- Open Access
RIG-I contributes to the innate immune response after cerebral ischemia
© Brand et al. 2015
- Received: 12 June 2015
- Accepted: 9 September 2015
- Published: 14 September 2015
Focal cerebral ischemia induces an inflammatory response that when exacerbated contributes to deleterious outcomes. The molecular basis regarding the regulation of the innate immune response after focal cerebral ischemia remains poorly understood.
In this study we examined the expression of retinoic acid-inducible gene (RIG)-like receptor-I (RIG-I) and its involvement in regulating inflammation after ischemia in the brain of rats subjected to middle cerebral artery occlusion (MCAO). In addition, we studied the regulation of RIG-I after oxygen glucose deprivation (OGD) in astrocytes in culture.
In this study we show that in the hippocampus of rats, RIG-I and IFN-α are elevated after MCAO. Consistent with these results was an increased in RIG-I and IFN-α after OGD in astrocytes in culture. These data are consistent with immunohistochemical analysis of hippocampal sections, indicating that in GFAP-positive cells there was an increase in RIG-I after MCAO. In addition, in this study we have identified n-propyl gallate as an inhibitor of IFN-α signaling in astrocytes.
Our findings suggest a role for RIG-I in contributing to the innate immune response after focal cerebral ischemia.
- Innate immunity
Stroke is a major problem affecting populations worldwide. It is the second most common cause of death in the world after heart disease. In the United States stroke is the fourth leading cause of death, and the societal costs are approximately 80 billion dollars, which are expected to double by the year 2030 . As a result, it is important to identify new and better therapies aimed at successfully treating this patient population.
Inflammation is a major contributor to the deleterious effects that present after an ischemic event such as stroke . A component of inflammation is the innate immune response, which is characterized by the activation of pattern recognition receptors (PRR) such as Toll-like receptors (TLRs), NOD-like receptors (NLRs) or RIG-like receptors (RLRs). TLRs have been previously studied in regards to their deleterious and beneficial effects following stroke [3–8]. Similarly NOD-like receptors (NLRs), such as NLRP1 or NLRP3, have been shown to contribute to the inflammatory response after stroke through the formation of inflammasomes [9–13]. However, not much is known about the contribution of RLR signaling to the pathology after focal cerebral ischemia.
RLR signaling is typically involved in the production of type I IFNs such as IFN-α following RNA viral infections. We have previously shown that in the central nervous system (CNS), RLR signaling contributes to the process of reactive astrogliosis following spinal cord injury , and that retinoic acid-inducible gene-I (RIG-I), an RLR PRR, is elevated in the cortex of patients with early signs of Alzheimer’s disease (mild cognitive impairment), contributing to the production of amyloid precursor protein and amyloid-β . However, the role of RIG-I to the regulation of inflammation after cerebral ischemia remains unexplored.
In the present project, we have studied the involvement of RIG-I after focal cerebral ischemia in rats. Here we also show the effects of oxygen glucose deprivation (OGD) on RIG-I signaling activation in astrocytes and identify n-propyl gallate (nPG) as an inhibitor of IFN-α in astrocytes.
Animals and focal cerebral ischemia
All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Miami (protocol number 12–200). To induce transient middle cerebral artery occlusion (MCAO), male Sprague–Dawley rats were anesthetized with 3 % isoflurane in a mixture of 70 % N2O/30 % O2 for 5 min. Rats were intubated and immobilized with pancuronium bromide (0.35 mg/kg, IV) and mechanically ventilated. Brain and body temperature were monitored and the animals were maintained at a normothermic (37 °C) temperature. A catheter was implanted into the tail artery for blood-gas monitoring during surgery. The right common carotid artery was exposed with a midline neck incision and dissected from the surrounding nerves. The external carotid artery and superior thyroid artery were then ligated. A 4-cm length of 3–0 suture that was heat-blunted with a 20 mm section coated with poly-l-lysine solution (0.1 % wt/vol) was used for the occlusion. This suture was inserted at the proximal external carotid artery into the internal carotid artery to the MCA. The MCA was occluded for 90 min, then released for reperfusion. The neck incision was closed with staples and the animals were allowed to wake-up from the anesthesia. The animal was then taken to a cage supplied with food and water until termination of the study. Animals that presented with difficulty eating, presented blood pressure abnormalities, respiratory difficulties or brains that were not well perfused were euthanized and excluded from the study.
