IL-10 regulates chronic demyelination by modulating microglia/macrophage and astrocyte activation. 1. IL-10 limits viral clearance without altering viral persistence by regulating effector T cell responses. 2. IL-10 reduces chronic demyelination by promoting early anti-inflammatory microglia/macrophage activation and limiting later pro-inflammatory activation. 3. IL-10 signals through STAT3 to regulate astrocyte numbers and process extension surrounding lesions, forming protective barriers around areas of damage.

1. Managing chronic demyelination :
cell type-specific regulation by Interleukin 10
(IL-10)

2. TNFα
iNOS etc
IFNγ
TNFα etc
By-stander damage /
Demyelination
• By stander damage is a consequence of the immune response to injury and /
or infection.
• Usually associated with pro-inflammation
• In the CNS : by-stander damage commonly manifests as demyelination.
- Multiple Sclerosis (MS), Spinal cord injury (SCI)
Macrophages
T cells
Introduction
Microglia Microglia /
Macrophages

3. The need for a balanced immune response
Pro-inflammation
Anti-inflammation
Pathogen clearance
Disease Resolution
Anti-inflammationPro-inflammation
Chronic inflammation
By-stander damage
By-stander
damage
Pathogen /
Disease persistence

4. JHMV infection of the CNS.
• Neurotropic strain of Mouse Hepatitis Virus : virus-induced encephalomyelitis.
• Ongoing demyelination – focal lesions primarily in spinal cord.
- Sub-lethal, acute infection : Infectious virus can be detected by plaque assay.
- Persistent, chronic infection in spinal cord.
No infectious virus ; Long-term persistence of viral Ag and RNA
Virus
Virus
Ag/RNA
5 10 14 21 60
Days post infection.
Arbitraryunits
Acute Chronic
Undetectable
Demyelination

5. Tropism in JHMV infection of the CNS.
Microglia
Astrocytes
Oligodendrocytes
Neurons
Ependymal cells
i.c. infection

6. Pro-inflammatory T cells control infectious virus
Oligodendrocytes
IFNγ
receptor
CD8
CD4
IFNγ
Astrocytes
Perforin
Granzyme

7. How is inflammation controlled?
• Fas – FasL
interactions
Induced death of
activated T cells
=
Loss of effector
T cells / responses
• Inhibitory ligand –
receptor interactions
T cell
“exhaustion”
= Effector responses
eg – cytolysis,
cytokine production
• Anti-inflammatory cytokines
• Regulation of microglia/
macrophage activation
• Suppression of effector T
cell responses
APCs T cells
PDL1 - PD1
APCs T cells
Eg – TGFb , Interleukin 10 (IL-10)

8. Interleukin-10 (IL-10) in viral infections
• A non-redundant, anti-inflammatory cytokine
• Produced by macrophages, microglia , T cells, B cells etc
• Inhibits activation of macrophages / microglia
(via regulation of MHC class II, co-stimulation etc)
• Up-regulated in infections such as influenza, HIV, LCMV
Suppression of
T cell activation
Virus infection Virus clearance Virus persistence Pathology
TMEV No effect
LCMV No effect ?

9. IL-10 in JHMV infection
CD4+ T cells are the major source of IL-10.
Puntambekar S.S et al ; J. Virol. 2011

10. Does IL-10 regulate JHMV pathogenesis?
Does IL-10 regulate :
1. Clearance of infectious virus?
3. Demyelination
1. Virus
2. Virus
Ag/RNA
5 10 14 21 60
Days post infection.
Arbitraryunits
Acute Chronic
Undetectable
2. Virus Persistence?
3. Demyelination?

11. 1. Does IL-10 regulate clearance of infectious virus?
i.c infection
7 10 14 21 31
Days post infection
Brain
Collect supernatent ;
Analyze for viral titers
by plaque assay.
Homogenize
Wt vs IL10 -/-
Spin down
tissue
homogenate
Acute Chronic

12. Faster virus clearance in IL10 -/- mice.
IL-10 limits virus clearance.

13. 2. Does IL-10 alter viral persistence?

