Toll-Like Receptor 4 Mediates Inflammatory Cytokine Secretion in Smooth Muscle Cells Induced by Oxidized Low-Density Lipoprotein

Oxidized low-density lipoprotein (oxLDL)-regulated secretion of inflammatory cytokines in smooth muscle cells (SMCs) is regarded as an important step in the progression of atherosclerosis; however, its underlying mechanism remains unclear. This study investigated the role of toll-like receptor 4 (TLR4) in oxLDL-induced expression of inflammatory cytokines in SMCs both in vivo and in vitro. We found that the levels of TLR4, interleukin 1-β (IL1-β), tumor necrosis factor-α (TNFα), monocyte chemoattractant protein 1 (MCP-1) and matrix metalloproteinase-2 (MMP-2) expression were increased in the SMCs of atherosclerotic plaques in patients with femoral artery stenosis. In cultured primary arterial SMCs from wild type mice, oxLDL caused dose- and time-dependent increase in the expression levels of TLR4 and cytokines. These effects were significantly weakened in arterial SMCs derived from TLR4 knockout mice (TLR4−/−). Moreover, the secretion of inflammatory cytokines was blocked by TLR4-specific antibodies in primary SMCs. Ox-LDL induced activation of p38 and NFκB was also inhibited in TLR4−/− primary SMCs or when treated with TLR4-specific antibodies. These results demonstrated that TLR4 is a crucial mediator in oxLDL-induced inflammatory cytokine expression and secretion, and p38 and NFκB activation.     

Whole Cigarette Smoke Increased the Expression of TLRs,HBDs, and Proinflammory Cytokines by Human Gingival Epithelial Cells through Different Signaling Pathways

The gingival epithelium is becoming known as a regulator of the oral innate immune responses to a variety of insults such as bacteria and chemicals, including those chemicals found in cigarette smoke. We investigated the effects of whole cigarette smoke on cell-surface-expressed Toll-like receptors (TLR)-2, −4 and −6, human β-defensin (HBD) and proinflammatory cytokine expression and production in primary human gingival epithelial cells. Whole cigarette smoke was shown to increase TLR2, TLR4 and TLR6 expression. Cigarette smoke led to ERK1/2, p38 and JNK phosphorylation in conjunction with nuclear factor-κB (NFκB) translocation into the nucleus. TLR expression following cigarette smoke exposure was down regulated by the use of ERK1/2, p38, JNK MAP kinases, and NFκB inhibitors, suggesting the involvement of these signaling pathways in the cellular response against cigarette smoke. Cigarette smoke also promoted HBD2, HBD3, IL-1β, and IL-6 expression through the ERK1/2 and NFκB pathways. Interestingly, the modulation of TLR, HBD, and cytokine expression was maintained long after the gingival epithelial cells were exposed to smoke. By promoting TLR, HBDs, and proinflammatory cytokine expression and production, cigarette smoke may contribute to innate immunity dysregulation, which may have a negative effect on human health.     

IL-1β Promotes Corneal Epithelial Cell Migration by Increasing MMP-9 Expression through NF-κB- and AP-1-Dependent Pathways

Interleukin-1β (IL-1β) plays a critical mediator in the pathogenesis of eye diseases. The implication of IL-1β in inflammatory responses has been shown to be mediated through up-regulation of inflammatory genes, including matrix metalloproteinase-9 (MMP-9). However, the detailed mechanisms of IL-1β-induced MMP-9 expression in Statens Seruminstitut Rabbit Corneal Cells (SIRCs) are largely unclear. Here, we demonstrated that in SIRCs, IL-1β induced MMP-9 promoter activity and mRNA expression associated with an increase in the secretion of pro-MMP-9. IL-1β-induced pro-MMP-9 expression and MMP-9 mRNA levels were attenuated by pretreatment with the inhibitor of MEK1/2 (U0126), JNK1/2 (SP600125), NF-κB (Bay11-7082), or AP-1 (Tanshinone IIA) and transfection with siRNA of p42 or JNK2. Moreover, IL-1β markedly stimulated p42/p44 MAPK and JNK1/2 phosphorylation in SIRCs. In addition, IL-1β also enhanced p42/p44 MAPK translocation from the cytosol into the nucleus. On the other hand, IL-1β induced c-Jun and c-Fos mRNA expression, c-Jun phosphorylation, and AP-1 promoter activity. NF-κB translocation, IκBα degradation, and NF-κB promoter activity were also enhanced by IL-1β. Pretreatment with U0126 or SP600125 inhibited IL-1β-induced AP-1 and NF-κB promoter activity, but not NF-κB translocation from the cytosol into the nucleus. Finally, we established that IL-1β could stimulate SIRCs migration via p42/p44 MAPK-, JNK1/2-, AP-1-, and NF-κB-dependent MMP-9 induction. These results suggested that NF-κB and AP-1 activated by JNK1/2 and p42/p44 MAPK cascade are involved in IL-1β-induced MMP-9 expression in SIRCs.     

The Nexus between VEGF and NFκB Orchestrates a Hypoxia-Independent Neovasculogenesis

Nuclear Factor-Kappa B [NFκB] activation triggers the elevation of various pro-angiogenic factors that contribute to the development and progression of diabetic vasculopathies. It has been demonstrated that vascular endothelial growth factor [VEGF] activates NFκB signaling pathway. Under the ischemic microenvironments, hypoxia-inducible factor-1 [HIF-1] upregulates the expression of several proangiogenic mediators, which play crucial roles in ocular pathologies. Whereas YC-1, a soluble guanylyl cyclase [sGC] agonist, inhibits HIF-1 and NFκB signaling pathways in various cell and animal models. Throughout this investigation, we examined the molecular link between VEGF and NFκB under a hypoxia-independent microenvironment in human retinal microvascular endothelial cells [hRMVECs]. Our data indicate that VEGF promoted retinal neovasculogenesis via NFκB activation, enhancement of its DNA-binding activity, and upregulating NFκB/p65, SDF-1, CXCR4, FAK, αVβ3, α5β1, EPO, ET-1, and MMP-9 expression. Conversely, YC-1 impaired the activation of NFκB and its downstream signaling pathways, via attenuating IκB kinase phosphorylation, degradation and activation, and thus suppressing p65 phosphorylation, nuclear translocation, and inhibiting NFκB-DNA binding activity. We report for the first time that the nexus between VEGF and NFκB is implicated in coordinating a scheme that upregulates several pro-angiogenic molecules, which promotes retinal neovasculogenesis. Our data may suggest the potential use of YC-1 to attenuate the deleterious effects that are associated with hypoxia/ischemia-independent retinal vasculopathies.     

Caspase-1/ASC Inflammasome-Mediated Activation of IL-1β–ROS–NF-κB Pathway for Control of Trypanosoma cruzi Replication and Survival Is Dispensable in NLRP3?/? Macrophages

In this study, we have utilized wild-type (WT), ASC^−/−, and NLRP3^−/− macrophages and inhibition approaches to investigate the mechanisms of inflammasome activation and their role in Trypanosoma cruzi infection. We also probed human macrophages and analyzed published microarray datasets from human fibroblasts, and endothelial and smooth muscle cells for T. cruzi-induced changes in the expression genes included in the RT Profiler Human Inflammasome arrays. T. cruzi infection elicited a subdued and delayed activation of inflammasome-related gene expression and IL-1β production in mφs in comparison to LPS-treated controls. When WT and ASC^−/− macrophages were treated with inhibitors of caspase-1, IL-1β, or NADPH oxidase, we found that IL-1β production by caspase-1/ASC inflammasome required reactive oxygen species (ROS) as a secondary signal. Moreover, IL-1β regulated NF-κB signaling of inflammatory cytokine gene expression and, subsequently, intracellular parasite replication in macrophages. NLRP3^−/− macrophages, despite an inability to elicit IL-1β activation and inflammatory cytokine gene expression, exhibited a 4-fold decline in intracellular parasites in comparison to that noted in matched WT controls. NLRP3^−/− macrophages were not refractory to T. cruzi, and instead exhibited a very high basal level of ROS (>100-fold higher than WT controls) that was maintained after infection in an IL-1β-independent manner and contributed to efficient parasite killing. We conclude that caspase-1/ASC inflammasomes play a significant role in the activation of IL-1β/ROS and NF-κB signaling of cytokine gene expression for T. cruzi control in human and mouse macrophages. However, NLRP3-mediated IL-1β/NFκB activation is dispensable and compensated for by ROS-mediated control of T. cruzi replication and survival in macrophages.     

