Figure 4
PG inhibits RLR signaling in primary astrocytes. a Bar-graph showing the results of a cell reporter assay for the activation of RLR signaling activation/production of IFN-α/β following stimulation of primary rat astrocytes with the RLR ligand poly(I:C)LMW at a concentration of 1, 3 or 6 μg/ml for 18 h. C = control (untreated cells). b Bar-graph showing the results of a cell reporter assay for the activation of RLR signaling activation/production of IFN-α/β following stimulation of primary rat astrocytes with the RLR ligand poly(I:C)LMW at a concentration of 6 μg/ml and treated with n-PG at a concentration of 5, 10, 50 μM for 18 h. C = control (untreated cells). c Representative immunoblot analysis of astrocyte lysates of cells in culture subjected to OGD (4 h) and astrocytes pretreated with 50 μM n-PG for 2 h and then subjected to OGD. Cell lysates were immunoblotted with an antibody against IFN-α. β-Actin was used as a standard and control for protein loading. Data presented as mean+/−SEM. *p < 0.05. N = 6 OGD and 8 OGD + nPG
Source publication
Background
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.
Methods
In this study we examined the expression of retinoic acid-inducible gene (RIG)-lik...
Contexts in source publication
Context 1
... identify a potential inhibitor of RLR signaling, we used a cell reporter assay that produces IFN-α/β after stimulation. We stimulated astrocytes in culture with the RLR ligand poly(I:C)LMW for 18 h at concentrations of 1, 3 and 6 μg/ml. Our findings indicate that at concentrations of 3 and 6 μg/ml of poly(I:C)LMW, RLR signaling increased (Fig. 4a). Then we stimulated cells in a similar manner with 6 μg/ml of poly(I:C)LMW com- bined with n-PG treatment at concentrations of 5, 10 and 50 μM. At all of these concentrations n-PG was able to lower RLR signaling activation when compared to the poly(I:C)LMW-treated/n-PG-untreated group, as deter- mined by the cell reporter assay (Fig. ...
Context 2
... (Fig. 4a). Then we stimulated cells in a similar manner with 6 μg/ml of poly(I:C)LMW com- bined with n-PG treatment at concentrations of 5, 10 and 50 μM. At all of these concentrations n-PG was able to lower RLR signaling activation when compared to the poly(I:C)LMW-treated/n-PG-untreated group, as deter- mined by the cell reporter assay (Fig. 4b). In addition, astrocytes were pretreated with 50 μM n-PG for 2 h prior to 4 h of OGD. The 50 μM, 2 h n-PG-treatment resulted in decreased IFN-α production (Fig. 4c); thus suggesting that n-PG can be used to inhibit RLR signal- ing in astrocytes after ...
Context 3
... all of these concentrations n-PG was able to lower RLR signaling activation when compared to the poly(I:C)LMW-treated/n-PG-untreated group, as deter- mined by the cell reporter assay (Fig. 4b). In addition, astrocytes were pretreated with 50 μM n-PG for 2 h prior to 4 h of OGD. The 50 μM, 2 h n-PG-treatment resulted in decreased IFN-α production (Fig. 4c); thus suggesting that n-PG can be used to inhibit RLR signal- ing in astrocytes after ...
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Citations
... Ifih1(MDA5) and Ddx58 (RIG-I) are robustly elevated following TBI and the expression levels of both genes are significantly greater in aged TBI mice. RIG-I has been shown to contribute to the inflammatory response in the brain following cerebral ischemia and Japanese encephalitis virus infection (87)(88)(89). This raises the intriguing possibility that RNA released from dying cells may also play a role in promoting inflammatory processes in the injured brain; here, we provide evidence that this may be exaggerated in the aged TBI brain. ...
