5. Mitochondria accumulate and ROS
levels increase in the absence of
autophagy
Mitochondrial ROS modulates cytosolic
antiviral signaling
Decreased autophagy with age impacts
ROS levels and cytosolic antiviral
signaling
Outline
16. Mitochondria accumulate in the
absence of autophagy
Relative mitochondrial
DNA copy number
Mitotracker Green
18SmtDNA
1.0 2.1
ATG5 +/+ -/-
(mito lipid)
(mito protein)
Miwa Sasai and Michal Tal
17. Redox balance
SODO2
-
CAT
GPx
H2O2 H2O
SOD - superoxide dismutase
CAT - catalase
Reduced glutathione (GSH) neutralizes peroxides using glutathione
peroxidase (GPx) and in this action is transformed into (GSSG).
Then glutathione reductase is used to reduce gluthione.
18. Oxidative stress
SODO2
- CAT
GPx
H2O2 H2O
Protein peroxidation
(change in protein activation or multimerization)
Lipid peroxidation
DNA oxidative damage
24. Mitochondrial accumulation and IFN
production
Increased ROS results in increased IFN
Measure ROS levels
Increase or decrease ROS levels and measure IFN
induction
Pro-
inflammatory
and Type I
IFN
30. Oh ROS, where art thou?
Intracellular ROS Mitochondria-associated ROS
NL
L
L
NL
L
L
H2O2 treatment to increase cellular
ROS
Cell permeable antioxidant
Rotenone treatment to increase
mitochondrial ROS
Catalase (antioxidant) targeted
specifically to the mitochondria
33. Cytokine mediated induction of
immune response
Insufficient recognition
and cytokine induction
to mount an effective
immune response
Sufficient cytokine
induction to elicit a
strong and effective
immune response
Immune
mediated
pathology
34. Implication for aging?
Dysfunctional autophagy
Increased load of defective mitochondria
Increased ROS
36. Age is the greatest risk factor in many diseases
ROS is implicated at the core of these late-onset
diseases
Late-onset diseases
AGE
(Modified from Finkel 2005)
Alzheimer’s Cardiovascular Cancer
Disease disease
37. Influenza and pneumonia deaths in 2006
90% of annual Flu related deaths in the USA
are in people over 65
Under1
year1-4
years
5-14
years
15-24
years
25-34
years
35-44
years
45-54
years
55-64
years
65-74
years
75-84
years
85
yearsand
over
0
100
200
300
400
500
600
deathratesper100,000
(based on CDC reported death rates)
38. innate immunity in aging
NKT cells – increased IL-17 in viral infection
DCs and pDCs have decreased TLR
responsiveness in general
Macrophages have decreased TLR4 signaling
and increased TLR3 signaling
Monocytes have decreased TLR signaling with
the exception of increased TLR5 signaling
39. Combining theories of the aging
immune system
Mitochondrial-lysosomal axis theory of aging
Molecular inflammatory theory of aging or
“Inflammaging”
Oxidative stress theory of aging
Oxi-inflammaging to
mitoxi-inflammaging?
41. Inflammaging
A strong immune response that provides
increased fitness will be evolutionarily selected
for, even if it causes pathology in old age.
Insufficient recognition
and cytokine induction
to mount an effective
immune response
Sufficient cytokine
induction to elicit a
strong and effective
immune response
Immune
mediated
pathology
42. Oxi
SODO2
-
CAT
GPx
H2O2 H2O
SODO2
- CAT
GPx
H2O2 H2O
Inflammatory skew of immune mediators
Correlations between high oxidative stress in
immune cells and poor function (i.e. chemo- taxis and
phagocytosis) as well as decreased longevity of the
individuals with those immune cells.
Pro Anti
Pro Anti
-Inflammaging
43. Mitochondrial accumulation and
cytokine production
Increased ROS results in increased IFN
Increased ROS levels with the absence of autophagy
Increasing ROS levels resulted in increased cytokine
production, while decreasing ROS levels resulted in
decreased cytokine production
Pro-
inflammatory
and Type I
IFN
(Tal et al PNAS 2009)
44. Is dysfunctional autophagy in aging causing a
hyperinflammatory state?
NFB
Inflammatory
Cytokines
IRF3
IFN
ISGs
Decreased mitophagy
Mitoxi-inflammaging?
46. Increased mitochondrial ROS with
age?
Young Elderly
NL
L
L
Are mitotic cells accumulating damaged mitochondria
with increased ROS production due to defects in
autophagic clearance?
NL
L
L
47. elderly MФ’s show defect in autophagy and
increased mtROS
Young (21-30) Elderly (>65)
Human Macrophages
0
20
40
60
80
100
mitosoxMFI
51. Why is seasonal Influenza potentially
deadly in the elderly?
Influenza induced primary viral pneumonia or
secondary bacterial pneumonia are one of the
most significant causes of infectious disease
related death in the elderly.
Severe flu infections in the elderly result in
increased lung inflammation reminiscent of the
increased inflammation and “cytokine storm”
induced by highly pathogenic flu strains which
result in inappropriate cellular response,
increased lung damage and inefficient viral
clearance.
