Potent Pro-inflammatory Factor Research Articles

Tumor necrosis factor-α induces proliferation and reduces apoptosis of colorectal cancer cells through STAT3 activation.

Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory factor that plays an important role in establishing a complicated connection between inflammation and cancer. TNF-α promotes tumor proliferation, migration, invasion, and angiogenesis according to numerous studies. Studies have shown the significant role of STAT3, a downstream transcription factor of another important inflammatory cytokine, IL-6 in the development and progression of different tumors especially colorectal cancer. In the present study, we investigated whether TNF-α has a role in proliferation and apoptosis of colorectal cancer cells through STAT3 activation. HCT116 cell line as human colorectal cancer cells was used in this study. Major assays were MTT assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, and ELISA. Results showed that TNF-α significantly increased the phosphorylation of STAT3 and expression of all the STAT3 target genes related to cell proliferation, survival, and metastasis compared with control. Moreover, our data showed that the STAT3 phosphorylation and expression of its target genes significantly were reduced in the presence of TNF-α + STA-21 compared with TNF-α-treated group demonstrating that the increase in genes expression partially was due to the TNF-α-induced STAT3 activation. On the other hand, STAT3 phosphorylation and mRNA levels of its target genes were partially decreased in the presence of TNF-α + IL-6R supporting the indirect pathway of STAT3 activation by TNF-α through inducing IL-6 production in cancer cells. Given the growing evidence for STAT3 as a key mediator of inflammation-induced colon cancer, our findings support further investigation of STAT3 inhibitors as potential cancer therapies.

Skeletal Lipocalin-2 Is Associated with Iron-Related Oxidative Stress in ob/ob Mice with Sarcopenia.

Obesity and insulin resistance accelerate aging-related sarcopenia, which is associated with iron load and oxidative stress. Lipocalin-2 (LCN2) is an iron-binding protein that has been associated with skeletal muscle regeneration, but details regarding its role in obese sarcopenia remain unclear. Here, we report that elevated LCN2 levels in skeletal muscle are linked to muscle atrophy-related inflammation and oxidative stress in leptin-deficient ob/ob mice. RNA sequencing analyses indicated the LCN2 gene expression is enhanced in skeletal muscle of ob/ob mice with sarcopenia. In addition to muscular iron accumulation in ob/ob mice, expressions of iron homeostasis-related divalent metal transporter 1, ferritin, and hepcidin proteins were increased in ob/ob mice compared to lean littermates, whereas expressions of transferrin receptor and ferroportin were reduced. Collectively, these findings demonstrate that LCN2 functions as a potent proinflammatory factor in skeletal muscle in response to obesity-related sarcopenia and is thus a therapeutic candidate target for sarcopenia treatment.

The Mechanism of Type I Interferon-Mediated Polarization of Tumor-Associated Neutrophils in Mice and Human