CCN2 Suppresses Catabolic Effects of Interleukin-1β through α5β1 and αVβ3 Integrins in Nucleus Pulposus Cells: IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION*

The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1β, and TNF-α and to determine whether CCN2 modulates IL-1β-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1β and TNF-α suppress CCN2 mRNA and protein expression in an NF-κB-dependent but MAPK-independent manner. The conserved κB sites located at −93/−86 and −546/−537 bp in the CCN2 promoter mediated this suppression. On the other hand, treatment of NP cells with IL-1β in combination with CCN2 suppressed the inductive effect of IL-1β on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4, and prolyl hydroxylase 3. Likewise, silencing of CCN2 in human NP cells resulted in elevated basal expression of several catabolic genes and inflammatory cytokines like IL-6, IL-4, and IL-12 as measured by gene expression and cytokine protein array, respectively. Interestingly, the suppressive effect of CCN2 on IL-1β was independent of modulation of NF-κB signaling. Using disintegrins, echistatin, and VLO4, peptide inhibitors to αvβ3 and α5β1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1β-induced catabolic gene expression. It is noteworthy that analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Taken together, these results suggest that CCN2 and inflammatory cytokines form a functional negative feedback loop in NP cells that may be important in the pathogenesis of disc disease.     

IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Th2-inducing pathological conditions such as parasitic diseases increase susceptibility to viral infections through yet unclear mechanisms. We have previously reported that IL-4, a pivotal Th2 cytokine, suppresses the response of murine bone-marrow-derived conventional dendritic cells (cDCs) and splenic DCs to Type I interferons (IFNs). Here, we analyzed cDC responses to TLR7 and TLR9 ligands, R848 and CpGs, respectively. We found that IL-4 suppressed the gene expression of IFNβ and IFN-responsive genes (IRGs) upon TLR7 and TLR9 stimulation. IL-4 also inhibited IFN-dependent MHC Class I expression and amplification of IFN signaling pathways triggered upon TLR stimulation, as indicated by the suppression of IRF7 and STAT2. Moreover, IL-4 suppressed TLR7- and TLR9-induced cDC production of pro-inflammatory cytokines such as TNFα, IL-12p70 and IL-6 by inhibiting IFN-dependent and NFκB-dependent responses. IL-4 similarly suppressed TLR responses in splenic DCs. IL-4 inhibition of IRGs and pro-inflammatory cytokine production upon TLR7 and TLR9 stimulation was STAT6-dependent, since DCs from STAT6-KO mice were resistant to the IL-4 suppression. Analysis of SOCS molecules (SOCS1, −2 and −3) showed that IL-4 induces SOCS1 and SOCS2 in a STAT6 dependent manner and suggest that IL-4 suppression could be mediated by SOCS molecules, in particular SOCS2. IL-4 also decreased the IFN response and increased permissiveness to viral infection of cDCs exposed to a HIV-based lentivirus. Our results indicate that IL-4 modulates and counteracts pro-inflammatory stimulation induced by TLR7 and TLR9 and it may negatively affect responses against viruses and intracellular parasites.     

