Blocking lymphocyte trafficking with FTY720 prevents inflammation-sensitized hypoxic-ischemic brain injury in newborns

Abstract

Intrauterine infection (chorioamnionitis) aggravates neonatal hypoxic-ischemic (HI) brain injury, but the mechanisms linking systemic inflammation to the CNS damage remain uncertain. Here we report evidence for brain influx of T-helper 17 (TH17)-like lymphocytes to coordinate neuroinflammatory responses in lipopolysaccharide (LPS)-sensitized HI injury in neonates. We found that both infants with histological chorioamnionitis and rat pups challenged by LPS/HI have elevated expression of the interleukin-23 (IL-23) receptor, a marker of early TH17 lymphocytes, in the peripheral blood mononuclear cells. Post-LPS/HI administration of FTY720 (fingolimod), a sphingosine-1-phosphate receptor agonist that blocks lymphocyte trafficking, mitigated the influx of leukocytes through the choroid plexus and acute induction of nuclear factor-κB signaling in the brain. Subsequently, the FTY720 treatment led to attenuated blood-brain barrier damage, fewer cluster of differentiation 4-positive, IL-17A-positive T-cells in the brain, less proinflammatory cytokine, and better preservation of growth and white matter functions. The FTY720 treatment also provided dose-dependent reduction of brain atrophy, rescuing >90% of LPS/HI-induced brain tissue loss. Interestingly, FTY720 neither opposed pure-HI brain injury nor directly inhibited microglia in both in vivo and in vitro models, highlighting its unique mechanism against inflammation-sensitized HI injury. Together, these results suggest that the dual hit of systemic inflammation and neonatal HI injury triggers early onset of the TH17/IL-17-mediated immunity, which causes severe brain destruction but responds remarkably to the therapeutic blockade of lymphocyte trafficking.

Keywords: FTY720; Fingolimod; Th17; adaptive immunity; chorioamnionitis; choroid plexus.

Copyright © 2014 the authors 0270-6474/14/3416467-15$15.00/0.

Figures

Figure 1.

Combined infection-HI induces early-stage TH17 cells in the blood and infiltration of CD43+ leukocytes in choroid plexus. A, The CBMCs of preterm neonates, with and without histological chorioamnionitis (CAM) (n = 8–9 for each group), were compared for the mRNA levels of IL-23R (normalized to CD3). Mononuclear cells in preterm neonates with severe chorioamnionitis had significant increase (p < 0.05) of IL-23R transcripts. B, The PBMCs of 7-d-old rat pups at 4 h after 0.3 mg/kg LPS challenge (n = 5), pure-HI insult (n = 6), and the combined LPS/HI insult with saline (PBS, n = 6) or FTY720 treatment (n = 7) were collected to compare the IL-23R mRNA levels (normalized to actin). LPS/HI injury significantly increased the expression of IL-23R mRNA, which was blocked by the FTY720 treatment. UN, Untouched. C, Flow cytometry showed preferential reduction of CD4+ T-cells in PBMCs by the FTY720 treatment at 16 h recovery (13%, n = 5) when compared with untouched (61%, n = 3), low-dose LPS exposed (72%, n = 3), pure-HI injured (66%, n = 5), or LPS/HI-injured (69%, n = 5) siblings. Flow cytometry also showed a small increase of CD4+ IL-17A+ T-cells after pure-HI (2.17%) and LPS/HI (4.92%) injury when compared with untouched (0.64%), low-dose LPS-exposed (1.04%), and LPS/HI-challenged but receiving the FTY720 treatment (0.12%) rat pups. D, Immunostaining showed intense ribbon-like anti-CD43 (leukosialin, W3/13) reactivity in the choroid plexus (CP) of unchallenged P7 rat pups, which was reduced at 4 h after LPS exposure (0.3 mg/kg), and in the contralateral hemisphere after LPS/HI insult. In contrast, the ribbon-like CD43 immunoreactivity was greatly reduced in the ipsilateral choroid plexus at 4 h after pure-HI or LPS/HI insult. CD43+ cells (arrows) scattered to the nearby subcortical WM were also detected in the ipsilateral LPS/HI-injured choroid plexus. The reduction of ribbon-like CD43 immunoreactivity in the LPS/HI-injured plexus was less pronounced after the FTY720 treatment (n = 5 for each condition). Scale bar, 30 μm.

Figure 2.

