Vitamin D supplementation decreases Aspergillus fumigatus specific Th2 responses in CF patients with aspergillus sensitization: a phase one open-label study
© Nguyen et al.; licensee BioMed Central. 2016
Received: 24 April 2014
Accepted: 7 May 2015
Published: 4 June 2015
Patients with cystic fibrosis (CF) complicated by allergic bronchopulmonary aspergillosis (ABPA) are vitamin D deficient and in vitro treatment with 1,25 (OH)2 vitamin D3 of CD4+ cells from CF patients with ABPA decreases Aspergillus fumigatus(Af)-induced Th2 responses. This Phase I clinical trial investigated the safety and effectiveness of daily vitamin D3 supplementation in CF patients with ABPA to reduce allergic responses and ABPA symptoms, and increase serum vitamin D levels.
Seven patients ages 12 years and older with a clinical diagnosis of CF and ABPA with current evidence of Af sensitization received 4000 IU vitamin D3 (cholecalciferol) daily for 24 weeks. The primary outcome of the study was safety followed by the Aspergillus induced IL-13 response in CD4+ T cells to test the hypothesis that vitamin D supplementation is safe and reduces Aspergillus induced IL-13 responses in CD4+ T cells. Secondary outcomes included total IgE, Aspergillus- specific IgE, vitamin D levels, FEV1, urinary calcium/creatinine ratio, and cytokine production by Aspergillus-stimulated peripheral blood T cells.
Six months of vitamin D3 supplementation resulted in significant increases in serum 25-(OH) vitamin D level, and the treatment was well tolerated without evidence of vitamin D toxicity or hypercalcemia. There were no serious adverse events. Daily vitamin D supplementation led to significantly decreased Aspergillus induced IL-13 responses between the baseline visit and that at 24 weeks (p = 0.04). Aspergillus-specific IgE level was also significantly decreased after 8 (p = 0.035) and 24 weeks of daily vitamin D supplementation (p = 0.04).
4000 IU vitamin D3 daily over a 24-week period is well tolerated in CF patients with a history ABPA and current evidence of Th2 immunity to Af. . Daily vitamin D supplementation was associated with reduced Aspergillus induced IL-13 responses from peripheral. . CD4+ T cells and Aspergillus-specific IgE levels, as well as increased serum vitamin D levels. This treatment was well tolerated and the study supports further investigation of the use of vitamin D supplementation in Th2 mediated diseases.
This trial was registered at www.clinicaltrials.gov as NCT01222273.
Cystic fibrosis (CF) is the most common severely life-shortening genetic disease in people of mixed European descent, with lower prevalence in Hispanics, African Americans, and Asians. Due to impaired mucociliary clearance, CF patients are susceptible to fungal colonization in the lungs, as up to 50 % of patients culture fungus from respiratory secretions . The most common fungal pathogen infecting CF patients is Aspergillus fumigatus. In one study, a striking 84 % of CF patients had Aspergillus fumigatus-specific IgG (Asp IgG) antibodies compared to 6 % in control subjects and 20 % in allergic asthmatics . While an extremely high percentage of CF patients have exposure to A. fumigatus, this may or may not result in clinical disease. Impaired clearance of Aspergillus fumigatus in the respiratory tract of immunocompromised individuals can lead to clinical manifestations that include invasive aspergillosis, mycetoma, fungal bronchitis, or allergic bronchopulmonary aspergillosis (ABPA) . In contrast to patients with clinically irrelevant colonization and other types of fungal disease, patients with CF and ABPA have allergic immunological responses to Aspergillus fumigatus antigens, which results in increased Th2 responses and increased IgE levels [4, 5].
Vitamin D deficiency is prevalent in the CF population due to inadequate absorption, impaired metabolism, and limited sun exposure . We have previously shown that patients in our CF center with CF and ABPA have significantly lower vitamin D levels than CF patients who do not have ABPA . Further, in vitro treatment with 1,25 (OH)2 vitamin D3 decreased Aspergillus induced IL-13 responses from CD4+ T cells from ABPA patients . In addition, it has been shown that a single, oral bolus of cholecalciferol (25,000 IU) increased serum-25 (OH) vitamin D concentrations and was associated with improved clinical outcomes in CF patients hospitalized with pulmonary exacerbations . Further, a large, single dose of vitamin D (25,000 IU) in CF patients during pulmonary exacerbations was associated with a decrease in the inflammatory cytokines IL-6 and TNFα . Taken together, these observations suggest that vitamin D plays a critical role in modulating immune responses. We therefore performed a Phase I, open label study to determine whether in CF patients with ABPA daily supplemental vitamin D is safe and reduces allergic response to A. fumigatus. Based on our prior work, we chose the Aspergillus specific IL-13 response as our primary immunologic outcome .
