Immunostimulating activity of maysin isolated from corn silk in murine RAW 264.7 macrophages

Corn silk (CS) has long been consumed as a traditional herb in Korea. Maysin is a major flavonoid of CS. The effects of maysin on macrophage activation were evaluated, using the murine macrophage RAW 264.7 cells. Maysin was isolated from CS by methanol extraction, and preparative C₁₈ reverse phase column chromatography. Maysin was nontoxic up to 100 μg/ml, and dose-dependently increased TNF-α secretion and iNOS production by 11.2- and 4.2-fold, respectively, compared to untreated control. The activation and subsequent nuclear translocation of NF-κB was substantially enhanced upon treatment with maysin (1-100 μg/ml). Maysin also stimulated the phosphorylation of Akt and MAPKs (ERK, JNK). These results indicated that maysin activates macrophages to secrete TNF-α and induce iNOS expression, via the activation of the Akt, NF-κB and MAPKs signaling pathways. These results suggest for the first time that maysin can be a new immunomodulator, enhancing the early innate immunity. [BMB Reports 2014; 47(7): 382-387]     

Tumor Necrosis Factor Receptor 2 (TNFR2)·Interleukin-17 Receptor D (IL-17RD) Heteromerization Reveals a Novel Mechanism for NF-κB Activation

TNF receptor 2 (TNFR2) exerts diverse roles in the pathogenesis of inflammatory and autoimmune diseases. Here, we report that TNFR2 but not TNFR1 forms a heteromer with interleukin-17 receptor D (IL-17RD), also named Sef, to activate NF-κB signaling. TNFR2 associates with IL-17RD, leading to mutual receptor aggregation and TRAF2 recruitment, which further activate the downstream cascade of NF-κB signaling. Depletion of IL-17RD impaired TNFR2-mediated activation of NF-κB signaling. Importantly, IL-17RD was markedly increased in renal tubular epithelial cells in nephritis rats, and a strong interaction of TNFR2 and IL-17RD was observed in the renal epithelia. The IL-17RD·TNFR2 complex in activation of NF-κB may explain the role of TNFR2 in inflammatory diseases including nephritis.     

High Glucose Induces CCL20 in Proximal Tubular Cells via Activation of the KCa3.1 Channel

Background

Inflammation plays a key role in the development and progression of diabetic nephropathy (DN). KCa3.1, a calcium activated potassium channel protein, is associated with vascular inflammation, atherogenesis, and proliferation of endothelial cells, macrophages, and fibroblasts. We have previously demonstrated that the KCa3.1 channel is activated by TGF-β1 and blockade of KCa3.1 ameliorates renal fibrotic responses in DN through inhibition of the TGF-β1 pathway. The present study aimed to identify the role of KCa3.1 in the inflammatory responses inherent in DN.

Methods

Human proximal tubular cells (HK2 cells) were exposed to high glucose (HG) in the presence or absence of the KCa3.1 inhibitor TRAM34 for 6 days. The proinflammatory cytokine chemokine (C-C motif) ligand 20 (CCL20) expression was examined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The activity of nuclear factor-κB (NF-κB) was measured by nuclear extraction and electrophoretic mobility shift assay (EMSA). In vivo, the expression of CCL20, the activity of NF-κB and macrophage infiltration (CD68 positive cells) were examined by real-time PCR and/or immunohistochemistry staining in kidneys from diabetic or KCa3.1-/- mice, and in eNOS-/- diabetic mice treated with the KCa3.1 channel inhibitor TRAM34.

Results

In vitro data showed that TRAM34 inhibited CCL20 expression and NF-κB activation induced by HG in HK2 cells. Both mRNA and protein levels of CCL20 significantly decreased in kidneys of diabetic KCa3.1-/- mice compared to diabetic wild type mice. Similarly, TRAM34 reduced CCL20 expression and NF-κB activation in diabetic eNOS-/- mice compared to diabetic controls. Blocking the KCa3.1 channel in both animal models led to a reduction in phosphorylated NF-κB.

Conclusions

Overexpression of CCL20 in human proximal tubular cells is inhibited by blockade of KCa3.1 under diabetic conditions through inhibition of the NF-κB pathway.     

