Clint Robbins Toronto General Research Institute Peter Munk Centre of Excellence in Aortic Disease Departments of Laboratory Medicine and Pathobiology and Immunology, University of Toronto

Ottawa Heart Conference: Inflammation in

Cardiometabolic disease March 31, 2017

No disclosures

Arterial macrophage responses in

cardiovascular health and disease

The normal arterial wall is densely populated by tissue MΦ

Ensan, Li, Besla et al. Nature Immunology 2016

The normal arterial wall is densely populated by tissue MΦ

50μm CD68 DAPI

arterial adventitia

10μm

CX3CL1 CD68 DAPI

MΦ occupy multiple arterial sites.

Spatial distribution of arterial MΦ

Ensan, Li, Besla et al. Nature Immunology 2016

DAPI

BrdU

merge

Mac3

Mac3

BrdU

MΦ dominate inflammation in cardiovascular disease

Robbins, Hilgendorf et al. Nature Medicine 2013

MΦ depletion ameliorates cardiovascular disease

Boring et al. Nature 1998

Are all macrophages created equal?

CCR2

Diversity among tissue macrophages

Diversity among tissue macrophages

Up-regulated Down-regulated

Ensan, Li, Besla et al. Nature Immunology 2016

arterial MΦ alveolar MΦ splenic MΦ microglia

ce

ll n

o.

Lyve-1

96% 12% 3% 1%

Macrophage Origins (beyond environmental influences)

J Exp Med.1968; 128:415-35.

Nature Reviews Immunology 14, 392–404 (2014)

The mouse monocyte compartment

Embryonic MΦ development

Ginhoux et al. Nat Rev Imm 14, 392–404 (2014).

Perdiguero and Geissmann CSH Symposia 2013

Functional differences between bone marrow dependent and independent macrophage networks

Arterial Macrophage

Origins

Embryonic origins of arterial macrophages

Ensan, Li, Besla et al. Nature Immunology 2016

Embryonic origins of macrophages: Lineage Tracing

• pulse-label CX3CR1-expressing progenitor cells in the yolk sac, fetal liver, etc.

• tamoxifen-induced expression of Cre recombinase

under control of CX3CR1 promoter

tamoxifen

X

CX3CR1creER R26Tomato CX3CR1creER: R26Tomato

Adapted from Hofer et al. Annu Rev Immunol. 2016

YS progenitor contribution to arterial MΦ

development

Ensan, Li, Besla et al. Nature Immunology 2016

tamoxifen E8.5

Contribution of YS-derived progenitors to

adult arterial MΦ pool

• YS-derived MΦ persist into adulthood

• what leads to

decline in YS MΦ population shortly after birth?

Ensan, Li, Besla et al. Nature Immunology 2016

Arterial MΦ colonization associates with a brief

post natal period of monocyte recruitment

Ensan, Li, Besla et al. Nature Immunology 2016

tamoxifen E18.5

Monocyte accumulation is associated with

increased arterial expression of chemotactic

factors and adhesion molecules

Ensan, Li, Besla et al. Nature Immunology 2016

Arterial MΦ colonization associates with a brief

post natal period of monocyte recruitment

Ensan, Li, Besla et al. Nature Immunology 2016

What maintains arterial

MΦ abundance in

adulthood?

Ensan, Li, Besla et al. Nature Immunology 2016

Maintenance of arterial MΦ in adulthood occurs

largely independent of circulating monocytes

Maintenance of arterial MΦ in adulthood occurs

largely independent of circulating monocytes

Ensan, Li, Besla et al. Nature Immunology 2016

8 months

parabiosis

C57BL6/J UBC-GFP

Arterial MΦ turnover kinetics

Ensan, Li, Besla et al. Nature Immunology 2016

DOX

H2B-GFP

chase

Tissue MΦ renew through local proliferation

Ensan, Li, Besla et al. Nature Immunology 2016

Arterial MΦ survival

CX3CR1 expression on arterial MΦ persists

into adulthood

Ensan, Li, Besla et al. Nature Immunology 2016

CX3CL1-CX3CR1 interactions maintain arterial

MΦ survival

CX3CL1-CX3CR1 interactions maintain arterial

MΦ survival: The tissue MΦ niche

Ensan, Li, Besla et al. Nature Immunology 2016

CX3CL1-CX3CR1 interactions maintain arterial

MΦ survival: arterial MΦ niche

Arterial MΦ responses

during inflammation

Arterial macrophage diversity during inflammation

Ensan, Li, Besla et al. Nature Immunology 2016

CD45.1 CD45.2

Arterial macrophage diversity during inflammation

Macrophage diversity during inflammation

Ensan, Li, Besla et al. Nature Immunology 2016

Macrophage diversity during inflammation

Ensan, Li, Besla et al. Nature Immunology 2016

Replenishment of arterial MΦ during inflammation

Ensan, Li, Besla et al. Nature Immunology 2016

Tissue resident (Lyve-1+) MΦ Bone marrow-derived

(Lyve-1–) MΦ

Fetal liver Yolk sac

CX3CR1+

precursor

CMP HSC

Bone marrow

Monocytes

Inflammation

Resolution

Flt3+

precursor Inflammation

Development and

homeostasis

Adapted from Klapproth et al. Nature Immunology 2016

• Resident arterial MΦ constitute a distinct population among tissue MΦ.

• Arterial MΦ developmental

pathway is unique - arise embryonically from CX3CR1+ precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth.

• Survival of arterial MΦ within

the vascular niche depends on a CX3CR1/CX3CL1 axis.

• In adulthood, proliferation

sustains arterial MΦ in the steady state and after severe depletion following sepsis.

• After infection, arterial MΦ

return to functional homeostasis rapidly.

Conclusions

Collaborators

Myron Cybulsky

Mansoor Husain

Gwendalyn Randolph

Slava Epelman

Cheolho Cheong

Marco Prinz

Ingo Hilgendorf

Filip Swirski

Peter Libby

Anthony Gramolini

Barry Rubin

Acknowledgements

Robbins Lab

Angela Li

Ricky Besla

Sherine Ensan

Eric Shikatani

Shaun Pacheco

Caleb Zavitz

Jake Cosme

Mark Roufaiel

Mahmoud El-Maklizi

Jesse Williams

Peter Wieghofer

Tae Jin Yun

Jun Seong Lee

Funding

•CIHR operating grant MOP133390

•Peter Munk Chair in Aortic Disease

Research