14. Summary 1
IL-10 limits viral clearance without altering viral persistence.
• IL-10 limits the clearance of infectious virus
- Faster virus clearance in IL10 -/- mice
- Increased IFNγ production by IL10 -/- effector T cells.
• IL-10 does not alter virus persistence.

15. Does IL-10 regulate JHMV pathogenesis?
1. Does IL-10 regulate clearance of infectious virus? - YES
2. Does IL-10 regulate viral persistence? - NO
3. Does IL-10 regulate pathology?
3. Demyelination
1. Virus
2. Virus
Ag/RNA
5 10 14 21 60
Days post infection.
Arbitraryunits
Acute Chronic
Undetectable

16. Approach
Days post infection
Wt vs IL10 -/-
14 21 31
Chronic
Collect
Spinal
Cords
Section
Stain with Myelin
Proteo- Lipid Protein.
(PLP)
Measure area of outlined lesion
using ImageJ
Freeze
In OCT

17. Increased lesion area in the absence of IL-10
n = 10 mice / group Puntambekar S.S et al ; Glia. 2015

18. Summary – 2
IL-10 limits chronic demyelination.
• In wt mice :
- Demyelination peaks at day 21 p.i.
- Lesion size remains constant at day 31 p.i.
• In IL-10 -/- mice :
- Demyelination is higher than wt at days 21 and 31 p.i.
- Lesion size progressively increases.

19. How does IL-10 regulate demyelination?
1. No IL-10 = increased pro-inflammation?
• Does IL-10 alter inflammation during chronic demyelination?

20. Does IL-10 alter chronic inflammation?
i.c infection
Days post infectionWt vs IL10 -/-
Homogenize
Harvest cells at interface ;
Stain and analyze by flow
cytometry
Spinal
Cord
7 10 14 21 31
Acute Chronic

21. CNS infection results in recruitment of inflammatory
infiltrates (CD45hi).
CD45- ; CNS
Resident cells ;
Astrocytes,
Oligodendrocytes.
CD45lo ;
microglia
CD45hi ;
Inflammatory
infiltrates
FSC
CD45
Naive 7 days post infection
CD45hi infiltrates = macrophages, monocytes, neutrophils, T
cells
CD45- ; CNS
Resident cells ;
Astrocytes,
Oligodendrocytes.
CD45lo ;
microglia

22. No difference in T cell inflammation in the absence of IL-10
Puntambekar S.S et al ; Glia. 2015

23. Transient increase in macrophages in infected spinal cord
Microglia
CD11b (+)
CD45 lo
CD45
CD11b
Macrophages
CD11b (+)
CD45 high
CD45
CD11b

24. Summary – 3
IL-10 does not alter chronic inflammation following JHMV infection.
• IL-10 does not alter the recruitment of T cells or macrophages.
• IL-10 does not alter chronic anti-viral T cell responses.
BUT
• IL-10 limits the retention of macrophages.
Does IL-10 regulate microglia / macrophage activation?

25. Pro-inflammatory vs. Anti-inflammatory microglia / macrophages
MHC Class II
Pro-inflammatory Anti-inflammatory / Repair
Promoting
MHC Class II
TNFa
iNOS
• Myelin stripping?
• By-stander tissue damage
• Tissue repair = Tissue damage
• Debris clearance?
Mannose
Receptor (MMR)

26. Microglia / Macrophages in demyelination
In MS, microglia / macrophages show a combination of pro- and anti-
inflammatory phenotypes
• Acute MS lesions –
 Activated microglia / macrophages present inside and surrounding lesions
 Active myelin phagocytosis = Myelin-laden microglia / macrophages?
• Chronic MS lesions –
 Rim of activated microglia / macrophages
 No myelin-laden microglia / macrophages
 Express MHC class II and co-stimulatory molecules
 Also express Arginase-1, Mannose receptor and other scavenger receptors

27. Reduced demyelination = Increased anti-inflammatory microglia /
macrophages?
MHC Class II
Mannose
Receptor (MMR)
=
day 14 p.i.

