Endogenous Proinflammatory Cytokines Research Articles

JAK1/2 Regulates Synergy Between Interferon Gamma and Lipopolysaccharides in Microglia.

Microglia, the resident immune cells of the brain, regulate neuroinflammation which can lead to secondary neuronal damage and cognitive impairment under pathological conditions. Two of the many molecules that can elicit an inflammatory response from microglia are lipopolysaccharide (LPS), a component of gram-negative bacteria, and interferon gamma (IFNγ), an endogenous pro-inflammatory cytokine. We thoroughly examined the concentration-dependent relationship between LPS from multiple bacterial species and IFNγ in cultured microglia and macrophages. We measured the effects that these immunostimulatory molecules have on pro-inflammatory activity of microglia and used a battery of signaling inhibitors to identify the pathways that contribute to the microglial response. We found that LPS and IFNγ interacted synergistically to induce a pro-inflammatory phenotype in microglia, and that inhibition of JAK1/2 completely blunted the response. We determined that this synergistic action of LPS and IFNγ was likely dependent on JNK and Akt signaling rather than typical pro-inflammatory mediators such as NF-κB. Finally, we demonstrated that LPS derived from Escherichia coli, Klebsiella pneumoniae, and Akkermansia muciniphila can elicit different inflammatory responses from microglia and macrophages, but these responses could be consistently prevented using ruxolitinib, a JAK1/2 inhibitor. Collectively, this work reveals a mechanism by which microglia may become hyperactivated in response to the combination of LPS and IFNγ. Given that elevations in circulating LPS and IFNγ occur in a wide variety of pathological conditions, it is critical to understand the pharmacological interactions between these molecules to develop safe and effective treatments to suppress this process.

The effect of human PBMCs immobilization on their Аβ42 aggregates-dependent proinflammatory state on a cellular model of Alzheimer's disease.

The leading pathological mechanisms of Alzheimer's disease are amyloidosis and inflammation. The presented work was aimed to study the effect of human peripheral blood mononuclear cells (hPBMcs) cells-matrix adhesion on their pro-inflammatory state in vitro. Although direct interaction of Аβ42 to PBMC is not a cellular model of Alzheimer's disease, PBMCs may serve as test cells to detect Аβ42-dependent molecular effects in monitoring disease progression. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or Alzheimer's disease-specific cytotoxic molecules such as Aβ42. The effect of recombinant amyloid β-peptide rАβ42 on the concentration of endogenous amyloid β-peptide Aβ40 and pro-inflammatory cytokines TNFα and IL-1β in human peripheral blood mononuclear cells that were cultured in suspension and immobilized in alginate microcarriers for 24 h were investigated. The localization and accumulation of Aβ40 and rAβ42 peptides in cells, as well as quantitative determination of the concentration of Aβ40 peptide, TNFα and IL-1β cytokines, was performed by intravital fluorescence imaging. The results were qualitatively similar for both cell models. It was determined that the content of TNFα and Aβ40 in the absence of rAβ42 in the incubation medium did not change for 24 h after incubation, and the content of IL-1β was lower compared to the cells that were not incubated. Incubation of cells in vitro with exogenous rAβ42 led to an increase in the intracellular content of TNFα and Aβ40, and no accumulation of IL-1β in cells was observed. The accumulation of Aβ40 in the cytoplasm was accompanied by the aggregation of rAβ42 on the outer surface of the cell plasma membrane. It was shown that the basic levels of indicators and the intensity of the response of immobilized cells to an exogenous stimulus were significantly greater than those of cells in suspension. To explore whether non-neuronal cells effects in alginate microcarriers were cell-matrix adhesion mediated, we tested the effect of blocking β1 integrins on proamyloidogenic and proinflammation cellular state. Immobilization within alginate hydrogels after incubation with the β1 integrins blocking antibodies showed a remarkable inhibition of TNFα and Aβ40 accumulation in rAβ42-treated cells. It can be concluded that activation of signal transduction and synthesizing activity of a portion of mononuclear cells of human peripheral blood is possible (can significantly increase) in the presence of cell-matrix adhesion.

Tripartite motif containing 59 mediates protective anti-oxidative effects in intestinal injury through Nrf2 signaling

