Vitamin D Regulates the Expression of Glucocorticoid Receptors in Blood of Severe Asthmatic Patients
Phase: NA
Conditions: Effect of Drug
Sponsor: Dubai Health Authority

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                                            To whom it may concern                                                                             
Date : 15/06/2020 
 
 
Subject : Vitamin D Regulates the Expression of Glucocorticoid Receptors in Blood of 
Severe Asthmatic Patients   
 
 
I am Dr.Bassam Mahboub ,Principle investigator for the study (Vitamin D Regulates the 
Expression of Glucocorticoid Receptors in Blood of Severe Asthmatic Patients) . I am 
confirming  that the document is relevant to the study in the record.   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Title :Vitamin D Regulates the Expression of Glucocorticoid Receptors in Blood of Severe 
Asthmatic Patients   
 
Introduction 
 
Vitamin D (VitD) deficiency is a public health concern affecting up to 80% of the 
population in certain countries and of all age groups 1. Hypovitaminosis D is 
prevalent in the Middle East and North Africa (MENA) 2, and VitD level is 
suboptimal in the residents of sunny United Arab Emirates (UAE) 3.  In addition 
to its well-established role in calcium and bone metabolism,  the potential roles 
of VitD in cancer development,  cardiovascular and chronic lung diseases are 
being increasingly appreciated 4. The increased awareness of VitD deficiency 
and related health problems made vitamin D supplementation a routine action 
plan even without laboratory confirmations leading to a higher risk of exogenous 
hypervitaminosis D 5. Black et al. were among the first to show a positive 
correlation between VitD status and pulmonary function 6. VitD deficiency is 
associated with features of asthma,  such as decreased lung function,  
increased airway remodeling,  and airway hyperresponsiveness 7. Several 
reports suggested the association of VitD levels with the severity of asthma 8.  
Different cross-sectional studies indicated that low serum VitD in patients with 
mild to moderate asthma is associated with poor asthma control,  more 
exacerbations,  reduced lung function, and increased medication use 9-11. 
Inverse relationships have also been reported between serum VitD levels and 
airway 
remodeling, 
IgE, 
eosinophil 
numbers, 
as 
well 
as 
airway 
hyperresponsiveness. Interestingly,  Gupta et al. demonstrated a role for active 
VitD in correcting IL-10 levels in a paediatric population of moderate and severe 
therapy-resistant asthma 
12. On the other hand, experimental studies 
demonstrated that vitamin D receptor (VDR)-deficient mice fail to develop 
airway inflammation (decreased infiltration of lymphocytes and eosinophils) and 
experimental allergic asthma 13. 
Steroid resistance, as documented in cases of severe asthma, is associated 
with increased expression of the glucocorticoid receptor (GR)-β,  which is an 
alternative splicing isoform and dominant-negative regulator of the GR-α 14. The 
abundance of GR-α mRNA is higher than GR-β mRNA in all normal tissues and 
cells. Normally, default splicing pathway leads to GR-α, while the alternative 

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splicing pathway that leads to GR-β is minimally activated 15. The latter 
pathway, however, seems to be upregulated in severe asthmatic patients 16. In 
chronic inflammatory diseases like asthma, upregulated blood levels of 
inflammatory cytokines can participate in glucocorticoid resistance and 
impaired GR function by modulating its translocation, DNA binding, and GR 
phosphorylation status [18].  
 
As data from observational studies suggested a better control of asthma 
following restoration of VitD levels. 
 
 
Hypothesis: 
 Correcting VitD levels in adult asthmatic patients could enhance steroid 
responsiveness by regulating blood GR-α/GR-β ratio levels.  
 
Primary Out comes: 
1.To find out the top pathways regulate Vitamin D 
identify VitD targeted enriched pathways, we analyzed Comparative 
Toxicogenomics .Enriched Ontology Clustering for the identified genes was 
performed using the Metascape (a web-based tool used for comprehensive 
gene list annotation and analysis resource)Database from thousands of 
experiments.[Time Frame: 8 weeks] 
2.To examine asthma severity in relation to steroid receptors genes [Time 
Frame: 8 weeks] 
 
3.study the effect of vitamin D on GR-α receptor  [Time Frame: 8 weeks] 
 
4. tudy the effects of Vitamin D on cytokines. [Time Frame: 8 weeks] 
 
