Nonclassical Monocytes Mediate Secondary Injury, Neurocognitive Outcome, and Neutrophil Infiltration after Traumatic Brain Injury

Abstract

Traumatic brain injury (TBI) results in rapid recruitment of leukocytes into the injured brain. Monocytes constitute a significant proportion of the initial infiltrate and have the potential to propagate secondary brain injury or generate an environment of repair and regeneration. Monocytes are a diverse population of cells (classical, intermediate, and nonclassical) with distinct functions, however, the recruitment order of these subpopulations to the injured brain largely remains unknown. Thus, we examined which monocyte subpopulations are required for the generation of early inflammatory infiltrate within the injured brain, and whether their depletion attenuates secondary injury or neurocognitive outcome. Global monocyte depletion correlated with significant improvements in brain edema, motor coordination, and working memory, and abrogated neutrophil infiltration into the injured brain. However, targeted depletion of classical monocytes alone had no effect on neutrophil recruitment to the site of injury, implicating the nonclassical monocyte in this process. In contrast, mice that have markedly reduced numbers of nonclassical monocytes (CX3CR1^-/-) exhibited a significant reduction in neutrophil infiltration into the brain after TBI as compared with control mice. Our data suggest a critical role for nonclassical monocytes in the pathology of TBI in mice, including important clinical outcomes associated with mortality in this injury process.

Figure 1

Severe TBI results in disruption of the structural architecture of the brain as well as infiltration of peripheral immune cells into the injured brain. Controlled cortical impact was performed at a velocity of 2.5m/s, a depth of 2mm and a dwell time of 0.2 seconds. Representative sections demonstrate (A, D) infiltration of CD45hi cells into the injured brain as compared to sham-injured animals. (B, E) Infiltration of Ly6G+ cells into the brain after injury compared to sham-injured animals. (C, F) Infiltration of F4/80+ cells into the brain after injury compared to sham-injured animals. All images are at 4× magnification.

Figure 2

Gating strategy for cells from brains of shielded CD45.1+CD45.2 mixed chimera mice: A) PBMCs from blood of chimeric mice 8 weeks after adoptive transfer, showing that all monocytes and neutrophils are CD45.1. (B) cells from brain of chimeric mice 24h post TBI, showing that resident microglia can be differentiated from infiltrating monocyte derived macrophages. Cells gated on singlets, then live cells then CD45.1 and CD45.2 populations. Arrow indicated directionality of gating. C) Histogram showing the CD45.2 expression between microglia and other peripheral cells compared to monocyte derived cells in a chimeric mouse.

Figure 3

Monocyte depletion inhibits neutrophil recruitment after TBI: (A–C) intravenous administration of one dose of clodronate does not affect neutrophil population whereas all monocyte subsets are depleted in circulation P ≤ 0.05. (D–F) Monocyte depletion abrogates neutrophil infiltration into the brain as well as monocyte derived macrophages. P ≤ 0.05 Mann Whitney test and n=15

Figure 4

Monocyte depletion improves functional outcome one month after TBI: (A–B) intravenous administration of clodronate improves the (A)time spent on the rotarod P ≤ 0.05and (B) the speed P ≤ 0.03. (C) Monocyte depletion improves spatial alternations in mice after TBI compared to control treatment P ≤ 0.05. (D) Monocyte depletion improves edema in mice brain via abrogation of neutrophil infiltration after TBI P ≤ 0.03. Mice n= 8. Mann whitney test.

Figure 5

Classical monocytes do not mediate neutrophil infiltration into the brain after TBI: (A–E) intravenous administration of two doses, 25ug each, of anti-CCR2 mAb completely eradicate the circulating classical monocytes P ≤ 0.002, but leaves the non-classical monocyte population unaffected. (F–G) Depletion of classical monocytes does not inhibit neutrophil infiltration into the brain. (H) Depletion of classical monocytes inhibit monocyte derived macrophage recruitment after TBI P ≤ 0.004. All tests are non-paired non parametric Mann-Whitney tests and mice n=15

Figure 6

Non-Classical monocytes mediate neutrophil infiltration into the brain after TBI: (A–E) CX3CR1^−/− mice have less non-classical monocytes than the CX3CR1^GFP/+ mice with no difference in other monocyte subsets or the circulating neutrophils. (F–G) Reduced non-classical monocytes results in inhibition of neutrophil infiltration into the brain P ≤ 0.05 Mann Whitney test. (H) Reduced non-classical monocytes have a dampening effect on the infiltrating monocyte derived macrophage population. N= 15