Chronic and Acute Markers of Interferon Stimulation · a8 (aB2) 6.84 2.95 9.87 3.74 2.81 0.82 a10...
Transcript of Chronic and Acute Markers of Interferon Stimulation · a8 (aB2) 6.84 2.95 9.87 3.74 2.81 0.82 a10...
Chronic and Acute Markers of
Interferon Stimulation
Thomas B. Lavoie
Product R&D and Assay Services (PRaDAS)
The Yin-Yang of the Interferon System Lorne, Victoria, Australia 2014
In
Acute and Chronic Markers of IFN/TLR
Stimulation
CXCL-10 ISG15 Others?
In
Why look for markers of IFN stimulation?
• IFN is implicated in a growing number of conditions.
– Lupus, Scleroderma, etc
• IFN can be difficult to measure in vivo.
– Not systemic
– Fast clearance
– Tools limited
• Markers allow inference of IFN activity and may offer
some discrimination.
• Our goal is to create a panel of IFN and Marker
assay(s) to perhaps differentiate diseases.
In
Why look for markers of IFN stimulation?
• Acute Markers
– Allow measurement of current IFN activity
– Disappear quickly when activity not present
• Intermediate markers
– Reveal recent IFN activity
– Fade slowly when activity not present
• Chronic Markers
– Reveal long term IFN activity
– Maintain levels in absence of IFN
In
Measurement of IFN
• Activity
– Cell based PCR assays (WISH assay)
• Indirect stimulation?
– Microarray (IFN signature)
• When, where, protein?
– Reporter Gene
• Specificity issues, indirect stimulation?
• Mass Based
– Immunoassays
• ELISAs prone to artifacts
• Sensitivity issues
In
Measurement of IFN
90% of Patients on IFN
Beta therapy have
quantifiable IFN Beta
2% of Healthy Donors have
1-2 pg/ml IFN-Beta
No elevation of IFN Beta
on other therapies (p=0.4)
IFN Beta in Normal and MS Patient Sera
Detected by ELISA
IFN
Bet
a Tre
ated
n=29
No
IFN
Tre
atm
ent n
-25
NH
S n=
193
0102030405060708090
100110120
IFN
-Bet
a (
pg
/m
l)
In
Measurement of IFN
Activity on average 30% of
Mass
Literature suggests that
there are inhibitors of
IFN-β in serum/plasma
Not correlated is sIFNAR2
Comparison of Reporter Gene
and ELISA Results
0 25 50 75 100 12501
23
4
56
7
89
r2=0.48
IFN- (pg/ml)
IFN
Act
ivit
y (
IU)
IFNAR2 vs Activity Observed
0 2000 4000 6000 80000.00.10.20.30.40.50.60.70.80.91.01.1
sIFNAR2 (pg/ml)
Act
ivit
y/P
red
icte
d
In
Measurement of IFN
41105 41110 New
DMEM Normal Hu Serum Normal Hu Serum
Subtype LLOQ LLOD LLOQ LLOD LLOQ LLOD
a1 (aD) 59.21 15.07 79.43 17.00 2.31 0.76
a2a (aA) 7.13 2.00 7.38 1.87 1.36 0.42
a4a (aM1) 123.68 39.52 220.99 64.91 1.38 0.43
a5 (aG) 40.63 11.39 83.55 20.79 1.70 0.51
a6 (aK) 10.02 3.14 8.04 1.89 1.13 0.33
a7 (aJ1) 8.57 2.96 10.56 2.96 2.09 0.59
a8 (aB2) 6.84 2.95 9.87 3.74 2.81 0.82
a10 (aC) 10.03 3.60 16.23 5.45 1.77 0.65
a14 (aH) 16.18 5.73 32.94 10.09 1.76 0.51
a16 (aWA) 162.98 49.17 519.16 192.07 2.01 0.68
a17 (aI) 6.06 2.07 11.96 4.32 1.36 0.50
a21 (aF) - - - - 6.18 1.67
500-12.5pg/mL 1000-12.5 pg/mL 250-3.9 pg/mL
New Assay sees all 12 subtypes well, more sensitive
In
Measurement of IFN
Most LLOQ’s in the 1-2 pg/ml range ( y axis reciprocal)
a1 (aD
)
a2a (a
A)
a4a (a
M1)
a5 (aG
)
a6 (aK
)
a7 (aJ
1)
a8 (aB
2)
a10 (a
C)
a14 (a
H)
a16 (a
WA)
a17 (a
I)
a21 (a
F)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.941105
41110
41120
1/
LL
OQ
a1 (aD
)
a2a (a
A)
a4a (a
M1)
a5 (aG
)
a6 (aK
)
a7 (aJ
1)
a8 (aB
2)
a10 (a
C)
a14 (a
H)
a16 (a
WA)
a17 (a
I)
a21 (a
F)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.541105
41110
41120
1/
LL
OD
In
Measurement of IFN
IFN-α Detected in 61% of Lupus samples 30% of samples have >1 pg/ml
Again activity by reporter gene assay ~30% of expected.
