Interleukin-12 P40 induces the expression of TNF-α in microglia and macrophages

Recently, it has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of a number of autoimmune inflammatory diseases such as multiple sclerosis. It is composed of two different subunits – p40 and p35. Expression of p40 mRNA but not that of p35 mRNA in excessive amount in the CNS of patients with Multiple Sclerosis (MS) suggests that IL-12 p40 may have a role in the pathogenesis of the disease. The present study was undertaken to explore the role of p40 in the expression of TNF-α in microglia. Interestingly, we have found that IL-12 p70, p402 (the p40 homodimer) and p40 (the p40 monomer) dose-dependently induced the production of TNF-α in BV-2 microglial cells. This induction of TNF-α production was accompanied by an induction of TNF-α mRNA. In addition to BV-2 glial cells, p70, p402 and p40 also induced the production of TNF-α in mouse primary microglia and peritoneal macrophages. Since the activation of both NF-κB and C/EBPb is important for the expression of TNF-α in microglial cells, we investigated the effect of p40 on the activation of NF-κB as well as C/EBPb. Activation of NF-κB as well as C/EBPb by p40 and inhibition of p40-induced expression of TNF-α by Dp65, a dominant-negative mutant of p65, and DC/EBPb, a dominant-negative mutant of C/EBPb, suggests that p40 induces the expression of TNF-α through the activation of NF-κB and C/EBPb. This study delineates a novel role of IL-12 p40 in inducing the expression of TNF-α in microglial cells which may participate in the pathogenesis of neuroinflammatory diseases.
Acknowledgements:   This study was supported by NIH grants (NS39940 and AG19487).     

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV‐2 Microglial Cells

Naegleria fowleri, a free‐living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV‐2 microglial cells induced by excretory and secretory proteins of N. fowleri (NfESP). Treatment of BV‐2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL‐1α and TNF‐α. NfESP‐induced IL‐1α and TNF‐α expression in BV‐2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP‐induced IL‐1α and TNF‐α production in BV‐2 cells were effectively downregulated by inhibition of NF‐kB and AP‐1. These results collectively suggest that NfESP stimulates BV‐2 cells to release IL‐1α and TNF‐α via NF‐kB‐ and AP‐1‐dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N. fowleri infection.     

Dl‐3‐n‐butylphthalide attenuates acute inflammatory activation in rats with spinal cord injury by inhibiting microglial TLR4/NF‐κB signalling

In this study, we examined the neuroprotective effects and anti‐inflammatory properties of Dl‐3‐n‐butylphthalide (NBP) in Sprague‐Dawley (SD) rats following traumatic spinal cord injury (SCI) as well as microglia activation and inflammatory response both in vivo and in vitro. Our results showed that NBP improved the locomotor recovery of SD rats after SCI an significantly diminished the lesion cavity area of the spinal cord, apoptotic activity in neurons, and the number of TUNEL‐positive cells at 7 days post‐injury. NBP inhibited activation of microglia, diminished the release of inflammatory mediators, and reduced the upregulation of microglial TLR4/NF‐κB expression at 1 day post‐injury. In a co‐culture system with BV‐2 cells and PC12 cells, NBP significantly reduced the cytotoxicity of BV‐2 cells following lipopolysaccharide (LPS) stimulation. In addition, NBP reduced the activation of BV‐2 cells, diminished the release of inflammatory mediators, and inhibited microglial TLR4/NF‐κB expression in BV‐2 cells. Our findings demonstrate that NBP may have neuroprotective and anti‐inflammatory properties in the treatment of SCI by inhibiting the activation of microglia via TLR4/NF‐κB signalling.     

Integrin‐linked kinase is involved in TNF‐α‐induced inducible nitric‐oxide synthase expression in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF‐α in cultured myoblasts. TNF‐α stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF‐α‐mediated iNOS expression was attenuated by integrin‐linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK) also inhibited the potentiating action of TNF‐α. Stimulation of cells with TNF‐α increased ILK kinase activity. TNF‐α also increased the Akt and mTOR phosphorylation. TNF‐α mediated an increase of NF‐κB‐specific DNA–protein complex formation, p65 translocation into nucleus, NF‐κB‐luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF‐α increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF‐κB signaling pathway. J. Cell. Biochem. 109: 1244–1253, 2010. © 2010 Wiley‐Liss, Inc.     