Inclusion Criteria: 
• Diagnosis of asthma 
• age group between 18 and 65 years 
• low level of 25-hydroxyvitamin D3 (25 D3) level less than 20ng/mL 

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Exclusion Criteria: 
• smoker patients 
• less than 18 years old 
• patient on Vitamin D supplementation already 
 
Materials and Methods  
 
In silico identification of top genes regulated by vitamin D supplementation 
Using  Comparative Toxicogenomics Database to identify the top genes influenced by vitamin D 
supplementation in thousands of experiments. One hundred five genes with at least five 
documented interactions with VitD were identified. Moroever, to explore if the identified genes 
are sharing common pathways, Enriched Ontology Clustering for the identified genes will 
performed using the Metascape (a web-based tool used for comprehensive gene list annotation and 
analysis resource) .  
 
 
In silico determination of effect of vitamin D treatment on the expression of NR3C1 and 
NR3C2 in monocytes, macrophages, and dendritic cells 
In order to investigate the effect of VitD supplementation on the mRNA expression of NR3C1,  
NR3C2 and VDR,  publicly available datasets (GSE52819,  GSE13762,  GSE56490) we will  
explored as they were designed to examine the global transcriptional effect of VitD 
supplementation on monocytes,  macrophages, and dendritic cells.  
 
In silico detemination of the mRNA expression levels, and correlations, of NR3C1, NR3C2, 
and VDR in the blood of asthmatic patients compared to healthy controls 
We will extract  from  publicly available datasets (GSE69683) with a large number of participants 
and their detailed clinical information. To find  Gene expression profiling of blood from these 
groups was analyzed, and the gene expression of GR (NR3C1 and NR3C2) which determined. 
 
Patients 
To do double-blinded, randomized, placebo-controlled study of VitD supplementation on 45 
asthmatics with VitD deficiency will performed. Moderate to severe asthmatics between 18 and 
65 years of age who had clinician-diagnosed asthma with 25-hydroxyvitamin D3 (25 D3) level 
less than 20ng/mL at the screening visit will be  recruited at the Rashid Hospital,  Dubai,  U.A.E.,  
and the Zayed Military Hospital,  Abu Dhabi,  U.A.E.  
 
mRNA expression and cytokine expression 
Whole blood will collected in PAXgene tubes (Qiagen, Germany) for the isolation of cellular 
RNA. Blood specimens will assessed for mRNA expression of Glucocorticoid receptor (GR-β and 

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GR-α) using qRT-PCR. Bioplex multiplex immunoassay (BioRad, CA,USA) will  use to assess 
the protein expression of IL-17A, IL-17-F, IFN-γ, IL-4, IL-5 and TNF-α in serum obtained from 
patients before and after treatment. 
 
Statistical Analysis 
Standard statistical t-tests and one-way ANOVA will be  performed to test for statistical 
significance between data groups using GraphPad Prism 8 (GraphPad, San Diego, CA, USA). p < 
0.05 was considered significant.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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                                            To whom it may concern                                                                             
Date : 15/06/2020 
 
 
Subject : Vitamin D Regulates the Expression of Glucocorticoid Receptors in Blood of 
Severe Asthmatic Patients   
 
 
I am Dr.Bassam Mahboub ,Principle investigator for the study (Vitamin D Regulates the 
Expression of Glucocorticoid Receptors in Blood of Severe Asthmatic Patients) . I am 
confirming  that the results  is relevant to the study in the record.   
 
 
Results : 
Top Vitamin D regulated pathways are involved in response to Steroids 
To identify VitD targeted enriched pathways, we analyzed Comparative Toxicogenomics Database 
from thousands of experiments. Interestingly, the top enriched pathways regulated by VitD were 
involved in several metabolic and inflammatory processes including response to steroids, 
lipopolysaccharide as well as Interleukin-4, Interleukin-13 and AGE-RAGE signalling (Suppl. 
Figure 1). Many of those VitD targeted pathways were also targets of dexamethasone. These 
include pathways involved in inflammation; proliferation; pro-apoptosis, beside others (Figure 
S1).  
 