Aging adversely affects inflammatory processes in the brain, which has important implications in the progression of neurodegenerative disease. Following traumatic brain injury (TBI), aged animals exhibit worsened neurological function and exacerbated microglial-associated neuroinflammation. Type I Interferons (IFN-I) contribute to the development of TBI neuropathology. Further, the Cyclic GMP-AMP Synthase (cGAS) and Stimulator of Interferon Genes (STING) pathway, a key inducer of IFN-I responses, has been implicated in neuroinflammatory activity in several age-related neurodegenerative diseases. Here, we set out to investigate the effects of TBI on cGAS/STING activation, IFN-I signaling and neuroinflammation in young and aged C57Bl/6 male mice. Using a controlled cortical impact model, we evaluated transcriptomic changes in the injured cortex at 24 hours post-injury, and confirmed activation of key neuroinflammatory pathways in biochemical studies. TBI induced changes were highly enriched for transcripts that were involved in inflammatory responses to stress and host defense. Deeper analysis revealed that TBI increased expression of IFN-I related genes (e.g. Ifnb1, Irf7, Ifi204, Isg15) and IFN-I signaling in the injured cortex of aged compared to young mice. There was also a significant age-related increase in the activation of the DNA-recognition pathway, cGAS, which is a key mechanism to propagate IFN-I responses. Finally, enhanced IFN-I signaling in the aged TBI brain was confirmed by increased phosphorylation of STAT1, an important IFN-I effector molecule. This age-related activation of cGAS and IFN-I signaling may prove to be a mechanistic link between microglial-associated neuroinflammation and neurodegeneration in the aged TBI brain.
... A permanent middle cerebral artery occlusion model of rats was established as introduced in detail in a previous study [15]. Briefly, rats were anesthetized with 10% hydrate chloral solution. ...
... RIG-I is traditionally considered as an intracellular molecule that responds to viral nucleic acids and triggers antiviral innate immunity. Recent studies indicated a role for RIG-I in managing inflammatory response after focal cerebral ischemia [15,17]. In this experiment, three genes involved in RIG-I signaling pathway, namely, Ddx58/RIG-I, Traf2, and Rel A/NF-κB (p65) were downregulated by PNS (Table 1). ...
... Increasing evidences suggest that RIG-I is also involved in non-viral infectious inflammatory processes such as atherosclerosis, rheumatoid arthritis, cancers [18,19]. Brand F et al [15] reported that the RIG-I protein expression increased in hippocampus after MCAO and involved in the inflammatory response. e regulatory functions of RIG-I , interferon regulatory factor (IRF3) and NF-κB, resulting in production of proinflammatory cytokines and chemokines [20,21]. ...
Panax notoginseng saponins (PNS), the main bioactive constituents of a traditional Chinese herb Panax notoginseng, were commonly used for ischemic stroke in China. However, the associated cellular and molecular mechanisms of PNS have not been well examined. This study aimed to decipher the underlying molecular target of PNS in the treatment of cerebral ischemia. The oxygen-glucose-deprived (OGD) model of rat brain microvascular endothelial cells (BMECs) was used in this study. The alteration of gene expression in rat BMECs after PNS treatment was measured by microarray and indicated that there were 38 signaling pathways regulated by PNS. Among them, RIG-I receptor and related signaling molecules TNF receptor-associated factor 2 (Traf2) and nuclear factor-kappa B (NF-κB) were significantly suppressed by PNS, which was verified again in OGD-induced BMECs measured by FQ-PCR and western blotting and in middle cerebral artery occlusion (MCAO) rats measured by immunohistochemistry. The levels of TNF-α, IL-8, and the downstream cytokines regulated by RIG-I receptor pathway were also decreased by PNS. Meanwhile, the neurological evaluation, hematoxylin and eosin (HE) staining, and Evans blue staining were conducted to evaluate the effect of PNS in MCAO rats. Results showed PNS significantly improved functional outcome and cerebral vascular leakage. Flow cytometry showed the number of the inflammatory cells infiltrated in brain tissue was decreased in PNS treatment. Our results identified that RIG-I signaling pathway mediated anti-inflammatory properties of PNS in cerebral ischemia, which provided the novel insights of PNS application in clinics.