Combined MMF and insulin therapy prevents renal injury in experimental diabetic rats

Conventional therapies for diabetic mellitus are not effective in preventing the progression from early diabetic nephropathy (DN) to end-stage renal disease. The role of inflammation in the pathogenesis of DN has been implicated both clinically and experimentally, which provides an alternative therapeutic target for DN. Anti-inflammatory impact of mycophenolate mofetil (MMF) alone and in combination with insulin had been observed in a rat model of experimental DN. In this study, the diabetic rats were subjected to different treatments. Compared to control, the expression levels of CD68, NGF, and NF-κB p65, as determined immunohistochemically, were elevated in diabetic rats. Treatment with combined MMF/insulin is associated with a significant reduction in renal tissue of NGF and NF-κB p65 expression, macrophage infiltration. It also partially improved the renal function and attenuated renal hypertrophy at early stage of DN. CD68 was found to positively correlate with urinary albumin excretion and NGF. The combined use of MMF/insulin seemed to offer more protections in rats with experimental diabetic renal injury, and the protective effects of MMF might be due to its anti-inflammatory actions through inhibition of NF-κB activation and reduction of T cells and macrophage infiltration and/or other kidney chemokine productions.     

TNF-α Decreases VEGF Secretion in Highly Polarized RPE Cells but Increases It in Non-Polarized RPE Cells Related to Crosstalk between JNK and NF-κB Pathways

Asymmetrical secretion of vascular endothelial growth factor (VEGF) by retinal pigment epithelial (RPE) cells in situ is critical for maintaining the homeostasis of the retina and choroid. VEGF is also involved in the development and progression of age-related macular degeneration (AMD). We studied the effect of tumor necrosis factor-α (TNF-α) on the secretion of VEGF in polarized and non-polarized RPE cells (P-RPE cells and N-RPE cells, respectively) in culture and in situ in rats. A subretinal injection of TNF-α caused a decrease in VEGF expression and choroidal atrophy. Porcine RPE cells were seeded on Transwell™ filters, and their maturation and polarization were confirmed by the asymmetrical VEGF secretion and trans electrical resistance. Exposure to TNF-α decreased the VEGF secretion in P-RPE cells but increased it in N-RPE cells in culture. TNF-α inactivated JNK in P-RPE cells but activated it in N-RPE cells, and TNF-α activated NF-κB in P-RPE cells but not in N-RPE cells. Inhibition of NF-κB activated JNK in both types of RPE cells indicating crosstalk between JNK and NF-κB. TNF-α induced the inhibitory effects of NF-κB on JNK in P-RPE cells because NF-κB is continuously inactivated. In N-RPE cells, however, it was not evident because NF-κB was already activated. The basic activation pattern of JNK and NF-κB and their crosstalk led to opposing responses of RPE cells to TNF-α. These results suggest that VEGF secretion under inflammatory conditions depends on cellular polarization, and the TNF-α-induced VEGF down-regulation may result in choroidal atrophy in polarized physiological RPE cells. TNF-α-induced VEGF up-regulation may cause neovascularization by non-polarized or non-physiological RPE cells.     

Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

The blockade of angiotensin II (Ang II) is a major therapeutic strategy for diabetic nephropathy. The main roles of Ang II in renal disease are mediated via the Ang type 1 receptor (AT1R). Upregulation of clusterin/apolipoprotein J has been reported in nephropathy models, suggesting it has a protective role in nephropathogenesis. Here, we studied how clusterin acts against Ang II-induced renal fibrosis. Levels of AT1R and fibrotic markers in clusterin-/- mice and Ang II infused rats transfected with an adenovirus encoding clusterin were evaluated by immunoblot analysis, real time RT-PCR, and immunohistochemical staining. The effect of clusterin on renal fibrosis was evaluated in NRK-52E cells, a cultured renal tubular epithelial cell line, using immunoblot analysis and real time RT-PCR. Nuclear localization of NF-κB was evaluated using immunofluorecence and co-immunoprecipitation. Renal fibrosis and expression of AT1R was higher in the kidneys of clusterin^-/- mice than in those of wild-type mice. Furthermore, loss of clusterin accelerated Ang II-stimulated renal fibrosis and AT1R expression. Overexpression of clusterin in proximal tubular epithelial cells decreased the levels of Ang II-stimulated fibrotic markers and AT1R. Moreover, intrarenal delivery of clusterin attenuated Ang II-mediated expression of fibrotic markers and AT1R in rats. Fluorescence microscopy and co-immunoprecipitation in conjunction with western blot revealed that clusterin inhibited Ang II-stimulated nuclear localization of p-NF-κB via a direct physical interaction and subsequently decreased the AT1R level in proximal tubular epithelial cells. These data suggest that clusterin attenuates Ang II-induced renal fibrosis by inhibition of NF-κB activation and subsequent downregulation of AT1R. This study raises the possibility that clusterin could be used as a therapeutic target for Ang II-induced renal diseases.     

