Interleukin-13 enhances cyclooxygenase-2 expression in activated rat brain microglia: implications for death of activated microglia

Brain inflammation has recently attracted widespread interest because it is a risk factor for the onset and progression of brain diseases. In this study, we report that cyclooxygenase-2 (COX-2) plays a key role in the resolution of brain inflammation by inducing the death of microglia. We previously reported that IL-13, an anti-inflammatory cytokine, induced the death of activated microglia. These results revealed that IL-13 significantly enhanced COX-2 expression and production of PGE(2) and 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in LPS-treated microglia. Two other anti-inflammatory cytokines, IL-10 and TGF-beta, neither induced microglial death nor enhanced COX-2 expression or PGE(2) or 15d-PGJ(2) production. Therefore, we hypothesized that the effect of IL-13 on COX-2 expression may be linked to death of activated microglia. We found that COX-2 inhibitors (celecoxib and NS398) suppressed the death of microglia induced by a combination of LPS and IL-13 and that exogenous addition of PGE(2) and 15d-PGJ(2) induced microglial death. Agonists of EP2 (butaprost) and peroxisome proliferator-activated receptor gamma (ciglitazone) mimicked the effect of PGE(2) and 15d-PGJ(2), and an EP2 antagonist (AH6809) and a peroxisome proliferator-activated receptor gamma antagonist (GW9662) suppressed microglial death induced by LPS in combination with IL-13. In addition, IL-13 potentiated LPS-induced activation of JNK, and the JNK inhibitor SP600125 suppressed the enhancement of COX-2 expression and attenuated microglial death. Taken together, these results suggest that IL-13 enhanced COX-2 expression in LPS-treated microglia through the enhancement of JNK activation. Furthermore, COX-2 products, PGE(2) and 15d-PGJ(2), caused microglial death, which terminates brain inflammation.     

An in vitro cell model to study microglia activation in diabetic retinopathy

Microglial activation has been studied extensively in diabetic retinopathy. We have previously detected activation and migration of microglia in 8-week-old diabetic rat retinas. It is widely acknowledged that microglia-mediated inflammation contributes to the progression of diabetic retinopathy. However, existing cell models do not explore the role of activated microglia in vitro. In this study, microglia were subject to various conditions mimicking diabetic retinopathy, including high glucose, glyoxal, and hypoxia. Under high glucose or glyoxal treatment, microglia demonstrated only partially functional changes, while under hypoxia, microglia became fully activated showing enlarged cell bodies, enhanced migration and phagocytosis as well as increased production of pro-inflammatory factors such as cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS). The data indicate that hypoxia-treated microglia is an optimal in vitro model for exploration of microglia activation in diabetic retinopathy.     

Human NK cells kill resting but not activated microglia via NKG2D- and NKp46-mediated recognition

Microglia are resident macrophage-like APCs of the CNS. To avoid escalation of inflammatory processes and bystander damage within the CNS, microglia-driven inflammatory responses need to be tightly regulated and both spatially and temporally restricted. Following traumatic, infectious, and autoimmune-mediated brain injury, NK cells have been found in the CNS, but the functional significance of NK cell recruitment and their mechanisms of action during brain inflammation are not well understood. In this study, we investigated whether and by which mechanisms human NK cells might edit resting and activated human microglial cells via killing in vitro. IL-2-activated NK cells efficiently killed both resting allogeneic and autologous microglia in a cell-contact-dependent manner. Activated NK cells rapidly formed synapses with human microglial cells in which perforin had been polarized to the cellular interface. Ab-mediated NKG2D and NKp46 blockade completely prevented the killing of human microglia by activated NK cells. Up-regulation of MHC class I surface expression by TLR4 stimulation protected microglia from NK cell-mediated killing, whereas MHC class I blockade enhanced cytotoxic NK cell activity. These data suggest that brain-infiltrating NK cells might restrict innate and adaptive immune responses within the human CNS via elimination of resting microglia.     

Prothrombin kringle-2 activates cultured rat brain microglia

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1beta, and TNF-alpha. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-kappaB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-kappaB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.     

Tenascin-R plays a role in neuroprotection via its distinct domains that coordinate to modulate the microglia function

Microglia are one of the main cell types activated by brain injury. In the present study, we have investigated how domains of the extracellular matrix molecule tenascin-R (TN-R) modulate microglia function. We found that epidermal growth factor-like repeats inhibited adhesion and migration of microglia via a protein kinase A-dependent mechanism. In contrast, fibronectin 6-8 repeats promoted adhesion and migration of the primary microglia via a protein kinase C-dependent mechanism. Both domains of TN-R induced an up-regulation in the secretion of cytokines, such as chemokine-induced cytokine 3 and tumor neurosis factor alpha. Interestingly, epidermal growth factor-like repeats and fibronectin 6-8 induced a dramatic up-regulation in the secretion of brain-derived neurotrophic factor/transforming growth factor-beta and nerve growth factor/transforming growth factor-beta, respectively, and conditioned medium from activated microglia was able to promote neurite outgrowth of N1E-115 cells and primary cortical neurons. These results suggest that TN-R plays a role in neuroprotection through distinct domains coordinating to modulate microglia function.     

