Spatio-temporal profile, phenotypic diversity, and fate of recruited monocytes into the post-ischemic brain.
A key feature of the inflammatory response after cerebral ischemia is the brain infiltration of blood monocytes. There are two main monocyte subsets in the mouse blood: CCR2+Ly6Chi"inflammatory" monocytes involved in acute inflammation, and CX3CR1+Ly6Clo"patrolling" monocytes, which may play a role in repair processes. We hypothesized that CCR2+Ly6Chiinflammatory monocytes are recruited in the early phase after ischemia and transdifferentiate into CX3CR1+Ly6Clo"repair" macrophages in the brain.



CX3CR1GFP/+CCR2RFP/+bone marrow (BM) chimeric mice underwent transient middle cerebral artery occlusion (MCAo). Mice were sacrificed from 1 to 28 days later to phenotype and map subsets of infiltrating monocytes/macrophages (Mo/MΦ) in the brain over time. Flow cytometry analysis 3 and 14 days after MCAo in CCR2-/-mice, which exhibit deficient monocyte recruitment after inflammation, and NR4A1-/-BM chimeric mice, which lack circulating CX3CR1+Ly6Clomonocytes, was also performed.



Brain mapping of CX3CR1GFP/+and CCR2RFP/+cells 3 days after MCAo showed absence of CX3CR1GFP/+Mo/MΦ but accumulation of CCR2RFP/+Mo/MΦ throughout the ischemic territory. On the other hand, CX3CR1+cells accumulated 14 days after MCAo at the border of the infarct core where CCR2RFP/+accrued. Whereas the amoeboid morphology of CCR2RFP/+Mo/MΦ remained unchanged over time, CX3CR1GFP/+cells exhibited three distinct phenotypes: amoeboid cells with retracted processes, ramified cells, and perivascular elongated cells. CX3CR1GFP/+cells were positive for the Mo/MΦ marker Iba1 and phenotypically distinct from endothelial cells, smooth muscle cells, pericytes, neurons, astrocytes, or oligodendrocytes. Because accumulation of CX3CR1+Ly6CloMo/MΦ was absent in the brains of CCR2 deficient mice, which exhibit deficiency in CCR2+Ly6ChiMo/MΦ recruitment, but not in NR4A1-/-chimeric mice, which lack of circulating CX3CR1+Ly6Clomonocytes, our data suggest a local transition of CCR2+Ly6ChiMo/MΦ into CX3CR1+Ly6CloMo/MΦ phenotype.



CX3CR1+Ly6Cloarise in the brain parenchyma from CCR2+Ly6ChiMo/MΦ rather than being de novo recruited from the blood. These findings provide new insights into the trafficking and phenotypic diversity of monocyte subtypes in the post-ischemic brain.
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Lidia
L
Garcia-Bonilla
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Giuseppe
G
Faraco
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Jamie
J
Moore
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Michelle
M
Murphy
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Gianfranco
G
Racchumi
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Jayashree
J
Srinivasan
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
David
D
Brea
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA.
Costantino
C
Iadecola
Feil Family Brain and Mind Research Institute, Weill Cornell Medicine, 407 East 61st Street RR409, New York, NY, 10065, USA. joa2006@med.cornell.edu.
Josef
J
Anrather
D000818:Animals; D001921:Brain / Q000473:pathology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002465:Cell Movement / Q000235:genetics / Q000502:physiology*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005786:Gene Expression Regulation / Q000502:physiology; D051272:Glucose Transporter Type 1 / Q000235:genetics / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000235:genetics / Q000473:pathology* / Q000503:physiopathology*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008840:Microfilament Proteins / Q000378:metabolism; D009000:Monocytes / Q000502:physiology*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism / Q000473:pathology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1 / Q000235:genetics / Q000378:metabolism; D054390:Receptors, CCR2 / Q000235:genetics / Q000378:metabolism; D023062:Receptors, Interleukin-8A / Q000235:genetics / Q000378:metabolism
C491049:Aif1 protein, mouse; D002135:Calcium-Binding Proteins; C515335:Ccr2 protein, mouse; D051272:Glucose Transporter Type 1; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; C539818:Nr4a1 protein, mouse; D057105:Nuclear Receptor Subfamily 4, Group A, Member 1; D054390:Receptors, CCR2; D023062:Receptors, Interleukin-8A; C495678:Slc2a1 protein, mouse
CCR2; CX3CR1; Cerebral ischemia; Ly6C; Macrophages; Monocytes