Cytokine Production In Astrocytes Research Articles

Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes.

Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain.

Differential production of interleukin‐6 and tumor necrosis factor‐α in primary rat astrocyte cultures using two distinct methods of microglia elimination

AbstractHepatic encephalopathy (HE) is a neurocognitive disorder due to acute or chronic liver failure as well as portosystemic bypass. Both neuroinflammation and oxidative stress have been strongly implicated in the pathogenesis of HE. An increasing body of evidence suggests that in the development of these mechanisms astrocytes may be involved. With regard of HE, this aspect of astrocytes is, however, neglected. While several studies focus on astrocytes related to the above‐mentioned pathological processes, the applied in vitro models are generated by diverse methods, which make the results difficult to compare with each other. In addition, these astrocyte cultures frequently contain microglia as well, complicating the interpretation of results. Therefore, we applied enriched primary rat astrocyte cultures generated by microglia elimination via shaking or the combination of cytosine β‐d‐arabinofuranoside hydrochloride (AraC) and l‐leucine methyl ester (LME). We examine whether some HE‐relevant pathological factors, such as ammonia, manganese (Mn), lipopolysaccharide (LPS), and H2O2 could elicit oxidative stress and cytokine production in astrocytes. Mn exposure increased the oxidative stress in astrocytes in a time‐dependent manner; furthermore, we could detect tumor necrosis factor (TNF)‐α production in both cultures at resting state. In addition, it was around 10‐fold higher in the cultures after shaking. LPS increased TNF‐α secretion in cultures after shaking, but not in cultures after AraC + LME treatment. In contrast, interleukin (IL)‐6 was detectable only in the AraC + LME‐treated cultures. In conclusion, we have demonstrated that HE‐relevant factors evoke oxidative stress and cytokine production, albeit depending on the elimination method used.

The role of TCPTP on leptin effects on astrocyte morphology

Leptin and LPS has been implicated in the development of hypothalamic astrogliosis in rodents. Astrocytes, which are interconnected by gap junction proteins, have emerged as important players in the control of energy homeostasis exerted by the hypothalamus. To investigate the hypothesis of action of T-cell protein tyrosine phosphatase (TCPTP) on the astrocyte morphology, astrocytes from the hypothalamus of one-day-old rats were stimulated with leptin and LPS (used as a positive control). Leptin and LPS induced a marked increase in astrocyte size, an increase in Ptpn2 (TCPTP gene) and gap junction alpha-1 protein, - Gja1 (connexin 43 - CX43 gene) mRNA expression and a decrease in gap junction protein, alpha 6 - Gja6 (CX30 gene) mRNA expression. Remarkably, these effects on astrocytes morphology and connexins were prevented by Ptpn2 siRNA. Astrocytes are known to produce cytokines; here we show that TCPTP acts as an important regulator of the cytokines and it possesses a reciprocal interplay with protein tyrosine phosphatase 1B (PTP1B). Our findings demonstrate that leptin and LPS alter astrocyte morphology by increasing TCPTP, which in turn modulates connexin 30 (CX30) and connexin 43 (CX43) expression. TCPTP and PTP1B seem to act in the regulation of cytokine production in astrocytes.

MiR-409-3p and MiR-1896 co-operatively participate in IL-17-induced inflammatory cytokine production in astrocytes and pathogenesis of EAE mice via targeting SOCS3/STAT3 signaling.

Th17 cells and interleukin-17 (IL-17) have been found to play an important role in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Response to IL-17, reactive astrocytes accompany with immune cells infiltration and axonal damage in MS/EAE. However, the role and the regulatory mechanism of IL-17-activated astrocytes in inflammation and in the EAE process still remain largely unknown. Here, we elucidated that miR-409-3p and miR-1896, as co-upregulated microRNAs in activated astrocytes and in EAE mice, targeted suppressor of cytokine signaling proteins 3 (SOCS3). Overexpression of miR-409-3p or miR-1896 significantly reduced SOCS3 expression and increased phosphorylation of STAT3 as well as induced the inflammatory cytokines production (IL-1β, IL-6, IP-10, MCP-1, and KC), CD4+ T cells migration and demyelination, in turn aggravating EAE development. Importantly, the effects of co-overexpression of miR-409-3p and miR-1896 in vitro or in vivo are strongly co-operative. In contrast, simultaneously silenced miR-409-3p and miR-1896 co-operatively ameliorates inflammation and demyelination in the central nervous system of EAE mice. Collectively, our findings highlight that miR-409-3p and miR-1896 co-ordinately promote the production of inflammatory cytokines in reactive astrocytes through the SOCS3/STAT3 pathway and enhance reactive astrocyte-directed chemotaxis of CD4+ T cells, leading to aggravate pathogenesis in EAE mice. Co-inhibition of miR-409-3p and miR-1896 may be a therapeutic target for treating MS and neuroinflammation.