... Correction of these inflammatory processes can decrease the post-ischemic neuronal damage and neurovascular injury (Abulafia et al., 2009;Yang et al., 2014). The evoked innate immune response was seen in the form of increased expression of retinoic acidinducible gene-I, AIM2-like receptors and IFN-α in rodent model of cerebral ischemia (Adamczak, 2012;Brand et al., 2015;Rathinam et al., 2010). Thus, inhibition of innate immune responses with the help of microbiota and its metabolite could be a significant therapeutic target for the treatment of ischemic brain injury (Jiao et al., 2020;Zheng et al., 2002). ...
Ischemic brain injury is a serious neurological complication, which accrues an immense activation of neuroinflammatory responses. Several lines of research suggested the interconnection of gut microbiota perturbation with the activation of proinflammatory mediators. Intestinal microbial communities also interchange information with the brain through various afferent and efferent channels and microbial by-products. Herein, we discuss the different microelements of gut microbiota and its connection with the host immune system and how change in immune-microbial signatures correlates with the stroke incidence and post-injury neurological sequelae. The activated inflammatory cells increase the production of proinflammatory cytokines, chemokines, proteases and adhesive proteins that are involved in the systemic inflammation, blood brain barrier disruption, gut dysbiosis and aggravation of ischemic brain injury. We suggest that fine-tuning of commensal gut microbiota (eubiosis) may regulate the activation of CNS resident cells like microglial, astrocytes, mast cells and natural killer cells.
... B cell depletion was also a promising therapy for DM [8,15,16]. DM is a chronic inflammatory disease, in which elevation of pro-inflammatory molecules can induce cell surface TLRs and retinoic acid-inducible gene I (RIG-I)-like receptors, resulting in inflammation [29][30][31]. Furthermore, the signaling pathway transduction by TLRs and RIG-I was crucial for innate immunity, and involved in both metabolic and cardiovascular diseases [32]. ...
Background Coronary artery disease patients with diabetes mellitus may have a higher risk for clinical and angiographic restenosis. Evidence has suggested that B cells play a functional role in the progression of atherosclerotic lesions. The purpose of this study was to investigate the clonal diversity of the B cell receptor (BCR) repertoire in patients with coronary in-stent restenosis (ISR) and diabetes mellitus.
Methods In the present study, we had enrolled 21 patients with ISR and DM or not and collected the peripheral blood mononuclear cell. The DNA was extrated and then we performed DNA-seq analysis to character the B-cell receptor profiles (BCR) of CAD patients with or without ISR. The BCR diversity and the overlap was evaluate by the bioinformatics base on the DNA-seq data.
Result Seq-data showed that the diversity of amino acids was altered in patients with ISR. Specifically, 6 V gene segments as well as 41 V/J pairs exhibited different frequencies in patients with ISR, with the altered common amino acid sequences reaching 0.1–1.01% in ISR patients.
Conclusion The present findings suggest that B cells may play a role in the occurrence of ISR and that further analysis of BCR profiles would enhance understanding of ISR.
... This possibility, however, remains to be experimentally validated. Similar consid erations apply to RIG I and cerebral stroke 264 and (we suspect) many other disorders with a hitherto poorly characterized inflammatory component. Elucidating the precise contribution of NAS signalling to human disease may foster the development of clinically useful NAS modulators. ...
Nucleic acid sensors, primarily TLR and RLR family members, as well as cGAS-STING signalling, play a critical role in the preservation of cellular and organismal homeostasis. Accordingly, deregulated nucleic acid sensing contributes to the origin of a diverse range of disorders, including infectious diseases, as well as cardiovascular, autoimmune and neoplastic conditions. Accumulating evidence indicates that normalizing aberrant nucleic acid sensing can mediate robust therapeutic effects. However, targeting nucleic acid sensors with pharmacological agents, such as STING agonists, presents multiple obstacles, including drug-, target-, disease- and host-related issues. Here, we discuss preclinical and clinical data supporting the potential of this therapeutic paradigm and highlight key limitations and possible strategies to overcome them.
