il10 in jhmv induced encephalomyelitis

Managing chronic demyelination :

cell type-specific regulation by Interleukin 10 (IL-10)

TNFαiNOS etc

IFNγTNFα 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

The need for a balanced immune response

Pro-inflammation

Anti-inflammation

Pathogen clearanceDisease Resolution

Anti-inflammationPro-inflammation

Chronic inflammationBy-stander damage

By-standerdamage

Pathogen / Disease persistence

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.

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Acute Chronic

Undetectable

Demyelination

Tropism in JHMV infection of the CNS.

Microglia

Astrocytes

Oligodendrocytes

Neurons

Ependymal cells

i.c. infection

Pro-inflammatory T cells control infectious virus

Oligodendrocytes

IFNγ receptor

IFNγ

CD8

CD4

IFNγ

Astrocytes

Perforin Granzyme

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)

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 ?

IL-10 in JHMV infectionCD4+ T cells are the major source of IL-10.

Puntambekar S.S et al ; J. Virol. 2011

Does IL-10 regulate JHMV pathogenesis?

Does IL-10 regulate :

1. Clearance of infectious virus?

3. Demyelination1. Virus

2. Virus Ag/RNA


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Acute Chronic

Undetectable

2. Virus Persistence?

3. Demyelination?

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

Faster virus clearance in IL10 -/- mice.

IL-10 limits virus clearance.

2. Does IL-10 alter viral persistence?

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.

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. Demyelination1. Virus

2. Virus Ag/RNA


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Acute Chronic

Undetectable

Approach

Days post infectionWt 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

Increased lesion area in the absence of IL-10

n = 10 mice / group Puntambekar S.S et al ; Glia. 2015

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.

How does IL-10 regulate demyelination?

1. No IL-10 = increased pro-inflammation?

• Does IL-10 alter inflammation during chronic demyelination?

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

SpinalCord

7 10 14 21 31

Acute Chronic

CNS infection results in recruitment of inflammatory infiltrates (CD45hi).

CD45- ; CNS Resident cells ;

Astrocytes,Oligodendrocytes.

CD45lo ;microglia

CD45hi ;Inflammatoryinfiltrates

FSC

CD45

Naive 7 days post infection

CD45hi infiltrates = macrophages, monocytes, neutrophils, T cells

CD45- ; CNS Resident cells ;

Astrocytes,Oligodendrocytes.

CD45lo ;microglia

No difference in T cell inflammation in the absence of IL-10

Puntambekar S.S et al ; Glia. 2015

Transient increase in macrophages in infected spinal cord

MicrogliaCD11b (+)CD45 lo

CD

45

CD11b

MacrophagesCD11b (+)CD45 high

CD

45

CD11b

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?

Pro-inflammatory vs. Anti-inflammatory microglia / macrophages

MHC Class II

Pro-inflammatory Anti-inflammatory / RepairPromoting

MHC Class II

TNFa

iNOS

• Myelin stripping?

• By-stander tissue damage

• Tissue repair = Tissue damage

• Debris clearance?

Mannose Receptor (MMR)

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

Reduced demyelination = Increased anti-inflammatory microglia /

macrophages?

MHC Class II

Mannose Receptor (MMR)=

day 14 p.i.

Does IL-10 regulate the anti-inflammatory microglia / macrophages

surrounding lesions?

i.c infection

Days post infectionWt vs IL10 -/- 21 31

Chronic Infection


Section Stain with Myelin Proteo-

Lipid Protein. (PLP) + Murine Mannose Receptor

(MMR)

Freeze In OCT

14

MMR(+) cells surrounding wild type lesions

Wt d14 p.i.MMR + PLP20X MMR + PLP

40X


MMR(+) cells surrounding IL10 -/- lesions

IL-10 d14 p.i.

MMR + PLP20X

1.

1.

2.

2.

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

Increased MMR(+) cells surrounding IL10-/- lesions at day 14 p.i.

n = 9 mice / groupND = Not Detected Puntambekar S.S et al ; Glia. 2015

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?

i.c infection

Wt vs IL10 -/-

Spinal Cord(d31)

Fix tissue+ Embed

In epoxy resinSection Stain with

Toluidine Blue

Are there activated macrophages in chronic IL10 -/- lesions?

Chronic IL10 -/- lesions – d31 p.i.