IFN- Specific Signal in Lupus
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 192021 22232425260
2
4
6
8
Lupus Sera Lupus Sera plus blocking PAb
Sample
IFN
- (
pg
/m
l)
In
Acute Markers
Good relationship between IFN
levels after injection,
temperature change, and 11-OH
corticosteriods.
Other Acute Markers.
IL-6/Fever with Consensus
IFN (Colter et al JICR 2001)
From Scott et al Antimicrob Agents Ther. 1983
Effect of IFN-Alpha 2b in Patients
0.0 2.5 5.0 7.5 10.0 12.50
5
10
15
20
25
30
35
40
45
IFN 11-OH Corticosteroids
Temp (delta)
0.0
0.5
1.0
1.5
2.0
20 30
Time (hrs)
IFN
U/
ml
uM
OL
/L
or D
eg C
In
Acute Markers
Good relationship between
IFN levels and CXCL-10
levels after injection.
2’5’OAS has prolonged
elevation.
Other Intermediate markers
might include β2-
Microglobulin and
Neopterin.
From McHutchinson et al Hepatology, 2007
IFN, CXCL-10 and 2'5'OAS induction by CPG
0 10 20 30 40 50 600
50
100
IFN- CXCL10
2'5' OAS
0
1000
2000
3000
4000
5000
6000
7000
100
200
300
Day
IFN
-(p
g/
ml)
or
2'5
'OA
S
fold
in
du
cti
on
CX
CL
-10(p
g/
ml)
In
Acute Markers
CXCL-10 is elevated in MS
patients on IFN therapy and
it’s levels correlate with
IFN-Beta levels
MS
No
IFN
MS
wIF
NN
HS
0
100
200
300
400 *p=0.019 *p=0.01
IP-1
0 (
pg
/m
l)
CXCL-10 vs IFN Beta in MS
0 20 40 60 80 100 1200
100
200
300
400
r = 0.7271
IFN-beta (pg/ml)
CX
CL
-10 (
pg
/m
l)
In
Acute Markers
C24 C48 T24 T48 Bl C20 T20 CSup TSup
A549 OvCAR
ISG-15 Western Blot
I
Acute Markers
A549 Daudi
OvCar U937
IFN-Beta 48 hours ISG-15 Western Blot
I
Acute Markers
ISG-15 is secreted from Daudi Cells stimulated with IFN-Beta
0 25 50 75 100
10
100
1000
100000.02 ng/ml
0.2 ng/ml
20 ng/ml
2 ng/ml
Hours
ISG
15 (
pg
/m
l)
In
Acute Markers
ISG15 is a good candidate
Acute/Intermediate marker
Serum ISG15 after 8 MU Betaseron Injection
1 2 30
500
1000
1500
2000
Time days
ISG
15 p
g/
ml
From D’Cunha et al, J.Immunol 1996
In
Acute Markers
IFN- mediated IFN- and IL-10
release from NK cell model
0 1 2 3 4 5 60
100
200
300
IFN-IL-10
Log [IFNa] pg/ml
An
aly
te (
pg
/m
l)
Previously IL-15 has been shown to induce IL-10 from NK cells
In
Acute Markers
CXCL-10 Induction by IFN- and IFN-
1 10 100 1000100
1000
10000
Beta 24 Beta 48 Beta 72
Gamma 48 Gamma 72Gamma 24
IFN U/ml
CX
CL
-10 (
pg
/m
l)
IL-8 Induction by IFN- and IFN-
1 10 100 1000100
1000
10000
Beta 24 Beta 48 Beta 72
Gamma 48 Gamma 72Gamma 24
IFN U/ml
CX
CL
-10 (
pg
/m
l)
IFN-β and IFN-γ differentially up-regulate CXCL-10 and IL-8 in OvCAR cells
In
Acute Markers
IFN- induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 80.9
9
90
900
Hepatocytes Monocytes
24
Lymphocytes
Hours
IFN
- (
pg
/m
l)
IFN- induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 810
100
1000
Hepatocytes Monocytes