... Unlike TLRs, the role of RLRs in cerebral ischemic stroke has been far less well studied. One previous report has indicated that RIG-I is upregulated in the hippocampus after focal cerebral ischemia (33); however, whether RLR signaling is beneficial or detrimental after stroke remains unclear. and NLRP3 are responsible for cerebral ischemic injury. ...
The innate immune system is an evolutionarily conserved system that senses and defends against infection and irritation. Innate immune signaling is a complex cascade that quickly recognizes infectious threats through multiple germline-encoded cell surface or cytoplasmic receptors and transmits signals for the deployment of proper countermeasures through adaptors, kinases, and transcription factors, resulting in the production of cytokines. As the first response of the innate immune system to pathogenic signals, inflammatory responses must be rapid and specific to establish a physical barrier against the spread of infection and must subsequently be terminated once the pathogens have been cleared. Long-lasting and low-grade chronic inflammation is a distinguishing feature of type 2 diabetes and cardiovascular diseases, which are currently major public health problems. Cardiometabolic stress-induced inflammatory responses activate innate immune signaling, which directly contributes to the development of cardiometabolic diseases. Additionally, although the innate immune elements are highly conserved in higher-order jawed vertebrates, lower-grade jawless vertebrates lack several transcription factors and inflammatory cytokine genes downstream of the Toll-like receptors (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) pathways, suggesting that innate immune signaling components may additionally function in an immune-independent way. Notably, recent studies from our group and others have revealed that innate immune signaling can function as a vital regulator of cardiometabolic homeostasis independent of its immune function. Therefore, further investigation of innate immune signaling in cardiometabolic systems may facilitate the discovery of new strategies to manage the initiation and progression of cardiometabolic disorders, leading to better treatments for these diseases. In this review, we summarize the current progress in innate immune signaling studies and the regulatory function of innate immunity in cardiometabolic diseases. Notably, we highlight the immune-independent effects of innate immune signaling components on the development of cardiometabolic disorders.
... Protein levels of inflammasome signaling proteins were determined in the cytosolic and mitochondrial fractions as described in [7]. Briefly, protein lysates were resolved in 10-20% Criterion TGX Stain-Free precasted gels (Bio-Rad), using antibodies (1:1000 dilution) to NLRC4 (Novus Biologicals, cat# NBP2-41124), caspase-1 (Novus Biologicals, cat# NB100-56565), caspase-11 (Novus Biologicals, cat# MAB8648), ASC (Santa Cruz, cat# sc-271054), IL-1β (Cell Signaling, cat# 12242S), IL-18 (Abcam, cat# ab71495), HSP60 (Cell Signaling, cat#12165) and β-actin (Sigma Aldric, cat# A5441). ...
Background
Inflammation is a natural part of the aging process. This process is referred to as inflammaging. Inflammaging has been associated with deleterious outcomes in the aging brain in diseases such as Alzheimer’s disease and Parkinson’s disease. The inflammasome is a multi-protein complex of the innate immune response involved in the activation of caspase-1 and the processing of the inflammatory cytokines interleukin (IL)-1β and IL-18. We have previously shown that the inflammasome plays a role in the aging process in the brain. In this study, we analyzed the brain of young (3 months old) and aged (18 months old) mice for the expression of inflammasome proteins.