CD43+ leukocytes enter neonatal brains after LPS/HI injury in distinct spatiotemporal routes. Immunocytochemistry was used to examine the influx of CD43+ leukocytes and their relationship to Iba1+ microglia/monocytes at 4 and 24 h after LPS/HI injury and the saline versus FTY720 treatment. A–C, At 4 h after LPS/HI injury, CD43+ leukocytes (green) mingled with Iba1+ ameboid microglia/monocytes (red) was detected in the ipsilateral subcortical WM but not in the contralateral hemisphere or after the FTY720 treatment (shown are typical results in >3 animals for each). D–I, CD43+ leukocytes were only accumulated in the ipsilateral leptomeningeal space of LPS/HI-injured and saline-treated rat pups at 24 h, but not 4 h, recovery. The FTY720 treatment also prevented the accumulation of CD43+ leukocytes in the leptomeningeal space. J–L, At 24 h recovery, CD43+ leukocytes mingled with round, activated Iba1+ microglia/monocytes were detected in the ipsilateral cortical gray matter (GM) of LPS/HI-injured rat pups. In contrast, CD43+ leukocytes were absent in the contralateral hemisphere or in pups that received FTY720 (shown are typical results in n > 6 animals). Scale bar, 20 μm. M, The NF-κB EMSA with the rat brain nuclear extract collected at 4 h recovery from LPS/HI insult and the saline versus FTY720 treatment. The FTY720 treatment prevented the high-order DNA-protein complex using a specific NF-κB probe (shown are the responses of 3 animals for each treatment). Mutant NF-κB probe was used as negative controls. N, At the same time, LPS/HI insult caused reduction of the cytoplasmic IκBα in the ipsilateral hemisphere (asterisks; right, R), which was also averted by the FTY720 treatment (shown are typical results in 2 subjects). L, Left; UN, untouched.

Figure 3.

FTY720 prevents LPS/HI-induced accumulation of CD4+ IL-17A+ lymphocytes and neuroinflammation in neonatal brains. A, Flow cytometry showed significant increase of CD4+ IL-17A+ cells in the brain of LPS/HI-injured rats (n = 7) at 24 h recovery, which was reduced by the FTY720 treatment (n = 8). In contrast, neither untouched (UN; n = 6), 0.3 mg/kg LPS-challenged (n = 4), or pure-HI injured (n = 5) rats showed >3% of CD4+ IL-17A+ T-cells among all CD4+ lymphocytes. B, The combined LPS/HI injury caused significantly increased RORc, IL-23R, and IL-17A mRNA, as well as a trend of increase in IL-22 mRNA (all TH17 markers) than pure-HI injury (n = 4 for each). The induction of Tbx21/T-bet (a TH1 and mixed TH1/TH17 cell marker), IFNγ (a TH1 cell marker), or Gata3 and IL-13 mRNA (TH2 cell markers) was similar between pure-HI and LPS/HI insults. The FTY720 treatment significantly decreased the LPS/HI-induced expression of RORc, IL-23 receptor, IL-17A, and Tbx21/T-bet mRNA but not in IFNγ, Gata3, or IL-13 mRNA (n = 4). C, Cytokine array showed a 9.7-fold increase of the brain MCP1 protein at 24 h after the LPS/HI insult, which was reduced to threefold in FTY720-treated rats (n = 4 for each). D, ELISA also showed significant attenuation of the LPS/HI-induced MCP1 expression in the brain, but not the plasma, of FTY720-treated animals (n = 4 for each). E, The FTY720 treatment also markedly reduced the post-LPS/HI induction of TSPO (translocation protein of 18 kDa, a marker for activated microglia) mRNA (n = 4 for each).

Figure 4.

FTY720 exerts no direct inhibition of microglial activation. A, FTY720 (1 μg/g, i.p.) was unable to prevent the induction of MCP1 after intracerebroventricular (ICV) injection of LPS (2 μg) in P7 rats, a model of direct microglial activation (n = 4 for each). B, Visualization of microglia and monocytes in P10 CX3CR1–GFP; CCR2–RFP mice and 24 h after intracerebroventricular injection of LPS (1 μg) with saline or FTY720 treatment (1 μg/g). In untouched mice, few GFP+ microglial processes extended into the pyramidal cell layer in the hippocampus (CA) and almost no RFP+ monocytes were detected in the brain. After intracerebroventricular LPS injection with either saline or FTY720 treatment, hypertrophic microglial processes extended into the pyramidal cell layer and a large number of monocytes were found in the brain (n = 3 for each). C, Application of pFTY720 (2–10 μm) failed to prevent LPS (5 ng/ml)-induced iNOS and COX2 expression in immortalized microglia SM826 cells (repeated 3 times). D, Similarly, pFTY720 failed to attenuate the LPS-mediated induction of inflammatory cytokines, including IL-1β and IL-6, in SM826 cells (repeated 3 times). E, RT-qPCR analysis showed high expression of the S1PR1 and S1PR2 isoforms in neonatal rat microglia. A previous study showed that pFTY720 lacks binding affinity to the S1PR2 isoform (Mandala et al., 2002). F, Neither pFTY720 nor S1P prevented LPS-induced IL-23 mRNA in SM826 microglial cells (shown are the mean and SD of three experiments). G, Immunoblot analysis of the effect of S1P and Tat-NBD peptide (5, 20 μm) on LPS (5 ng/ml)-induced NF-κB signaling activation in SM826 microglial cells. The Tat-NBD peptide showed dose-dependent attenuation of IκBα degradation in the cytosol and nuclear accumulation of NF-κB/RelA at 30 min after LPS-exposure, whereas S1P lacked this effect (shown are the typical result in 3 experiments). H, Eight hours exposure to S1P produced dose-dependent reduction of the S1PR1 mRNA level in SM826 cells (shown are the mean and SD in three experiments). UN, Untouched.