For detection of RLR proteins after MCAO, protein lysates ipsilateral to the ischemic side, of hippocampal brain areas were obtained and resolved by immunoblotting. We did not detect any effects of MCAO on RIG-I signaling on the contralateral side (data not shown). Sham animals were used as controls. Animals were sacrificed at different time points after ischemia (1 h, 4 h, 1d and 3d after MCAO) and then lysates were prepared and resolved by immunoblotting as described in de Rivero Vaccari et al.  using antibodies against RIG-I (1:1000, Anaspec), IFN-α (1:1000, Abcam) and β-actin (1:5000, Sigma). Data was normalized to β-actin.
For immunohistochemical analysis rats were anesthetized and perfusion-fixed with 4 % paraformaldehyde (PFA) as described in de Rivero Vaccari et al. .
Immunohistochemistry and confocal microscopy
Immunostained brain sections of sham and 4 h MCAO animals were examined with an Olympus FV-1000 Laser Scanning Confocal Microscope (Olympus). Rats were perfused with 4 % paraformaldehyde as described  and processed for cryostat sectioning (Leica SM 2000R Sliding Microtome). Sections (50 μm) were prepared for immunohistochemical analysis as described in de Rivero Vaccari et al.  with a primary antibody to RIG-I (1:500, Abcam) and with the astrocytic marker anti-glial fibrillary acidid protein (GFAP) (Millipore Bioscience Research Reagents). Alexa-Fluor secondary antibody conjugates (1:500, Invitrogen) were used. Secondary antibodies alone were used as control for antibody specificity.
Astrocyte culture preparation
Primary rat astrocyte cells (Lonza) were plated and grown in culture for 7 days prior to experimentation at a density of 4×104 cells/9.5 cm2 as described in de Rivero Vaccari et al. . Cells were grown in Astrocyte Basic Medium (Lonza).
Oxygen Glucose Deprivation (OGD)
Astrocyte cultures were grown as described above. For OGD, cultures were exposed to 95 % nitrogen and 5 % CO2 atmosphere maintained by constant gas flow at 37 °C in all experiments. Oxygen tension was kept at 5 % during the duration of the experiment. The Nitrogen/CO2 content was controlled with the ProOx P110 Oxygen Controller with an E702 Oxygen Sensor (model #P-110-E70) with single set point controller (#P110) (Biospherix). The media used in these OGD experiments consisted of hypoglycemic medium containing normal salts, 1 mg/mL bovine serum albumin (BSA) and 55 μM glucose (1 % of the normal glucose concentration). Control groups were grown under normal culture conditions (5 % CO2 and 95 % Oxygen).
NF-κB activation assay
To test for NF-κB activation, protein lysates (Control, 2, 3 and 4 h after OGD) were obtained from rat astrocytes and assayed with the PathScan Phospho-NF-κB p65 (Ser536) Sandwich Elisa kit (Cell Signaling) according to manufacturer’s instructions. Briefly, 100 μl of cell lysate were added and sealed to the appropriate wells for a 2 h incubation period at 37 °C. Each well was washed 4 times with 1X Wash Buffer. A detection antibody was added to each well and incubated at 37 °C for 1 h. Then 100 μl of reconstituted HRP-Linked secondary antibody were added to each well and incubated for 30 min followed by adding 100 μl of TMB Substrate to each well for 10 min and a STOP solution. The plate was read using a spectrophotometer (Victor3 1420 multilabel counter, Perkin Elmer) at 450 nm.
Cell reporter assay for RLR signaling activation in astrocytes
Astrocytes were grown in culture as described above in a 96-well plate. RLR signaling was stimulated in cells with poly(I:C)LMW (low molecular weight) at 3 different concentrations (1, 3 and 6 μg/ml, dissolved in sterile water) for 24 h. On day 2, 150 μl per well of B16-BLUE IFN-α/β cells (Invivogen) in suspension (50,000 cells) were added and incubated overnight at 37 °C in 5 % CO2. On day 3, 50 μl per well of the supernatant were added to 150 μl of QUANTI-Blue (Invivogen). The plate was incubated for 2 h at 37 °C and read at 600 nm in a spectrophotometer (Victor3 1420 multilabel counter, Perkin Elmer). A similar procedure was carried to test n-propyl-gallate (n-PG, dissolved in dimethyl sulfoxide (DMSO), MP Biomedicals) as a blocker of type I IFN signaling at different concentrations (5, 10 and 50 μM).
