Organic dust was collected from settled dust (~3 feet above floor level) at local swine confinement feeding operations housing more than 500–700 hogs as previously described (11). Briefly, aqueous dust extracts were prepared by incubating 1g of the dust in 10ml of Hank’s Balanced Salt Solution (Life Technologies, Grand Island, NY) at room temperature for one hour. Mixture was centrifuged twice at 500g and final supernatant was filter (0.22 µm) sterilized, which is a process that also removes coarse particles. Batches of stock (100%) aqueous extracts (ODE) were frozen at −80°C, and ODE was diluted (vol/vol) in growth media or sterile phosphate buffered saline (PBS) for in vitro and in vivo experimental studies, respectively. The diluted 1%–5% ODE has previously been determined to elicit optimal pro-inflammatory chemokine release from cultured cells (13,21), and the diluted 12.5% ODE has been shown to elicit maximal airway inflammation in mice and is well tolerated (10). Complete analysis of the dust extract has been previously published (14). Briefly, stock (100%) ODE contains 23.3–31.1 mg/ml of total protein as measured by nanodrop spectrophotometry (NanoDrop Technologies, Wilmington, DE). Endotoxin levels in stock ODE range from 184 to 760 EU/ml as assayed using the limulus amebocyte lysate assay according to manufacturer’s instruction (Sigma, St. Louis, MO).

BEAS-2B cells (American Tissue Culture Collection, Manassas, VA), an SV40-transformed human bronchial epithelia cell line, were plated on type I collagen-coated (Celtrix Laboratories (Palo Alto, USA)) 100mm×155mm dishes and maintained in serum-free LHC-9-RPMI (Sigma-Aldrich, St. Louis, MO) supplemented with Fungizone and Penicillin/Streptomycin (Invitrogen, Grand Island, NY) at 37°C in 5% CO₂. Confluent monolayers (~80% confluency) were pretreated with or without 1,25-(OH)₂D₃ (100 nM) (Sigma-Aldrich) for 18 hours. Next, cells were washed and fresh control or vitamin D-supplemented media was reapplied and epithelial cells were simulated with 5% ODE or saline for 24 hours. Cell-free supernatants were collected and frozen at −80°C for later chemokine analysis. Cells were harvested to determine protein concentrations by a nanodrop spectrophotometer. Viability of cell cultures was assured by lactate dehydrogenase assay (Sigma-Aldrich).

Human peripheral blood monocytes were collected from volunteer donors at the institution’s Elutriation Core Facility as previously described (21). Briefly, monocytes were isolated by countercurrent centrifugal elutriation of mononuclear leukocyte-rich fractions and purity of monocyte populations confirmed by determination of CD14 cell surface expression by flow cytometry (>99%). Peripheral blood was taken with written informed consent, and studies were approved by the University of Nebraska Medical Center Institutional Review Board. Cells (1×10⁶/ml) were cultured in complete RPMI supplemented with Fungizone (Invitrogen) and penicillin/streptomycin (Invitrogen). Consistent with epithelial cell experimental studies for chemokine production assessment, monocytes were pretreated with or without 100nM of 1,25-(OH)₂D₃ for 18 hours, whereupon cells were washed, and stimulated with 1% ODE in the presence or absence of freshly supplemented vitamin D media for 24 hours. Cell-free supernatants were collected and frozen at −80°C until later chemokine analysis. Cell viability was assured by trypan blue exclusion method.

Precision-cut murine lung slices from control C57BL/6 mice were prepared in cultures as previously described (22). Lung slices were pre-treated with 1,25-(OH)₂D₃ (100nM) for 18 hours, and subsequently re-stimulated with or without 5% ODE for 24 hours. Cell-free culture supernatants were collected and stored at −80°C for later analysis.

