Download A Role for Immature Myeloid Cells in Immune Senescence

A Role for Immature Myeloid Cells in
Immune Senescence
Elena Y. Enioutina Diana Bareyanand Raymond A. Daynes
Introduction
 Immune senescence
• Causes and consequences of immune system aging in mammals
• Compromised innate & adaptive immune responses
• Oxidative stress & inflammatory cytokines
 Myeloid cells
• Possessing immune suppressive activities
• NO-dependent mechanism
• Abnormal accumulation->cancers, persistent bacterial or viral infections,
or after surgical trauma or thermal injury
• Gr1+CD11b+ cells as myeloid-derived suppressor cells (MDSCs)
 MDSCs
• suppress the proliferation of both CD4+ and CD8+ T cells
• increased activities of arginine metabolizing enzymes, inducible NO
synthase (iNOS), and/or arginase 1 (ARG-1)
Review
Molecular mechanisms regulating myeloid-derived suppressor cell
differentiation and function
Trends in Immunology
Volume 32, Issue 1 January 2011, Pages 19-25
Introduction
I.
immune-compromising activities of Gr1+CD11b+ cells contribute to
immune senescence?
II. characterize the signaling defects responsible for their immune
suppressive activities in aged hosts?
The blood, secondary lymphoid organs, and BM of aged mice possess
elevated numbers of myeloid cells bearing the Gr1+CD11b+ phenotype
 Gr1+CD11b+ cells residing in multiple lymphoid organs increased in aged mice
BALB/c or DO11.10
18~22 mo
3 mo
similar
5-fold
ring shape
: immature
CD11b+Ly6CintLy6Gint
-> CD31+(PECAM-1)
CD11b+
18~22 mo
3 mo
CD11b+
 BALB/c-Tg(DO11.10)10Loh/J
• Mice carrying the MHC class II restricted rearranged T cell receptor
transgene, Tg(DO11.10)10Dlo, react to ovalbumin (OVA) peptide antigen
• Intraperitoneal administration of OVA to transgenic mice results in a rapid
deletion of the immature CD4+ CD8+ TCRlo thymocytes with progression to
mature thymocytes
• increased accumulation of immature myeloid cells in multiple secondary
lymphoid organs of aged mice
-> orchestrate some of the immune dysfunctions associated with advanced age
Stimulation of aged splenocytes with TLR4 ligand results in a significant
increase in the percentage of Gr1+CD11b+ and CD4+ T cells producing
proinflammatory cytokines
 Gr1+CD11b+ cells and CD4+ T cells in SPLs of aged mice produce proinflammatory cytokines
Splenocytes : 1 × 106 cells/ml in the presence of 0, 10, or 100 ng/ml LPS
A. Gr1+CD11b+ cells of BALB/c
3 mo
22 mo
6h
24 h
24 h
24 h
Neutralizing
24 h
B. CD4+ cells of DO11.10
48 h
Sup.-> ELISA
C. Splenocytes culture of BALB/c
Gr1+CD11b+ cells from aged mice suppress CD4+ T cell responses, markedly
increase iNOS or ARG-1 expression after activation, and produce elevated
levels of proinflammatory cytokines
 Gr1+CD11b+ cells isolated from the SPLs of aged mice produce proinflammatory cytokines,
increased activity of iNOS and ARG-1, suppressed CD4+ T cell proliferation
OVA-specific CD4+ T cell proliferation
B. Gr1+CD11b+ cells isolated from the SPLs
Sup.
48 h
24 h
4d
Gr1+CD11b+ cells from
SPLs or BM of BALB/c
:
Splenocytes DO11.10 mice (3 mo)
2 × 106 cells/ml+100 μg/ml OVA
ELISA
Sup.
24 h
48 h
C. Gr1+CD11b+ cells isolated from the SPLs
Sup.
48 h
Sup.
