il10 in jhmv induced encephalomyelitis
TRANSCRIPT
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Managing chronic demyelination :
cell type-specific regulation by Interleukin 10 (IL-10)
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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
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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
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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.
Arb
itrar
y un
its
Acute Chronic
Undetectable
Demyelination
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Tropism in JHMV infection of the CNS.
Microglia
Astrocytes
Oligodendrocytes
Neurons
Ependymal cells
i.c. infection
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Pro-inflammatory T cells control infectious virus
Oligodendrocytes
IFNγ receptor
IFNγ
CD8
CD4
IFNγ
Astrocytes
Perforin Granzyme
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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)
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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 ?
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IL-10 in JHMV infectionCD4+ T cells are the major source of IL-10.
Puntambekar S.S et al ; J. Virol. 2011
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Does IL-10 regulate JHMV pathogenesis?
Does IL-10 regulate :
1. Clearance of infectious virus?
3. Demyelination1. Virus
2. Virus Ag/RNA
5 10 14 21 60 Days post infection.
Arb
itrar
y un
its
Acute Chronic
Undetectable
2. Virus Persistence?
3. Demyelination?
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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
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Faster virus clearance in IL10 -/- mice.
IL-10 limits virus clearance.
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2. Does IL-10 alter viral persistence?
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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.
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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
5 10 14 21 60 Days post infection.
Arb
itrar
y un
its
Acute Chronic
Undetectable
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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
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Increased lesion area in the absence of IL-10
n = 10 mice / group Puntambekar S.S et al ; Glia. 2015
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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.
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How does IL-10 regulate demyelination?
1. No IL-10 = increased pro-inflammation?
• Does IL-10 alter inflammation during chronic demyelination?
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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
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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
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No difference in T cell inflammation in the absence of IL-10
Puntambekar S.S et al ; Glia. 2015
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Transient increase in macrophages in infected spinal cord
MicrogliaCD11b (+)CD45 lo
CD
45
CD11b
MacrophagesCD11b (+)CD45 high
CD
45
CD11b
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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?
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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)
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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
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Reduced demyelination = Increased anti-inflammatory microglia /
macrophages?
MHC Class II
Mannose Receptor (MMR)=
day 14 p.i.
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Does IL-10 regulate the anti-inflammatory microglia / macrophages
surrounding lesions?
i.c infection
Days post infectionWt 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
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MMR(+) cells surrounding wild type lesions
Wt d14 p.i.MMR + PLP20X MMR + PLP
40X
Puntambekar S.S et al ; Glia. 2015
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MMR(+) cells surrounding IL10 -/- lesions
IL-10 d14 p.i.
MMR + PLP20X
1.
1.
2.
2.
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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
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Increased MMR(+) cells surrounding IL10-/- lesions at day 14 p.i.
n = 9 mice / groupND = Not Detected Puntambekar S.S et al ; Glia. 2015
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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?
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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?
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Chronic IL10 -/- lesions – d31 p.i.
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Pro-inflammatory macrophages and chronic demyelination.
Pro-inflammatory macrophages
MHC class II
Increased effector T cell responses
iNos
Oxidative damage, cell death
Demyelination
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Does IL-10 regulate the pro-inflammatory activation of lesion-
associated microglia / macrophages?
i.c infection
Days post infectionWt 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.
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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
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Increased numbers of MHC class II (+) microglia/macrophages
associated with IL-10 -/- lesions
Puntambekar S.S et al ; Glia. 2015
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Does IL-10 regulate the pro-inflammatory activation of lesion-
associated microglia / macrophages?
i.c infection
Days post infectionWt 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.
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Iba1 iNos Merge
Iba1 iNos MergeG HG
Lesion associated iNos(+) microglia / macrophages
d31 d31 d31
Wt
IL10-/-
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Increased numbers of iNos (+) microglia/macrophages associated with
IL-10 -/- lesions
Puntambekar S.S et al ; Glia. 2015
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Summary
IL-10 “de-activates” lesion-associated microglia / macrophages
Anti-inflammatoryPro-inflammatory De-activated
d14 d21 d31
Wt
IL10 -/-
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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
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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
31Chronic Infection
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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
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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
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Compact “glial scar”
Severe“astrogliosis”
Hypertrophy
Diffuse “astrogliosis”Naïve GFAP+
GFAP
Proliferation
Process extensionLesion associated astrocytes
Lesion associated astrocytes
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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
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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
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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 infection21 31
Chronic
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IL-10 -/- lesions are associated with reduced number of astrocytes
n = 6-10 mice / group / time point
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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
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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
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Final Summary
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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