Roser Gorina

Astrocyte TLR4 activation induces a proinflammatory environment through the interplay between MyD88-dependent NFκB signaling, MAPK, and Jak1/Stat1 pathways

There is increasing evidence that astrocytes play important roles in immune regulation in the brain. Astrocytes express toll‐like receptors (TLR) and build up responses to innate immune triggers by releasing proinflammatory molecules. We investigate signaling pathways and released molecules after astrocyte TLR4 activation. Purified rodent brain astrocyte cultures were treated with the TLR4 activator bacterial lipopolysaccharide (LPS). Tools used to interfere with this system include small interference RNA, inhibitory drugs, and MyD88 or Stat1 deficient mice. LPS induced early activation of the transcription factor NFκB, through the MyD88 adaptor, and expression of TNF‐α, VCAM‐1, IL‐15, and IL‐27. LPS also induced delayed Jak1/Stat1 activation, which was MyD88‐independent but was not mediated by IFN‐β. Jak1/Stat1 activation induced the expression of negative cytokine regulator SOCS‐1 and CXCL10 chemokine (IP‐10). Mitogen‐activated protein kinases (MAPK) were also involved in TLR4 signaling in a MyD88‐independent fashion. p38 exerted a strong influence on LPS‐induced gene expression by regulating the phosphorylation of Stat1 and the transcriptional activity of NFκB, while JNK regulated the Jak1/Stat1 pathway, and ERK1/2 controlled the expression of Egr‐1 and influenced MyD88‐dependent MMP‐9 expression. Interplay between these signals was evidenced by the increased induction of MMP‐9 in Stat1‐deficient cells challenged with LPS, suggesting that Stat1 negatively regulates the expression of MMP‐9 induced by LPS. Therefore, astrocytes are responsive to TLR4 activation by inducing a complex set of cell‐dependent molecular reactions mediated by NFκB, MAPK and Jak1/Stat1 signaling pathways. Here we identified cross‐talking signals generating a proinflammatory environment that will modulate the response of surrounding cells.

Induction of COX-2 Enzyme and Down-regulation of COX-1 Expression by Lipopolysaccharide (LPS) Control Prostaglandin E2 Production in Astrocytes

Background: The relative contribution of COX-2 and COX-1 to prostanoid formation under neuroinflammation is complex. Results: LPS induced COX-2 and mPGES1 but down-regulated COX-1 and TS in astroglia. These effects accounted for the high production of PGE2. Conclusion: PGE2 after LPS results from the coordinated COX-2 up-regulation and COX-1 down-regulation in astrocytes. Significance: Changes in COX-2 and COX-1 expression mediate astroglial PGE2 generation in neuroinflammation. Pathological conditions and pro-inflammatory stimuli in the brain induce cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism mediating the production of prostanoids that, among other actions, have strong vasoactive properties. Although low basal cerebral COX-2 expression has been reported, COX-2 is strongly induced by pro-inflammatory challenges, whereas COX-1 is constitutively expressed. However, the contribution of these enzymes in prostanoid formation varies depending on the stimuli and cell type. Astrocyte feet surround cerebral microvessels and release molecules that can trigger vascular responses. Here, we investigate the regulation of COX-2 induction and its role in prostanoid generation after a pro-inflammatory challenge with the bacterial lipopolysaccharide (LPS) in astroglia. Intracerebral administration of LPS in rodents induced strong COX-2 expression mainly in astroglia and microglia, whereas COX-1 expression was predominant in microglia and did not increase. In cultured astrocytes, LPS strongly induced COX-2 and microsomal prostaglandin-E2 (PGE2) synthase-1, mediated by the MyD88-dependent NFκB pathway and influenced by mitogen-activated protein kinase pathways. Studies in COX-deficient cells and using COX inhibitors demonstrated that COX-2 mediated the high production of PGE2 and, to a lesser extent, other prostanoids after LPS. In contrast, LPS down-regulated COX-1 in an MyD88-dependent fashion, and COX-1 deficiency increased PGE2 production after LPS. The results show that astrocytes respond to LPS by a COX-2-dependent production of prostanoids, mainly vasoactive PGE2, and suggest that the coordinated down-regulation of COX-1 facilitates PGE2 production after TLR-4 activation. These effects might induce cerebral blood flow responses to brain inflammation.