Elevated evidence has reported the important role of oxidative stress injury and inflammatory response in the progression of colitis. Tumor Suppressor TSBF1, TRIM59, is a ubiquitin E3 ligase and mediates immune response. However, the underlying molecular function of TRIM59 on regulation of colitis is still not understood. In the current study, we identify the TRIM59 as a critical and novel endogenous suppressor of kelch-like ECH-associated protein 1 (KEAP1), and we also determine that TRIM59 is a KEAP1-interacting partner protein that catalyses its ubiquitination and degradation in intestinal epithelial cells (IEC). Moreover, IEC-specific loss of the Trim59 disrupts colon metabolic homeostasis, accompanied by intestinal oxidative stress injury, elevated endogenous reactive oxygen species (ROS) production and pro-inflammatory cytokines release, significantly promotes acute or chronic colitis progression. Conversely, transgenic mice with Trim59 overexpression by adeno-associated virus (AAV)-induced Trim59 gene therapeutics mitigates colitis in acute or chronic colitis rodent models and in vitro experiments. Mechanistically, in response to onset of colitis, TRIM59 directly interacts with KEAP1 and promotes ubiquitin–proteasome degradation, thus results in NRF2 activation and its downstream cascade anti-oxidative stress-related pathway activation, which facilitates anti-oxidant defense and reduces tissue damage. All the findings elucidated the potential role of TRIM59 in colitis progression by mediating KEAP1 deactivation and degradation, and could be considered as a therapeutic target for the treatment of such disease.

Myrtenal improves memory deficits in mice exposed to radiofrequency-electromagnetic radiation during gestational and neonatal development via enhancing oxido-inflammatory, and neurotransmitter functions

ObjectiveRadiofrequency-electromagnetic radiation (RF-EMR) exposure during gestational and neonatal development may interact with the foetus and neonate considered hypersensitive to RF-EMR, consequently resulting in developmental defects associated with neuropsychological and neurobehavioral disorders, including learning and memory impairment. This study assessed the potential of Myrtenal (Myrt) to improve memory deficits in C57BL/6 mice exposed to RF-EMR during gestational and neonatal development. MethodThirty-five male mice were randomly allocated into 5 cohorts, each comprising of 7 mice. Group I was administered vehicle, Group II: RF-EMR (900 MHz); Group III: RF-EMR (900 MHz) + 100 mg/kg Myrt; Group IV: RF-EMR (900 MHz) + 200 mg/kg Myrt; and Group V: RF-EMR (900 MHz) + donepezil 0.5 mg/kg. ResultsMyrt treatment improved short-term memory performance in RF-EMR (900 MHz)-exposed mice by augmenting activities of endogenous antioxidant enzymes and proinflammatory cytokines, thereby protecting the brain from oxido-inflammatory stress. Additionally, Myrt restored the homeostasis of neurotransmitters in RF-EMR-exposed animals. ConclusionResults from the present study shows that exposure to RF-EMR impaired short-term memory in animals and altered the response of markers of oxido-inflammatory stress, and neurotransmitters. It is therefore conceivable that the recommendation of Myrt-enriched fruits may offer protective benefits for foeti and neonates prone to RF-EMR exposure.

Reduced dopaminergic neuron degeneration and global transcriptional changes in Parkinson's disease mouse brains engrafted with human neural stems during the early disease stage

IntroductionCurrent stem cell therapies for Parkinson's disease (PD) focus on a neurorestorative approach that aims to repair the CNS during the symptomatic phase. However, the pleiotropic and supportive effects of human neural stem cells (hNSCs) may make them effective for PD treatment during the disease's earlier stages. In the current study, we investigated the therapeutic effects of transplanting hNSCs during the early stages of PD development when most dopaminergic neurons are still present and before symptoms appear. Previous studies on hNSCs in Parkinson's disease focus on the substantia nigra and its immediate surroundings, but other brain structures are affected in PD as well. Here, we investigated the therapeutic effects of hNSCs on the entire PD-afflicted brain transcriptome using RNA sequencing (RNA-seq). MethodsPD was induced with a single intranasal infusion of 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and hNSCs were transplanted unilaterally into the striatum one week later. The timepoint for hNSC transplantation coincided with upregulation of endogenous proinflammatory cytokines in the CNS, which play a role in stem cell migration. At 3 weeks post-transplantation (4 weeks post-MPTP), we assessed motor symptoms through behavioral tests, quantified dopaminergic neurons in the substantia nigra, and performed global transcriptional profiling to understand the mechanism underlying the effect of hNSCs on dopaminergic neuron degeneration. ResultsWe found that early hNSC engraftment mitigated motor symptoms induced by MPTP, and also reduced MPTP-induced loss of dopaminergic neurons. In this study, we uniquely presented the first comprehensive analysis of the effect of hNSC transplantation on the transcriptional profiling of PD mouse brains showing decreased expression of 249 and increased expression of 200 genes. These include genes implicated in mitochondrial bioenergetics, proteostasis, and other signaling pathways associated with improved PD outcome following hNSC transplantation. ConclusionThese findings indicate that NSC transplantation during the asymptomatic phase of PD may limit or halt the progression of this neurodegenerative disorder. Transcriptional profiling of hNSC-engrafted PD mouse brains provides mechanistic insight that could lead to novel approaches to ameliorating degeneration of dopaminergic neurons and improving behavioral dysfunction in PD.