Statistical comparisons between the different groups were done using a two-tailed Student’s t-test or a one-way ANOVA followed by Dunnett’s multiple comparison tests. P-values of significance used were P < 0.05.
RLR signaling protein expression is altered in the hippocampus of rats after MCAO
MCAO induces alterations in RIG-I protein expression in astrocytes
OGD induces RLR signaling components in astrocytes
Propyl Gallate (n-PG) inhibits RLR signaling in primary astrocytes
Worldwide, stroke is the second most common cause of death after heart disease. Despite its prevalence, the therapies available for stroke patients remain limited and in most instances do not offer significant improvements. As a result, it is important to identify new targets that can be used for the development of treatments for cerebral ischemia. A promising target for the treatment of stroke is the inflammatory innate immune response.
The involvement of PRR such as TLR and NLR in brain ischemia has been previously described [11, 13, 19–23]. In the present study we extend the knowledge regarding the role of PRR in brain ischemia by studying the effects of focal cerebral ischemia on RIG-I expression after MCAO. RIG-I is a RLR involved in the process of reactive astrogliosis after spinal cord injury . In this study we have identified RIG-I as a PRR altered by focal cerebral ischemia in the hippocampus of rats after MCAO. When looking at whole hippocampal proteins lysates, RIG-I protein expression increased after MCAO. The increase in RIG-I expression was consistent with the increase in IFN-α. When analyzing the hippocampus of rats in immunohistochemical sections after MCAO, we detected increased immunoreactivity of RIG-I in astrocytes, suggesting that indeed RIG-I is increased after MCAO in the hippocampus. Moreover, our data in culture indicate that in astrocytes, RIG-I expression increased after OGD, which is consistent with our immunohistochemistry findings in GFAP-positive cells in vivo. This increase in RIG-I by OGD in cultured astrocytes was consistent with an increase in IFN-α expression, which is produced in a NF-κB dependent manner. In addition, in vivo, RIG-I expression was more prevalent in astrocytes.
Propyl gallate has been previously shown to be beneficial in the treatment of dilated cardiomyopathy in mice , and it is usually found as a food additive to prevent oxidation due to its strong anti-oxidant effects . At the same time, n-PG has anti-inflammatory effects by down-regulating NF-κB or by suppressing the phosphorylation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) [26, 27]. Consistent with these findings, in the present study, we have identified n-PG as an inhibitor of IFN-α signaling in astrocytes. Future studies should also look into more specific blockers of RIG-I or should use RIG-I knockout animals to better elucidate the contribution of RIG-I to the pathology of brain ischemia.
To the best of our knowledge, this is the first report to indicate an involvement of RIG-I in the inflammatory response after stroke, and we have identified n-PG as a potential anti-inflammatory drug after cerebral ischemia. Whether RLR signaling is beneficial or detrimental after stroke is under investigation. However, it has been suggested that inhibition of type I IFN signaling is beneficial in reducing hypoxia-induced neuroinflammation . In contrast, other reports indicate that delivery of IFN-β has anti-inflammatory properties after ischemic stroke in rats by decreasing infiltration of neutrophils and monocytes into the brain . As a result, the role of RLRs and type I IFN signaling after cerebral ischemia needs further investigation in order to obtain a better idea regarding the potential therapeutic potential of targeting RIG-I signaling under these conditions.