Brain-Derived Antigens in Lymphoid Tissue of Patients with Acute Stroke

In experimental animals, the presence of brain-derived constituents in cervical lymph nodes has been associated with the activation of local lymphocytes poised to minimize the inflammatory response after acute brain injury. In this study, we assessed whether this immune crosstalk also existed in stroke patients. We studied the clinical course, neuroimaging, and immunoreactivity to neuronal derived Ags (microtubule-associated protein-2 and N-methyl d-aspartate receptor subunit NR-2A), and myelin-derived Ags (myelin basic protein and myelin oligodendrocyte glycoprotein) in palatine tonsils and cervical lymph nodes of 28 acute stroke patients and 17 individuals free of neurologic disease. Stroke patients showed greater immunoreactivity to all brain Ags assessed compared with controls, predominantly in T cell zones. Most brain immunoreactive cells were CD68+ macrophages expressing MHC class II receptors. Increased reactivity to neuronal-derived Ags was correlated with smaller infarctions and better long-term outcome, whereas greater reactivity to myelin basic protein was correlated with stroke severity on admission, larger infarctions, and worse outcome at follow-up. Patients also had more CD69+ T cells than controls, indicative of T cell activation. Overall, the study showed in patients with acute stroke the presence of myelin and neuronal Ags associated with lymph node macrophages located near activated T cells. Whether the outcome of acute stroke is influenced by Ag-specific activation of immune responses mediated by CD69 lymphocytes deserves further investigation.

AG490 prevents cell death after exposure of rat astrocytes to hydrogen peroxide or proinflammatory cytokines: involvement of the Jak2/STAT pathway

Janus kinases/STAT pathway mediates cellular responses to certain oxidative stress stimuli and cytokines. Here we examine the activation of Stat1 and Stat3 in rat astrocyte cultures and its involvement in cell death. H2O2, interferon (INF)‐γ and interleukin (IL)‐6 but not IL‐10 caused cell death. Stat1 was phosphorylated on tyrosine (Tyr)‐701 after exposure to H2O2, INF‐γ or IL‐6 but not IL‐10. Tyr‐705 pStat3 was observed after H2O2, IL‐6 and IL‐10. Also, H2O2 induced serine (Ser)‐727 phosphorylation of Stat1 but not Stat3. The degree of Tyr‐701 pStat1 by the different treatments positively correlated with the corresponding reduction of cell viability. AG490, a Jak2 inhibitor, prevented Tyr‐701 but not Ser‐727, Stat1 phosphorylation. Also, AG490 inhibited Tyr‐705 Stat3 phosphorylation induced by H2O2 and IL‐6 but did not prevent that induced by IL‐10. Furthermore, AG490 conferred strong protection against cell death induced by INF‐γ, IL‐6 and H2O2. These results suggest that Jak2/Stat1 activation mediates cell death induced by proinflammatory cytokines and peroxides. However, we found evidence suggesting that AG490 reduces oxidative stress induced by H2O2, which further shows that H2O2 and/or derived reactive oxygen species directly activate Jak2/Stat1, but masks the actual involvement of this pathway in H2O2‐induced cell death.

Astrocytes are very sensitive to develop innate immune responses to lipid-carried short interfering RNA.

Short interfering RNA (siRNA) inhibits the synthesis of specific proteins through RNA interference (RNAi). However, siRNA can induce innate immune responses that are mediated by toll‐like receptors (TLRs) in cells of the immune system. Here, we sought to evaluate whether siRNA can induce such responses in glial cells. We examined the effects of various siRNA sequences prepared with lipids (oligofectamine). Lipid‐siRNA induced variable degrees of silencing‐independent nonspecific effects, e.g. increased Stat1 and Cox‐2 expression and release of IL‐6 and IP‐10 in primary astroglia. This was prevented through chemical modification of siRNA by nucleoside 2′‐O‐methylation, without impairing specific gene silencing. Lipid‐siRNA also induced nonspecific responses in purified astroglia, but not in microglia, or 3T3 cells. The highest TLR7 and TLR3 mRNA expression was found in microglia and purified astroglia, respectively. Accordingly, the TLR3 agonist poly(I:C) (PIC) induced higher release of IFN‐β in primary and purified astroglia than in microglia. As siRNA, PIC induced IP‐10, Stat1, VCAM‐1, and Cox‐2 and increased TLR3 mRNA expression. The effects of lipid‐siRNA in purified astrocytes were attenuated after silencing TLR3 or TLR7 expression, and by the PKR inhibitor 2‐aminopurine. Furthermore, lipid‐siRNA induced the expression of RIG‐I. In contrast, siRNA devoid of lipids did not enter the astrocytes, did not silence gene expression, and did not induce Stat1 or Cox‐2. The results show that, in astroglia, lipid‐siRNA induces innate immune responses that are mediated, at least in part, by intracellular mechanism dependent on TLR7, TLR3, and helicases.

Exposure of glia to pro‐oxidant agents revealed selective Stat1 activation by H2O2 and Jak2‐independent antioxidant features of the Jak2 inhibitor AG490

The JAK/STAT pathway is activated in response to cytokines and growth factors. In addition, oxidative stress can activate this pathway, but the causative pro‐oxidant forms are not well identified. We exposed cultures of rat glia to H2O2, FeSO4, nitroprussiate, or paraquat. We assessed oxidative stress by measuring reactive oxygen species (ROS) and oxidated proteins, we determined phosphorylated Stat1 (pStat1), and we evaluated the effect of antioxidants (trolox, propyl gallate, and N‐acetylcysteine) and of Jak2 (Janus tyrosine kinases) inhibitors (AG490 and Jak2‐Inhibitor‐II). Pro‐oxidant agents induced ROS and protein oxidation, excluding nitroprussiate that induced protein nitrosylation. H2O2, and to a lesser extent FeSO4, increased the level of pStat1, whereas nitroprussiate and paraquat did not. Trolox and propyl gallate strongly prevented ROS formation but they did not abolish H2O2‐induced pStat1. In contrast, NAC did not reduce the level of ROS but it prevented the increase of pStat1 induced by H2O2, evidencing a differential effect on ROS formation and on Stat1 phosphorylation. H2O2 induced pStat1 in mixed glia cultures and, to a lesser extent, in purified astroglia, but not in microglia. Jak2 inhibitors reduced H2O2‐induced pStat1, suggesting the involvement of this kinase in the increased phosphorylation of Stat1 by peroxide. Unexpectedly, AG490, but not Jak2‐Inhibitor‐II, reduced ROS formation, and it abrogated lipid peroxidation in microsomal preparations. Furthermore, AG490 reduced ROS in glial cells that were transfected with siRNA to silence Jak2 expression. These findings reveal previously unrecognized Jak2‐independent antioxidant properties of AG490, and show that Jak2‐dependent Stat1 activation by peroxide is dissociated from ROS generation.

Antioxidant CR-6 protects against reperfusion injury after a transient episode of focal brain ischemia in rats

Oxidative and nitrosative stress are targets for intervention after ischemia/reperfusion. The aim of this study was to explore the effect of CR-6, a vitamin-E analogue that is antioxidant and scavenger of nitrogen-reactive species. Sprague–Dawley rats had the middle cerebral artery (MCA) occluded either for 90 mins or permanently. Cortical perfusion was continuously monitored by laser–Doppler flowmetry. CR-6 (100 mg/kg) was administered orally either at 2 and 8 h after MCA occlusion, or at 2 h only. Infarct volume, neurological deficit, and signs of reperfusion injury were evaluated. CR-6 was detected in plasma and brain by HPLC. CR-6 reduced glutathione consumption in the ischemic brain and superoxide generation in the isolated MCA. CR-6 decreased infarct volume and attenuated the neurological deficit at 1 and 7 days after ischemia/reperfusion, but not after permanent ischemia. Immediately after reperfusion, cortical blood flow values returned to their baseline (±20%) in several animals, whereas others showed hyper-perfusion (>20% of baseline). Reactive hyperemia was associated with adverse events such as increased cortical BBB leakage, edema, protein nitrotyrosination, COX-2 expression, and neutrophil accumulation; and with a poorer outcome, and CR-6 attenuated these effects. In conclusion, oral CR-6 administration after transient ischemia protects the brain from reperfusion injury.

Induction of hemeoxygenase-1 expression after inhibition of hemeoxygenase activity promotes inflammation and worsens ischemic brain damage in mice.

Hemeoxygenase (HO) is an enzymatic system that degrades heme. HO-1 is an inducible isoform whereas HO-2 is constitutive. Stroke strongly induces HO-1 expression but the underlying mechanisms are not fully elucidated. Cytokines that are up-regulated after ischemia, like interleukin (IL)-10, can induce HO-1 gene expression, which is positively regulated by the transcriptional activator nuclear factor erythroid 2-related factor 2 (Nrf2) and negatively regulated by the transcriptional repressor breast cancer type 1 susceptibility protein (BRCA1) associated C-terminal helicase 1 (Bach-1). While Nrf2 is activated after ischemia and drugs promoting Nrf2 activation increase HO-1 and are beneficial, the involvement of Bach-1 is unknown. Here we investigated mechanisms involved in HO-1 induction and evaluated the effects of HO activity inhibition in mouse permanent middle cerebral artery occlusion (pMCAO). HO-1 was induced after ischemia in IL-10-deficient mice suggesting that post-ischemic HO-1 induction was IL-10-independent. Attenuation of Bach-1 gene repression after ischemia was associated to enhanced HO-1 induction. Administration of the HO activity inhibitor zinc proto-porphyrin IX (ZnPP) i.p. 24h before pMCAO exacerbated ischemia-induced tumor necrosis factor-α (TNF-α) and IL-1β, nitro-oxidative stress, and the presence of neutrophils at 8h, and increased infarct volume at day 4. However, ZnPP did not worsen ischemic damage when given 30min before pMCAO. ZnPP induced HO-1 expression in the cerebral vasculature at 24h, when it was still detected by high-performance liquid chromatography (HPLC) in plasma. While ZnPP was not found in brain tissue extracts of controls, it could be detected after ischemia, supporting that a small fraction of the injected drug can reach the tissue following blood-brain barrier breakdown. The deleterious effect of inhibiting HO activity in ischemia became apparent in the presence of ZnPP-induced HO-1, which is known to exert effects independent of its enzymatic activity. In conclusion, HO-1 induction after ischemia was associated to down-regulation of transcriptional repressor Bach-1, and induction of HO-1 when HO enzymatic activity was inhibited was related to worst outcome after brain ischemia.