28. Does IL-10 regulate the anti-inflammatory microglia / macrophages
surrounding lesions?
i.c infection
Days post infection
Wt vs IL10 -/- 21 31
Chronic Infection
Collect
Spinal
Cords
Section
Stain with Myelin Proteo-
Lipid Protein. (PLP) +
Murine Mannose Receptor
(MMR)
Freeze
In OCT
14

29. MMR(+) cells surrounding wild type lesions
Wt d14 p.i.
MMR + PLP
20X MMR + PLP
40X
Puntambekar S.S et al ; Glia. 2015

30. MMR(+) cells surrounding IL10 -/- lesions
IL-10 d14 p.i.
MMR + PLP
20X
1.
1.
2.
2.

31. 4. # MMR (+) cells / lesion area
1. Stain with PLP (myelin) + MMR (anti-
inflammatory microglia / macrophages)
Measure area of outlined lesion using
ImageJ
2.
3.
Count MMR(+) cells in
outlined lesion.
Quantitation of MMR(+) cells around lesion – Approach

32. Increased MMR(+) cells surrounding IL10-/- lesions at day 14 p.i.
n = 9 mice / group
ND = Not Detected Puntambekar S.S et al ; Glia. 2015

33. Summary - 4
IL-10 regulates early, anti-inflammatory activation
• IL-10 does not alter the distribution of anti-inflammatory microglia /
macrophages
• IL-10 limits the early anti-inflammatory activation of microglia /
macrophages during chronic demyelination
Does IL-10 influence pro-inflammatory microglia / macrophage
activation?

34. i.c infection
Wt vs IL10 -/-
Spinal
Cord
(d31)
Fix tissue
+ Embed
In epoxy resin
Section Stain with
Toluidine Blue
Are there activated macrophages in chronic IL10 -/- lesions?

35. Chronic IL10 -/- lesions – d31 p.i.

36. Pro-inflammatory macrophages and chronic demyelination.
Pro-inflammatory
macrophages
MHC class II
Increased effector T cell
responses
iNos
Oxidative damage,
cell death
Demyelination

37. Does IL-10 regulate the pro-inflammatory activation of lesion-
associated microglia / macrophages?
i.c infection
Days post infection
Wt vs IL10 -/- 21 31
Chronic Infection
Collect
Spinal
Cords
Section
Stain with Myelin
Proteo- Lipid Protein.
(PLP) + MHC class II +
Iba1
Freeze
In OCT
• Quantify area of outlined lesion
using ImageJ
• Quantify cells within outlined lesion
• # of cells / lesion area.

38. Lesion associated MHC class II (+) microglia / macrophages
Iba1
Iba1
MHC
class II
MHC
class II
Merge
Merge
d31 d31 d31
Wt
IL10
-/-
Puntambekar S.S et al ; Glia. 2015

39. Increased numbers of MHC class II (+) microglia/macrophages
associated with IL-10 -/- lesions
Puntambekar S.S et al ; Glia. 2015

40. Does IL-10 regulate the pro-inflammatory activation of lesion-
associated microglia / macrophages?
i.c infection
Days post infection
Wt vs IL10 -/- 21 31
Chronic Infection
Collect
Spinal
Cords
Section
Stain with Myelin
Proteo- Lipid Protein.
(PLP) + iNos + Iba1
Freeze
In OCT
• Quantify area of outlined lesion
using ImageJ
• Quantify cells within outlined lesion
• # of cells / lesion area.

41. Iba1 iNos Merge
Iba1 iNos MergeG HG
Lesion associated iNos(+) microglia / macrophages
d31 d31 d31
Wt
IL10
-/-

42. Increased numbers of iNos (+) microglia/macrophages associated with
IL-10 -/- lesions
Puntambekar S.S et al ; Glia. 2015

43. Summary
IL-10 “de-activates” lesion-associated microglia / macrophages
Anti-inflammatoryPro-inflammatory De-activated
d14 d21 d31
Wt
IL10 -/-

44. Do lesion-associated microglia/macrophages express the
IL-10R?
d31 CD11b d31 IL-10Rα d31 Merge
40X 40X 40X
Collect
Spinal
Cords
Section
Stain with Myelin Proteolipid
Protein + Iba1 +
IL-10Rα
Freeze
In OCT
Puntambekar S.S , in preparation

