FTY720 Reduces Post-Ischemic Brain Lymphocyte Influx but Does Not Improve Outcome in Permanent Murine Cerebral Ischemia

BACKGROUND: The contribution of neuroinflammation and specifically brain lymphocyte invasion is increasingly recognised as a substantial pathophysiological mechanism after stroke. FTY720 is a potent treatment for primary neuroinflammatory diseases by inhibiting lymphocyte circulation and brain immigration. Previous studies using transient focal ischemia models showed a protective effect of FTY720 but did only partially characterize the involved pathways. We tested the neuroprotective properties of FTY720 in permanent and transient cortical ischemia and analyzed the underlying neuroimmunological mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: FTY720 treatment resulted in substantial reduction of circulating lymphocytes while blood monocyte counts were significantly increased. The number of histologically and flow cytometrically analyzed brain invading T- and B lymphocytes was significantly reduced in FTY720 treated mice. However, despite testing a variety of treatment protocols, infarct volume and behavioural dysfunction were not reduced 7d after permanent occlusion of the distal middle cerebral artery (MCAO). Additionally, we did not measure a significant reduction in infarct volume at 24 h after 60 min filament-induced MCAO, and did not see differences in brain edema between PBS and FTY720 treatment. Analysis of brain cytokine expression revealed complex effects of FTY720 on postischemic neuroinflammation comprising a substantial reduction of delayed proinflammatory cytokine expression at 3d but an early increase of IL-1β and IFN-γ at 24 h after MCAO. Also, serum cytokine levels of IL-6 and TNF-α were increased in FTY720 treated animals compared to controls. CONCLUSIONS/SIGNIFICANCE: In the present study we were able to detect a reduction of lymphocyte brain invasion by FTY720 but could not achieve a significant reduction of infarct volumes and behavioural dysfunction. This lack of neuroprotection despite effective lymphopenia might be attributed to a divergent impact of FTY720 on cytokine expression and possible activation of innate immune cells after brain ischemia

DAMP Signaling is a Key Pathway Inducing Immune Modulation after Brain Injury

Acute brain lesions induce profound alterations of the peripheral immune response comprising the opposing phenomena of early immune activation and subsequent immunosuppression. The mechanisms underlying this brain-immune signaling are largely unknown. We used animal models for experimental brain ischemia as a paradigm of acute brain lesions and additionally investigated a large cohort of stroke patients. We investigated the inflammatory potency of HMGB1 and its signaling pathways by immunological in vivo and in vitro techniques. Features of the complex behavioral sickness behavior syndrome were characterized by homecage behavior analysis. HMGB1 downstream signaling, particularly with RAGE, was studied in various transgenic animal models and by pharmacological blockade. Our results indicate that HMGB1 was released from the ischemic brain in the hyperacute phase of stroke in mice and patients. Cytokines secreted in the periphery in response to brain injury induced sickness behavior, which could be abrogated by inhibition of the HMGB1-RAGE pathway or direct cytokine neutralization. Subsequently, HMGB1-release induced bone marrow egress and splenic proliferation of bone marrow-derived suppressor cells, inhibiting the adaptive immune responses in vivo and vitro. Furthermore, HMGB1-RAGE signaling resulted in functional exhaustion of mature monocytes and lymphopenia, the hallmarks of immune suppression after extensive ischemia. This study introduces the HMGB1-RAGE-mediated pathway as a key mechanism explaining the complex postischemic brain-immune interactions

Neuroinflammation after intracerebral hemorrhage

Spontaneous intracerebral hemorrhage (ICH) is a particularly severe type of stroke for which no specific treatment has been established yet. Although preclinical models of ICH have substantial methodological limitations, important insight into the pathophysiology has been gained. Mounting evidence suggests an important contribution of inflammatory mechanisms to brain damage and potential repair. Neuroinflammation evoked by intracerebral blood involves the activation of resident microglia, the infiltration of systemic immune cells and the production of cytokines, chemokines, extracellular proteases and reactive oxygen species (ROS). Previous studies focused on innate immunity including microglia, monocytes and granulocytes. More recently, the role of adaptive immune cells has received increasing attention. Little is currently known about the interactions among different immune cell populations in the setting of ICH. Nevertheless, immunomodulatory strategies are already being explored in ICH. To improve the chances of translation from preclinical models to patients, a better characterization of the neuroinflammation in patients is desirable

Flow cytometric analysis of brain immune cells at 5d after permanent MCAO.

<p>Cells per one hemisphere (Mean±SD). N = 6 mice per group. Data of 2 individual experiments.</p

FTY720 changes serum cytokine levels.

<p>Serum cytokine concentrations of the pro-inflammatory cytokines IL-6, IFN-γ and TNF-α (<b>A–C</b>) and anti-inflammatory cytokines TGF-β and IL-10 (<b>D,E</b>) were measured in naïve mice and at 24 h and 5d after FTY720 or control treatment. Each assay was performed in duplicate (n = 5, serum sampling as 2–3 individual experiments, assays as one experiment). * P<0.05 between treatment groups at the respective time point.</p

FTY720 does not influence brain edema and blood-brain-barrier permeability.

<p>(<b>A</b>) Brain water content was analyzed as the percentage water content per hemisphere by the “wet/dry weight” method and is shown as the water content ratio of ischemic/non-ischemic hemispheres. Open bars represent water content ratios in animals at 3d after Sham operation (“Sham”) and black bars in mice at 3d after permanent coagulation MCAO (“MCAO”) receiving FTY720 or PBS treatment by oral gavage (n = 5 per group, 2 individual experiments). (<b>B</b>) Blood-brain-barrier permeability was tested using the Evans blue assay for dye extravasation at 3d after permanent coagulation MCAO in mice receiving FTY720 or PBS treatment by oral gavage. Data is shown as the ratio for ischemic/non-ischemic hemisphere fluorescence intensity at 680 nm (n = 5, one experiment).</p

Mean values and standard deviations of data shown in Figure 1b.

<p>* = p<0.05 between Naïve mice and respective time point after FTY720 (n = 5 per group).</p

FTY720 does not reduce infarct volume after cortical permanent ischemia.

<p>Animals in the treatment groups (FTY720) received daily administrations of 1 mg/kg FTY720 by oral gavage starting at 48 h before infarct induction.I Infarct volumes were determined (<b>A</b>) at 3d (n = 8 per group, p = 0.73, 2 individual experiments) and (<b>B</b>) at 7d (n = 17, 0.54, 4 individual experiments) after infarct induction. Control animals received daily PBS injections. (<b>C</b>) Animals were treated daily with either FTY720 or PBS, starting from 3 h after MCAO and infarct volumes were determined at 7d after brain ischemia (n = 10, p = 0.43, 2 individual experiments). (<b>D</b>) Mice received a single dose of FTY720 or PBS at 48 h before brain ischemia and infarct volumetry was performed at day 7 (n = 10, p = 0.27). Behavioural dysfunction and recovery after experimental stroke was assessed in FTY720 pretreated animals (daily treatment starting 48 h before MCAO) or in control animals by the (<b>E</b>) “cylinder test” (n = 12, 3 individual experiments) and the (<b>F</b>) “corner test” (n = 12, 3 individual experiments).</p