Cytokine production and inflammation drive autophagy in the tumor microenvironment: Role of stromal caveolin-1 as a key regulator

Recently, we proposed a new paradigm for understanding the role of the tumor microenvironment in breast cancer onset and progression. In this model, cancer cells induce oxidative stress in adjacent fibroblasts. This, in turn, results in the onset of stromal autophagy, which produces recycled nutrients to “feed” anabolic cancer cells. However, it remains unknown how autophagy in the tumor microenvironment relates to inflammation, another key driver of tumorigenesis. To address this issue, here we employed a well-characterized co-culture system in which cancer cells induce autophagy in adjacent fibroblasts via oxidative stress and NFκB-activation. We show, using this co-culture system, that the same experimental conditions that result in an autophagic microenvironment, also drive in the production of numerous inflammatory mediators (including IL-6, IL-8, IL-10, MIp1α, IFNγ, RANTES (CCL5) and GMCSF). Furthermore, we demonstrate that most of these inflammatory mediators are individually sufficient to directly induce the onset of autophagy in fibroblasts. To further validate the in vivo relevance of these findings, we assessed the inflammatory status of Cav-1 (−/−) null mammary fat pads, which are a model of a bonafide autophagic microenvironment. Notably, we show that Cav-1 (−/−) mammary fat pads undergo infiltration with numerous inflammatory cell types, including lymphocytes, T-cells, macrophages and mast cells. Taken together, our results suggest that cytokine production and inflammation are key drivers of autophagy in the tumor microenvironment. These results may explain why a loss of stromal Cav-1 is a powerful predictor of poor clinical outcome in breast cancer patients, as it is a marker of both (1) autophagy and (2) inflammation in the tumor microenvironment. Lastly, hypoxia in fibroblasts was not sufficient to induce the full-blown inflammatory response that we observed during the co-culture of fibroblasts with cancer cells, indicating that key reciprocal interactions between cancer cells and fibroblasts may be required.Key words: caveolin-1, oxidative stress, cytokine production, inflammation, tumor microenvironment, autophagy, breast cancer     

CFTR Is a Negative Regulator of NFκB Mediated Innate Immune Response

Background

Dysfunctional CFTR in the airways is associated with elevated levels of NFκB mediated IL-8 signaling leading to neutrophil chemotaxis and chronic lung inflammation in cystic fibrosis. The mechanism(s) by which CFTR mediates inflammatory signaling is under debate.

Methodology/Principal Findings

We tested the hypothesis that wt-CFTR down-regulates NFκB mediated IL-8 secretion. We transiently co-expressed wt-CFTR and IL-8 or NFκB promoters driving luciferase expression in HEK293 cells. Wt-CFTR expression in HEK293 cells suppresses both basal and IL1β induced IL-8, and NFκB promoter activities as compared to the control cells transfected with empty vector (p<0.05). We also confirmed these results using CFBE41o- cells and observed that cells stably transduced with wt-CFTR secrete significantly lower amounts of IL-8 chemokine as compared to non-transfected control cells. To test the hypothesis that CFTR must be localized to cell surface lipid rafts in polarized airway epithelial cells in order to mediate the inflammatory response, we treated CFBE41o- cells that had been stably transduced with wt-CFTR with methyl-β-cyclodextrin (CD). At baseline, CD significantly (p<0.05) induced IL-8 and NFκB reporter activities as compared to control cells suggesting a negative regulation of NFκB mediated IL-8 signaling by CFTR in cholesterol-rich lipid rafts. Untreated cells exposed to the CFTR channel blocker CFTR-172 inhibitor developed a similar increase in IL-8 and NFκB reporter activities suggesting that not only must CFTR be present on the cell surface but it must be functional. We verified these results in vivo by comparing survival, body weight and pro-inflammatory cytokine response to P. aeruginosa LPS in CFTR knock out (CFKO) mice as compared to wild type controls. There was a significant (p<0.05) decrease in survival and body weight, an elevation in IL-1β in whole lung extract (p<0.01), as well as a significant increase in phosphorylated IκB, an inducer of NFκB mediated signaling in the CFKO mice.

