Mitochondrial accumulation and increased ROS levels in the absence of autophagy modulates cytosolic antiviral signaling. Decreased autophagy with age impacts ROS levels and antiviral signaling. Specifically: 1) In the absence of autophagy, mitochondria accumulate and ROS levels increase. This leads to increased cytosolic antiviral signaling and cytokine production. 2) Autophagy deficient cells have increased mitochondrial ROS, which modulates cytosolic antiviral signaling pathways and results in increased type I interferon and inflammatory cytokine production. 3) Decreased autophagy with age impacts mitochondrial and ROS homeostasis, which affects antiviral signaling. This may contribute to increased susceptibility to viral infection

1. AUTOPHAGY, ROS AND AGING
IMPACT CYTOSOLIC ANTIVIRAL
IMMUNITY
Michal Caspi Tal
Iwasaki Lab

2. Clearance is a critical part of the
recycling process

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

6. IRF3
IFN
viral recognition
 Endosomal  Cytosolic
DNA
sensor?
IRF7
IFN
TLR7,8,9
MyD88

7. Autophagy
Isolation
Membrane
Autophagosome Autolysosome
Lysosome
P62
ATG5
Rapamycin
LC3

8. Autophagy in disease and
immunity
 Antigen Presentation
 Pathogen Clearance
 Pathogen Recognition
 Late-onset diseases
 Cancer
 Neuro-degenerative
diseases
 Inflammatory
diseases
 Crohn’s

9. Autophagy delivers viral ligands to
signaling endosome
Endosome
IRF7
IFN
TLR7,8,9
MyD88
Autophagosome

10. IRF3
IFN
Autophagy and viral recognition
 Endosomal  Cytosolic ???
IRF7
IFN
TLR7,8,9
MyD88

11. What happens in the absence of
autophagy?
WT ATG5-/-
NL
L
L
NL
L
L

12. mitochondrial membrane potential
matrix
IMS
IMM
H+ H+ H+
H+

13. Affecting ROS levels
Rotenone ROS
ROS
NAC
PG
MitoSOX
matrix
IMS
IMM
O2
-
NAC – N-acetylcystein is a
precursor in the
formation of the
antioxidant glutathione
PG - propyl gallate

14. Mitochondria accumulate and
ROS levels increase in the
absence of autophagy

15. “Defective” mitochondria accumulate
without autophagy
(mito lipid)
(mitopotential)

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

19. Mitochondrial accumulation and
increased levels of ROS
 Increased ROS results in increased IFN
 Measure ROS levels

20. Autophagy deficient MEFs have an increase
of mitochondrial ROS
100
101
102
103
104
0
20
40
60
80
100
%ofmax
RelativeMFI
MitoSOX

21. Mitochondrial ROS modulates
cytosolic antiviral signaling

22. Autophagy in cytosolic viral
recognition

23. RLR signaling
IPS-1
NFB
Inflammatory
Cytokines
IRF3
IFN
VSV, Flu
tPoly (I:C)

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

25. Autophagy deficient MEFs show increased
cytokine production
VSV - - + +
VSV - - + + tPoly I:C - - + +

26. Autophagy deficient MEFs are less
susceptibile to VSV infection

27. ATG5 Rescue

28. ROS indcuction augments IFN
production
Rotenone ROS
Poly I:C + + +
+
Rotenone - - +
+

29. NAC
PG
ROS
Antioxidant reduces IFN production
in ATG5 KO

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

31. Mitochondrial ROS levels control
IFN induction
0
50
100
150
200
IFNrelativequantity
0
50
100
150
200
IFNrelativequantity
0
600
1200
IFNrelativequantity
0
600
1200
IFNrelativequantity
PIC - - + + + + + + + + + +
Rot µM - - - - 10 1 0.1 - -
NAC - - - - - - - - - - + +
PIC + + + + + + + + + + + +
H2O2 - -
10µM 1nM
IFN α
mRNA
IFN β
mRNA
IFN α
mRNA
IFN β
mRNA
WT
MCAT

32. Mitochondria-associated ROS are integral in IFN
signlaing
NFB
Inflammatory
Cytokines
IRF3
IFN
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

35. Decreased autophagy with age
impacts ROS levels and
cytosolic antiviral signaling

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?

40. The mitochondrial-lysosomal axis
theory of aging
Lipofuscin
Organelle turnover
-Free radicals
Protein aggregates
Accumulation of
garbage over time
Lysosome

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?
NFB
Inflammatory
Cytokines
IRF3
IFN
ISGs
Decreased mitophagy
Mitoxi-inflammaging?

45. young elderly
0
2
4
6
P62MFInormalizedtocontrol
young elderly
0.0
0.5
1.0
1.5
2.0
LC3IIlevelsnormalizdtob-actin
Autophagic clearance is defective in elderly
human cells
Lysosome
P62
P62
LC3

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

48. 0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
2 0
2 2
2 4
2 6
2 8
2 0
2 5
2 1 0
2 1 5
Young (21-30) Elderly (>65)
PIC + + + + + +
Rot - + - - + -
NAC - - + - - +
RLR signaling is significantly increased in
elderly macrophages
Rotenone NAC ROS
ROS
IL-6pg/ml

49. 100
101
102
103
100
101
102
103
104
IL-6 IFNβ
FoldchangemRNA
Young Elderly
PIC - + + + - + + +
Young Elderly
PIC - + + + - + + +
RLR signaling dichotomy in elderly
human cells

50. Pro-caspase-1
(Inactive)
ASC
Mitochondria
IFNs
IL-6
IL-1β
IL-1βPro
NLRP3
inflammasome
IPS-1
Influenza
RIG-I
NOD2
IPS-1
mtROS
MDA5
Caspase-1
LRR
CARD
Helicase
NACHT
PYD
Caspase1
Viral RNA
NLRP3
Inactive
NLRP3
H+
H+
TGN (pH 6.0-6.5)
H+
CGN (pH 7.2) M2 protein
ASC
H+H+
Disturbance in
intracellular
ionic concentration
vRNA
Inactive
NLRP3Nucleus
Recognition of Influenza

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-1pg/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-1pg/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

56. MX1
MX2 IRF7IRF3
IFITM3IFITM2
P<0.001 P<0.005
elderlyyoung elderlyyoung elderlyyoung
FLU effector ISGs are basally increased
in elderly humans
Juilee Thackar

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
IFNunits/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
NFB
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.

66. Our aging world
Japan
USA
China
India
Kenya
Japan
USA
China
India
Kenya
Old-age dependency Median age of population
UN world population projections, 2010
ratio of individuals over 65yrs /
individuals 20-65yrs in a country

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.