FAK activation is required for TNF‐α‐induced IL‐6 production in myoblasts

Tumor necrosis factor‐α (TNF‐α) is a pleiotropic cytokine produced by activated macrophages. IL‐6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL‐6 production stimulated by TNF‐α in cultured myoblasts. TNF‐α caused concentration‐dependent increases in IL‐6 production. TNF‐α‐mediated IL‐6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3‐kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF‐κB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IκB protease inhibitor (L ‐1‐tosylamido‐2‐phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF‐α. TNF‐α increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF‐α activated IκB kinase α/β (IKKα/β), IκBα phosphorylation, p65 phosphorylation, and κB‐luciferase activity. TNF‐α mediated an increase of κB‐luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF‐α increased IL‐6 production in myoblasts via the FAK/PI3K/Akt and NF‐κB signaling pathway. J. Cell. Physiol. 223: 389–396, 2010. © 2010 Wiley‐Liss, Inc.     

Role of PPARγ in the salutary effects of 17β‐estradiol on kupffer cell cytokine production following trauma‐hemorrhage

Studies have shown that administration of 17β‐estradiol prevents trauma‐hemorrhage‐induced increase in proinflammatory cytokine production by Kupffer cells and associated multiple organ injury. Since activation of peroxisome proliferator‐activated receptor γ (PPARγ) following ischemic conditions has been shown to be protective, we examined if PPARγ plays any role in the salutary effects of 17β‐estradiol on Kupffer cell cytokine production following trauma‐hemorrhage. Male mice underwent trauma‐hemorrhage (mean blood pressure 40 mmHg for 90 min, then resuscitation). 17β‐estradiol (50 µg/kg) or vehicle with or without PPARγ antagonist GW9662 was injected subcutaneously at the middle of resuscitation. At 2 h after trauma‐hemorrhage, plasma interleukin (IL)‐6 and tumor necrosis factor (TNF)‐α levels, Kupffer cell IL‐6 and TNF‐α production and mRNA expression, and PPARγ, nuclear factor (NF)‐κB and activator protein (AP)‐1 DNA binding activity were determined. Kupffer cell IL‐6 and TNF‐α production, as well as plasma IL‐6 and TNF‐α levels, increased following trauma‐hemorrhage. Moreover, NF‐κB and AP‐1 DNA binding activity and IL‐6 and TNF‐α mRNA expression were also enhanced under such conditions. However, 17β‐estradiol administration normalized all these parameters. Although PPARγ activity decreased after trauma‐hemorrhage, administration of 17β‐estradiol following trauma‐hemorrhage elevated PPARγ activity above the normal level. Inhibition of PPARγ by co‐administration of GW9662, however, abolished the salutary effects of 17β‐estradiol on plasma cytokine and Kupffer cells. Thus, activation of PPARγ appears to play an important role in mediating the salutary effects of 17β‐estradiol on plasma cytokine levels and Kupffer cell cytokine production after trauma‐hemorrhage, which are likely mediated via NF‐κB and AP‐1. J. Cell. Physiol. 226: 205–211, 2010. © 2010 Wiley‐Liss, Inc.     

miR‐124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast‐like synoviocytes via targeting PIK3/NF‐κB pathway

Abnormal hyperplasia of fibroblast‐like synoviocytes (FLS) leads to the progression of rheumatoid arthritis (RA). This study aimed to investigate the role of miR‐124a in the pathogenesis of RA. The viability and cell cycle of FLS in rheumatoid arthritis (RAFLS) were evaluated by Cell Counting Kit 8 and flow cytometry assay. The expression of PIK3CA, Akt, and NF‐κB in RAFLS was examined by real‐time PCR and Western blot analysis. The production of tumour necrosis factor (TNF)‐α and interleukin (IL)‐6 was detected by ELISA. The joint swelling and inflammation in collagen‐induced arthritis (CIA) mice were examined by histological and immunohistochemical analysis. We found that miR‐124a suppressed the viability and proliferation of RAFLS and increased the percentage of cells in the G1 phase. miR‐124a suppressed PIK3CA 3'UTR luciferase reporter activity and decreased the expression of PIK3CA at mRNA and protein levels. Furthermore, miR‐124a inhibited the expression of the key components of the PIK3/Akt/NF‐κB signal pathway and inhibited the expression of pro‐inflammatory factors TNF‐α and IL‐6. Local overexpression of miR‐124a in the joints of CIA mice inhibited inflammation and promoted apoptosis in FLS by decreasing PIK3CA expression. In conclusion, miR‐124a inhibits the proliferation and inflammation in RAFLS via targeting PIK3/NF‐κB pathway. miR‐124a is a promising therapeutic target for RA.     