48 h
Gr1+ myeloid cells residing in the lymphoid organs of aged mice negatively
affect Ag-induced CD4+ T cell proliferation in vitro and the ability to mount
T cell-dependent Ab responses in vivo
 Depletion of Gr1+ cells from the secondary lymphoid organs of aged mice normalizes their
immune responsiveness
A. Splenocytes DO11.10 mice (3 mo , 20 mo) 2 × 106 cells/ml+100 μg/ml OVA or Gr1 depleted splenocytes ( 20 mo )
Sup.
In vivo Gr1 depletion of Gr1 by i.p. with anti-Gr1 mAb to aged
DO11.10 mice on days −6, −3, and 0
Sup.
Sup.
s.c with 50 μl of vaccine containing 50 μg OVA in Alum
T cell-dependent Ab
responses
Day 0
OVA-specific IgG Abs by ELISA
Activated Gr1+CD11b+ cells from aged donors express decreased levels of
phosphorylated Akt and fail to inactivate GSK3β effectively
Review
An Overview of Stress Response and Hypometabolic Strategies in Caenorhabditis elegans: Conserved and Contrasting Signals
with the Mammalian System
Int J Biol Sci 2010; 6:9-50
 Purified Gr1+CD11b+cells from aged donors have a reduced ability to
phosphorylate Akt and are incapable of effectively inactivating GSK3β
Gr1+CD11b+ cells isolated from SPLs of BALB/c mice
cocultured with LPS (10 ng/ml) for 30 and 60 min
decreased
decreased
Total cellular proteins prep.
->Western blot
SHIP1 is only expressed by hematopoietic cells and inhibits
the downstream activation of Akt by enzymatically converting
PI3K-induced PIP3 to PIP2
Total cellular proteins prep.
->Western blot
 The inhibition of GSK3β activity in Gr1+CD11b+ cells from aged mice ameliorates the
proinflammatory condition and reduces inducible iNOS activity.
Gr1+CD11b+ cells isolated from SPLs of BALB/c mice
cocultured with LPS (10 ng/ml) + IFN-γ or IL-4
48 h -> ELISA
reduced
reduced
reduced
GSK3 inhibitor
No change
 Treatment of activated CD4+ T cells isolated from aged donors with a specific GSK3 inhibitor
enhances T cell proliferation, increases IL-2, and reduces IFN-γ production
Splenocytes from DO11.10 mice were cultured 5 × 106cells/ml with OVA (100 μg/ml).
4 d -> ELISA
24 h -> ELISA
CD4+ T cells isolated from SPLs of DO11.10 mice (5 × 106 cells/ml)
-> anti-CD3ε (2 μg/ml) coated tissue culture plates -> Adding anti-CD28 Abs (2 μg/ml)
Discussion
• Multiple lymphoid organs of healthy aged mice harbor increased numbers of Gr1+CD11b+ cells
• CD11b+Ly6CintLy6Gint -> suppressive activities of splenic Gr1+CD11b+ cells in aged mice ( Vitamin E -> enhancement )
• MDSC -> ↑iNOS (Salmonella infected) , ↑ ARG-1(transplantable tumor bearing)
• Monocytes undergo a microenvironment-dependent polarization process
• LPS + IFN-γ -> M1 macrophages -> iNOS
• IL-4, IL-13, IL-10 -> M2 macrophages -> ARG-1
• immature Gr1+CD11b+ cells
• LPS + IFN-γ -> iNOS
• LPS + IL-4 -> ARG-1
• Many other substances (e.g., GM-CSF, G-CSF, PGs, S100A8/A9 proteins, IL-1β, IL-6, and IL-12) have been implicated in
