data_sheet_1_Peripheral T Cell Depletion by FTY720 Exacerbates Hypoxic-Ischemic Brain Injury in Neonatal Mice.pdf

<p>Hypoxic-ischemic injury to the developing brain remains a major cause of significant long-term morbidity and mortality. Emerging evidence from neonatal brain injury models suggests a detrimental role for peripheral lymphocytes. The immunomodulatory substance FTY720, a sphingosine-1-phosphate receptor agonist, was shown to reduce adult ischemia-induced neurodegeneration through its lymphopenic mode of action. In the present study, we hypothesized that FTY720 promotes neuroprotection by reducing peripheral lymphocytes and their infiltration into the injured neonatal brain. Term-born equivalent postnatal day 9 C57BL/6 mice were exposed to hypoxia ischemia (HI) followed by a single injection of 1 mg/kg FTY720 or vehicle (0.9% sodium chloride). Brain injury, microglia, and endothelial activation were assessed 7 days post HI using histology and western blot. Peripheral and cerebral leukocyte subsets were analyzed by multichannel flow cytometry. Whether FTY720s’ effects could be attributed to its lymphopenic mode of action was determined in T cell-depleted mice. In contrast to our hypothesis, FTY720 exacerbated HI-induced neuropathology including loss of gray and white matter structures. While microglia and endothelial activation remained unchanged, FTY720 induced a strong and sustained depletion of peripheral T cells resulting in significantly reduced cerebral infiltration of CD4 T cells. CD4 T cell subset analysis revealed that circulating regulatory and effector T cells counts were similarly decreased after FTY720 treatment. However, since neonatal HI per se induces a selective infiltration of Foxp3 positive regulatory T cells compared to Foxp3 negative effector T cells effects of FTY720 on cerebral regulatory T cell infiltration were more pronounced than on effector T cells. Reductions in T lymphocytes, and particularly regulatory T cells coincided with an increased infiltration of innate immune cells, mainly neutrophils and inflammatory macrophages. Importantly anti-CD3-mediated T cell depletion resulted in a similar exacerbation of brain injury, which was not further enhanced by an additional FTY720 treatment. In summary, peripheral T cell depletion by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T cell infiltration and exacerbation HI-induced brain injury. This study indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention.</p

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<p>Prematurely born infants are highly susceptible to various environmental factors, such as inflammation, drug exposure, and also high environmental oxygen concentrations. Hyperoxia induces perinatal brain injury affecting white and gray matter development. It is well known that mitogen-activated protein kinase signaling is involved in cell survival, proliferation, and differentiation. Therefore, we aim to elucidate cell-specific responses of neuronal overexpression of the small GTPase Ras on hyperoxia-mediated brain injury. Six-day-old (P6) synRas mice (neuronal Ras overexpression under the synapsin promoter) or wild-type littermates were kept under hyperoxia (80% oxygen) or room air (21% oxygen) for 24 h. Apoptosis was analyzed by Western blot of cleaved Caspase-3 and neuronal and oligodendrocyte degeneration via immunohistochemistry. Short-term differentiation capacity of oligodendrocytes was assessed by quantification of myelin basic protein expression at P11. Long-lasting changes of hyperoxia-induced alteration of myelin structures were evaluated via transmission electron microscopy in young adult animals (P42). Western blot analysis of active Caspase-3 demonstrates a significant upregulation in wild-type littermates exposed to hyperoxia whereas synRas mice did not show any marked alteration of cleaved Caspase-3 protein levels. Immunohistochemistry revealed a protective effect of neuronal Ras overexpression on neuron and oligodendrocyte survival. Hyperoxia-induced hypomyelination in wild-type littermates was restored in synRas mice. These short-term protective effects through promotion of neuronal survival translated into long-lasting improvement of ultrastructural alterations of myelin sheaths in mice with neuronal overexpression of Ras compared with hyperoxic wild-type mice. Our data suggest that transgenic increase of neuronal Ras activity in the immature brain results in secondary protection of oligodendrocytes from hyperoxia-induced white matter brain injury.</p

Additional file 1 of Hypothermia combined with extracellular vesicles from clonally expanded immortalized mesenchymal stromal cells improves neurodevelopmental impairment in neonatal hypoxic-ischemic brain injury

Additional file 1. Supplementary figures and tables

T cell-specific ablation of Nrp-1 expression in Nrp-1KO mice.

