Macrophages prevent hemorrhagic infarct transformation in murine stroke models.

OBJECTIVE: Inflammation is increasingly viewed as a new therapeutic target in subacute stages of brain infarction. However, apart from causing secondary damage, inflammation could equally promote beneficial lesion remodeling and repair. Distinct subpopulations of monocytes/macrophages (MOs/MPs) may critically determine the outcome of lesion-associated inflammation. METHODS: We addressed the role of bone marrow-derived MOs/MPs in 2 different mouse models of ischemic stroke using a combined cell-specific depletion, chemokine receptor knockout, bone marrow chimeric, and pharmacological approach. RESULTS: Starting within 24 hours of stroke onset, immature Ly6c(hi) monocytes infiltrated into the infarct border zone and differentiated into mature Ly6c(lo) phagocytes within the lesion compartment. MO/MP infiltration was CCR2-dependent, whereas we did not obtain evidence for additional recruitment via CX3CR1. Depletion of circulating MOs/MPs or selective targeting of CCR2 in bone marrow-derived cells caused delayed clinical deterioration and hemorrhagic conversion of the infarctions. Bleeding frequently occurred around thin-walled, dilated neovessels in the infarct border zone and was accompanied by decreased expression of transforming growth factor (TGF)-ß1 and collagen-4, along with diminished activation of Smad2. Injection of TGF-ß1 into the lesion border zone greatly reduced infarct bleeding in MO/MP-depleted mice. INTERPRETATION: Bone marrow-derived MOs/MPs recruited via CCR2 and acting via TGF-ß1 are essential for maintaining integrity of the neurovascular unit following brain ischemia. Future therapies should be aimed at enhancing physiological repair functions of CCR2(+) MOs/MPs rather than blocking their hematogenous recruitment.

Effects of dimethyl fumarate on neuroprotection and immunomodulation.

BACKGROUND: Neuronal degeneration in multiple sclerosis has been linked to oxidative stress. Dimethyl fumarate is a promising novel oral therapeutic option shown to reduce disease activity and progression in patients with relapsing-remitting multiple sclerosis. These effects are presumed to originate from a combination of immunomodulatory and neuroprotective mechanisms. We aimed to clarify whether neuroprotective concentrations of dimethyl fumarate have immunomodulatory effects. FINDINGS: We determined time- and concentration-dependent effects of dimethyl fumarate and its metabolite monomethyl fumarate on viability in a model of endogenous neuronal oxidative stress and clarified the mechanism of action by quantitating cellular glutathione content and recycling, nuclear translocation of transcription factors, and the expression of antioxidant genes. We compared this with changes in the cytokine profiles released by stimulated splenocytes measured by ELISPOT technology and analyzed the interactions between neuronal and immune cells and neuronal function and viability in cell death assays and multi-electrode arrays. Our observations show that dimethyl fumarate causes short-lived oxidative stress, which leads to increased levels and nuclear localization of the transcription factor nuclear factor erythroid 2-related factor 2 and a subsequent increase in glutathione synthesis and recycling in neuronal cells. Concentrations that were cytoprotective in neuronal cells had no negative effects on viability of splenocytes but suppressed the production of proinflammatory cytokines in cultures from C57BL/6 and SJL mice and had no effects on neuronal activity in multi-electrode arrays. CONCLUSIONS: These results suggest that immunomodulatory concentrations of dimethyl fumarate can reduce oxidative stress without altering neuronal network activity.

Platelet depletion does not alter long-term functional outcome after cerebral ischaemia in mice.

Platelets are key mediators of thrombus formation and inflammation during the acute phase of ischaemic stroke. Particularly, the platelet glycoprotein (GP) receptors GPIbα and GPVI have been shown to mediate platelet adhesion and activation in the ischaemic brain. GPIbα and GPVI blockade could reduce infarct volumes and improve functional outcome in mouse models of acute ischaemic stroke, without concomitantly increasing intracerebral haemorrhage. However, the functional role of platelets during long-term stroke recovery has not been elucidated so far. Thus, we here examined the impact of platelet depletion on post-stroke recovery after transient middle cerebral artery occlusion (tMCAO) in adult male mice. Platelet depleting antibodies or isotype control were applied from day 3-28 after tMCAO in mice matched for infarct size. Long-term functional recovery was assessed over the course of 28 days by behavioural testing encompassing motor and sensorimotorical functions, as well as anxiety-like or spontaneous behaviour. Whole brain flow cytometry and light sheet fluorescent microscopy were used to identify resident and infiltrated immune cell types, and to determine the effects of platelet depletion on the cerebral vascular architecture, respectively. We found that delayed platelet depletion does not improve long-term functional outcome in the tMCAO stroke model. Immune cell abundance, the extent of thrombosis and the organisation of the cerebral vasculature were also comparable between platelet-depleted and control mice. Our study demonstrates that, despite their critical role in the acute stroke setting, platelets appear to contribute only marginally to tissue reorganisation and functional recovery at later stroke stages.

Intracerebral granulocyte-macrophage colony-stimulating factor induces functionally competent dendritic cells in the mouse brain.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor and a proinflammatory cytokine. While GM-CSF is lacking in normal brain tissue, it is expressed under pathological conditions and correlates with the presence of dendritic cells (DC). However, the role of GM-CSF for the onset of immune responses in the brain is still unclear. To analyze the role of GM-CSF for the induction and functional activity of immune cells in the brain, we performed chronic intracerebroventricular administration of GM-CSF to the brains of adult mice. After GM-CSF administration, intracerebral leukocytes (ICL) were characterized by means of flow cytometry, immunohistochemistry, and an ex vivo functional assay. GM-CSF treatment significantly increased the number of leukocytes expressing high levels of CD45, indicative of peripheral, blood-derived cells. The infiltrating cells were preferentially DC of the myeloid lineage (CD45(high) CD11c+ CD11b+) with an activated phenotype characterized by upregulated expression of MHCII and the costimulatory ligand CD80. Furthermore, DC from GM-CSF treated mice were fully competent to activate naive allogeneic T cells in a mixed leukocyte reaction. In contrast, intracerebroventricular IFN-gamma administration stimulated MHCII expression on cells resembling resident microglia, but did not induce comparable presence of DC. Taken together, intracerebroventricular GM-CSF treatment results in high numbers of DC in the brain. Moreover, these GM-CSF-induced DC display an activated phenotype and exhibit the capacity to act as fully competent DC even without a further inflammatory stimulus.