Inclusion and exclusion criteria for vitamin D supplementation trial
● ≥12 years male or female
● Systemic corticosteroids (1 mg/kg if <20 kg or >20 mg of prednisone per day)
● Confirmed CF diagnosis
1. One or more clinical features consistent with CF phenotype AND (2 or 3)
● Investigational drug use within 30 days of screening
2. Positive sweat chloride >60 mEq/L
● Laboratory abnormalities at screening
3. Two identifiable mutations consistent with CF
● Serum calcium > 11 mg/dL
● Written informed consent (or assent)
● 25 (OH) D > 50 ng/mL at screening
● Clinically stable at enrollment as assessed by site investigator
● Creatinine ≥ 1.5 or estimated GFR < 60 by Cockcroft-Gault or MDRD equation
● Past or present respiratory culture positive for Aspergillus fumigatus
● LFT ≥ 3x ULN
● IgE ≥ 250 and/or presence of Class II or higher Aspergillus specific IgE on enrollment
● History or transplantation or currently on transplant list
● Ability to comply with medication use, study visits and study procedures as judged by site investigator
● Positive serum pregnancy test at screening
● Pregnant, breastfeeding, or if post-menarche female, unwilling to practice birth control during participation in study
● Presence of a condition or abnormality that would compromise safety of subject or quality of data
● Diagnosis of HIV and a CD4+ T cell count < 500 cells/mL or active hepatitis C infection
● Undergoing therapy for non-tuberculosis mycobacterial infection
Timeline of clinical trial visits and assessments
Day-7 to 28
Day 55 ± 7
Day 112 ± 7
Day 182 ± 7
Day 196 ± 7
Abbreviated physical exam
Clinical laboratory assessments
Blood collection for CF genotype
Blood collection for HLA typing
Blood for banking (immune monitoring)
Adverse event assessments
Medication and diary drug
Review study drug count and diary
The Institutional Review Board at the University of Pittsburgh approved the trial, and patients provided written informed consent and were enrolled in the trial by one of the primary investigators.
Vitamin D supplementation
After meeting enrollment criteria, patients were given the study drug. Vitamin D3 (cholecalciferol) was dispensed as 1000 IU tablets, and patients were instructed to take four tablets daily.
Human CD4+ and CD11c + cells were obtained from whole blood using Vacutainer CPT tubes (BD Pharmingen, Franklin Lakes, NJ) and re-suspended in cell buffer comprised of EDTA (Gibco, Grand Island, NY), BSA (Sigma, St. Louis, MO), and 1X PBS (Fisher, Waltham, MA). All cells were isolated by magnetic bead activated sorting using microbeads and MidiMacs (Miltenyi, Auburn, CA). The CD4+ cells were first isolated by positive separation in MS columns using CD4 Microbeads (Miltenyi). CD11c + cells were subsequently isolated from the negative fraction of the CD4+ isolation using Anti APC Microbeads then CD11c-APC microbeads for the final magnetic separation. DCs were then plated in flat bottom 96 well plates at a density of 5×105 and 5×104 DC cells per well in medium containing RPMI, L-glutamine, pen strep, FBS (Gibco), and Human AB serum (Atlanta Biologicals, Lawrenceville, GA). Cells in the plate were then stimulated with the following stimulators: TSLP (5 ng/mL, R and D Systems, Minneapolis, MN) and/or Aspergillus extract (ASPEXT, 1 μg/ml) (Holister-stier, Spokane, WA). One well in the plate was left un-stimulated as a control. 5×105 CD4+ cells were then added. Control wells received CD4+ T-cells that were cultured in media or stimulated with CD3/CD28 beads (Dynal/Invitrogen, Grand Island, NY). Additionally, recombinant IL-2 was added to all wells on the plate (7.5 ng/ml). Medium containing RPMI, 5 % L-glutamine, 5 % pen strep, 10 % FBS (Gibco), and 5 % Human AB serum (Atlanta Biologicals) was then added to each well to reach final volume. All cells were then incubated at 37 °C and 5 % CO2 for 96 h. In some experiments, anti-human IL-10 (1 μg/ml final concentration) or recombinant human TGF-B sRII Fc Chimera (10 μg/ml final concentration) were added.