Conclusions/Significance

Our data suggest that CFTR is a negative regulator of NFκB mediated innate immune response and its localization to lipid rafts is involved in control of inflammation.     

Bindarit: An anti-inflammatory small molecule that modulates the NFκB pathway

The activation of nuclear factor (NF)κB pathway and its transducing signaling cascade has been associated with the pathogenesis of many inflammatory diseases. The central role that IκBα and p65 phosphorylation play in regulating NFκB signalling in response to inflammatory stimuli made these proteins attractive targets for therapeutic strategies. Although several chemical classes of NFκB inhibitors have been identified, it is only for a few of those that a safety assessment based on a comprehensive understanding of their pharmacologic mechanism of action has been reported. Here, we describe the specific anti-inflammatory effect of bindarit, an indazolic derivative that has been proven to have anti-inflammatory activity in a variety of models of inflammatory diseases, including lupus nephritis, arthritis and pancreatitis. The therapeutic effects of bindarit have been associated with its ability to selectively interfere with monocyte recruitment and the “early inflammatory response,” although its specific molecular mechanisms have remained ill-defined. For this purpose, we investigated the effect of bindarit on the LPS-induced production of inflammatory cytokines (MCP-1 and MCPs, IL-12β/p40, IL-6 and IL-8/KC) in both a mouse leukaemic monocyte-macrophage cell line and bone marrow-derived macrophages (BMDM). Bindarit inhibits the LPS-induced MCP-1 and IL-12β/p40 expression without affecting other analyzed cytokines. The effect of bindarit is mediated by the downregulation of the classical NFκB pathway, involving a reduction of IκBα and p65 phosphorylation, a reduced activation of NFκB dimers and a subsequently reduced nuclear translocation and DNA binding. Bindarit showed a specific inhibitory effect on the p65 and p65/p50 induced MCP-1 promoter activation, with no effect on other tested activated promoters. We conclude that bindarit acts on a specific subpopulation of NFκB isoforms and selects its targets wihtin the whole NFκB inflammatory pathway. These findings pave the way for future applications of bindarit as modulator of the inflammatory response.Key words: inflammation, NFκB, MCP-1, IL-12β/p40, macrophages, lipopolysaccharide, bindarit     

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway.     

Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia,HIF1 induction and NFκB activation in the tumor stromal microenvironment

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB—another inducer of autophagy—prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knockdown of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (−/−) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the “autophagic tumor stroma model of cancer metabolism”, and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a “lethal” tumor microenvironment.Key words: caveolin-1, autophagy, BNIP3, cancer-associated fibroblasts, HIF1, hypoxia, LC3, mitophagy, NFκB, oxidative stress, predictive biomarker, TIGAR, tumor stroma     

Regulation of ΔNp63α by NFκB

ΔNp63α, the dominant negative isoform of the p63 family is an essential survival factor in head and neck squamous cell carcinoma. This isoform has been shown to be downregulated in response to several DNA damaging agents, thereby enabling an effective cellular response to genotoxic agents. Here, we identify a key molecular mechanism underlying the regulation of ΔNp63α expression in response to extrinsic stimuli, such as chemotherapeutic agents. We show that ΔNp63α interacts with NFκB in presence of cisplatin. We find that NFκB promotes ubiquitin-mediated proteasomal degradation of ΔNp63α. Chemotherapy-induced stimulation of NFκB leads to degradation of ΔNp63α and augments trans-activation of p53 family-induced genes involved in the cellular response to DNA damage. Conversely, inhibition of NFκB with siRNA-mediated silencing NFκB expression attenuates chemotherapy induced degradation of ΔNp63α. These data demonstrate that NFκB plays an essential role in regulating ΔNp63α in response to extrinsic stimuli. Our findings suggest that the activation of NFκB may be a mechanism by which levels of ΔNp63α are reduced, thereby rendering the cells susceptible to cell death in the face of cellular stress or DNA damage.Key words: ΔNp63α, NFκB, ubiquitination, cisplatin, head and neck cancer