monocyte innate immune response to vitamin d...2012/08/09 · objectives •background on vitamin d...
TRANSCRIPT
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SIAM Life Science 2012Session MS40Signaling: Vitamin D in Monocytes andReceptor Clustering in Mast Cells
Hewison and Adams laboratoryUCLA-Orthopaedic Hospital
Research CenterRene Chun
August 9, 2012
Monocyte Innate Immune Response toVitamin D
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Objectives
• Background on vitamin D and humanhealth
• Overview of vitamin D metabolism andaction, in particular, the role of serumvitamin D binding protein (DBP)
• Some findings on vitamin D and immunefunction and DBP
• Development of mathematical models• Implications from mathematically analysis
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Vitamin D Supplements
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Historical: Childhood Rickets Due toLow Vitamin D
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Hewison and Adams labs at UCLA
Vitamin D
Public health significance:Some (many?) may have vitamin D levels too low to actualizeits full benefits
Current: Areas of Interest
bonecomplications
duringpregnancy
inflammatory bowel
disease
kidney disease
immune responseheart disease
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Forms of vitamin D
OH
OH
1,25(OH)2D3
1α-hydroxylase
OH
OH
OH
1,24,25(OH)3D3
24-hydroxylase
OHOH
24,25(OH)2D3
1α-hy
droxyl
ase
24-hydroxylase
OH
25(OH)D3
25-hydroxylase
vitamin D3
7-dehydrocholesterolU.V.B
photolysis
(CYP2R1)
(CYP27B1) (CYP24A1)
Bone healthImmune regulationAnti-proliferation
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Vitamin D and Bacterial Killing
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DBP polymorphisms
GC-‐1S
GC-‐1F
GC-‐2
glu-‐ala-‐thr-‐pro-‐thr
asp-‐ala-‐thr-‐pro-‐thr
asp-‐ala-‐thr-‐pro-‐lys
1F > 1S > 2 affinity for 25D
Arnaud J, et al., Hum Genet. 1993 Sep;92(2):183-8
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White GC-1S > GC-1FBlack GC-1F > GC-1SWhite GC-2 > Black GC-2USA Blacks
GC-1F 70%GC-1S16%GC-2 11%
USA WhitesGC-1F 17%GC-1S55%GC-2 26%
Since affinity for 25D: GC-1F > GC-1S > GC-2 Perhaps a basis for some differences between whites and blacks in vitamin D influenced health outcomes?
DBP and Ethnic VariationsKamboh, Human Genetics (1986) 72:281-293
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JCEM 95:3368 (2010)
Questions
Did bound or free-ligand entry drive response?
How did DBP polymorphisms impact response?
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JCEM 95:3368 (2010)
Findings
Did bound or free-ligand entry drive response?Free-ligand entry
How did DBP polymorphisms impact response?Lower affinity DBP yielded higher response
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How much free ligand?
25D + DBP
25D + ALB
1,25D + DBP
1,25D + ALB
25D/DBP
25D/ALB
1,25D/DBP
1,25D/ALB
Ka (M-‐1)
7x108
4x107
6x105
5.4x104
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How much free ligand?
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Mathematical Analysis
Collaborators:• Brad Peercy, Dept of Mathematics & Statistics,
Univ of Maryland, Baltimore County• Arthur Sherman, Lab of Biological Modeling,
NIDDK, NIH
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Mathematical Analysis
Expand existing mathematical model by adding ….• Multiple genotypes for DBP (model #1)• In vitro experimental data (model #2)
To ask ….Using model #1• How DBP genotype effects free levels of vitamin D ligands?
Using model #2• Does the intracrine or endocrine mechanism account for vitamin D
action in adherent monocytes?