the expansion and activation of MDSCs
• S100A8 and A9 are increased 1.5- to 2-fold in healthy aged mice
• NO produced by splenic Gr1+ cells is responsible for suppressing CD4+ T cell activities with aging
• Stimulated Gr1+CD11b+ cells from the SPLs of aged animals had a diminished capacity to activate Akt, resulting in a
reduced ability to inactivate the downstream enzyme, GSK3β
• SHIP-1 and PTEN are enyzmes that inhibit the Akt activation
• Inhibition of GSK3 activity in stimulated Gr1+CD11b+ cells from aged mice
• decreased the production of proinflammatory cytokines and iNOS activity
• no effect on inducible ARG-1 activity
• Increase T cell proliferation
Discussion
5th IDO GVHD day 49
B6 vs IDO KO -> BALB.B
B6 vs IDO KO -> B6
)
)
1
1
b
B6 vs IDO KO -> BALB.B
4th IDO GVHD day 70
b
3rd IDO GVHD day 94
0
0
10
20
30
40
50
60
70
80
90
D ay
B 6 T C D B M + B 6 T c e lls - > B . B
I D O K O T C D B M + B 6 T c e lls - > B . B
100
t
n
(
D
i
1
t
6
%
t
G
3
9
n
1
r
6
i
n
C
-
9
12
C D 1 1 b
in t
C h a n g e
15
12
9
6
i
12
15
in t
3
3
G
15
G r-1
1
Gr-1int CD11bint Change
C h an g e
C
-
in t
r
C D 11b
Gr-1int CD11bint(%)
t
n
in t
i
(
D
%
1
G r-1
0
0
0
10 20 30 40 50 60 70 80 90 100
Day
0
10
20
30
40
50
60
70
80
90
D a y
B6 TCD BM+ B6 T cells -> B.B
B 6 T C D B M + B 6 T c e lls - > B 6
IDO KO TCD BM + B6 T cells -> B.B
I D O K O T C D B M + B 6 T c e lls - > B 6
100
Discussion
5th IDO GVHD day 49
B6 vs IDO KO -> BALB.B
B6 vs IDO KO -> B6
0
0
10
20
30
40
50
60
70
80
90
D ay
B 6 T C D B M + B 6 T c e lls - > B . B
I D O K O T C D B M + B 6 T c e lls - > B . B
100
h
%1
g
i
D
h
10
C D 1 1 b
h ig h
C h a n g e
25
1
10
15
t
15
20
in t
30
20
C
-
20
25
G r-1
15
r
25
30
n
30
5
G
Gr-1int CD11bhigh Change
C h an g e
10
i
h ig h
5
5
G
C D 11b
Gr-1int CD11bhigh (%)
%
1
(
i
h
t
C
r
in t
i
n
1
D
g
h
G r-1
(
)
1
)
1
b
B6 vs IDO KO -> BALB.B
b
4th IDO GVHD day 70
3rd IDO GVHD day 94
0
0
10 20 30 40 50 60 70 80 90 100
Day
0
0
10
20
30
40
50
60
70
80
90
D a y
B6 TCD BM+ B6 T cells -> B.B
B 6 T C D B M + B 6 T c e lls - > B 6
IDO KO TCD BM + B6 T cells -> B.B
I D O K O T C D B M + B 6 T c e lls - > B 6
100
Discussion
4th IDO GVHD day 70
3rd IDO GVHD day 94
Gr-1high CD11bhigh(%)
Gr-1
high
CD11b
Change
100
90
80
70
60
50
40
30
20
10
0
0
10 20 30 40 50 60 70 80 90 100
Day
B6 TCD BM+ B6 T cells -> B.B
IDO KO TCD BM + B6 T cells -> B.B
B6 vs IDO KO -> BALB.B
B6 vs IDO KO -> B6
Gr-1high CD11bhigh Change
Gr-1high CD11bhigh Change
100
90
80
70
60
50
40
30
20
10
0
Gr-1high CD11b high (%)
high
Gr-1high CD11bhigh (%)
B6 vs IDO KO -> BALB.B
5th IDO GVHD day 49
0
10 20 30 40 50 60 70 80 90 100
Day
100
90
80
70
60
50
40
30
20
10
0
0
10 20 30 40 50 60 70 80 90 100
Day
B6 TCD BM+ B6 T cells -> B.B
B6 TCD BM+ B6 T cells -> B6
IDO KO TCD BM + B6 T cells -> B.B
IDO KO TCD BM + B6 T cells -> B6