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.