Neutrophils are the most abundant cells of all white blood cells and play a key role in host inflammatory responses. Importantly, inflammation has been associated with increased susceptibility for cancer and neutrophils, as a crucial component of this process, play essential role in inflammation-driven tumorigenesis. Neutrophils also represent an independent prognostic marker in a broad variety of neoplasias. The tumor microenvironment represents a special niche that is extremely influencing infiltrating immune cells. The concept of immune cell polarization was first described for macrophages (anti-tumor M1/pro-tumor M2). Recently neutrophil polarization has been postulated. Neutrophils appear to have diverse phenotypes in the tumor microenvironment i.e. tumor promoting (N2) or inhibiting (N1).Previously, we could show that significantly elevated numbers of neutrophils accumulate in tumors of mice that lack endogenous type I IFNs (Ifnb1-/-). Such tumor associated neutrophils (TANs) do not only efficiently support tumor angiogenesis and growth by up-regulating pro-angiogenic molecules (VEGF and MMP9), but also secrete higher amounts of neutrophil-attracting chemokines and display prolonged survival, compared to their WT counterparts thus representing pro-tumor N2 phenotype. Moreover, we could show that these N2 neutrophils efficiently support metastatic processes, due to up-regulation of pro-metastatic proteins, like Bv8, MMP9, S100A8 and S100A9, and due to impaired tumor cell killing. Treatment of such cells with rmIFNb reversed such an effect leading to anti-tumor N1 polarization.Here, we add further evidence emphasizing the importance of type I IFNs for neutrophil polarization in tumor microenvironment and reveal possible mechanism responsible for this phenomenon. In Ifnb1-/- mice, we observe a significant down-regulation of anti-tumor neutrophil markers, like ICAM1 and TNF-α. Moreover, neutrophils show reduced formation of NETs, accompanied by lower tumor killing capacity. Under these conditions, massively enhanced neutrophil turnover in combination with accumulation of immature neutrophils is observed. Importantly, therapeutic intervention in both Ifnb1-/- and WT mice using low dose IFN-β, induced anti-tumor activation of neutrophils. Correspondingly, in human melanoma patients undergoing type I IFN therapy, neutrophil anti-tumor characteristics were augmented, compared to untreated patients, suggesting effective outcome of this therapy.Further, we evaluated the mechanism of prolonged neutrophil survival in the absence of endogenous IFN-β. Importantly, we found that G-CSF mRNA expression levels in Ifnb1-/- neutrophils from different anatomical compartments as well as G-CSF blood serum levels were markedly up-regulated in such mice. G-CSF-expression levels were strongly reduced when the Ifnb1-/- neutrophils were incubated with rmIFN-β suggesting involvement of type I interferons in G-CSF down-regulation. Notably, we could recently show that G-CSF induces synthesis of enzyme Nicotinamide phosphoribosyltransferase (NAMPT), which is a rate-limiting enzyme converting nicotinamide (NA) into NAD+ that in turn activates NAD+ -dependent protein deacetylases sirtuins (SIRTs). NAMPT serves as an inhibitor of neutrophil apoptosis and as neutrophil chemoattractant by upregulation of CXCL8. It is a potent pro-inflammatory factor (upregulation of ROS release) and pro-angiogenic factor (smooth muscle maturation). At the same time, NAMPT was found to be strongly overexpressed in tumors and serum of leukemia patients. Analysis of NAMPT and SIRTs levels in tumor bearing Ifnb1-/- mice revealed highly upregulated levels of NAMPT and SIRT1 in blood neutrophils of these animals, in comparison to WT mice, which was in line with elevated levels of G-CSF. It also correlates with enhanced tumor angiogenesis, growth and metastasis. Based on these observations, we identified a new mechanism of interferon-mediated activation of pro-tumor neutrophils. Since tumor associated neutrophils represent a highly potent therapeutic target, these data highlight the therapeutic potential of interferons and NAMPT inhibitors, suggesting optimization of their clinical use as potent anti-tumor agent. DisclosuresNo relevant conflicts of interest to declare.

Trace Levels of Staphylococcal Enterotoxin Bioactivity Are Concealed in a Mucosal Niche during Pulmonary Inflammation.

Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. The localization of these factors, their bioactivity and natural countermeasures remain unclear. This concept was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. The Inflammatory and inhibitory potentials of the identified proteins were measured using T cells assays. A potent pro-inflammatory factor was detected in BALF, and we hypothesized SEA could be recovered with its biological activity. Highly purified BALF fractions with bioactivity were subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. Inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show a fraction of bioactive SEA resided in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail.