Results
Our findings indicate that the inflammasome proteins NLRC4, caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and IL-18 are elevated in the cytosol of cortical lysates in aged mice when compared to young. In addition, in the cytosolic fraction of hippocampal lysates in aged mice, we found an increase in NLRC4, caspase-1, caspase-11, ASC and IL-1β. Moreover, we found higher levels of ASC in the mitochondrial fraction of aged mice when compared to young, consistent with higher levels of the substrate of pyroptosis gasdermin-D (GSDM-D) and increased pyroptosome formation (ASC oligomerization). Importantly, in this study we obtained fibroblasts from a subject that donated his cells at three different ages (49, 52 and 64 years old (y/o)) and found that the protein levels of caspase-1 and ASC were higher at 64 than at 52 y/o. In addition, the 52 y/o cells were more susceptible to oxidative stress as determined by lactose dehydrogenase (LDH) release levels. However, this response was ameliorated by inhibition of the inflammasome with Ac-Tyr-Val-Ala-Asp-Chloromethylketone (Ac-YVAD-CMK). In addition, we found that the protein levels of ASC and IL-18 are elevated in the serum of subjects over the age of 45 y/o when compared to younger subjects, and that ASC was higher in Caucasians than Blacks and Hispanics, whereas IL-18 was higher in Caucasians than in blacks, regardless of age.
Conclusions
Taken together, our data indicate that the inflammasome contributes to inflammaging and that the inflammasome-mediated cell death mechanism of pyroptosis contributes to cell demise in the aging brain.
... The innate immune response is a well-known contributor to the inflammatory response after stroke (Xu and Jiang, 2014;Brand et al., 2015;Neumann et al., 2015). We have previously shown that the inflammasome, an arm of the innate immune response involved in the activation of caspase-1 and the pro-inflammatory cytokines interleukin (IL)-1β and IL-18, contributes to the pathology of stroke (Abulafia et al., 2009). ...
The inflammasome is a key contributor to the inflammatory innate immune response after stroke. We have previously shown that inflammasome proteins are released in extracellular vesicles (EV) after brain and spinal cord injury. In addition, we have shown that inflammasome proteins offer great promise as biomarkers of central nervous system (CNS) injury following brain trauma. In the present study, we used a Simple Plex Assay (Protein Simple), a novel multi-analyte automated microfluidic immunoassay platform, to analyze serum and serum-derived EV samples from stroke patients and control subjects for inflammasome protein levels of caspase-1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), Interleukins (IL)-1β, and (IL)-18. Receiver operator characteristic (ROC) curves with associated confidence intervals obtained from the analysis of serum samples revealed that the area under the curve (AUC) for ASC was 0.99 with a confidence interval between 0.9914 and 1.004, whereas the AUC for caspase-1, IL-1β, and IL-18 were 0.75, 0.61, and 0.67, respectively. Thus, these data indicate that ASC is a potential biomarker of stroke and highlight the role of the inflammasome in the inflammatory response after brain ischemia.
... Our results show that the overexpression of RIG-I triggers IRF3 phosphorylation and IFN-β transcription per se. Interestingly, it has been reported that RIG-I is also overexpressed in the hippocampus after focal cerebral hypoxia 25 . ...
Dermatomyositis is an inflammatory myopathy characterized by symmetrical proximal muscle weakness and skin changes. Muscle biopsy hallmarks include perifascicular atrophy, loss of intramuscular capillaries, perivascular and perimysial inflammation and the overexpression of IFN-inducible genes. Among them, the retinoic-Acid inducible gene 1 (RIG-I) is specifically overexpressed in perifascicular areas of dermatomyositis muscle. The aim of this work was to study if RIG-I expression may be modulated by hypoxia using an in vitro approach. We identified putative hypoxia response elements (HRE) in RIG-I regulatory regions and luciferase assays confirmed that RIG-I is a new HIF-inducible gene. We observed an increase expression of RIG-I both by Real time PCR and Western blot in hypoxic conditions in human muscle cells. Cell transfection with a constitutive RIG-I expression vector increased levels of phospho-IRF-3, indicating that RIG-I promotes binding of transcription factors to the enhancer sequence of IFN. Moreover, release of IFN-β was observed in hypoxic conditions. Finally, HIF-1α overexpression was confirmed in the muscle biopsies and in some RIG-I positive perifascicular muscle fibres but not in controls. Our results indicate that hypoxia triggers the production of IFN-I in vitro, and may contribute to the pathogenesis of DM together with other inflammatory factors.