15-deoxy-Delta12,14-prostaglandin J2 inhibits IFN-inducible protein 10/CXC chemokine ligand 10 expression in human microglia: mechanisms and implications

Regulation of cytokine and chemokine expression in microglia may have implications for CNS inflammatory disorders. In this study we examined the role of the cyclopentenone PG 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) in microglial inflammatory activation in primary cultures of human fetal microglia. 15d-PGJ(2) potently inhibited the expression of microglial cytokines (IL-1, TNF-alpha, and IL-6). We found that 15d-PGJ(2) had differential effects on the expression of two alpha-chemokines; whereas the Glu-Lys-Arg (ELR)(-) chemokine IFN-inducible protein-10/CXCL10 was inhibited, the ELR(+) chemokine IL-8/CXCL8 was not inhibited. These findings were shown in primary human microglia and the human monocytic cells line THP-1 cells, using diverse cell stimuli such as bacterial endotoxin, proinflammatory cytokines (IL-1 and TNF-alpha), IFN-beta, and HIV-1. Furthermore, IL-8/CXCL8 expression was induced by 15d-PGJ(2) alone or in combination with TNF-alpha or HIV-1. Combined results from EMSA, Western blot analysis, and immunocytochemistry showed that 15d-PGJ(2) inhibited NF-kappaB, Stat1, and p38 MAPK activation in microglia. Adenoviral transduction of super-repressor IkappaBalpha, dominant negative MKK6, and dominant negative Ras demonstrated that NF-kappaB and p38 MAPK were involved in LPS-induced IFN-inducible protein 10/CXCL10 production. Interestingly, although LPS-induced IL-8/CXCL8 was dependent on NF-kappaB, the baseline or 15d-PGJ(2)-mediated IL-8/CXCL8 production was NF-kappaB independent. Our results demonstrate that 15d-PGJ(2) has opposing effects on the expression of two alpha-chemokines. These data may have implications for CNS inflammatory diseases.     

Burning down the house: IRF7 makes the difference for microglia

The endogenous microenvironment of the brain is an essential watchdog to guard over myeloid cell function during diseases. Limiting inflammatory reactions of activated microglia and blood‐derived monocytes is a key prerequisite for the resolution of tissue insults. So far, however, it was unknown why monocytes but not microglia are able to shift to an anti‐inflammatory state during inflammation. In this issue of The EMBO Journal, Cohen and colleagues identified the molecular switch underlying this fundamental functional change. The authors found that the transforming growth factor‐β1 (TGFβ1) prevents activated microglia to switch to an anti‐inflammatory state by regulating the expression of Irf7.     

Apolipoprotein J (clusterin) activates rodent microglia in vivo and in vitro

Apolipoprotein J (apoJ; also known as clusterin and sulfated glycoprotein (SGP)-2) is associated with senile plaques in degenerating regions of Alzheimer's disease brains, where activated microglia are also prominent. We show a functional link between apoJ and activated microglia by demonstrating that exogenous apoJ activates rodent microglia in vivo and in vitro. Intracerebroventricular infusion of purified human plasma apoJ ( approximately 4 microg over 28 days) activated parenchymal microglia to a phenotype characterized by enlarged cell bodies and processes (phosphotyrosine immunostaining). In vitro, primary rat microglia were also activated by apoJ, with changes in morphology and induction of major histocompatibility complex class II (MHCII) antigen. ApoJ increased the secretion of reactive nitrogen intermediates in a dose-dependent manner (EC(50) 112 nm), which was completely blocked by aminoguanidine (AG), a nitric oxide synthase inhibitor. However, AG did not block the increased secretion of tumor necrosis factor-alpha by apoJ (EC(50) 55 nm). Microglial activation by apoJ was also blocked by an anti-apoJ monoclonal antibody (G7), and by chemical cleavage of apoJ with 2-nitro-5-thiocyanobenzoate. The mitogen-activated protein kinase kinase and protein kinase C inhibitors PD98059 and H7 inhibited apoJ-mediated induction of reactive nitrogen intermediate secretion from cultured microglia. As a functional measure, apoJ-activated microglia secreted neurotoxic agents in a microglia-neuron co-culture model. We hypothesize that ApoJ contributes to chronic inflammation and neurotoxicity through direct effects on microglia.     

BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats

The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro‐inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF‐κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro‐inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro‐inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI.     