Antioxidant, Anti-inflammatory, and Anti-arthritic Effect of Thymoquinone-rich Black Cumin (Nigella sativa) oil (BlaQmax®) on Adjuvant-induced Arthritis

Rheumatoid Arthritis (RA) is a complex autoimmune disorder involving chronic and persistent inflammation, principally influencing the synovial joints which further prompting the obliteration of articular cartilage. Although black cumin (Nigella sativa) oil has already studied for its anti-arthritic properties, the current study was focused on the comparative evaluation of the antioxidant and anti-inflammatory properties of a thymoquinone (TQ)-rich (5% w/v) black cumin oil (BQ) with the commonly available standard black cumin oil (BM) containing 0.4% (w/v) TQ, and subsequent investigation on the potential application of BQ in the management of RA. Adjuvant-induced arthritis (AA) was instigated by a single intradermal infusion of 0.1 mL of Complete Freund's adjuvant (CFA) on the paw of adult Wistar rats. Based on the primary dose-response study using the carrageenan-induced paw edema model, 50 mg/kg b.wt. of BQ was employed for the treatment. The endogenous antioxidants (SOD, Catalase, GPx, and GSH), pro-inflammatory cytokines (COX-2, Nitrate, iNOS, TNF-α, IL-6), lipid peroxidation, and histopathology were evaluated to monitor the influence of BQ in AA rats. Adjuvant-induced animals showed a critical downregulation in antioxidant status with elevated levels of pro-inflammatory cytokines and lipid peroxidation. But, the treatment with BQ significantly reversed the antioxidant and inflammatory markers with downregulation of the pro-inflammatory gene expressions. Histopathology showed a significant reduction in the massive cell infiltration and epidermal edema of the paw tissue in AA rats when administered with BQ and indicated its potential effect to alleviate RA conditions in experimental rats.

Royal Jelly (Bee Product) Decreases Inflammatory Response in Wistar Rats Induced with Ultraviolet Radiation.

BACKGROUND:Ultraviolet (UV) radiation damages human skin by triggering various types of cellular damage, several main factors involved are nuclear-related factor 2 (Nrf2), nuclear factor kappa-light-chain-enhancer of activated B cells (nF-kB) and pro-inflammatory cytokine, TNF alpha. Royal jelly (RJ) possesses the effect of protecting DNA and tissue against oxidative damage.AIM:This study aimed to assess the efficacy of RJ as a protector of ultraviolet radiation, by assessing endogenous anti-oxidant expression (Nrf2), transcription factors (Nf-kB) and proinflammatory cytokines (TNF alpha).METHODS:This study was an experimental study with post-test control group design. Thirty Wistar rats were induced by exposing 40 Watt UV-B lamps for 2 hours/day in 14 days. The rats were grouped into groups with RJ cream application with doses of 2.5%, 5%, and 10%, negative control with vaseline, and normal control. Examination of Nrf2 and NF-kB levels was carried out by ELISA. Quantitative analysis to obtain the percentage of TNF alpha expression on the tissue was entered into the ImageJ® program. Bivariate analysis was carried out by the T-test.RESULTS:Nrf2 levels elevated following the increase of RJ dose, with the highest level was at RJ 10%. Nf-kB levels decreased following the increase of RJ dose, with the lowest level was at RJ 10%. TNF alpha expression was reduced in groups of RJ in various doses. Increased dose resulted in a more diminished level of TNF alpha.CONCLUSION:Royal jelly cream application protected the skin from UV radiation by increasing cellular antioxidants and suppressing inflammatory cascade.

Epigallocatechin gallate attenuates mitochondrial DNA-induced inflammatory damage in the development of ventilator-induced lung injury