52. Increased flu induced IL-6 and IL-1β
production in elderly MФ’s
0
200
400
600
800
1000
IL-6pg/ml
0
10
20
30
40
50
60
70
80
IL-1pg/ml
Young (21-30) Elderly (>65)
5 MOI
FLU
- + + - + +
RAP - - + - - +
Young (21-30) Elderly (>65)
5 MOI
FLU
- + + - + +
RAP - - + - - +
10 individuals per group
PR8 NS-1/GFP virus hMDMs
In vitro infection
53. Increased flu induced IL-6 and IL-1β
production in elderly MФ’s
0
100
200
300
400
IL-6pg/ml
0
10
20
30
40
50
60
70
80
IL-1pg/ml
Young (21-30) Elderly (>65)
1 MOI
FLU
- + + - + +
RAP - - + - - +
Young (21-30) Elderly (>65)
1 MOI
FLU
- + + - + +
RAP - - + - - +
10 individuals per group
PR8 NS-1/GFP virus hMDMs
In vitro infection
54. Elderly human cells have a ROS dependent
resistance to flu infection
0
10
20
30
40
50
60
70
80
%infectedCD14+cells
age young elderly young elderly
1 MOI
FLU
+ + + +
NAC - - + +
55. Decreased IFN-I production and yet
increased resistance to flu
Elderly cells may not be capable of supporting
maximal viral replication.
Increased cell death
Blockage in autophagic clearance
ATP levels
Direct antiviral protection through pro-
inflammatory cytokines?
Increased basal levels of IFNs or ISGs?
Increased production of other types of IFN?
IFNγ has been shown to be increased with age,
correlates with viral titer in humans, and can induce a
subset of ISGs
57. Are the elderly more susceptible to flu
infection?
Increased hospitalizations of flu infected
elderly
When flu infections don’t show increased
morbidity in the elderly, this is attributed to pre-
existing immunity
58. Are the elderly more susceptible to flu
infection?
10 PFU A/PR8 (i.n)
Day 9 PI collect
bronchoalveolar
lavage and lungs
WT
ATG5+/-
59. 6 wk old WT
2 yr old WT
1 yr old WT
1 yr old Atg5+/-
Iris Pang
Old mice and ATG5 HETs are resistant to 10 PFU flu infection
60. Iris Pang and Michal Tal
The old mice and ATG5 HETs are
resistant to 10pfu of flu
102
103
104
105
ND ND
PFU/ml
0
5
10
15
20
25
TotalCellNumber/BAL
(x105
)
6wk WT 1yr WT 1yr ATG5 HET 2yr WT
BAL cellularity BAL lung titer
6wk WT 1yr WT 1yr ATG5 HET 2yr WT
61. Increase the exposure
1000 PFU A/PR8 (i.n) Day 3
and 6 PI collect
bronchoalveolar
lavage and lungs
WT ATG5+/-
62. Reduced flu replication in old mice and ATG5
+/- upon 1000pfu of flu
Iris Pang
10 5
10 6
10 7
10 8
Pulmonaryviraltiter(PFU/ml)
6wk WT 6wk ATG5+/- 8mth ATG5+/- 2yr WT
Day PI 3 6 3 6 3 6 3 6
63. Cytokine dichotomy in lung after
1000pfu flu infection
6w
k
W
T
6w
k
ATG
5
HET
8m
th
ATG
5
HET
2yrW
T
0
2 0
4 0
6 0
8 0
IFNunits/mlBAL
6w
k
W
T
6w
k
ATG
5
HET
8m
th
ATG
5
HET
2yrW
T
0
2 0 0 0
4 0 0 0
6 0 0 0
IL-6pg/mlBAL
IFNα IL-6
Iris Pang and Michal Tal
64. Is dysfunctional autophagy in aging causing a
hyperinflammatory state?
ISGs
P62
NFB
Inflammatory
Cytokines
65. Future directions
Identify the target of oxidation by which
mitochondrial ROS modulates cytosolic
antiviral signaling.
Investigate the IL-6/IFN dichotomy with aging
and long term defects in mitochondrial
clearance.
Assess levels of other IFNs and validate IFITM
expression from elderly cells and old mice.
67. Implications
Autophagic control of mitochondrial number and
integrity modulates cytosolic antiviral responses
Defects in autophagy can lead to a hyper-
inflammatory state and impact many of the chronic
diseases associated with aging
“Feed a cold, starve a fever” – viral anorexia to
induce autophagy?
Modulating autophagy and inflammatory responses
could possibly reduce the morbidity and mortality of
flu infection in the aged population
68. Lung pathology at 6 days post infection
with 1000pfu PR8
peribronchiolar
inflammation
Perivascular
inflammation
alveolitis Lymphoid
aggregates
6wk WT ++ ++ +++ -
6wk ATG5
HET
+ ++ ++ -
8mth ATG5
HET
++ + + -
2yr WT +++ ++ ++ ++++
6wk WT 6wk ATG5 HET 8mth ATG5 HET 2yr WT
uninfected
69. Lung pathology at 6 days post infection
with 1000pfu PR8
peribronchiolar
inflammation
Perivascular
inflammation
alveolitis Lymphoid
aggregates
6wk WT ++ ++ +++ -
6wk ATG5
HET
+ ++ ++ -
8mth ATG5
HET
++ + + -
2yr WT +++ ++ ++ ++++
6wk WT 6wk ATG5 HET 8mth ATG5 HET 2yr WT
uninfected
70. Lessons from in vivo intranasal flu
infections
Decreased mitophagy may contribute to flu
resistance with age, but reduced tolerance to
flu infection with age is likely leading to the
increased morbidity.
