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
Disclosures Neither I nor Dr. Benjamin Segal have any conflicts of interest to disclose regarding the research herein described.
Most T Cell Associated Factors are Dispensable in EAE DC Naïve CD4 + T Cell TGF- β + IL-6 Polarizing Factors IL-12, IFN- γ IL-4 TGF- β + RA IL-23 Tbet Gata3 ROR γ t FoxP3 T H 1 T H 2 T H 17 T reg Effector IFN- γ , TNF- α , GM-CSF IL-4, IL-5, IL-13 IL-17, IL-17F, IL-21, IL-22, TGF- β , IL-10, IL-35 Cytokines TNF- α , IL-6, GM-CSF
Experimental Autoimmune Encephalomyelitis: Adoptive Transfer Score Scale: 1 – Limp Tail 2 – Mouse can be flipped onto back 3 – Hind Limb Weakness 4 – Full Hind Limb Paralysis 4 4 4 3 3 3 2 2 2 Harvest Purify CD4+ T donor LNs Cells Transfer to 1 1 1 Recipients -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20 -18 -18 -18 -18 -16 -16 -16 -16 -14 -14 -14 -14 -12 -12 -12 -12 -10 -10 -10 -10 -8 -8 -8 -8 -6 -6 -6 -6 -4 -4 -4 -4 -2 -2 -2 -2 0 0 0 0 2 2 2 2 4 4 4 4 6 6 6 6 8 8 8 8 10 10 10 10 12 12 12 12 14 14 14 14 16 16 16 16 18 18 18 18 20 20 20 20 Onset Peak Late/Chronic Culture under polarizing conditions Prime Donor Mice: 4 days MOG 35-55 /CFA(s.c.)
WT Recipients of GM-CSF -/- T Cells Undergo Remission WT or WT GM-CSF -/- Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol :ji1701484.
GM-CSFR -/- Recipients of WT T cells Undergo Remission Congenic WT or GM-CSFR -/- (CD45.1) WT Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol :ji1701484.
CNS-Infiltrating Immune Cells are Altered in GM-CSFR -/- Recipients WT GM-CSFR -/- Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol :ji1701484.
GM-CSFR -/- Recipients Have Decreased CCL6 in the CNS CCL2 WT 1000 GM-CSFR -/- pg/mg Total Protein 800 600 CCL6 **** 4000 400 200 3000 0 2000 Naive Onset Peak Late * CXCL2 1000 100 0 80 Naive Onset Peak Late 60 40 20 0 Naive Onset Peak Late
Which Cells Are Expressing CCR1 Ligands, and is the Expression Driven by GM-CSF Signaling? Bone Marrow WT(RFP/CD45.2) Harvest CNS Adoptive Transfer 5x10 6 WT T H 17 cells Flow Sort cell populations for RNA 8 Wk 0 9 Wk WT (CD45.1) Bone Marrow GM-CSFR -/- (CD45.2)
Profiling the Transcriptome of WT vs GM-CSFR -/- Myeloid Cells Myeloid-Derived DCs (CD45 hi CD11b + CD11c + ) CCL6 CCL7 CCR1 ligands CCL8 CCL9 CCR1 CCR1 400 400 WT Relative mRNA Expression Relative mRNA Expression RFP + ** GM-CSFR -/- Csf2R β c -/- 300 300 * Monocytes/Macrophages (CD45 hi CD11b + CD11c - ) 200 200 100 100 0 0 Granulocytes Mono/Macro mDCs WT 1 WT 2 WT 3 WT 4 GMR 1 GMR 2 GMR 3 GMR 4
CCL6 Is Expressed by CNS-Infiltrating Myeloid Cells CD45 hi CD11b + CCL6 + Gated on CD45 hi Gated on CD45 hi CD45 hi CD11b + CCL6 + Ly6G - CD11b CCR1 - Isotype CCL6 Ly6G CD11c % CCL6+ of CD45hi CCL6+ Subsets 100 15 % CCL6 + Cells of CD45 hi % Indicated Cells 80 of CD45 hi CCL6 + 10 60 40 5 20 0 0 Peak adoptive transfer EAE Gran Mono/Macro mDCs
CCR1 is Expressed by CNS-Infiltrating Myeloid Cells CD45 hi CD11b + CCR1 + CD45 hi CD11b + CCR1 + Ly6G - Gated on CD45 hi Gated on CD45 hi CD11b CCR1 - Isotype CCR1 Ly6G CD11c % CCR1+ of CD45hi CCR1+ Subsets 20 100 % CCR1 + Cells of CD45 hi % Indicated Cells 80 of CD45 hi CCR1 + 15 60 10 40 5 20 0 0 Peak adoptive transfer EAE Gran Mono/Macro mDCs
Does CCR1 Blockade Impact Adoptive Transfer EAE? CCL6 **** 4000 WT J113863 GM-CSFR -/- 3000 Selective CCR1 Antagonist pg/ml 2000 * 1000 0 Naive Onset Peak Late CCR1 Antagonist 4 Vehicle CCR1antag 3 Clinical Score 2 1 0 0 5 10 15 20 25 30 Days Post Transfer Duncker PC, Stoolman JS, Huber AK, Segal BM (2017). J Immunol :ji1701484.
Therapeutic CCR1 Antagonism Decreases Total Cells and Granulocytes in the CNS SC Granulocytes h i S C # o f C D 4 5 25 1 . 0 × 1 0 * 6 * * 8 . 0 × 1 0 20 % CD11b + Ly6G + 5 of CD45 hi h i 6 . 0 × 1 0 15 5 # C D 4 5 4 . 0 × 1 0 10 5 2 . 0 × 1 0 5 5 0 0 Vehicle CCR1 e 1 l c R i C h e C V WT WT adoptive transfers treated with CCR1 antagonist d6-10 and euthanized d11 (peak)
Conclusions Naive Preclinical Onset Peak Chronic ↑ CCR1 Expression Blood CNS CCL6 GM-CSF CCL2 CXCL2
Acknowledgements Neuroimmunology Research Group Irani Lab Segal Lab David Irani, MD Benjamin Segal, MD Amanda Huber, PhD Alina Monteagudo Caballero, PhD Ashley Munie Laura Riley Andrew Sas, MD, PhD Jesse Washnock-Schmid Mao-Draayer Lab Ying-Jian Zhang Yang Mao-Draayer, MD Former Segal Lab Members Catherine Dowling, MD Qin Wang, MD/PhD Joshua Stoolman, PhD Immunology Program Funding Qi Wu, PhD David Giles, PhD • Zarinah Aquil • NIH - 1RO1NS057670 • Beth Moore, PhD • NIH – R21NS103215 • Yasmina Laouar, PhD • NIH Training Grant - T32AI007413 • Gabriel Nuñez, MD • ACTRIMS Travel Grant • Michal Olszewski, DVM, PhD
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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