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Affinity differences(published):GC1F/GC1FGC1F/GC1SGC1F/GC2GC1S/GC1SGC1S/2GC2/2
Affinity(published):DBP for 25DDBP for 1,25DALB for 25DALB for 1,25D
Concentrations(user input):25D1,25DDBPALB
Variables and parameters
Model #1
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Feldman, Rodbard & Levine, AnalyHcal Biochemistry, 1972Dunn, Annals of NY Academy of Sciences, 1988
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Free Vitamin Levels Based on GenotypicVariation in DBP
PLoS One (2012) 7:e30773
In vitro (5% serum) In vivo
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Free Vitamin Levels Based on GenotypicVariation in DBP
PLoS One (2012) 7:e30773
“Sufficiency” as described in Nov 2010report by Institute of Medicine (IOM):20 nanograms per milliliter (50 nM) serum25D is needed for good bone health forpractically all individuals
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Model #2
v1 = 25Dv2 = 1,25D
v1
v2
CYP27B1:v1
Albumin:v1
Albumin:v2
DBP:v2
DBP:v1
CYP27B1
VDR:v1
VDR:v2
CAMPDNACAMP
Ka=700µM-‐1
Ka=0.6µM-‐1
Ka=40µM-‐1
Ka=0.054µM-‐1
Ka=5µM-‐1
Km=1µM
Ka=104µM-‐1
intracellular
extracellular
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Some knowledge exists …From model #1•Affinity of DBP for 25D & 1,25D•Affinity differences in DBP genotypes•Amounts of DBP in differing genotypes•Affinity of ALB for 25D & 1,25D
Needed for model #2•Affinity of VDR for 1,25D (much less known for 25D)•Amount of VDR in cells (has been estimated in some cell types)•Permeability of cells for 1,25D (none for 25D)•Affinity of CYP27B1 for 25D (reconstitution studies)•Rate of conversion to 1,25D by CYP27B1 (reconstitution studies)
Guesstimated …•Amount of CYP27B1•Amount of CYP27B1 and VDR in activated immune cell•Affinity of 25D/VDR binding to VDRE•Affinity of 1,25D/VDR binding to VDRE (EMSA data exists)•Effectiveness of 25D/VDR/VDRE complex on transcription of CAMP•Effectiveness of 1,25D/VDR/VDRE complex on transcription CAMP•Extracellular volume >> intracellular volume
Parameters and variables for model #2
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VDR/Ligand Interactions
25D VDR/25D 1,25D VDR/1,25D
r1 r2
r2 > r1
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r1 =Kr 2
ppv1cmmRT
Kr 2ppv1
cmm + Kr1mmv2
cpp + Kr1mmKr 2
pp, r2 =
K1mmv2
cppRTKr 2
ppv1cmm + Kr1
mmv2cpp + Kr1
mmKr 2pp,
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Active Transcription Complexes
o1 = active complex containing 25D
o2 = active complex containing 1,25D
o2 > o1
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o1 =Kcc2
p r1m
Kcc1m r2
p + Kcc2p r1
m + Kcc1m Kcc2
p , o2 =Kcc1
m r2p
Kcc1m r2
p + Kcc2p r1
m + Kcc1m Kcc2
p
Kidney International 56:S46-S51 (1999)
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ΔΔCT=log2(CAMP/CAMP0)
CAMP production
…. solve computationallyand then express graphically ….
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CAMP = " o1 + o2( ) + CAMP0
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r1 =Kr 2
ppv1cmmRT
Kr 2ppv1
cmm + Kr1mmv2
cpp + Kr1mmKr 2
pp, r2 =
K1mmv2
cppRTKr 2
ppv1cmm + Kr1
mmv2cpp + Kr1
mmKr 2pp,
!
o1 =Kcc2
p r1m
Kcc1m r2
p + Kcc2p r1
m + Kcc1m Kcc2
p , o2 =Kcc1
m r2p
Kcc1m r2
p + Kcc2p r1
m + Kcc1m Kcc2
p
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In vitro (5% serum) data and model fit
Then adjust parameters to project in vivo responses …
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Intracrine vs. Endocrine?
Basal = 1x VDR, 1x CYP27B1Activated = 5x VDR, 10x CYP27B1
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Summary
Local production of 1,25D from 25D is aplausible mechanism to account for vitamin Daction in immune cells.
DBP genotype, DBP amount and total 25Dinteract to determine free 25D levels which impactsvitamin D action.
Thus, one fixed amount of total 25D to definesufficiency may be an inadequate clinicalguideline.