Splenocytes from Nrp-1fl/fl x CD4crewt (WT, black bars) and Nrp-1fl/fl x CD4cretg (KO, white bars) littermates were cultured in vitro (unstimulated) and stimulated with αCD3 and αCD28 (stimulated) for 48 hours. Successful ablation of Nrp-1 expression in (A) Foxp3-CD4+ conventional T cells, (B) Foxp3+CD4+ Tregs and (C) CD8+ T cells was verified by flow cytometry and is shown as mean ± SD. Representative FACS plots including FMO controls are shown in the upper panels. Data from one experiment with n = 2 mice is depicted. (D) The percentages of CD8+ and CD4+ T cells in spleen of naïve Nrp-1fl/fl x CD4crewt (WT) and Nrp-1fl/fl x CD4cretg (KO) littermates were determined by flow cytometry. Data from 2–3 independent experiments with n = 9–12 mice in total are shown as mean ± SD. Each dot represents one animal. (TIF)</p

Cytokine secretion of sorted Nrp-1⁺CD8⁺ and Nrp-1^-CD8⁺ T cells re-stimulated with low concentrations of αCD3/αCD28.

MACS-sorted CD8+ T cells from spleen of C57BL/6 mice were stimulated in vitro with 1 μg/ml αCD3/αCD28 for 48h. Nrp-1+CD8+ and Nrp-1-CD8+ T cells were sorted by FACS and re-stimulated with 0.5 μg/ml or 0.1 μg/ml αCD3 plate-bound/αCD28 soluble for another 48h. The concentration of cytokines in the supernatant was determined by Luminex technology. Data from 2 independent experiments with n = 3 mice per experiment are summarized as mean ± SD. Statistical analysis was performed with One-Way ANOVA. *, p (TIF)</p

<i>Plasmodium berghei ANKA</i> infection induces Nrp-1 expression on CD8⁺ T cells that correlates with disease severity.

Nrp-1 expression on CD8+ T cells was analyzed by flow cytometry in naïve (0 days post infection) and PbA-infected C57BL/6 mice at day 0, 3, 4, 5 and 6 post infection. (A) A representative gating strategy, including fluorescence minus one (FMO) control, is shown for immune cells in the brain 5 days after PbA infection or from non-infected (d0) and PbA-infected mice (d6). The frequency of Nrp-1-expressing CD8+ T cells was analyzed in (B) spleen, (C) blood and (D, left) brain. (D, right) Absolute numbers of Nrp-1+CD8+ T cells are shown for the brain. (E) The percentages of Nrp-1 expressing cells among gated CD8+CD11+ antigen-experienced T cells were analyzed in spleen, blood and brain 6 days post PbA infection by flow cytometry. (F) Correlation was calculated between the frequency of cerebral CD8+ T cells (left) or Nrp-1+CD8+ T cells (right) during the course of PbA infection and the severity of experimental cerebral malaria (ECM) pathology assessed by the Rapid-Murine-Coma-and-Behavior-Scale (RMCBS) score. ECM pathology characterized by neurologic deficits results in a decline in score with increasing severity. (B-D) Results from one (day 3, 4, 5) or two (day 0 and 6) experiments with n = 5–11 mice per time point are summarized as mean (± SD). (E) Results from two experiments with n = 8–14 mice in total are shown as mean. Each dot represents one animal. Statistical analysis was performed with (C, D, E) nonparametric Kruskal-Wallis test and Dunn’s multiple comparisons test and with (B) ordinary one-way ANOVA and Holm-Sidak’s multiple comparisons test. *, p<0.05; **, p<0.01; ***, p<0.001, ****, p<0.0001. (F) Correlation is shown based on n = 26 mice and statistical significance was calculated using nonparametric Spearman correlation test. P and r values are displayed in the graphs.</p

Antigen-specific Nrp-1⁺CD8⁺ T cells exhibit a more activated phenotype than Nrp-1^-CD8⁺ counterparts.

MACS-sorted CD8+ T cells isolated from spleen of OT-I mice were stimulated with indicated concentration of OVA in the presence of irradiated splenocytes as APCs for 48h. The expression of (A) Nrp-1 and (B) CD69, PD1 and GzmB on gated Nrp-1+CD8+ T cells and Nrp-1-CD8+ T cells was analyzed by flow cytometry. Results from three independent experiments with cells from n = 6–8 mice in total are summarized as mean ± SD. Statistical significance was calculated with One-Way ANOVA and Tukey`s multiple comparisons test. **, p (TIF)</p

T cell numbers and T cell activation in spleen and blood are not altered in Nrp-1KO mice upon PbA infection.