24
Lymphocytes
Hours
IFN
- (
pg
/m
l)
IFN- induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 80.01
0.1
1
10
100
1000
10000
Hepatocytes Monocytes
24
Lymphocytes
Hours
IFN
- (
pg
/m
l)
Tissue dependent regulation of IFN expression
May give indications of tissue involved in activation
In
Acute Markers
Tissue dependent regulation of marker expression
May give indications of tissue of activation
CXCL-10 induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 81
10
100
1000
10000
100000
Hepatocytes Monocytes
24
Lymphocytes
Hours
CX
CL
-10 (
pg
/m
l)
IL-6 induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 810
100
1000
10000
100000
Hepatocytes Monocytes
24
Lymphocytes
Hours
IL-6
(p
g/
ml)
TNF induction by poly I:C in Hepatocytes and Monocytes
0 2 4 6 81
10
100
1000
10000
Hepatocytes Monocytes
24
Lymphocytes
Hours
TN
F
(p
g/
ml)
In
Chronic Markers
In
Chronic Markers
Soluble Type I and Type II Receptors are elevated in Males
In
Chronic Markers
Soluble Type I and Type II Receptors are not
coordinately controlled
sIFNGR1 may be elevated in RA (Bello et al Biotherapy 1998)
In
Chronic Markers
From Gilli et al Neurology 2008
In
Chronic Markers
sIFNAR2 Levels in Autoimmunity
Normal n
=59
SLE n=111
RA n=16
Sjogren's n=11
Scleroderma n=10
0
2500
5000
7500
100001000035000
p=0.008
sIF
NA
R2 p
g/
ml
sIFNAR2 Elevated in Lupus
Not in RA, Sjogren’s, Scleroderma
Percent IFN positive?
Strength of signal?
Duration of signal?
In
Chronic Markers
sIFNAR2 does not correlate with IFN detected in samples
sIFNAR2 vs IFN- in MS
0 20 40 60 80 100 1200
2000
4000
6000
r = 0.05367
IFN- pg/mL
sIF
NA
R2 p
g/
mL
In
Chronic Markers
From Miyake et al Cytokine 2009
Chronic IFN stimulation in Cancer
Stage dependent?
In
Chronic Markers
From Muzugoshi et al Hepatology 1999
and Hep C
In
Why look for markers of IFN stimulation?
• Summary and Conclusions
– Acute Markers
• 11-OH Steroids CXCL-10 (9, 11?)
• IL-6 Fever
• ISG15?
– Intermediate Markers
• Neopterin 2’5’OAS
• β2-Microgloblulin
• ISG15?
– Chronic Markers
• sIFNAR2
• sIFNGR1?
In
Why look for markers of IFN stimulation?
• Summary and Conclusions
– Good panel of Acute and Intermediate markers
• May provide Flare/Exacerbation Information
– Limited panel of Chronic Markers
• May be suitable for patient stratification/prognosis
– Most useful panel will be a mixture of both
• Provides real time and long term information.
In
Acute and Chronic Markers of IFN-Stimulation
PRaDAS Quality Control
Taher Fatakdawala* Sehrish Ajmal*
Robert Niemczyk* Karen Zipf*
Xiao-Hong Lin*
Tara Stauffer* Production Team
Michael Skawinski*
Lara Izotova *
Eric Zhu*
Barbara Schwartz Ray Donnelly (FDA/NIH)*
Nirupama Putcha
Sanjna Sanghvi
Katherine Carrino-Bednarski
Jonathon Ferreira