Suppressing NF-κB and NKRF Pathways by Induced Pluripotent Stem Cell Therapy in Mice with Ventilator-Induced Lung Injury

Background

High-tidal-volume mechanical ventilation used in patients with acute lung injury (ALI) can induce the release of inflammatory cytokines, as macrophage inflammatory protein-2 (MIP-2), recruitment of neutrophils, and disruption of alveolar epithelial and endothelial barriers. Induced pluripotent stem cells (iPSCs) have been shown to improve ALI in mice, but the mechanisms regulating the interactions between mechanical ventilation and iPSCs are not fully elucidated. Nuclear factor kappa B (NF-κB) and NF-κB repressing factor (NKRF) have been proposed to modulate the neutrophil activation involved in ALI. Thus, we hypothesized intravenous injection of iPSCs or iPSC-derived conditioned medium (iPSC-CM) would decrease high-tidal-volume ventilation-induced neutrophil infiltration, oxidative stress, and MIP-2 production through NF-κB/NKRF pathways.

Methods

Male C57BL/6 mice, aged between 6 and 8 weeks, weighing between 20 and 25 g, were exposed to high-tidal-volume (30 ml/kg) mechanical ventilation with room air for 1 to 4 h after 5×10⁷ cells/kg mouse iPSCs or iPSC-CM administration. Nonventilated mice were used as control groups.

Results

High-tidal-volume mechanical ventilation induced the increases of integration of iPSCs into the injured lungs of mice, microvascular permeability, neutrophil infiltration, malondialdehyde, MIP-2 production, and NF-κB and NKRF activation. Lung injury indices including inflammation, lung edema, ultrastructure pathologic changes and functional gas exchange impairment induced by mechanical ventilation were attenuated with administration of iPSCs or iPSC-CM, which was mimicked by pharmacological inhibition of NF-κB activity with SN50 or NKRF expression with NKRF short interfering RNA.

Conclusions

Our data suggest that iPSC-based therapy attenuates high-tidal-volume mechanical ventilation-induced lung injury, at least partly, through inhibition of NF-κB/NKRF pathways. Notably, the conditioned medium of iPSCs revealed beneficial effects equal to those of iPSCs.     

Neurotropin Suppresses Inflammatory Cytokine Expression and Cell Death through Suppression of NF-κB and JNK in Hepatocytes

Inflammatory response and cell death in hepatocytes are hallmarks of chronic liver disease, and, therefore, can be effective therapeutic targets. Neurotropin® (NTP) is a drug widely used in Japan and China to treat chronic pain. Although NTP has been demonstrated to suppress chronic pain through the descending pain inhibitory system, the action mechanism of NTP remains elusive. We hypothesize that NTP functions to suppress inflammatory pathways, thereby attenuating disease progression. In the present study, we investigated whether NTP suppresses inflammatory signaling and cell death pathways induced by interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) in hepatocytes. NTP suppressed nuclear factor-κB (NF-κB) activation induced by IL-1β and TNFα assessed by using hepatocytes isolated from NF-κB-green fluorescent protein (GFP) reporter mice and an NF-κB-luciferase reporter system. The expression of NF-κB target genes, Il6, Nos2, Cxcl1, ccl5 and Cxcl2 induced by IL-1β and TNFα was suppressed after NTP treatment. We also found that NTP suppressed the JNK phosphorylation induced by IL-1β and TNFα. Because JNK activation contributes to hepatocyte death, we determined that NTP treatment suppressed hepatocyte death induced by IL-1β and TNFα in combination with actinomycin D. Taken together, our data demonstrate that NTP attenuates IL-1β and TNFα-mediated inflammatory cytokine expression and cell death in hepatocytes through the suppression of NF-κB and JNK. The results from the present study suggest that NTP may become a preventive or therapeutic strategy for alcoholic and non-alcoholic fatty liver disease in which NF-κB and JNK are thought to take part.     