This work was supported by a grant to JPdRV from the American Heart Association 12SDG11970010 and The Miami Project to Cure Paralysis.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Corbyn Z. Statistics: a growing global burden. Nature. 2014;510(7506):S2–3. doi:10.1038/510S2a.View ArticlePubMedGoogle Scholar
- Danton GH, Dietrich WD. Inflammatory mechanisms after ischemia and stroke. J Neuropathol Exp Neurol. 2003;62(2):127–36.PubMedGoogle Scholar
- Brea D, Blanco M, Ramos-Cabrer P, Moldes O, Arias S, Perez-Mato M, et al. Toll-like receptors 2 and 4 in ischemic stroke: outcome and therapeutic values. J Cereb Blood Flow Metab. 2011;31(6):1424–31. doi:10.1038/jcbfm.2010.231.PubMed CentralView ArticlePubMedGoogle Scholar
- Brea D, Blanco M, Sobrino T, Ramos-Cabrer P, Castillo J. The levels of expression of toll-like receptors 2 and 4 in neutrophils are associated with the prognosis of ischaemic stroke patients. Rev Neurol. 2011;52(1):12–9.PubMedGoogle Scholar
- Brea D, Sobrino T, Rodriguez-Yanez M, Ramos-Cabrer P, Agulla J, Rodriguez-Gonzalez R, et al. Toll-like receptors 7 and 8 expression is associated with poor outcome and greater inflammatory response in acute ischemic stroke. Clin Immunol. 2011;139(2):193–8. doi:10.1016/j.clim.2011.02.001.View ArticlePubMedGoogle Scholar
- Downes CE, Crack PJ. Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS? Br J Pharmacol. 2010;160(8):1872–88. doi:10.1111/j.1476-5381.2010.00864.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Fadakar K, Dadkhahfar S, Esmaeili A, Rezaei N. The role of Toll-like receptors (TLRs) in stroke. Rev Neurosci. 2014. doi:10.1515/revneuro-2013-0069.PubMedGoogle Scholar
- Leung PY, Packard AE, Stenzel-Poore MP. It's all in the family: multiple Toll-like receptors offer promise as novel therapeutic targets for stroke neuroprotection. Future Neurol. 2009;4(2):201–8. doi:10.2217/14796708.4.2.201.PubMed CentralView ArticlePubMedGoogle Scholar
- Fann DY, Santro T, Manzanero S, Widiapradja A, Cheng YL, Lee SY, et al. Intermittent fasting attenuates inflammasome activity in ischemic stroke. Exp Neurol. 2014;257:114–9. doi:10.1016/j.expneurol.2014.04.017.View ArticlePubMedGoogle Scholar
- de Rivero Vaccari JP, Dietrich WD, Keane RW. Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury. J Cereb Blood Flow Metab. 2014;34(3):369–75. doi:10.1038/jcbfm.2013.227.PubMed CentralView ArticlePubMedGoogle Scholar
- Fann DY, Lee SY, Manzanero S, Tang SC, Gelderblom M, Chunduri P, et al. Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke. Cell Death Dis. 2013;4:e790. doi:10.1038/cddis.2013.326.View ArticlePubMedGoogle Scholar
- Fann DY, Lee SY, Manzanero S, Chunduri P, Sobey CG, Arumugam TV. Pathogenesis of acute stroke and the role of inflammasomes. Ageing Res Rev. 2013;12(4):941–66. doi:10.1016/j.arr.2013.09.004.View ArticlePubMedGoogle Scholar
- Abulafia DP, de Rivero Vaccari JP, Lozano JD, Lotocki G, Keane RW, Dietrich WD. Inhibition of the inflammasome complex reduces the inflammatory response after thromboembolic stroke in mice. J Cereb Blood Flow Metab. 2009;29(3):534–44. doi:10.1038/jcbfm.2008.143.View ArticlePubMedGoogle Scholar
- de Rivero Vaccari JP, Minkiewicz J, Wang X, De Rivero Vaccari JC, German R, Marcillo AE, et al. Astrogliosis involves activation of retinoic acid-inducible gene-like signaling in the innate immune response after spinal cord injury. Glia. 2012;60(3):414–21. doi:10.1002/glia.22275.PubMed CentralView ArticlePubMedGoogle Scholar
- de Rivero Vaccari JP, Brand 3rd FJ, Sedaghat C, Mash DC, Dietrich WD, Keane RW. RIG-1 receptor expression in the pathology of Alzheimer's disease. J Neuroinflammation. 2014;11:67. doi:10.1186/1742-2094-11-67.PubMed CentralView ArticlePubMedGoogle Scholar