α1 adrenoceptor activation by norepinephrine inhibits LPS‐induced cardiomyocyte TNF‐α production via modulating ERK1/2 and NF‐κB pathway

Cardiomyocyte tumour necrosis factor α (TNF‐α) production contributes to myocardial depression during sepsis. This study was designed to observe the effect of norepinephrine (NE) on lipopolysaccharide (LPS)‐induced cardiomyocyte TNF‐α expression and to further investigate the underlying mechanisms in neonatal rat cardiomyocytes and endotoxaemic mice. In cultured neonatal rat cardiomyocytes, NE inhibited LPS‐induced TNF‐α production in a dose‐dependent manner. α₁‐ adrenoceptor (AR) antagonist (prazosin), but neither β₁‐ nor β₂‐AR antagonist, abrogated the inhibitory effect of NE on LPS‐stimulated TNF‐α production. Furthermore, phenylephrine (PE), an α₁‐AR agonist, also suppressed LPS‐induced TNF‐α production. NE inhibited p38 phosphorylation and NF‐κB activation, but enhanced extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation and c‐Fos expression in LPS‐treated cardiomyocytes, all of which were reversed by prazosin pre‐treatment. To determine whether ERK1/2 regulates c‐Fos expression, p38 phosphorylation, NF‐κB activation and TNF‐α production, cardiomyocytes were also treated with U0126, a selective ERK1/2 inhibitor. Treatment with U0126 reversed the effects of NE on c‐Fos expression, p38 mitogen‐activated protein kinase (MAPK) phosphorylation and TNF‐α production, but not NF‐κB activation in LPS‐challenged cardiomyocytes. In addition, pre‐treatment with SB202190, a p38 MAPK inhibitor, partly inhibited LPS‐induced TNF‐α production in cardiomyocytes. In endotoxaemic mice, PE promoted myocardial ERK1/2 phosphorylation and c‐Fos expression, inhibited p38 phosphorylation and IκBα degradation, reduced myocardial TNF‐α production and prevented LPS‐provoked cardiac dysfunction. Altogether, these findings indicate that activation of α₁‐AR by NE suppresses LPS‐induced cardiomyocyte TNF‐α expression and improves cardiac dysfunction during endotoxaemia via promoting myocardial ERK phosphorylation and suppressing NF‐κB activation.     

TNF‐α regulates the early development of avascular necrosis of the femoral head by mediating osteoblast autophagy and apoptosis via the p38 MAPK/NF‐κB signaling pathway

Previous studies have shown that the tumor necrosis factor‐α (TNF‐α) levels in serum and bone tissues formed in avascular necrosis of femoral head (ANFH) patients were higher than those of normal individuals, indicating TNF‐α might play a role in the pathogenesis of ANFH. However, the underlying mechanisms remain unclear. Hematoxylin and eosin staining was performed to show the pathological changes of ANFH bone tissues. TNF‐α expression in normal and ANFH tissues was examined by quantitative real‐time polymerase chain reaction and western blot analyses. Osteoblast autophagy and apoptosis, as well as signaling pathways activation, were measured by their corresponding marker proteins. Osteoblast proliferation, autophagy, and apoptosis were evaluated using cell counting kit‐8, transmission electron microscopy, and flow cytometry. The structures of bone tissues of ANFH were obviously damaged. TNF‐α expression was significantly upregulated in ANFH bone tissues compared to normal tissues. Autophagy and apoptosis were remarkably promoted, and p38 mitogen‐activated protein kinase (MAPK)/nuclear factor‐κB (NF‐κB) signaling pathways were markedly activated in ANFH. Suppression of the p38 MAPK/NF‐κB pathway significantly attenuated the TNF‐α‐induced autophagy, however, enhanced the TNF‐α‐induced apoptosis in osteoblasts. Increased TNF‐α in ANFH regulated osteoblast autophagy and apoptosis by p38 MAPK/NF‐κB signaling pathways, blocking the pathway by inhibitors exacerbated TNF‐α‐induced apoptosis through impairing autophagy flux.     