Trace levels of staphylococcal enterotoxin bioactivity are concealed in a mucosal niche during pulmonary inflammation (MUC1P.917)

Abstract Pathogen and cellular by-products released during infection or trauma are critical for initiating mucosal inflammation. This was studied in mice undergoing pulmonary inflammation after Staphylococcal enterotoxin A (SEA) inhalation. A potent pro-inflammatory factor was detected using a sensitive bioassay suggesting the presence of an alarmin, a danger-associated molecular pattern (DAMP), and/or a cytokine. Highly purified bronchoalveolar lavage fluid (BALF) fractions obtained by sequential chromatography were screened for bioactivity and subjected to mass spectrometry. SEA was the only identified protein with known inflammatory potential, and unexpectedly, it co-purified with immunosuppressive proteins. Among them was lactoferrin, which inhibited SEA and anti-CD3/-CD28 stimulation by promoting T cell death and reducing TNF synthesis. Higher doses of lactoferrin were required to inhibit effector compared to resting T cells. This inhibition relied on the continual presence of lactoferrin rather than a programming event. The data show that bioactive SEA is constituted in a mucosal niche within BALF even after the initiation of inflammation. These results may have clinical value in human diagnostic settings since traces levels of SEA can be detected using a sensitive bioassay, and may help pinpoint potential mediators of lung inflammation when molecular approaches fail. Submitted to American Journal of Respiratory Cell and Molecular Biology.

TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity.

Microglia-mediated neuroinflammation has been reported as a common feature of familial and sporadic forms of Parkinson's disease (PD), and a growing body of evidence indicates that onset and progression of PD correlates with the extent of neuroinflammatory responses involving Interferon γ (IFNγ). Transforming growth factor β1 (TGFβ1) has been shown to be a major player in the regulation of microglia activation states and functions and, thus, might be a potential therapeutic agent by shaping microglial activation phenotypes during the course of neurodegenerative diseases such as PD. In this study, we demonstrate that TGFβ1 is able to block IFNγ-induced microglia activation by attenuating STAT1 phosphorylation and IFNγRα expression. Moreover, we identified a set of genes involved in microglial IFNγ signaling transduction that were significantly down-regulated upon TGFβ1 treatment, resulting in decreased sensitivity of microglia toward IFNγ stimuli. Interestingly, genes mediating negative regulation of IFNγ signaling, such as SOCS2 and SOCS6, were up-regulated after TGFβ1 treatment. Finally, we demonstrate that TGFβ1 is capable of protecting midbrain dopaminergic (mDA) neurons from IFNγ-driven neurotoxicity in mixed neuron-glia cultures derived from embryonic day 14 (E14) midbrain tissue. Together, these data underline the importance of TGFβ1 as a key immunoregulatory factor for microglia by silencing IFNγ-mediated microglia activation and, thereby, rescuing mDA neurons from IFNγ-induced neurotoxicity. Interferon γ (IFNγ) is a potent pro-inflammatory factor that triggers the activation of microglia and the subsequent release of neurotoxic factors. Transforming growth factor β1 (TGFβ1) is able to inhibit the IFNγ-mediated activation of microglia, which is characterized by the release of nitric oxide (NO) and tumor necrosis factor α (TNFα). By decreasing the expression of IFNγ-induced genes as well as the signaling receptor IFNγR1, TGFβ1 reduces the responsiveness of microglia towards IFNγ. In mixed neuron-glia cultures, TGFβ1 protects midbrain dopaminergic (mDA) neurons from IFNγ-induced neurotoxicity.

High‐Mobility Group Box 1 Inhibition Alleviates Lupus‐Like Disease in BXSB Mice

High-mobility group box 1 protein (HMGB1), a ubiquitous nuclear DNA-binding protein, functions as a potent proinflammatory factor. In this study, we evaluated the effects of HMGB1 inhibition on murine lupus using the lupus-prone model. We treated male BXSB mice with neutralizing anti-HMGB1 monoclonal antibody (HMGB1 mAb) from age 16 weeks to 26 weeks. The control group received the same amount of control IgG. Lupus-prone male BXSB mice treated with HMGB1mAb showed attenuated proteinuria, glomerulonephritis, circulating anti-dsDNA and immune complex deposition. Levels of serum IL-1β, IL-6, IL-17 and IL-18 were also significantly decreased by administration of HMGB1mAb in lupus-prone BXSB mice. HMGB1mAb treatment also decreased the caspase-1 activity in the kidneys of BXSB mice and reduced the mouse mortality. Our study supports that HMGB1 inhibition alleviates lupus-like disease in BXSB mice and might be a potential treatment option for human SLE.