Male C57BL/6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 3–4 weeks of age. Upon arrival, mice were randomly assigned to a specially ordered rodent chow (Land O’ Lakes-Purina; Minneapolis, MN) diet of standard soy-based (soy naturally contains small amounts of vitamin D) rodent chow supplemented with 0 IU/g or 5 IU/g of 1,25 hydroxyvitamin D. Manufacturer-provided samples of the diet were independently tested by N-P Analytical Laboratories (St. Louis, MO), which reported concentrations of 2.3 IU/gm and 5.55 IU/gm of 1,25-(OH)₂D₃ in the low and high concentration diets, respectively. Mice were weighed twice weekly to assure proper and equal growth between groups. At age 8 weeks (chosen because the half-life of the active form 1,25-(OH)₂D₃ is approximately 7 days) (23) mice were anesthetized by isoflurane and treated with 50 µl of ODE (12.5%) or sterile saline via intranasal route as previously described (11). All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center.

Five hours post-exposure, animals were euthanized by intraperitoneal injection of 50 mg/kg of sodium pentobarbital (Nembutal; Abbott Labs, Chicago, IL). The trachea was exposed and a cannula was intra-luminally placed just below the larynx. Bronchoalveolar lavage fluid (BALF) was collected by whole lung lavage with 3×1 ml of sterile PBS as previously described (11). Cell-free BALF supernatant from first lavage was frozen at −80°C for later chemokine analysis and cells from all three lavages were pooled. Total cells were enumerated and spun onto slides with Cytopro cytocentrifuge (Wescor, Logan, UT) and stained with DiffQuick (Dade Behring, Newar, DE). Counts of the cells determined the differential ratio of cell types in 200 cells per slide per mouse.

Murine neutrophil chemoattractants, KC/CXCL1 (cytokine-induced neutrophil-attracting chemokine) and MIP-2/CXCL2 (macrophage inflammatory protein 2-alpha), were quantitated in cell-free BALF supernatants according to the manufacturer’s instructions using commercially available ELISA kits (R&D Systems, Minneapolis, MN) with sensitivities of 15.6 pg/ml and 7.8 pg/ml, respectively. The human neutrophil chemoattractant, interleukin-8 (IL-8)/CXCL8, was quantitated from cell culture supernatant by sandwich ELISA as previously described (12) with sensitivity of 21 pg/ml.

Protein kinase C (PKC) isoform activity from murine tracheal epithelial cells following in vivo stimulation with ODE and saline was conducted. Whole tracheas were snap frozen in cell lysis buffer and stored at −80°C until later PKCε and PKCα activity assays could be performed as previously described (11). Prior to assay, the tracheae are thawed and the epithelial cells are extracted using a sterile cell scraper. The collected epithelial cell fraction in lysis buffer is then sonicated and assayed for kinase activity. PKC activity was expressed in relation to the total amount of cellular protein assayed as picomoles of phosphate incorporated per minutes per milligram. PKC activity is reported as fold-increase from baseline: dust-induced kinase activity divided by saline-alone kinase activity.

Whole blood was collected from mice at time of sacrifice, and serum 25(OH) vitamin D levels were quantified by the institutions’ core clinical standard high performance liquid chromatography-tandem mass spectrometry methods.

Human peripheral blood monocytes were stimulated with 1% ODE or saline for 24 hours whereupon RNA was extracted from cell pellets using the Magmax 96 kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. For lung tissue, after BALF was removed, the right ventricle was perfused with 10 ml of sterile PBS with heparin to remove blood from the pulmonary vasculature. Whole lung tissues were harvested and stored in RNA later (Applied Biosystems) until RNA extraction could be performed by using TRIzol reagent (Invitrogen). RNA concentration and purity was determined by NanoDrop spectrophotometer and samples had A260/A280 ratio of 1.9–2.0. cDNA was synthesized as previously described (24). Real-time PCR reactions were prepared in triplicate using 1x TaqMan Master Mix (Applied Biosystems) and primers and probed for TLR2 (Applied Biosystems; human: Hs00152932_m1; mouse: Mm00442346_m1) and TLR4 (human: HS00152939_m1; mouse: Mm00442346_m1). Ribosomal (18s) RNA was used as an endogenous control. PCR was performed using an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). Threshold values were normalized to the expression of ribosomal RNA. Real-time-PCR results are either expressed as the percent fold-increase in induction (normalized copy number of stimulated cells divided by normalized copy number of unstimulated cells×100) or as values normalized to expression of ribosomal RNA.