Folic acid deficiency enhanced microglial immune response via the Notch1/nuclear factor kappa B p65 pathway in hippocampus following rat brain I/R injury and BV2 cells

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.     

Acetate reduces microglia inflammatory signaling in vitro

Acetate supplementation increases brain acetyl‐CoA and histone acetylation and reduces lipopolysaccharide (LPS)‐induced neuroglial activation and interleukin (IL)‐1β expression in vivo. To determine how acetate imparts these properties, we tested the hypothesis that acetate metabolism reduces inflammatory signaling in microglia. To test this, we measured the effect acetate treatment had on cytokine expression, mitogen‐activated protein kinase (MAPK) signaling, histone H3 at lysine 9 acetylation, and alterations of nuclear factor‐kappa B (NF‐κB) in primary and BV‐2 cultured microglia. We found that treatment induced H3K9 hyperacetylation and reversed LPS‐induced H3K9 hypoacetylation similar to that found in vivo. LPS also increased IL‐1β, IL‐6, and tumor necrosis factor‐alpha (TNF‐α) mRNA and protein, whereas treatment returned the protein to control levels and only partially attenuated IL‐6 mRNA. In contrast, treatment increased mRNA levels of transforming growth factor‐β1 (TGF‐β1) and both IL‐4 mRNA and protein. LPS increased p38 MAPK and JNK phosphorylation at 4 and 2–4 h, respectively, whereas treatment reduced p38 MAPK and JNK phosphorylation only at 2 h. In addition, treatment reversed the LPS‐induced elevation of NF‐κB p65 protein and phosphorylation at serine 468 and induced acetylation at lysine 310. These data suggest that acetate metabolism reduces inflammatory signaling and alters histone and non‐histone protein acetylation.     

Essential involvement of cross‐talk between IFN‐γ and TNF‐α in CXCL10 production in human THP‐1 monocytes

Interferon (IFN)‐γ‐induced protein 10 (IP‐10/CXCL10), a CXC chemokine, has been documented in several inflammatory and autoimmune disorders including atopic dermatitis and bronchial asthma. Although CXCL10 could be induced by IFN‐γ depending on cell type, the mechanisms regulating CXCL10 production following treatment with combination of IFN‐γ and TNF‐α have not been adequately elucidated in human monocytes. In this study, we showed that TNF‐α had more potential than IFN‐γ to induce CXCL10 production in THP‐1 monocytes. Furthermore, IFN‐γ synergistically enhanced the production of CXCL10 in parallel with the activation of NF‐κB in TNF‐α‐stimulated THP‐1 cells. Blockage of STAT1 or NF‐κB suppressed CXCL10 production. JAKs inhibitors suppressed IFN‐γ plus TNF‐α‐induced production of CXCL10 in parallel with activation of STAT1 and NF‐κB, while ERK inhibitor suppressed production of CXCL10 as well as activation of NF‐κB, but not that of STAT1. IFN‐γ‐induced phosphorylation of JAK1 and JAK2, whereas TNF‐α induced phosphorylation of ERK1/2. Interestingly, IFN‐γ alone had no effect on phosphorylation and degradation of IκB‐α, whereas it significantly promoted TNF‐α‐induced phosphorylation and degradation of IκB‐α. These results suggest that TNF‐α induces CXCL10 production by activating NF‐κB through ERK and that IFN‐γ induces CXCL10 production by increasing the activation of STAT1 through JAKs pathways. Of note, TNF‐α‐induced NF‐κB may be the primary pathway contributing to CXCL10 production in THP‐1 cells. IFN‐γ potentiates TNF‐α‐induced CXCL10 production in THP‐1 cells by increasing the activation of STAT1 and NF‐κB through JAK1 and JAK2. J. Cell. Physiol. 220: 690–697, 2009. © 2009 Wiley‐Liss, Inc.