VDR, NR3C1 and NR3C2 expression levels in different types of immune cells 
The level of expression of GR coding gene (NR3C1), mineralosteroid receptor coding gene 
(NR3C2) 17, and VDR were then determined in different immune cells. NR3C1 had a significantly 
higher level of expression compared to VDR and NR3C2 in different types of  immune cells 
(Figure 1). Among all cells, its expression was significantly higher on  Th2 CD4 cells (215.9 TPM). 
Classical (94.5 TPM) and non-classical monocytes (46.7 TPM) had the highest VDR expression 
among all inflammatory cells. Moroever, the expression of all three genes was higher in non-
classical monocytes compared to classical ones (2.0, 1.49, and 1.21 folds, respectively). Naïve 
CD8 T (74.1 TPM) cells and Th1/17 CD4 cells on the other hand (64.7 TPM) had the highest 
NR3C2 mRNA expression among all immune cells. On the other hand, activation of T cells seems 
to induce the expression of VDR and NR3C1, but not NR3C2 genes. Activated CD8 T cells express 
higher VDR and NR3C1 (10.6 and 1.47 respectively) and lower NR3C2 (0.12-fold), compared to 
naïve cells. Similarly, activated CD4 T cells express higher VDR, and NR3C1 (3.95 and 1.25 
respectively) and lower NR3C2 (0.11-fold), compared to naïve CD4 T cells (Figure 1).  
 

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Figure 1: NR3C1, NR3C2 and VDR mRNA expression level in immune cells. Data was 
extracted using publicly available “Database of Immune Cell Expression, Expression 
quantitative trait loci (eQTLs) and Epigenomics”.   
 
VDR and steroid receptors genes are downregulated in asthma relative to severity 
Activated T cells was shown to upregulate VDR, while treatment with VitD suppressed the 
proliferation of these cells, as well as their ability to produce IFN-γ  and IL-17 cytokines. 18. The 
level of expression of VDR in the blood of asthmatic and its association with the expression of 
steroid receptors relative to disease severity was not determined. To investigate that, we analyzed 
the expression profile of publicly available datasets (GSE69683) which has a large number of 
participants and detailed clinical information. A total number of 498 participants, of which 87 
healthy controls, 77 moderate asthma, and 246 non-smokers severe asthma were included in the 
U-BIOPRED study. The differential expression of NR3C1, NR3C2, and VDR genes in the blood 
of patients from all groups was then determined. Patients with moderate asthma had higher levels 
of NR3C1 mRNA expression compared to health controls, although not to a significant level (log2 
intensity 10.17±0.29; p=0.07) (Figure 2A). When compared to moderate asthmatics, the 
expression of this gene, however, was significantly downregulated in severe asthmatic (log2 
intensity 10.14±0.23 versus 10.26±0.21; p<0.01)). NR3C2 mRNA expression was significantly 
downregulated in severe asthmatic compared to moderate asthma (log2 intensity 5.397±0.727 
versus 5.62±0.5011, p<0.01), and to healthy controls (log2 intensity 5.575±0.5894 versus 
5.397±0.727, p=0.03) (Figure 2B). Moderate asthmatics showed higher NR3C2 mRNA expression 
than healthy controls, although the difference was not significant. On the other hand, VDR mRNA 
expression was significantly upregulated in severe asthmatics compared to healthy controls (log2 
intensity 8.386±0.46 versus 8.206±0.44 p<0.01), but not to moderate asthmatics (Figure 2C). 
Interestingly, NR3C1 mRNA expression correlated positively (Spearman r =0.11, 95% confident 
interval=0.02007 to 0.1988 and p = 0.01) (Figure 2D), while NR3C2 expression correlated 

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negatively (Spearman r =-0.55, 95% confident interval=-0.5724 to -0.438 and p <0.0001) with the 
levels of VDR expression in blood (Figure 2E). 
 
 
Figure 2: NR3C1, NR3C2 and VDR mRNA expression level in the blood of subjects with 
severe asthma, moderate asthma, and non-asthmatics. Data was collected in the U-
BIOPRED study (GSE69683).  
 
