Figure legends
Figure 1: Higher serum levels of 1,25(OH) VitaminD3(=VitD3) are
associated with lower CRP levels in the blood of control pre-school
children. a: Experimental design of the study. b,c Linear
regression analysis of serum levels of CRP and 25(OH)VitD3 in control
and asthmatic children (d-f).
Fig 2: VitD3 supplementation is correlated with less
exacerbations in preschool asthma children and PD1 is decreased
in control children with higher serum levels of 1,25 (OH) Vitamin D3.(a) Exacerbations in asthmatic children during last 6 months
(B0) depending on Vitamin D3 supplement intake (0=no Vitamin D3
supplement, 1= VitaminD3 supplement) as infant (No VitaminD3 supplement
n=16,Vitamin D3 supplement n=5). (b) C-Reactive protein (CRP)
measured in serum of asthmatic children at B0, in relation to number of
exacerbations during the last 6 months retrospective from B0 (n= 8 in
both groups). (c) CRP in same children related to their FEV1%,
investigated at B0 (n=3-16 per group). p=0,0059. Two-tailed Student’s T
Test. d. Experimental design. b. Total blood cells
(tempus tube) RNA isolation for qPCR from healthy and asthmatic
children. e .PD1/HPRT mRNA expression in control children (CN)
and asthmatic children (A), considering the serum level of 25 (OH) VitD3
lower than 20 ng/ml (low) and higher than 20 ng/ml (high). (N=6-6-7-5;
CN low 25(OH)VitD3 versus CN high 25(OH)VitD3: p=0.001; CN low
25(OH)VitD3 versus A low 25(OH)VitD3: p= 0.0033. Two-tailed Students
T-test).
Figure 3. VitD3 given intranasally limits airways inflammation.
a. Experimental design. b. Representative histological
sections of the lung after Giemsa staining (n=4 per group) and lung
inflammation score (PBS versus 1x and 2xOVA p=0.079;1xOVA versus
VitD3+1xOVA p= 0.028 after Mann Whithney).c. BALF was collected
and Eosinophils (into the red square) were stained by FACS staining (n=4
or 3 per group as indicated) and analysed by using Two tailed T test
analysis (PBS versus 2xVitD3 p= 0.029 ; PBS versus 2xOVA p=0.066).
Figure 4. Airway challenge with 1,25 (OH) Vitamin D3 inhibited
IL-33, Ror-alpha in the airways. a. Experimental design of airway
tolerance. b. AREGB/HPRT mRNA expression in αCD3/CD28
stimulated lung cells. p=0,0038. Two-tailed T-test.c. IL-33/HPRT mRNA expression in αCD3/CD28 stimulated lung
cells. P=0,035. Two-tailed T-test. d. ST2/HPRT mRNA expression
in αCD3/CD28 stimulated lung cells. e. ICOS/HPRT mRNA
expression in αCD3/CD28 stimulated lung cells. p=0,042. p=0,015.f. RORa/HPRT mRNA expression in αCD3/CD28 stimulated lung
cells. PBS vs. 2xVitD3: p=0,0005. 2xVitD3 vs 2xOVA: p=0,011. 2xOVA vs
2xVitD3: p=0,026. g. IL-5 ELISA; p=0.047. Two-Tailed T-test.h. IL-13 ELISA. PBS vs. VitD3: p=0.0188; PBS vs. 2xOVA+VitD3:
p=0.0109. Both two-tailed T-test.
Figure 5: Analysis of the mechanism of airway tolerance: OVA
intranasal challenge induced T regulatory cells associated markers and
VitD3 reduced PD1mRNA . a. Experimental design. Balb/c n=3 per
group; VitD3=1a,25 DihodroxyvitaminD3 (Sigma Aldrich); 10 ng
intranasal/mouse; OVA = OVA Texas Red; 500 ug/mouse. b.Analysis of CD4+ lymphocytes in unstimulated, 1,25(OH)-Vitamin-D3
(=VitD3) and/or OVA in vivo treated wild-type mice. p=0,049. Two-tailed
T-test c. CD25+ Foxp3+CD4+: n per group=3; PBS vs 2xVitD3
p=0,025,p=0,0504; PBS vs 2xOVA p=0,003; PBS vs 2xOVA+VitD3, p= 0,034;
2xOVA vs 2xOVA+VitD3, p=0,032; 2x VitD3 vs 2xOVA, p=0,008. d.Foxp3/HPRT mRNA expression in total lung cells by qPCR: PBS vs 2xOVA
>p= 0,00069; 2x VitD3 vs 2xOVA p=0,0015; 2xOVA vs
2xOVA+VitD3 p= 0,0073.Two-tailed T test. e. IL10 measured by
Elisa in the supernatants of αCD3 antibodies stimulated lung cells. mIL
10 Elisa: PBS vs 2xOVA p=0,035; PBS vs 2xVitD3+OVA p=0,0149; 2xVitD3 vs
2x OVA p=0,0207 ; 2x OVA vs 2xOVA+VitD3 p=0,0042; two-tailed T test.f. Lung PD1/HPRT p=0.0099 n=3,3,2,3, respectively. Two tailed T
test. Balb/c n=3 per group; VitD3=1α,25 DihodroxyvitaminD3 (Sigma
Aldrich)10 ng intranasal/mouse; OVA = OVA Texas Red > 500
ug/mouse 20ug/ul -> 25 ul. Two-tailed Student’s T test.