IL-4, IL-5, IL-10, IL-13, and IL-17 were measured by Luminex (Millipore, Billerica, MA) according to manufacturer’s instructions. The data was analyzed with Bio-Plex Manager software (Bio-Rad, Hercules, CA). To control for percentage of Aspergillus specific CD4+ T-cell in peripheral blood we normalized the APSEXT response to the CD3/CD8 stimulated response as this represents the maximal cytokine response in the culture system.
Clinical laboratory assessment
Serum was collected and 25-OH vitamin D, IgE, Asp IgE, and calcium levels were determined by Sunquest Clinical Labs. Baseline total IgE and Asp IgE levels are reported from the screening visit. In addition, we measured Ca, Phosphorous, ALT, AST, total bilirubin, alkaline phosphatase, BUN, Cr, serum HCG, and IgG in all or a subset of the study participants. Urinary calcium and creatinine were measured at each time point and urinary calcium:creatinine ratio was calculated to assess for potential hypercalciuria (an early sign of vitamin D toxicity).
Lung function was measured using standard pulmonary function tests according to American Thoracic Society guidelines. Forced expiratory volume in 1 s (FEV1) was measured and the percent of predicted FEV1 (FEV1 % predicted) was calculated based on patient’s age and height using NHANES-3 reference values 25.
All statistical analyses were performed using a one-tailed paired T-test or one-way ANOVA with Prism software (GraphPad).
Demographics of patients enrolled in vitamin D supplementation trial
22.5 ± 10.68 (17.25–30.75)
21.91 ± 2.69 (13.10–30.83)
4 female; 3 male
344.6 ± 284.9 (142–835)
Asp IgE (kUA/I)
18.4 ± 14.7 (4.24–37.5)
ΔF508/R553X; ΔF508/612+ 1G-T; neg/ΔF508; ΔF508/ΔF508; MEG/G542X; ΔF508/1213delT; ΔF508/R1162X
Vitamin D supplementation in CF patients with ABPA is well tolerated and safe
Compliance by pill count
Should have taken
Compliance by pill count (%)
Vit D dose at enrollment
ABDEK 2/day plus vitamin D 1000 units and 400 units once daily
Effect of vitamin D supplementation on aspergillus-induced IL-13 responses
We have also previously shown that stimulating CD11c + DCs with TSLP increases Th2 response to A. fumigatus in CF patients with ABPA via upregulation of OX40L . After 24 but not 8 weeks of vitamin D supplementation, TSLP-CD11c + DCs, co-cultured with autologous CD4+ T cells, also showed a significant reduction in the A. fumigatus IL-13 response (Fig. 2).
Effect of vitamin D supplementation on secondary outcomes
In most individuals, when benign antigens are inhaled, the lung responds with a tolerogenic immunological response [13–15]. However, in diseases such as ABPA, tolerance is lost or not established, and patients become sensitized to the inhaled fungal antigen Aspergillus fumigatus. We have previously shown that peripheral CD11c + DCs and TSLP-DCs from ABPA patients induce robust Th2 cytokine responses from autologous CD4+ T-cells in an OX40L-dependent manner . In addition, in our CF cohort, vitamin D deficiency was associated with ABPA. In vitro treatment with 1,25-(OH)2 vitamin D3 reduced the robust Th2 response in patients with ABPA . Based on data from our observational study, we initiated a clinical trial to assess the safety and the immunological effects of supplemental vitamin D3 (cholecalciferol) in patients with CF and ABPA.