Nrp-1fl/fl x CD4crewt (WT, black bars) and Nrp-1fl/fl x CD4cretg (KO, white bars) littermates were infected i.v. with 105 PbA-infected red blood cells (iRBCs) at day 0. Absolute numbers of CD8+ T cells, CD11a+CD8+ T cells and GzmB+CD8+ T cells in (A) spleen and (B) blood were determined at day 6 or 7 after infection by flow cytometry. Data from 2–4 independent experiments with n = 13–22 mice in total (A) or from 2 experiments with n = 7–8 mice in total (B) are depicted as mean ± SD. Each dot represents one animal. (TIF)</p

Nrp-1 expression defines a highly activated CD8⁺ T cell phenotype.

Splenic CD8+ T cells from naïve C57BL/6 mice were stimulated with αCD3 and αCD28 in vitro. (A) Nrp-1, CD69 and PD-1 expression of CD8+ T cells was analyzed by flow cytometry after 0, 24, 48, 72 or 96 hours of in vitro stimulation with 1 μg/ml αCD3/αCD28. (B) Nrp-1 expression on gated CD8+ T cells (left) and the expression of activation-associated molecules was analyzed by flow cytometry on Nrp-1+ (black bars) and Nrp-1- CD8+ T cells (white bars) 48 hours after in vitro stimulation with 1 μg/ml, 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 and is summarized as mean ± SD. Experiments have been performed as technical duplicates of stimulated T cells from one mouse. Mean values of duplicates have been included as one data point that represents one biological replicate (cells from one mouse). (C) The stability of Nrp-1 expression after in vitro induction was analyzed by isolating Nrp-1+CD8+ T cells using FACS after 48 hours of stimulation. The cells were re-cultured with IL-2 and Nrp-1 expression was analyzed at 0, 24, 48, 72 and 96 hours after re-cultivation by flow cytometry as shown by exemplary contour plots. Data from one to two independent experiments with (A) n = 8 mice in total and (B) n = 3–8 mice in total and (C) one experiment with n = 3 mice in total are presented as mean values with SD. (A, B) Ordinary one-way ANOVA with Holm-Sidak’s or Tukey’s multiple comparisons test or (C) Kruskal-Wallis test with Dunn’s multiple comparisons test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p

Re-stimulated Nrp-1⁺CD8⁺ T cells maintained their high level of T cell activation.

CD8+ T cells from Nrp-1tdTomato reporter mice or from C57BL/6 mice were separated into Nrp-1tdtomato+ or Nrp-1+ fluorochrome-labeled antibody-stained (represented as black bars) and Nrp-1- not endogenously expressing tdTomato or negative for Nrp-1 fluorochrome-labeled antibody staining (white bars) by FACS sorting 48 hours after in vitro stimulation. Cells were then stimulated again with αCD3 and αCD28 for 48 hours and analyzed by flow cytometry. (A) Nrp-1 expression of Nrp-1+ or Nrp-1- CD8+ T cells after sorting (post-sort) and re-stimulation with indicated concentrations of αCD3 and αCD28 was measured by flow cytometry. (B and C) The activation state (CD44, PD-1), effector function (GzmB) and proliferation (Ki-67) of CD8+ T cells that were either Nrp-1+ or Nrp-1- prior to re-stimulation was analyzed 48 hours after re-stimulation with (B) 1 μg/ml, (C) 0.5 μg/ml or 0.1 μg/ml αCD3/αCD28 on gated CD8+ T cells by flow cytometry. (D) The supernatants of this re-cultivation (with 1 ug/ml αCD3/αCD28) were collected and the concentrations of cytokines were determined using Luminex technology. Data from two to three independent experiments with n = 5–8 mice in total are shown as mean ± SD. Depending on the sorted cell number of Nrp-1+ or Nrp-1- CD8+ T cells, some of the experiments have been performed as technical duplicates. In this case, the mean was calculated and plotted as one dot. (A, C) ordinary one-way ANOVA with Holm-Sidak’s multiple comparisons test, (B) Student’s t-test or (D) Mann-Whitney test were used to test significance. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.</p