KSRP: A checkpoint for inflammatory cytokine production in astrocytes

Chronic inflammation in the central nervous system (CNS) is a central feature of many neurodegenerative and autoimmune diseases. As an immunologically competent cell, the astrocyte plays an important role in CNS inflammation. It is capable of expressing a number of cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) that promote inflammation directly and through the recruitment of immune cells. Checkpoints are therefore in place to keep tight control over cytokine production. Adenylate/uridylate-rich elements (ARE) in the 3' untranslated region of cytokine mRNAs serve as a major checkpoint by regulating mRNA stability and translational efficiency. Here, we examined the impact of KH-type splicing regulatory protein (KSRP), an RNA binding protein which destabilizes mRNAs via the ARE, on cytokine expression and paracrine phenotypes of primary astrocytes. We identified a network of inflammatory mediators, including TNF-α and IL-1β, whose expression increased 2 to 4-fold at the RNA level in astrocytes isolated from KSRP(-/-) mice compared to littermate controls. Upon activation, KSRP(-/-) astrocytes produced TNF-α and IL-1β at levels that exceeded control cells by 15-fold or more. Conditioned media from KSRP(-/-) astrocytes induced chemotaxis and neuronal cell death in vitro. Surprisingly, we observed a prolongation of half-life in only a subset of mRNA targets and only after selective astrocyte activation. Luciferase reporter studies indicated that KSRP regulates cytokine gene expression at both transcriptional and post-transcriptional levels. Our results outline a critical role for KSRP in regulating pro-inflammatory mediators and have implications for a wide range of CNS inflammatory and autoimmune diseases.

Morphine Exacerbates HIV-1 Tat-Induced Cytokine Production in Astrocytes through Convergent Effects on [Ca2+]i, NF-κB Trafficking and Transcription

Astroglia are key cellular sites where opiate drug signals converge with the proinflammatory effects of HIV-1 Tat signals to exacerbate HIV encephalitis. Despite this understanding, the molecular sites of convergence driving opiate-accelerated neuropathogenesis have not been deciphered. We therefore explored potential points of interaction between the signaling pathways initiated by HIV-1 Tat and opioids in striatal astrocytes. Profiling studies screening 152 transcription factors indicated that the nuclear factor-kappa B (NF-κB) subunit, c-Rel, was a likely candidate for Tat or Tat plus opiate-induced increases in cytokine and chemokine production by astrocytes. Pretreatment with the NF-κB inhibitor parthenolide provided evidence that Tat±morphine-induced release of MCP-1, IL-6 and TNF-α by astrocytes is NF-κB dependent. The nuclear export inhibitor, leptomycin B, blocked the nucleocytoplasmic shuttling of NF-κB; causing p65 (RelA) accumulation in the nucleus, and significantly attenuated cytokine production in Tat±morphine exposed astrocytes. Similarly, chelating intracellular calcium ([Ca2+]i) blocked Tat±morphine-evoked MCP-1 and IL-6 release, while artificially increasing the concentration of extracellular Ca2+ reversed this effect. Taken together, these results demonstrate that: 1) exposure to Tat±morphine is sufficient to activate NF-κB and cytokine production, 2) the release of MCP-1 and IL-6 by Tat±morphine are highly Ca2+-dependent, while TNF-α appears to be less affected by the changes in [Ca2+]i, and 3) in the presence of Tat, exposure to opiates augments Tat-induced NF-κB activation and cytokine release through a Ca2+-dependent pathway.

Eicosapentaenoic acid inhibits interleukin-6 production in interleukin-1 β-stimulated C6 glioma cells through peroxisome proliferator-activated receptor-gamma

Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimer's disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1 β-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1 β-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) γ antagonists abolished the inhibitory effect of EPA on IL-1 β-induced IL-6 production, whereas PPAR α antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPAR γ. Among the PPAR γ ligands tested in this study, ciglitazone, a synthetic agonist of PPAR γ, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Δ 12,14-prostaglandin J 2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.

SunghyangJungki-San Ga Pogongyoung inhibits IL-1 mediated tumour necrosis factor-alpha secretion in astrocytes.

Astrocytes play an important role in initiating and modulating inflammatory responses within the central nervous system (CNS). Extensive studies in rodents have shown that substance P induces inflammatory cytokine production in astrocytes. In this study we have examined whether an aqueous extract of SunghyangJungki-San Ga Pogongyoung (SSGP) inhibits the secretion of TNF-alpha from primary cultures of rat astrocytes. SSGP (10-1,000 microg/mL) significantly inhibited the TNF-alpha secretion by astrocytes stimulated with lipopolysaccharide (LPS) and substance P (SP). Interleukin-1 (IL-1) has been shown to elevate TNF-alpha secretion from LPS-stimulated astrocytes while having no effect on astrocytes in the absence of LPS. We therefore examined whether IL-1 mediated inhibition of TNF-alpha secretion from primary astrocytes by SSGP. Treatment with SSGP (10-1,000 microg/mL) to astrocytes stimulated with both LPS and SP decreased IL-1 secretion significantly. Moreover, the secretion of TNF-alpha by LPS and SP in astrocytes was progressively inhibited with an increasing amount of IL-1 neutralizing antibody. Our results suggest that SSGP may inhibit TNF-alpha secretion by inhibiting IL-1 secretion and that SSGP has an antiinflammatory activity in the CNS.