Ezrin-Radixin-Moesin-binding Phosphoprotein 50 (EBP50) and Nuclear Factor-κB (NF-κB): A FEED-FORWARD LOOP FOR SYSTEMIC AND VASCULAR INFLAMMATION*

The interaction between vascular cells and macrophages is critical during vascular remodeling. Here we report that the scaffolding protein, ezrin-binding phosphoprotein 50 (EBP50), is a central regulator of macrophage and vascular smooth muscle cells (VSMC) function. EBP50 is up-regulated in intimal VSMC following endoluminal injury and promotes neointima formation. However, the mechanisms underlying these effects are not fully understood. Because of the fundamental role that inflammation plays in vascular diseases, we hypothesized that EBP50 mediates macrophage activation and the response of vessels to inflammation. Indeed, EBP50 expression increased in primary macrophages and VSMC, and in the aorta of mice, upon treatment with LPS or TNFα. This increase was nuclear factor-κB (NF-κB)-dependent. Conversely, activation of NF-κB was impaired in EBP50-null VSMC and macrophages. We found that inflammatory stimuli promote the formation of an EBP50-PKCζ complex at the cell membrane that induces NF-κB signaling. Macrophage activation and vascular inflammation after acute LPS treatment were reduced in EBP50-null cells and mice as compared with WT. Furthermore, macrophage recruitment to vascular lesions was significantly reduced in EBP50 knock-out mice. Thus, EBP50 and NF-κB participate in a feed-forward loop leading to increased macrophage activation and enhanced response of vascular cells to inflammation.     

Hyperinvasive Meningococci Induce Intra-nuclear Cleavage of the NF-κB Protein p65/RelA by Meningococcal IgA Protease

Differential modulation of NF-κB during meningococcal infection is critical in innate immune response to meningococcal disease. Non-invasive isolates of Neisseria meningitidis provoke a sustained NF-κB activation in epithelial cells. However, the hyperinvasive isolates of the ST-11 clonal complex (ST-11) only induce an early NF-κB activation followed by a sustained activation of JNK and apoptosis. We show that this temporal activation of NF-κB was caused by specific cleavage at the C-terminal region of NF-κB p65/RelA component within the nucleus of infected cells. This cleavage was mediated by the secreted 150 kDa meningococcal ST-11 IgA protease carrying nuclear localisation signals (NLS) in its α-peptide moiety that allowed efficient intra-nuclear transport. In a collection of non-ST-11 healthy carriage isolates lacking NLS in the α-peptide, secreted IgA protease was devoid of intra-nuclear transport. This part of iga polymorphism allows non-invasive isolates lacking NLS, unlike hyperinvasive ST-11 isolates of N. meningitides habouring NLS in their α-peptide, to be carried asymptomatically in the human nasopharynx through selective eradication of their ability to induce apoptosis in infected epithelial cells.     

Shigella IpaH0722 E3 Ubiquitin Ligase Effector Targets TRAF2 to Inhibit PKC–NF-κB Activity in Invaded Epithelial Cells

NF-κB plays a central role in modulating innate immune responses to bacterial infections. Therefore, many bacterial pathogens deploy multiple mechanisms to counteract NF-κB activation. The invasion of and subsequent replication of Shigella within epithelial cells is recognized by various pathogen recognition receptors as pathogen-associated molecular patterns. These receptors trigger innate defense mechanisms via the activation of the NF-κB signaling pathway. Here, we show the inhibition of the NF-κB activation by the delivery of the IpaH E3 ubiquitin ligase family member IpaH0722 using Shigella''s type III secretion system. IpaH0722 dampens the acute inflammatory response by preferentially inhibiting the PKC-mediated activation of NF-κB by ubiquitinating TRAF2, a molecule downstream of PKC, and by promoting its proteasome-dependent degradation.     

Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Activates the JNK Pathway through Its Extreme C Terminus via a Mechanism Involving TRADD and TRAF2

The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-κB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-κB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-κB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro³⁷⁹, Glu³⁸¹, Ser³⁸³, or Tyr³⁸⁴ diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-κB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-κB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-κB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-κB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.     

TGFβ-activated Kinase 1 (TAK1) Inhibition by 5Z-7-Oxozeaenol Attenuates Early Brain Injury after Experimental Subarachnoid Hemorrhage

Accumulating evidence suggests that activation of mitogen-activated protein kinases (MAPKs) and nuclear factor NF-κB exacerbates early brain injury (EBI) following subarachnoid hemorrhage (SAH) by provoking proapoptotic and proinflammatory cellular signaling. Here we evaluate the role of TGFβ-activated kinase 1 (TAK1), a critical regulator of the NF-κB and MAPK pathways, in early brain injury following SAH. Although the expression level of TAK1 did not present significant alternation in the basal temporal lobe after SAH, the expression of phosphorylated TAK1 (Thr-187, p-TAK1) showed a substantial increase 24 h post-SAH. Intracerebroventricular injection of a selective TAK1 inhibitor (10 min post-SAH), 5Z-7-oxozeaenol (OZ), significantly reduced the levels of TAK1 and p-TAK1 at 24 h post-SAH. Involvement of MAPKs and NF-κB signaling pathways was revealed that OZ inhibited SAH-induced phosphorylation of p38 and JNK, the nuclear translocation of NF-κB p65, and degradation of IκBα. Furthermore, OZ administration diminished the SAH-induced apoptosis and EBI. As a result, neurological deficits caused by SAH were reversed. Our findings suggest that TAK1 inhibition confers marked neuroprotection against EBI following SAH. Therefore, TAK1 might be a promising new molecular target for the treatment of SAH.     

TRIF Mediates Toll-like Receptor 5-induced Signaling in Intestinal Epithelial Cells

Toll-like receptors (TLRs) associate with adaptor molecules (MyD88, Mal/TIRAP, TRAM, and TRIF) to mediate signaling of host-microbial interaction. For instance, TLR4 utilizes the combination of both Mal/TIRAP-MyD88 (MyD88-dependent pathway) and TRAM-TRIF (MyD88-independent pathway). However, TLR5, the specific receptor for flagellin, is known to utilize only MyD88 to elicit inflammatory responses, and an involvement of other adaptor molecules has not been suggested in TLR5-dependent signaling. Here, we found that TRIF is involved in mediating TLR5-induced nuclear factor κB (NFκB) and mitogen-activated protein kinases (MAPKs), specifically JNK1/2 and ERK1/2, activation in intestinal epithelial cells. TLR5 activation by flagellin permits the physical interaction between TLR5 and TRIF in human colonic epithelial cells (NCM460), whereas TLR5 does not interact with TRAM upon flagellin stimulation. Both primary intestinal epithelial cells from TRIF-KO mice and TRIF-silenced NCM460 cells significantly reduced flagellin-induced NFκB (p105 and p65), JNK1/2, and ERK1/2 activation compared with control cells. However, p38 activation by flagellin was preserved in these TRIF-deficient cells. TRIF-KO intestinal epithelial cells exhibited substantially reduced inflammatory cytokine (keratinocyte-derived cytokine, macrophage inflammatory protein 3α, and IL-6) expression upon flagellin, whereas control cells from TRIF-WT mice showed robust cytokine expression by flagellin. Compare with TRIF-WT mice, TRIF-KO mice were resistant to in vivo intestinal inflammatory responses: flagellin-mediated exacerbation of colonic inflammation and dextran sulfate sodium-induced experimental colitis. We conclude that in addition to MyD88, TRIF mediates TLR5-dependent responses and, thereby regulates inflammatory responses elicited by flagellin/TLR5 engagement. Our findings suggest an important role of TRIF in regulating host-microbial communication via TLR5 in the gut epithelium.     

RIP4 (DIK/PKK), a novel member of the RIP kinase family,activates NF-kappa B and is processed during apoptosis

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals.