CCL6 Is Induced in CNS-Infiltrating Myeloid Cells By a GM- CSF - - PowerPoint PPT Presentation

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CCL6 Is Induced in CNS-Infiltrating Myeloid Cells By a GM- CSF - - PowerPoint PPT Presentation

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CCL6 Is Induced in CNS-Infiltrating Myeloid Cells By a GM- CSF Dependent Pathway, Driving Chronic Disability During Experimental Autoimmune Demyelination Patrick Duncker Graduate Student Laboratory of Dr. Benjamin Segal University of Michigan

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SLIDE 1

CCL6 Is Induced in CNS-Infiltrating Myeloid Cells By a GM- CSF Dependent Pathway, Driving Chronic Disability During Experimental Autoimmune Demyelination

Patrick Duncker

Graduate Student Laboratory of Dr. Benjamin Segal University of Michigan ACTRIMS 2018

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SLIDE 2

Disclosures

Neither I nor Dr. Benjamin Segal have any conflicts of interest to disclose regarding the research herein described.

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SLIDE 3

Most T Cell Associated Factors are Dispensable in EAE

Tbet

TH1 IL-12, IFN-γ IFN-γ, TNF-α, GM-CSF

RORγt

TH17

TGF-β + IL-6

IL-23 IL-17, IL-17F, IL-21, IL-22, TNF-α, IL-6, GM-CSF TH2 IL-4 IL-4, IL-5, IL-13 Treg

TGF-β + RA

TGF-β, IL-10, IL-35

Naïve CD4+ T Cell DC Gata3 FoxP3 Polarizing Factors Effector Cytokines

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SLIDE 4

Experimental Autoimmune Encephalomyelitis: Adoptive Transfer

Prime Donor Mice: MOG35-55/CFA(s.c.)

  • 18
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  • 2

2 4 6 8 10 12 14 16 18 20

Harvest donor LNs Culture under polarizing conditions 4 days

  • 18
  • 16
  • 14
  • 12
  • 10
  • 8
  • 6
  • 4
  • 2

2 4 6 8 10 12 14 16 18 20

Purify CD4+ T Cells Transfer to Recipients

  • 18
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  • 10
  • 8
  • 6
  • 4
  • 2

2 4 6 8 10 12 14 16 18 20 1 2 3 4

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2 4 6 8 10 12 14 16 18 20 1 2 3 4

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2 4 6 8 10 12 14 16 18 20 1 2 3 4

Onset Peak Late/Chronic

Score Scale: 1 – Limp Tail 2 – Mouse can be flipped onto back 3 – Hind Limb Weakness 4 – Full Hind Limb Paralysis

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SLIDE 5

WT Recipients of GM-CSF-/- T Cells Undergo Remission

WT WT or GM-CSF-/-

Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol:ji1701484.

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SLIDE 6

GM-CSFR-/-Recipients of WT T cells Undergo Remission

Congenic (CD45.1) WT WT or GM-CSFR-/-

Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol:ji1701484.

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SLIDE 7

CNS-Infiltrating Immune Cells are Altered in GM-CSFR-/- Recipients

Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol:ji1701484.

WT GM-CSFR-/-

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SLIDE 8

GM-CSFR-/-Recipients Have Decreased CCL6 in the CNS

200 400 600 800 1000

CCL2

pg/mg Total Protein

Naive Onset Peak Late

20 40 60 80 100

CXCL2 Naive Onset Peak Late

1000 2000 3000 4000

CCL6

**** *

Naive Onset Peak Late

WT GM-CSFR-/-

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SLIDE 9

WT (CD45.1) GM-CSFR-/- (CD45.2)

Adoptive Transfer

5x106 WT TH17 cells

8 Wk 9 Wk

Harvest CNS

Flow Sort cell populations for RNA Bone Marrow Bone Marrow WT(RFP/CD45.2)

Which Cells Are Expressing CCR1 Ligands, and is the Expression Driven by GM-CSF Signaling?

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SLIDE 10

Myeloid-Derived DCs (CD45hiCD11b+CD11c+) Monocytes/Macrophages (CD45hiCD11b+CD11c-)

CCL6 CCL7 CCL8 CCL9 CCR1 ligands

Profiling the Transcriptome of WT vs GM-CSFR-/- Myeloid Cells

WT 1 WT 2 WT 3 WT 4 GMR 1 GMR 2 GMR 3 GMR 4

Granulocytes 100 200 300 400

CCR1

Relative mRNA Expression **

Mono/Macro mDCs 100 200 300 400

CCR1

Relative mRNA Expression * RFP+ Csf2Rβc-/-

WT GM-CSFR-/-

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SLIDE 11

CCR1 - Isotype CD11b CCL6 Ly6G CD11c

CCL6 Is Expressed by CNS-Infiltrating Myeloid Cells

Gran Mono/Macro mDCs 20 40 60 80 100

CCL6+ Subsets

% Indicated Cells

  • f CD45hiCCL6+

Gated on CD45hi Gated on CD45hi CD45hiCD11b+CCL6+ CD45hiCD11b+CCL6+Ly6G-

5 10 15

% CCL6+ of CD45hi

% CCL6+ Cells of CD45hi

Peak adoptive transfer EAE

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SLIDE 12

CCR1 is Expressed by CNS-Infiltrating Myeloid Cells

Peak adoptive transfer EAE Gated on CD45hi Gated on CD45hi CD45hiCD11b+CCR1+ CD45hiCD11b+CCR1+Ly6G-

Gran Mono/Macro mDCs 20 40 60 80 100

CCR1+ Subsets

% Indicated Cells

  • f CD45hiCCR1+

CCR1 - Isotype CD11b CCR1 Ly6G CD11c

5 10 15 20

% CCR1+ of CD45hi

% CCR1+ Cells of CD45hi

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SLIDE 13

Does CCR1 Blockade Impact Adoptive Transfer EAE?

5 10 15 20 25 30 1 2 3 4

CCR1 Antagonist

Days Post Transfer Clinical Score Vehicle CCR1antag

J113863 Selective CCR1 Antagonist

1000 2000 3000 4000

CCL6

pg/ml **** * WT GM-CSFR-/-

Naive Onset Peak Late Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol:ji1701484.

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SLIDE 14

Therapeutic CCR1 Antagonism Decreases Total Cells and Granulocytes in the CNS

V e h i c l e C C R 1 2 . 0 × 1 0

5

4 . 0 × 1 0

5

6 . 0 × 1 0

5

8 . 0 × 1 0

5

1 . 0 × 1 0

6

S C # o f C D 4 5

h i

# C D 4 5

h i

* *

Vehicle CCR1 5 10 15 20 25

SC Granulocytes

% CD11b+Ly6G+

  • f CD45hi

*

WTWT adoptive transfers treated with CCR1 antagonist d6-10 and euthanized d11 (peak)

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SLIDE 15

Conclusions

GM-CSF CCL2 CXCL2

Naive Preclinical Onset Peak Chronic ↑ CCR1 Expression

CCL6

Blood CNS

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SLIDE 16

Acknowledgements

Irani Lab

David Irani, MD Amanda Huber, PhD

Mao-Draayer Lab

Yang Mao-Draayer, MD Catherine Dowling, MD Qin Wang, MD/PhD Qi Wu, PhD

Segal Lab

Benjamin Segal, MD Alina Monteagudo Caballero, PhD Ashley Munie Laura Riley Andrew Sas, MD, PhD Jesse Washnock-Schmid Ying-Jian Zhang

Former Segal Lab Members

Joshua Stoolman, PhD David Giles, PhD

Neuroimmunology Research Group

Funding

  • NIH - 1RO1NS057670
  • NIH – R21NS103215
  • NIH Training Grant - T32AI007413
  • ACTRIMS Travel Grant

Immunology Program

  • Zarinah Aquil
  • Beth Moore, PhD
  • Yasmina Laouar, PhD
  • Gabriel Nuñez, MD
  • Michal Olszewski, DVM, PhD
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SLIDE 17

CCL6 Is Induced in CNS-Infiltrating Myeloid Cells By a GM- CSF Dependent Pathway, Driving Chronic Disability During Experimental Autoimmune Demyelination

Patrick Duncker

Graduate Student Laboratory of Dr. Benjamin Segal University of Michigan ACTRIMS 2018