21–28. doi: 10.1002/bies.10368
- 4. Beard JA, Bearden A, Striker R (2011) Vitamin D and the anti-viral state. J Clin Virol 50: 194–200. doi: 10.1016/j.jcv.2010.12.006
- 5. Schwalfenberg GK (2011) A review of the critical role of vitamin D in the functioning of the immune system and the clinical implications of vitamin D deficiency. Mol Nutr Food Res 55: 96–108. doi: 10.1002/mnfr.201000174
- 6. Ginde AA, Mansbach JM, Camargo CA Jr (2009) Association between serum 25-hydroxyvitamin D level and upper respiratory tract infection in the Third National Health and Nutrition Examination Survey. Arch Intern Med 169: 384–390. doi: 10.1001/archinternmed.2008.560
- 7. Bartley J (2010) Vitamin D, innate immunity and upper respiratory tract infection. J Laryngol Otol 124: 465–469. doi: 10.1017/s0022215109992684
- 8. Jakiela B, Brockman-Schneider R, Amineva S, Lee WM, Gern JE (2008) Basal cells of differentiated bronchial epithelium are more susceptible to rhinovirus infection. Am J Respir Cell Mol Biol 38: 517–523. doi: 10.1165/rcmb.2007-0050oc
- 9. Schroth MK, Grimm E, Frindt P, Galagan DM, Konno SI, et al. (1999) Rhinovirus replication causes RANTES production in primary bronchial epithelial cells. Am J Respir Cell Mol Biol 20: 1220–1228. doi: 10.1165/ajrcmb.20.6.3261
- 10. Mosser AG, Vrtis R, Burchell L, Lee WM, Dick CR, et al. (2005) Quantitative and qualitative analysis of rhinovirus infection in bronchial tissues. Am J Respir Crit Care Med 171: 645–651. doi: 10.1164/rccm.200407-970oc
- 11. Konno S, Grindle KA, Lee WM, Schroth MK, Mosser AG, et al. (2002) Interferon-gamma enhances rhinovirus-induced RANTES secretion by airway epithelial cells. Am J Respir Cell Mol Biol 26: 594–601. doi: 10.1165/ajrcmb.26.5.4438
- 12. Hu R, Wu R, Deng J, Lau D (1998) A small proline-rich protein, spr1: specific marker for squamous lung carcinoma. Lung Cancer 20: 25–30. doi: 10.1016/s0169-5002(97)00097-4
- 13. Lau D, Guo L, Chan A, Wu R (2003) SPR1. An early molecular marker for bronchial carcinogenesis. Methods Mol Med 75: 397–403. doi: 10.1385/1-59259-324-0:397
- 14. Li S, Nikulina K, DeVoss J, Wu AJ, Strauss EC, et al. (2008) Small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia in dry eye disease. Invest Ophthalmol Vis Sci 49: 34–41. doi: 10.1167/iovs.07-0685
- 15. Yim S, Dhawan P, Ragunath C, Christakos S, Diamond G (2007) Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D(3). J Cyst Fibros 6: 403–410. doi: 10.1016/j.jcf.2007.03.003
- 16. Rohde CM, Manatt M, Clagett-Dame M, DeLuca HF (1999) Vitamin A antagonizes the action of vitamin D in rats. J Nutr 129: 2246–2250.
- 17. Rohde CM, DeLuca HF (2005) All-trans retinoic acid antagonizes the action of calciferol and its active metabolite, 1,25-dihydroxycholecalciferol, in rats. J Nutr 135: 1647–1652.
- 18. Gray TE, Guzman K, Davis CW, Abdullah LH, Nettesheim P (1996) Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 14: 104–112. doi: 10.1165/ajrcmb.14.1.8534481
- 19. Hansdottir S, Monick MM, Lovan N, Powers L, Gerke A, et al. (2010) Vitamin D decreases respiratory syncytial virus induction of NF-kappaB-linked chemokines and cytokines in airway epithelium while maintaining the antiviral state. J Immunol 184: 965–974. doi: 10.4049/jimmunol.0902840
- 20. Igarashi Y, Skoner DP, Doyle WJ, White MV, Fireman P, et al. (1993) Analysis of nasal secretions during experimental rhinovirus upper respiratory infections. J Allergy Clin Immunol 92: 722–731. doi: 10.1016/0091-6749(93)90016-9
- 21. Banerjee A, Panettieri R Jr (2012) Vitamin D modulates airway smooth muscle function in COPD. Curr Opin Pharmacol 12: 266–274. doi: 10.1016/j.coph.2012.01.014
- 22. Zosky GR, Berry LJ, Elliot JG, James AL, Gorman S, et al. (2011) Vitamin d deficiency causes deficits in lung function and alters lung structure. Am J Respir Crit Care Med 183: 1336–1343. doi: 10.1164/rccm.201010-1596oc
- 23. Weiss ST, Litonjua AA (2011) The In Utero Effects of Maternal Vitamin D Deficiency: How it Results in Asthma and Other Chronic Diseases. Am J Respir Crit Care Med 183: 1286–1287. doi: 10.1164/rccm.201101-0160ed
- 24. Binkley N, Krueger D (2000) Hypervitaminosis A and bone. Nutr Rev 58: 138–144. doi: 10.1111/j.1753-4887.2000.tb01848.x
- 25. Feskanich D, Singh V, Willett WC, Colditz GA (2002) Vitamin A intake and hip fractures among postmenopausal women. JAMA 287: 47–54. doi: 10.1001/jama.287.1.47
- 26. Herfs M, Hubert P, Poirrier AL, Vandevenne P, Renoux V, et al. (2012) Proinflammatory cytokines induce bronchial hyperplasia and squamous metaplasia in smokers: implications for chronic obstructive pulmonary disease therapy. Am J Respir Cell Mol Biol 47: 67–79. doi: 10.1165/rcmb.2011-0353oc
- 27. Sabetta JR, DePetrillo P, Cipriani RJ, Smardin J, Burns LA, et al. (2010) Serum 25-hydroxyvitamin d and the incidence of acute viral respiratory tract infections in healthy adults. PLoS One 5: e11088. doi: 10.1371/journal.pone.0011088
- 28. Hayes DP (2010) Influenza pandemics, solar activity cycles, and vitamin D. Med Hypotheses. 74: 831–834. doi: 10.1016/j.mehy.2009.12.002
- 29. Bals R, Wang X, Zasloff M, Wilson JM (1998) The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci U S A 95: 9541–9546. doi: 10.1073/pnas.95.16.9541
- 30. Wang TT, Nestel FP, Bourdeau V, Nagai Y, Wang Q, et al. (2004) Cutting edge: 1,25-dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression. J Immunol 173: 2909–2912. doi: 10.4049/jimmunol.173.5.2909
- 31. Brogden KA (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol 3: 238–250. doi: 10.1038/nrmicro1098
- 32. Henzler Wildman KA, Lee DK, Ramamoorthy A (2003) Mechanism of lipid bilayer disruption by the human antimicrobial peptide, LL-37. Biochemistry 42: 6545–6558. doi: 10.1021/bi0273563
- 33. Lachowicz-Scroggins ME, Boushey HA, Finkbeiner WE, Widdicombe JH (2010) Interleukin-13-induced mucous metaplasia increases susceptibility of human airway epithelium to rhinovirus infection. Am J Respir Cell Mol Biol 43: 652–661. doi: 10.1165/rcmb.2009-0244oc
- 34. Wang JH, Kwon HJ, Jang YJ (2009) Rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously. Laryngoscope 119: 1406–1411. doi: 10.1002/lary.20498