Pro-Stimulatory Role of Methemoglobin in Inflammation Through Hemin Oxidation and Polymerization

Inflammation or vascular occlusion by parasitized red blood cell contributes to the pathogenesis of cerebral malaria. The current study aimed to characterize the role of major pro-oxidant factor methemoglobin present in the malaria culture supernatant contributing in inflammation during malaria. Heme and heme polymer stimulate macrophage to secrete large amount of reactive oxygen species into the external micro-environment. The addition of methemoglobin along with heme or heme polymer amplifies production of ROS from macrophages several folds. Methemoglobin mediated stimulatory effect is not due to release of iron, enhanced production of H2O2 or mutual interaction of reaction components. Spectroscopic studies show that methemoglobin accepts heme as a substrate and oxidizes it through a single electron transfer mechanism. Heme oxidation product is a heme polymer with similar chemical and structural properties to synthetic β-hematin. Phenyl N-t-butylnitrone inhibits heme polymerization (IC50=30 nM) and indicates the absolute necessity of heme oxidation and heme free radical generation for heme polymerization. Methemoglobin produced heme polymer is a potent pro-inflammatory factor to release ROS into external microenvironment. Interestingly, methemoglobin not only produces pro-inflammatory heme polymer, but it also amplifies the potential of heme or preformed heme polymer (haemozoin or β-hematin) to produce several folds high ROS production from macrophages. This study illustrates the pro-inflammatory effect of methemoglobin, the underlying novel mechanism by which this occurs and a possible clinical intervention. Based on the results, we recommend methemoglobin directed peroxidase inhibitors as an adjuvant therapy during malaria.

THE PROTECTIVE EFFECTS OF X-BOX BINDING PROTEIN 1 ON TUMOUR NECROSIS FACTOR-ALPHA INDUCED PRO-INFLAMMATORY RESPONSE

ObjectivesTumour necrosis factor alpha (TNF-α) is a potent pro-inflammatory factor playing a critical role in the initiation and progression of atherosclerosis. Exposure of vascular endothelial cells to TNF-α is known...

Peroxiredoxin I null mice exhibits reduced acute lung inflammation following ozone exposure

Acute ozone (O(3)) exposure causes oxidative stress leading inflammation in the lung. However, its precise mechanisms are not fully elucidated. Here, we examined the role of peroxiredoxin I (PrxI) in O(3)-induced pulmonary inflammation using PrxI null (PrxI(-/-)) and wild-type (WT) mice. PrxI is known as an antioxidant and also emerged as a potent proinflammatory factor that activates toll-like receptor 4/nuclear factor-kappa B signalling. Both mice were exposed to 2 ppm O(3) for 6 h and their responses to oxidative stress and acute inflammation in the lung were evaluated after 18 h. The O(3) inhalation activated the transcription factor nuclear factor-erythroid 2-related factor 2 and upregulated heme oxygenase-1 mRNA, the typical makers of oxidative stress, to similar extent in both lungs observed after 0 and 4 h, respectively. O(3) exposure induced significantly less pulmonary inflammation in PrxI(-/-) than in WT mice judging from the reduced infiltrations of neutrophils into the lung and the suppressed production of proinflammatory mediators, such as interleukin-6 and keratinocyte chemoattractant in the bronchoalveolar lavage fluids. Our results suggest that PrxI is not an effective protector against O(3)-induced oxidative damages reportedly caused by harmful lipid metabolites but plays a positive role in the initiation of lung inflammation following O(3) exposure.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1β production

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.