Human peripheral blood monocytes were evaluated for cell-surface molecule expression by means of flow cytometry for TLR2 and TLR4 after incubation with and without vitamin D (100 nM) for 24 and 48 hours. Cells (5 x10⁵) were stained in a standard procedure with antibodies against TLR2, TLR4 (BioLegend, San Diego, CA), and irrelevant isotype control antibodies (to account for nonspecific binding) in PBS containing 0.1% bovine serum albumin. Flow cytometry analyses were performed with the FACSCalibur dual-laser cytometers available in the UNMC Flow Cytometry core (Becton-Dicksinson, Lincoln Park, NJ).

Data are presented as the mean ± standard error of mean (SEM). Statistics were performed using a two-tailed, non-paired t-test to determine significant differences between treatment groups. One-way analysis of variance (ANOVA) with Tukey multi-comparison post-test was employed to compare differences among three or more treatment groups. In all analyses, GraphPad (version 5.01) software was used, and p values less than 0.05 were considered statistically significant.

Because our group and others have demonstrated an important role for TLR2 and TLR4 receptor signaling pathways in mediating ODE-induced airway disease (14,15), we first sought to confirm recent reports that vitamin D reduces TLR2 and TLR4 monocyte cell surface expression (19). Treatment with vitamin D reduces monocyte TLR2 and TLR4 mRNA expression as assessed by quantitative real-time PCR and cell surface protein expression as assessed by flow cytometry (Figure 1A–B). This was not an immediate response because significant suppression of monocyte mRNA and protein were not observed until 24 hours following treatment with vitamin D (data not shown and Figure 1), suggesting that prolonged pretreatment with vitamin D would be required to determine chemokine responses.

We next sought to determine if vitamin D could down-regulate ODE-induced human neutrophil chemoattractant, IL-8, release. ODE-induced IL-8 production was reduced with vitamin D pretreatment (18 hour) in both human epithelial cells and monocytes (Figure 2A–B). Consistent with single cell culture studies, vitamin D pretreatment resulted in reduced ODE-stimulated CXCL1 and CXCL2 (murine neutrophil chemokine homologs) production from lung slices, a representation of tissue-structured multicellular lung responses.

Consistent with previous studies (11), acute ODE intranasal inhalation resulted in significant (p<0.05) increases in neutrophil influx and release of murine neutrophil chemoattractants, CXCL1 and CXCL2, in BALF at 5 hours post-exposure (Figure 3A–B). However, the increase in total leukocytes and CXCL1 and CXCL2 release following in vivo ODE challenge was significantly reduced (p<0.05) in mice fed a relatively high vitamin D supplemented diet as compared to mice fed a relatively low vitamin D diet (Figure 3A–B). Moreover, serum 25(OH) vitamin D levels were significantly reduced (p<0.05) by approximately 25% in mice given the low vitamin D diet (mean ± SEM: 16.6 ng/ml ± 1.231) as compared to high vitamin D diet (20.5 ng/ml ± 1.009).

In our previous work, we reported that PKCα and PKCε activity was important in mediating human bronchial epithelial cell ODE-induced IL-8 release (12), and that tracheal epithelial cell PKCα and PKCε were activated in vivo following organic dust exposure (11). In this study, there was activation of tracheal epithelial cell PKCα and PKCε in the low vitamin D treatment group following in vivo ODE challenge, which was nearly absent in the high vitamin D + ODE treatment group (Figure 4).

To determine whether dietary vitamin D supplementation was important in lung TLR2 and TLR4 expression following ODE exposure, whole lung mRNA expression of TLR2 and TLR4 was investigated following ODE exposure in mice fed a low vs. high vitamin D diet for 5 weeks. Mice fed a low vitamin D diet had significantly increased TLR2 and TLR4 expression following ODE exposure as compared to saline exposure (Figure 5). Interestingly, this ODE-induced up-regulation in TLR2 and TLR4 mRNA was not observed in mice fed a relatively high vitamin D diet (Figure 5).