Pro-inflammatory macrophages and chronic demyelination.

Pro-inflammatory macrophages

MHC class II

Increased effector T cell responses

iNos

Oxidative damage, cell death

Demyelination

Does IL-10 regulate the pro-inflammatory activation of lesion-

associated microglia / macrophages?

i.c infection


Chronic Infection



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.

Lesion associated MHC class II (+) microglia / macrophages

Iba1

Iba1

MHC class II

MHC class II

Merge

Merge

d31 d31 d31

Wt

IL10-/-


Increased numbers of MHC class II (+) microglia/macrophages

associated with IL-10 -/- lesions


Does IL-10 regulate the pro-inflammatory activation of lesion-

associated microglia / macrophages?

i.c infection


Chronic Infection



Proteo- Lipid Protein. (PLP) + iNos + Iba1

Freeze In OCT

• Quantify area of outlined lesion

using ImageJ

• Quantify cells within outlined lesion


Iba1 iNos Merge

Iba1 iNos MergeG HG

Lesion associated iNos(+) microglia / macrophages

d31 d31 d31

Wt

IL10-/-

Increased numbers of iNos (+) microglia/macrophages associated with

IL-10 -/- lesions


Summary

IL-10 “de-activates” lesion-associated microglia / macrophages

Anti-inflammatoryPro-inflammatory De-activated

d14 d21 d31

Wt

IL10 -/-

Do lesion-associated microglia/macrophages express the IL-10R?

d31 CD11b d31 IL-10Rα d31 Merge

40X 40X 40X


Section Stain with Myelin Proteolipid

Protein + Iba1 + IL-10Rα

Freeze In OCT

Puntambekar S.S , in preparation

Do astrocytes respond to IL-10?

i.c infection

WT


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

31Chronic Infection

GFAP+ astrocytes surrounding chronic lesions express IL-10Rαd31 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 40XRepresentative of 3-6 animals / group

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 diffuseAstrogliosis

Compact Glial Scar Formation

Less SevereInjury

More SevereInjury

Time post injury

Compact “glial scar”

Severe“astrogliosis”

Hypertrophy

Diffuse “astrogliosis”Naïve GFAP+

GFAP

Proliferation

Process extensionLesion associated astrocytes

Lesion associated astrocytes

STAT3 controls astrogliosis

XSTAT3 - 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

LFB

GFA

PControl GFAP-STAT3-KO

Hermann J.E. et al ; J. Neurosci. 2008

IL-10 signals through STAT3

IL-1

0Rα

IL-1

0Rα

IL-1

0Rβ

IL-1

0Rβ

Jak-1 Jak-1Tyk-2 Tyk-2

P

STAT3

STAT3

P

IL-1

0

IL-1

0

STAT

3

STAT

3

PP

Nucleus

Cytoplasm

1. Does IL-10 regulate astrocyte numbers in chronic lesions?

i.c infection

Wt vs IL10 -/-



Proteo- Lipid Protein. (PLP) + GFAP

Freeze In OCT

• Quantify area of outlined

lesion using ImageJ

• Quantify GFAP+ DAPI+

cells within outlined lesion


Days post infection21 31

Chronic

IL-10 -/- lesions are associated with reduced number of astrocytes

n = 6-10 mice / group / time point

Does IL-10 regulate formation of overlapping astrocyte networks?

i.c infection

Wt vs IL10 -/-



Proteo- Lipid Protein. (PLP) + GFAP

Freeze In OCT

Days post infection21 31

Chronic

Impaired astrocyte process extension in the absence of IL-10 d21 d31PLP / GFAP PLP / GFAPWT WT

IL-10-/- IL-10-/-

40X 40X

40X 40X

d21 PLP / GFAPWT

40Xd21 d31

Representative of 3-6 animals / group

Inset = 100X

In preparation

Final Summary

Thank you.Stephen A. StohlmanCornelia C. BergmannDavid R. HintonBruce Trapp.

Stohlman / Bergmann Lab

Carine SavarinMihyun HwangAlice Valentin TorresKrista DisanoJeff Atkinson

Natasha TowneKate Stenson

Jennifer Powers

Xinghua Yin (Andy)

Past Lab Members

Parul KapilTeresa AquinoMi WidnessTimothy PharesNiranjan Butchi

il10 in jhmv induced encephalomyelitis

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