This Phase I trial was implemented to test the safety and efficacy of vitamin D supplementation in CF patients with documented evidence of Af seinsitization. From a clinical perspective, the addition of daily vitamin D supplementation of 4000 IU significantly increased serum 25-OH vitamin D levels without vitamin D toxicity or hypercalcemia over the 24-week clinical trial period in CF patients with ABPA. It is important to note that this dose of vitamin D was prescribed on top of the patients regular vitamin D schedule (Table 4). Vitamin D dosing has moved beyond the typical 400 IU per day (Table 1) and this may explain why baseline vitamin D levels were higher in this cohort than our prior study . As further evidence of compliance urine calcium:creatinine ratios increased to above 0.2 in 3 out of 7 patients at the 24-week time point. Typically, ratios above 0.2 may be indicative of hypercalciuria . However, urine samples were collected at random during clinic visits. A 24-h urine collection would be more accurate at determining urine calcium excretion in our patients [16, 17]. After 24 weeks on daily vitamin D supplementation, patients had 25-OH vitamin D levels of 44.86 ± 8.630 ng/mL. These values are considered to be slightly above the minimal threshold (>30 ng/mL) of preferred 25-OH vitamin D concentrations recommended by the Cystic Fibrosis Foundation (CFF) .
The primary immunological outcome measure was the A. fumigatus specific IL-13 response in peripheral CD4+ T-cells. Over the 24-week period, IL-13 responses to A. fumigatus from DC/T-cell co-cultures significantly decreased. We have previously shown that in vitro 1,25-(OH)2 vitamin D3 increased CD4 + CD25 + TGFβ + Tregs  and others have shown that in vitro 1,25 dihydroxyvitamin D3 synergized to increase IL-10 in Tregs . However, in this vitamin D supplementation study, blockade of TGFβ or IL-10 did not reverse inhibitory effects of vitamin D, suggesting that the decreased Th2 response in peripheral CD4+ T-cells after 24-weeks of vitamin D is not dependent on TGFβ or IL-10 in the ex vivo culture. We did not assess the frequency of FoxP3+ CD4+ T-cells in peripheral blood in this study and thus we cannot exclude an affect of vitamin D on the in vivo Treg population. It was recently shown that 1,25-(OH)2 vitamin D3 increased the expression the anti-microbial peptide LL-37 in CF epithelial cells along with decreasing inflammatory cytokines [20, 21]. LL-37 has been shown to have antimicrobial activity against microbes as well as fungi and viruses  and it has immunomodulatory effects on both epithelial cells and dendritic cell differentiation . In addition to decreasing Th2 responses, one explanation for the decreased response to A. fumigatus with vitamin D supplementation may be due to an increase in antimicrobial peptides such as LL-37. While our clinical study had only 7 CF patients with ABPA who completed the 24-week vitamin D supplementation trial, we demonstrated that vitamin D supplementation reduced A. fumigatus specific IL-13 responses.
There are several limitations to the current study. First, prior to this vitamin D supplementation trial, the guidelines for 25-OH vitamin D concentrations were modified by the CFF. This may have contributed to the baseline Vitamin D levels being higher in this cohort prior to supplementation particularly in comparison to 25-OH vitamin D levels of 26.80 ± 15.32 ng/mL than in the initial observational study7, which were well below the current clinically recommended serum concentrations . This may have limited our ability to detect an immunomodulatory effect of Vitamin D on Aspergillus responses. Despite the higher mean vitamin D levels in this cohort, Aspergillus-specific IgE levels decreased over the 24-week period. Second, the limited size of the study population did not permit us to track changes in other therapies for ABPA, such as prednisone and antifungal use that may have affected lung function and in vitro responses. Further studies are necessary to clarify this.
Others have shown that vitamin D deficiency is significantly and inversely associated with total IgE and Asp IgE [12, 24]. Additionally the presence of Aspergillus colonization itself has been shown to impair vitamin D receptor expression in CF . Furthermore a recent clinical trial of vitamin D in asthma patients who were vitamin D deficient was negative in terms of vitamin D supplementation reducing time to first exacerbation or affecting FEV1 . However Th2 immunity was not assessed as part of this trial. Moreover there was only measure of serum vitamin D levels which may not accurately measure VDR function which can be regulated by Aspergillus . There is evidence that we impacted VDR function in this trial as measured by the increase in the urine calcium:creatinine ratio.
Taken together with the results from this Phase I study, vitamin D supplementation may be beneficial in decreasing these allergic responses. These data provide rationale for increased vitamin D supplementation of CF patients with ABPA in addition to supporting further study of the effects of vitamin D supplementation to treat or prevent ABPA. Moreover fungal specific IL-13 responses may be a useful biomarker for future studies in patients with ABPA.
This work was supported by grants from NHLBI (R37HL079142 and P50 HL084932).
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