Figure 5.

FTY720 markedly reduces LPS/HI brain injury but is ineffective against pure HI injury. Because 80 min pure-HI insult caused little brain damage (Yang et al., 2013a), we used a longer duration of pure-HI (90 min) to compare with the combined LPS/HI (80 min) insult. A, Representative brain photographs of P8 rats that were treated by the indicated condition 24 h earlier and received an intraperitoneal injection of NaF at 2 h before transcardial perfusion; the residual NaF fluorescence in the HI-injured hemisphere (asterisks) indicates BBB damage and increased permeability. B, Quantification of NaF fluorescence in the LPS-, HI-, or LPS/HI-treated hemisphere compared with untouched (UN) brains. LPS/HI injury markedly induced BBB permeability, which was significantly reduced by the FTY720 treatment (n = 3 for untouched, 4 for LPS-alone, and 5 for HI, LPS/HI–PBS, and LPS/HI–FTY720 groups). C, MMP zymogram of the ipsilateral rat cortical hemisphere collected at 24 h after the indicated treatment. Shown are three typical samples in each condition. Asterisk indicates nonspecific band. Note the induction of MMP9 by HI or LPS/HI insult, additional processing of MMP9 in LPS/HI insult, and the absence of MMP9 induction in FTY720-treated animals. D, Representative brains of the LPS/HI-injured and saline or FTY720 (1 μg/g body × 2)-treated rat pups at 7 d recovery. Red arrows indicate brain atrophy. E, MMP zymogram of rat brains subjected to HI injury and the saline versus FTY720 treatment at 24 h recovery. Shown are two representative samples in each treatment. The FTY720 therapy failed to prevent HI-induced MMP9 activation in the ipsilateral (right, R) cortical hemisphere. L, Left. F, Typical brains of HI-injured rat pups that received either saline or FTY720 (1 μg/g body × 2) treatment at 7 d recovery. G, Quantification of tissue loss in the cerebral cortex, hippocampus, and striatum after the indicated injury and treatment at 7 d recovery (n = 10–17 in each group as indicated). The FTY720 treatment conferred dose-dependent reduction of LPS/HI-induced tissue loss in all regions but resulted in a similar extent of brain atrophy after pure-HI injury.

Figure 6.

FTY720 protects WM and motor development after neonatal LPS/HI injury. A, MEMRI comparison at 24 h recovery. Note the disappearance of cytoarchitectural distinctions in the ipsilateral hemisphere (asterisk) in saline-treated, but not FTY720-treated, rats (n = 4–5 for each group). B, Comparison of ADC at 24 h after LPS/HI. Note the reduction of ADC signals in the ipsilateral hemisphere in saline-treated animals (asterisk). Sodium MRI acquisition and quantification showed spatial correlation between increased sodium concentration and reduced ADC signal. The LPS/HI injury also caused shifting of sodium toward the ipsilateral hemisphere. C, Representative in vivo DTI image of saline- and FTY720-treated animals at 24 h recovery of LPS/HI injury. The TBSS detected regions with p < 0.05 difference in mean diffusivity (in gray matter) and FA (WM) between saline-treated (n = 5) and FTY720-treated (n = 4) animals at 24 h after LPS/HI injury. D, Representative in vivo diffusion tensor image of saline- and FTY720-treated rat pups at 14 d recovery. E, Quantification showed significant preservation of ADC signals in the cerebral cortex of FTY720-treated rat pups (n = 8) when compared with those receiving the saline treatment (n = 9). F, Comparison of the latency to fall (seconds) on rotarods in untouched (UN), LPS/HI-injured and saline- or FTY720-treated rat pups at 24 d of age (n = 3 for untouched, 8 each for saline- and FTY720-treated animals). G, Comparison of the body weight of untouched, LPS/HI-injured, and saline- or FTY720-treated rats pups at 24 d of age (n = 3 for untouched, 8 for saline- or FTY720-treated animals). Cont, Contralateral; Ipsi, ipsilateral.

Figure 7.

Divergent pathogenic pathways in pure versus inflammation-sensitized HI injury in neonatal brains. Our results suggest that the combined insult of fetal/peripheral inflammation and HI induces early-stage TH17 cells to enter neonatal brains through the choroid plexus to amplify microglial activation, which plays a preferential role in infection-sensitized over pure-HI brain injury. Our results also indicate that the benefits of FTY720 against LPS-sensitized HI injury mainly derive from blocking the brain influx of early-onset T-cells through the choroid plexus. Whether microglia feedback to promote TH17 cell maturation and the cytokines involved in the reciprocal activation is yet to determined.