45. Do astrocytes respond to IL-10?
i.c infection
WT
Collect
Spinal
Cords
Section
Stain with Myelin Proteolipid
Protein + Glial Fibrillary
Acidic Protein (GFAP) +
IL-10Rα
Freeze
In OCT
• Identify lesion based on loss of PLP
• GFAP + IL-10Rα+ cells surrounding
lesion
Days post infection
31
Chronic Infection

46. GFAP+ astrocytes surrounding chronic lesions express IL-10Rα
d31 d31 d31GFAP IL-10Rα Merge
WT Lesion WT Lesion WT Lesion
40X 40X 40X
WT Non-Lesioned
WM
WT Non-Lesioned
WM
WT Non-Lesioned
WM
40X 40X 40X
Representative of 3-6 animals / group

47. Reactive Astrocytes and CNS pathology
• Astrogliosis : “ a finely graded continuum of changes in astrocyte gene
expression and cellular functions”
• Degree of astrogliosis depends on the severity and length of injury /
disease.
Naïve
Mild – Moderate
Astrogliosis
Severe diffuse
Astrogliosis
Compact Glial
Scar Formation
Less Severe
Injury
More Severe
Injury
Time post injury

48. Compact
“glial scar”
Severe
“astrogliosis”
Hypertrophy
Diffuse “astrogliosis”
Naïve
GFAP+
GFAP
Proliferation
Process extensionLesion associated
astrocytes
Lesion associated
astrocytes

49. STAT3 controls astrogliosis
X
STAT3 - flox GFAP-Cre GFAP-STAT3-KO
STAT3 signaling is
deleted specifically in
GFAP+ astrocytes
GFAP-STAT3-KO
Spinal Cord
Injury
• Impaired formation of overlapping, astrocyte structures / barriers
• Spread of demyelination = increased lesion size
LFBGFAP
Control GFAP-STAT3-KO
Hermann J.E. et al ; J. Neurosci. 2008

50. IL-10 signals through STAT3
IL-10Rα
IL-10Rα
IL-10Rβ
IL-10Rβ
Jak-1 Jak-1
Tyk-2 Tyk-2
P
STAT3
STAT3
P
IL-10
IL-10
STAT3
STAT3
PP
Nucleus
Cytoplasm

51. 1. Does IL-10 regulate astrocyte numbers in chronic lesions?
i.c infection
Wt vs IL10 -/-
Collect
Spinal
Cords
Section
Stain with Myelin
Proteo- Lipid Protein.
(PLP) + GFAP
Freeze
In OCT
• Quantify area of outlined
lesion using ImageJ
• Quantify GFAP+ DAPI+
cells within outlined lesion
• # of cells / lesion area.
Days post infection
21 31
Chronic

52. IL-10 -/- lesions are associated with reduced number of
astrocytes
n = 6-10 mice / group / time point

53. Does IL-10 regulate formation of overlapping astrocyte
networks?
i.c infection
Wt vs IL10 -/-
Collect
Spinal
Cords
Section
Stain with Myelin
Proteo- Lipid Protein.
(PLP) + GFAP
Freeze
In OCT
Days post infection21 31
Chronic

54. Impaired astrocyte process extension in the absence of IL-10
d21 d31PLP / GFAP PLP / GFAP
WT WT
IL-10-/- IL-10-/-
40X 40X
40X 40X
d21 PLP / GFAP
WT
40X
d21 d31
Representative of 3-6
animals / group
Inset = 100X
In preparation

55. Final Summary

56. Thank you.
Stephen A. Stohlman
Cornelia C. Bergmann
David R. Hinton
Bruce Trapp.
Stohlman / Bergmann Lab
Carine Savarin
Mihyun Hwang
Alice Valentin Torres
Krista Disano
Jeff Atkinson
Natasha Towne
Kate Stenson
Jennifer Powers
Xinghua Yin (Andy)
Past Lab Members
Parul Kapil
Teresa Aquino
Mi Widness
Timothy Phares
Niranjan Butchi