Hypoxia-inducible factor 1 alpha expression increases during colorectal carcinogenesis and tumor progression

BackgroundHypoxia-inducible factor 1 alpha (HIF-1α) is involved in processes promoting carcinogenesis of many tumors. However, its role in the development of colorectal cancer is unknown. To investigate the significance of HIF-1α during colorectal carcinogenesis and progression we examined its expression in precursor lesions constituting the conventional and serrated pathways, as well as in non-metastatic and metastatic adenocarcinomas.MethodsImmunohistochemistry and Western blot is used to analyse HIF-1α expression in normal colonic mucosa, hyperplastic polyps (HPP), sessile serrated adenomas (SSA), low-grade (TA-LGD) and high-grade (TA-HGD) traditional adenomas as well as in non-metastatic and metastatic colorectal adenocarcinomas. Eight colorectal carcinoma cell lines are tested for their HIF-1α inducibility after lipopolysaccharide (LPS) stimulation using western blot and immunocytochemistry.ResultsIn normal mucosa, HPP and TA-LGD HIF-1α was not expressed. In contast, perinuclear protein accumulation and nuclear expression of HIF-1α were shown in half of the examined SSA and TA-HGD. In all investigated colorectal carcinomas a significant nuclear HIF-1α overexpression compared to the premalignant lesions was observed but a significant correlation with the metastatic status was not found. Nuclear HIF-1α expression was strongly accumulated in perinecrotic regions. In these cases HIF-1α activation was seen in viable cohesive tumor epithelia surrounding necrosis and in dissociated tumor cells, which subsequently die. Enhanced distribution of HIF-1α was also seen in periiflammatory regions. In additional in vitro studies, treatment of diverse colorectal carcinoma cell lines with the potent pro-inflammatory factor lipopolysaccharide (LPS) led to HIF-1α expression and nuclear translocation.ConclusionWe conclude that HIF-1α expression occurs in early stages of colorectal carcinogenesis and achieves a maximum in the invasive stage independent of the metastatic status. Perinecrotic activation of HIF-1α in invasive tumors underlines a dual role of HIF-1α by regulating both pro-survival and pro-death processes. HIF-1α up-regulation in response to LPS-mediated stimulation and periinflammatory expression in invasive carcinomas suggest its involvement in inflammatory events. These patterns of HIF-1α inducibility could contribute indirectly to the acquisition of a metastatic phenotype.

Bordetella bronchisepticaFlagellin Is a Proinflammatory Determinant for Airway Epithelial Cells

Motility is an important virulence phenotype for many bacteria, and flagellin, the monomeric component of flagella, is a potent proinflammatory factor. Of the three Bordetella species, Bordetella pertussis and Bordetella parapertussis are nonmotile human pathogens, while Bordetella bronchiseptica expresses flagellin and causes disease in animals and immunocompromised human hosts. The BvgAS two-component signal transduction system regulates phenotypic-phase transition (Bvg+, Bvg-, and Bvg(i)) in bordetellae. The Bvg- phase of B. bronchiseptica is characterized by the expression of flagellin and the repression of adhesins and toxins necessary for the colonization of the respiratory tract. B. bronchiseptica naturally infects a variety of animal hosts and constitutes an excellent model to study Bordetella pathogenesis. Using in vitro coculture models of bacteria and human lung epithelial cells, we studied the effects of B. bronchiseptica flagellin on host defense responses. Our results show that B. bronchiseptica flagellin is a potent proinflammatory factor that induces chemokine, cytokine, and host defense gene expression. Furthermore, we investigated receptor specificity in the response to B. bronchiseptica flagellin. Our results show that B. bronchiseptica flagellin is able to signal effectively through both human and mouse Toll-like receptor 5.