Supplementation of vitamin D selectively favours the upregulation of GR-α receptor 
To validate the in silico findings, a double-blinded, randomized, placebo-controlled study was 
performed to test the effect of VitD supplementation on 45 vitamin D deficient asthmatics. 
Moderate to severe asthmatics between 18 and 65 years of age who were diagnosed with asthma 
and VitD (25D3) levels less than 20ng/mL at the screening visit were recruited at two hospitals in 
UAE (Rashid Hospital, Dubai, U.A.E. and Zayed Military Hospital, Abu Dhabi, U.A.E.). The 
patients were divided into two groups, an experimental group and a placebo group. The baseline 
serum levels of VitD in the experimental and placebo groups were comparable (11.74 ± 0.77 
ng/mL, 12.96 ± 1.29 ng/mL, respectively). The experimental group received 50, 000 IU of VitD 

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oral supplementation weekly for eight weeks 19. Following treatement, a 2.84±0.01-fold increase 
in blood VitD D levels (33.34 ± 2.04 ng/mL) was observed (Figure 3). The level of VitD in the 
placebo group did not change (14.45 ± 1.61 ng/mL) (Table1 and Figure 3). This result indicated 
that the regimen used was effective to correct VitD deficiency.  
 
Table 1: Clinical characteristics of patients received vitamin D or placebo for eight weeks. 
Variable 
Total 
Placebo 
Vitamin D 
Total 
54 
20 
34 
Male 
19 (35%) 
4 (20%) 
15 (44%) 
Female 
35 (64.8%) 
16 (80%) 
19 (55.8%) 
Age (years) 
38.64±1.90 
40.60±2.97 
37.45±2.48 
Body Mass Index 
32.27±0.92 
32.32±5.52 
32.32±1.28 
Pre-treatment 25D3 levels (ng/mL) 
  
12.96±1.29 
11.74±0.77 
Post treatment 25D3 levels (ng/mL) 
  
14.45±1.61 
33.34±2.04 
 
 
Figure 3: Pre-treatment and post-treatment levels of serum vitamin D (25D3) in patients 
who received vitamin D or placebo for eight weeks. Severe asthmatic patients with 
vitamin deficiency were divided into 2 groups. 23 patients received 50000 IV of Vitamin 
D orally over 8 weeks, while 22 patients received placebo. Vitamin D levels were 
measured in the blood of these patients before and after the treatment.  
 
We next determined whether VitD supplementation affects the gene expression of steroid receptor 
GR-α or GR-β. Correction of VitD levels lead to a significant increase in the expression of GR-α 
0
1 0
2 0
3 0
4 0
2 5 D 3  le v e ls
2 5 D 3  le v e ls  (n g /m L )
P re  tre a tm e n t
P o s t tre a tm e n t
P la c e b o
V ita m in  D
0.03
n s
n s

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when compared to baseline level. A significant increase was observed following treatment with 
VitD compared to pre-treatment levels (1.89 ± 0.56, 5.34 ± 1.40, respectively; p<0.05) (Figure 
4A). Moroever, there was a significant increase in GR-α expression in patients who received VitD 
compared to those who received the placebo control (5.34 ± 1.40 versus 1.64 ± 0.76-fold; p<0.05). 
Interestingly, no change in GR-β expression was observed following treatment with VitD (1.49 ± 
0.27 versus1.30 ± 0.25) (Figure 4B). Relatively, the GR-α /GR-β ratio increased significantly 
following VitD supplementation compared to pre-treatment levels (3.58 ± 0.83 folds; p<0.05); or 
patients who received a placebo (Figure 4C).  
 
 
Figure 4: Vitamin D treatment increases the expression of GR- α with no effect on GR- β. 
Asthmatic subjects were treated with 50, 000 IU of vitamin D orally or placebo for eight 
weeks. Blood specimens obtained following 8 weeks of treatment were analyzed for 
mRNA expression of GR-α (A), GR-β (B) using RT qPCR and the ratio of GR-α /GR-β 
was calculated (C). *p<0.05 
 

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Vitamin D supplementation downregulated the blood levels of IL-17 and IL-4 cytokines in 
asthmatic patients  
The effect of VitD supplementation on the blood levels of asthma related pro-inflammatory 
cytokines was then determined (Figure 5)20. Blood levels of IL-17A, IL-17F, IFN-γ, IL-4, IL-5, 
and TNF-α were determined before and after VitD supplementation using ELISA assay (Figure 
5). VitD preferentially suppressed the blood levels of IL-17F cytokine (p=0.04) without affecting 
IL-17A; while there was no significant difference in the placebo group (p=0.17). A decrease in 
blood IL-4 cytokine levels was obsereved following treatment, however, not to a significant level 
(p=0.07). No significant effect of VitD supplementation was observed on the rest of the cytokines 
tested. 
 
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