- de Rivero Vaccari JP, Lotocki G, Alonso OF, Bramlett HM, Dietrich WD, Keane RW. Therapeutic neutralization of the NLRP1 inflammasome reduces the innate immune response and improves histopathology after traumatic brain injury. J Cereb Blood Flow Metab. 2009;29(7):1251–61. doi:10.1038/jcbfm.2009.46.PubMed CentralView ArticlePubMedGoogle Scholar
- Clemens JA, Stephenson DT, Smalstig EB, Dixon EP, Little SP. Global ischemia activates nuclear factor-kappa B in forebrain neurons of rats. Stroke. 1997;28(5):1073–80. discussion 80–1.View ArticlePubMedGoogle Scholar
- Terai K, Matsuo A, McGeer EG, McGeer PL. Enhancement of immunoreactivity for NF-kappa B in human cerebral infarctions. Brain Res. 1996;739(1–2):343–9.View ArticlePubMedGoogle Scholar
- Hua F, Ma J, Ha T, Kelley JL, Kao RL, Schweitzer JB, et al. Differential roles of TLR2 and TLR4 in acute focal cerebral ischemia/reperfusion injury in mice. Brain Res. 2009;1262:100–8. doi:10.1016/j.brainres.2009.01.018.PubMed CentralView ArticlePubMedGoogle Scholar
- Bohacek I, Cordeau P, Lalancette-Hebert M, Gorup D, Weng YC, Gajovic S, et al. Toll-like receptor 2 deficiency leads to delayed exacerbation of ischemic injury. J Neuroinflammation. 2012;9:191. doi:10.1186/1742-2094-9-191.PubMed CentralView ArticlePubMedGoogle Scholar
- Cao CX, Yang QW, Lv FL, Cui J, Fu HB, Wang JZ. Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun. 2007;353(2):509–14. doi:10.1016/j.bbrc.2006.12.057.View ArticlePubMedGoogle Scholar
- Caso JR, Pradillo JM, Hurtado O, Lorenzo P, Moro MA, Lizasoain I. Toll-like receptor 4 is involved in brain damage and inflammation after experimental stroke. Circulation. 2007;115(12):1599–608. doi:10.1161/CIRCULATIONAHA.106.603431.View ArticlePubMedGoogle Scholar
- Pradillo JM, Fernandez-Lopez D, Garcia-Yebenes I, Sobrado M, Hurtado O, Moro MA, et al. Toll-like receptor 4 is involved in neuroprotection afforded by ischemic preconditioning. J Neurochem. 2009;109(1):287–94. doi:10.1111/j.1471-4159.2009.05972.x.View ArticlePubMedGoogle Scholar
- Du CK, Zhan DY, Morimoto S. In vivo effects of propyl gallate, a novel Ca(2+) sensitizer, in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation. Life Sci. 2014;109(1):15–9. doi:10.1016/j.lfs.2014.06.004.View ArticlePubMedGoogle Scholar
- Final report on the amended safety assessment of Propyl Gallate. Int J Toxicol. 2007;26 Suppl 3:89–118. doi:10.1080/10915810701663176.
- Hsu HC, Lin WC, Chang PJ, Hong CZ, Chen CH. Propyl gallate inhibits TPA-induced inflammation via the nuclear factor-kappaB pathway in human THP-1 monocytes. Exp Ther Med. 2013;5(3):964–8. doi:10.3892/etm.2013.896.PubMed CentralPubMedGoogle Scholar
- Jung HJ, Kim SJ, Jeon WK, Kim BC, Ahn K, Kim K, et al. Anti-inflammatory activity of n-propyl gallate through down-regulation of NF-kappaB and JNK pathways. Inflammation. 2011;34(5):352–61. doi:10.1007/s10753-010-9241-0.View ArticlePubMedGoogle Scholar
- Minter MR, Zhang M, Ates RC, Taylor JM, Crack PJ. Type-1 interferons contribute to oxygen glucose deprivation induced neuro-inflammation in BE(2)M17 human neuroblastoma cells. J Neuroinflammation. 2014;11:43. doi:10.1186/1742-2094-11-43.PubMed CentralView ArticlePubMedGoogle Scholar
- Veldhuis WB, Derksen JW, Floris S, Van Der Meide PH, De Vries HE, Schepers J, et al. Interferon-beta blocks infiltration of inflammatory cells and reduces infarct volume after ischemic stroke in the rat. J Cereb Blood Flow Metab. 2003;23(9):1029–39. doi:10.1097/01.WCB.0000080703.47016.B6.View ArticlePubMedGoogle Scholar