Requirement of HMGB1 and RAGE for the maturation of human plasmacytoid dendritic cells

Dendritic cells (DC) are key components of innate and adaptive immune responses. Plasmacytoid DC (PDC) are a specialized DC subset that produce high amounts of type I interferons in response to microbes. High mobility group box 1 protein (HMGB1) is an abundant nuclear protein, which acts as a potent pro-inflammatory factor when released extracellularly. We show that HMGB1 leaves the nucleus of maturing PDC following TLR9 activation, and that PDC express on the plasma membrane the best-characterized receptor for HMGB1, RAGE. Maturation and type I IFN secretion of PDC is hindered when the HMGB1/RAGE pathway is disrupted. These results reveal HMGB1 and RAGE as the first known autocrine loop modulating the maturation of PDC, and suggest that antagonists of HMGB1/RAGE might have therapeutic potential for the treatment of systemic human diseases.

Potent pro-inflammatory actions of leukemia inhibitory factor in the spinal cord of the adult mouse

Injury in the peripheral or central nervous systems causes a significant rise in the levels of the pleiotropic cytokine leukemia inhibitory factor (LIF). This increase influences cell survival, reactive gliosis and inflammatory responses. Since prior work has focused primarily on peripheral nerve and brain, little is known about the role of LIF in the spinal cord injury response. We address this issue by examining the effects of injury in the LIF knockout (KO) mouse, as well as using an adenoviral vector to over-express LIF in the spinal cord of adult mice. We find that LIF over-expression results in a dramatic rise in cell proliferation, primarily in microglia/macrophages. Astrocytes are not stimulated to proliferate but are activated by the elevated LIF. LIF over-expression also causes the development of severe hindlimb motor dysfunction, an effect mediated by the enhanced activation of microglia/macrophages, as inhibiting microglial activation with minocycline attenuates these motor deficits. Conversely, proliferation is significantly diminished and the microglial/macrophage response to spinal cord injury is much less in the LIF KO compared to wild type (WT). Thus, LIF is a potent pro-inflammatory factor in the adult spinal cord and represents a potential target for the manipulation of inflammatory reactions after spinal cord injury.

Corticotropin-releasing hormone deficiency unmasks the proinflammatory effect of epinephrine.

Traditionally, the adrenal gland has been considered an important endocrine component of the pathway to inhibit acute inflammation via hypothalamic corticotropin-releasing hormone (CRH)-mediated secretion of glucocorticoid. Immunoreactive CRH found in inflamed tissues is a potent proinflammatory factor. Using genetic and pharmacological models of CRH deficiency, we now show that CRH deficiency unmasks a major proinflammatory effect of epinephrine secreted from the adrenal medulla. Together, epinephrine and peripheral CRH stimulate inflammation, and glucocorticoid acts as a counterbalancing force in this regard. Our findings suggest that stimulation of the acute inflammatory response should be included with the other "fight-or-flight" actions of epinephrine.

Amelioration of lupus-like autoimmune disease in NZB/WF1 mice after treatment with a blocking monoclonal antibody specific for complement component C5.

New Zealand black x New Zealand white (NZB/W) F1 mice spontaneously develop an autoimmune syndrome with notable similarities to human systemic lupus erythematosus. Female NZB/WF1 mice produce high titers of antinuclear antibodies and invariably succumb to severe glomerulonephritis by 12 months of age. Although the development of the immune-complex nephritis is accompanied by abundant local and systemic complement activation, the role of proinflammatory complement components in disease progression has not been established. In this study we have examined the contribution of activated terminal complement proteins to the pathogenesis of the lupus-like autoimmune disease. Female NZB/W F1 mice were treated with a monoclonal antibody (mAb) specific for the C5 component of complement that blocks the cleavage of C5 and thus prevents the generation of the potent proinflammatory factors C5a and C5b-9. Continuous therapy with anti-C5 mAb for 6 months resulted in significant amelioration of the course of glomerulonephritis and in markedly increased survival. These findings demonstrate an important role for the terminal complement cascade in the progression of renal disease in NZB/W F1 mice, and suggest that mAb-mediated C5 inhibition may be a useful approach to the therapy of immune-complex glomerulonephritis in humans.