Activated microglia are less vulnerable to hemin toxicity due to nitric oxide-dependent inhibition of JNK and p38 MAPK activation

In intracerebral hemorrhage, microglia become rapidly activated and remove the deposited blood and cellular debris. To survive in a harmful hemorrhagic or posthemorrhagic condition, activated microglia must be equipped with appropriate self-defensive mechanism(s) to resist the toxicity of hemin, a component released from damaged RBCs. In the current study, we found that activation of microglia by pretreatment with LPS markedly reduced their vulnerability to hemin toxicity in vitro. Similarly, intracorpus callosum microinjection of LPS prior to hemin treatment reduced the brain tissue damage caused by hemin and increased microglial density in the penumbra in rats. LPS induced the expressions of inducible NO synthase (iNOS) and heme oxygenase (HO)-1, the rate-limiting enzyme in heme degradation in microglia. The preventive effect by LPS was significantly diminished by an iNOS inhibitor, L-N(6)-(1-iminoethyl)lysine, whereas it was mimicked by a NO donor, diethylamine-NONOate, both suggesting the crucial role of NO in the modulation of hemin-induced toxicity in activated microglia. We further found that NO reduced hemin toxicity via inhibition of hemin-induced activation of JNK and p38 MAPK pathways in microglia. Whereas HO-1 expression in LPS-stimulated microglia was markedly blocked by L-N(6)-(1-iminoethyl)lysine, the HO-1 inhibitor, tin protoporphyrin, increased iNOS expression and decreased the susceptibility of LPS-activated microglia to hemin toxicity. The data indicate that the mutual interaction between NO and HO-1 plays a critical role in modulating the adaptive response of activated microglia to hemin toxicity. Better understanding of the survival mechanism of activated microglia may provide a therapeutic strategy to attenuate the devastating intracerebral hemorrhagic injury.     

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.     

Role of cyclophilin A from brains of prion-infected mice in stimulation of cytokine release by microglia and astroglia in vitro

Prion diseases or transmissible spongiform encephalopathy diseases are typically characterized by deposition of abnormally folded partially protease-resistant host-derived prion protein (PrPres), which is associated with activated glia and increased release of cytokines. This neuroinflammatory response may play a role in transmissible spongiform encephalopathy pathogenesis. We previously reported that brain homogenates from prion-infected mice induced cytokine protein release in primary astroglial and microglial cell cultures. Here we measured cytokine release by cultured glial cells to determine what factors in infected brain contributed to activation of microglia and astroglia. In assays analyzing IL-12p40 and CCL2 (MCP-1), glial cells were not stimulated in vitro by either PrPres purified from infected mouse brains or prion protein amyloid fibrils produced in vitro. However, significant glial stimulation was induced by clarified scrapie brain homogenates lacking PrPres. This stimulation was greatly reduced both by antibody to cyclophilin A (CyPA), a known mediator of inflammation in peripheral tissues, and by cyclosporine A, a CyPA inhibitor. In biochemical studies, purified truncated CyPA fragments stimulated a pattern of cytokine release by microglia and astroglia similar to that induced by scrapie-infected brain homogenates, whereas purified full-length CyPA was a poor stimulator. This requirement for CyPA truncation was not reported in previous studies of stimulation of peripheral macrophages, endothelial cell cardiomyocytes, and vascular smooth muscle cells. Therefore, truncated CyPA detected in brain following prion infection may have an important role in the activation of brain-derived primary astroglia and microglia in prion disease and perhaps other neurodegenerative or neuroinflammatory diseases.     

Dietary Iron Enhances Colonic Inflammation and IL-6/IL-11-Stat3 Signaling Promoting Colonic Tumor Development in Mice

Chronic intestinal inflammation and high dietary iron are associated with colorectal cancer development. The role of Stat3 activation in iron-induced colonic inflammation and tumorigenesis was investigated in a mouse model of inflammation-associated colorectal cancer. Mice, fed either an iron-supplemented or control diet, were treated with azoxymethane and dextran sodium sulfate (DSS). Intestinal inflammation and tumor development were assessed by endoscopy and histology, gene expression by real-time PCR, Stat3 phosphorylation by immunoblot, cytokines by ELISA and apoptosis by TUNEL assay. Colonic inflammation was more severe in mice fed an iron-supplemented compared with a control diet one week post-DSS treatment, with enhanced colonic IL-6 and IL-11 release and Stat3 phosphorylation. Both IL-6 and ferritin, the iron storage protein, co-localized with macrophages suggesting iron may act directly on IL-6 producing-macrophages. Iron increased DSS-induced colonic epithelial cell proliferation and apoptosis consistent with enhanced mucosal damage. DSS-treated mice developed anemia that was not alleviated by dietary iron supplementation. Six weeks post-DSS treatment, iron-supplemented mice developed more and larger colonic tumors compared with control mice. Intratumoral IL-6 and IL-11 expression increased in DSS-treated mice and IL-6, and possibly IL-11, were enhanced by dietary iron. Gene expression of iron importers, divalent metal transporter 1 and transferrin receptor 1, increased and iron exporter, ferroportin, decreased in colonic tumors suggesting increased iron uptake. Dietary iron and colonic inflammation synergistically activated colonic IL-6/IL-11-Stat3 signaling promoting tumorigenesis. Oral iron therapy may be detrimental in inflammatory bowel disease since it may exacerbate colonic inflammation and increase colorectal cancer risk.