ObjectiveWe aim to investigate the role of mitochondrial DNA (mtDNA), a novel endogenous pro-inflammatory cytokine, in the development of ventilator-induced lung injury (VILI). Moreover, the protective effect of epigallocatechin gallate (EGCG) on VILI through inhibiting local mtDNA release was examined. MethodsFrom March 2015 to March 2016, bronchoalveolar lavage fluid (BALF) from 36 patients with VILI and well-matched 36 patients without VILI after major surgery were consecutively collected. The expression levels of mtDNA and inflammatory cytokines in BALF were tested. SD rats were divided into five groups: control, low tidal volume (7 ml/kg) group, high tidal volume (HTV, 40 ml/kg) group, HTV+low dose EGCG and HTV+high dose EGCG groups. BALF were collected to examine the expression levels of mtDNA and several inflammatory cytokines and the lung tissue was harvested for pathological examinations. In addition, cyclic stretch cell culture was used and culture media was collected to analyze expressions of inflammatory cytokines. Administration of mtDNA in a rat model and in vitro cell culturing were used to confirm its pro-inflammatory properties in the development of inflammatory lung injury. ResultsA Significant elevation of mtDNA was detected in BALF from patients with VILI (581 ± 193 vs. 311 ± 137, p < 0.05) and also in rats ventilated with HTV. EGCG could significantly inhibit HTV-induced local mtDNA release and attenuate the level of inflammatory lung injuries (reduced infiltration of local inflammatory cells, lower lung wet/dry ratio and expression levels of inflammatory cytokines). The beneficial effects of EGCG on preventing inflammatory lung injuries were in a concentration-dependent manner. Meanwhile, higher expression levels of mtDNA and inflammatory cytokines were observed in the media of cyclic stretched cell culture compared to those in the control group (p < 0.05). Furthermore, intra-tracheal administration of mtDNA in rats could lead to a marked increase of local inflammatory cytokines and subsequent inflammatory lung injuries (p < 0.05). And by adding mtDNA into the cell culture, higher level of inflammatory cytokines in the media was detected (p < 0.05). EGCG also showed preventive effects on inflammatory responses on a concentration-dependent manner (p < 0.05). ConclusionThe increased expression level of mtDNA and subsequent inflammatory cytokines overproduction may play an important role in the development of VILI. EGCG may be a potential novel therapeutic candidate for protection against VILI by inhibiting the local release of mtDNA.

Senescent Cells Impair Erectile Function through Induction of Endothelial Dysfunction and Nerve Injury in Mice.

Erectile dysfunction (ED) is a major health problem, particularly in the elderly population, which is rapidly increasing. It is necessary to elucidate the mechanism by which ED occurs in the elderly. Cellular senescence is commonly detected in old tissues, and it is well known that senescent cells not only withdraw from the cell cycle but also remain viable and actively produce a variety of cytokines. We examined the effect of senescent cells on erectile function after injection of senescent cells into the penises of mice. Human umbilical vein endothelial cells were infected with an adenovirus expressing a constitutively active mutant of Ras to induce senescence, and were injected into the penises of nude mice. These senescent cells expressed proinflammatory cytokines such as interleukin-1β (IL-1β). Injection of senescent cells impaired erectile function, as assessed by the measurement of intracavernous pressure. Although the structure of the cavernous body did not remarkably change, expression of the catalytically active form of endothelial nitric oxide synthase and that of total neural nitric oxide synthase significantly decreased after injection. The penises injected with the senescent cells expressed human IL-1β and subsequently endogenous proinflammatory cytokines such as mouse IL-1β and tumor necrosis factor-α. These results suggested that senescent cells impaired erectile function through induction of endothelial dysfunction and nerve injury. These effects may be mediated by proinflammatory cytokines produced by senescent cells.

Human neural stem cells rapidly ameliorate symptomatic inflammation in early-stage ischemic-reperfusion cerebral injury.

IntroductionClinically, a good deal of injury from stroke results from ischemic-reperfusion. There is a loss of cerebral parenchyma and its associated cells, disruption of neuronal connections, compromise of the blood-brain barrier, and inflammation. We tested whether exogenously engrafted human neural stem cells could migrate rapidly and extensively to damaged regions, following transplantation into a neurogenic site where migration cues are already underway during stroke onset, then counteract a number of these pathological processes.MethodsOne day post-injury, we injected human neural stem cells (hNSCs) into the ipsilesional hippocampus of a mouse model of stroke with middle cerebral artery occlusion to induce focal ischemia followed by reperfusion (MCAO/R). The time frame for hNSC transplantation corresponded to upregulation of endogenous proinflammatory cytokines. We examined the effect of hNSC transplantation on pathological processes and behavioral dysfunction 48 hours post-injury.ResultsTwenty-four hours after transplantation, engrafted hNSCs had migrated extensively to the lesion, and infarct volume was reduced relative to MCAO/R controls. The behavioral deficits seen in MCAO/R controls were also significantly improved. Given this rapid response, we hypothesized that the mechanisms underlying therapeutic activity were anti-inflammatory rather than due to cell replacement. In support of this idea, in hNSC-transplanted mice we observed reduced microglial activation, decreased expression of proinflammatory factors (tumor necrosis factor-α, interleukin (IL)-6, IL-1β, monocyte chemotactic protein-1, macrophage inflammatory protein-1α) and adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), and amelioration of blood-brain barrier damage.ConclusionsWhile long-term effects of engrafted hNSCs on the amelioration of ischemic stroke-induced behavioral dysfunction in a rodent model have been reported, our study is the first to show rapid, beneficial impacts on behavioral function (within 24 hours) upon early delivery of hNSCs into the hippocampus.Electronic supplementary materialThe online version of this article (doi:10.1186/scrt519) contains supplementary material, which is available to authorized users.

Scaffold structure and fabrication method affect proinflammatory milieu in three-dimensional-cultured chondrocytes.

Cartilage tissue engineering has emerged as an attractive therapeutic option for repairing damaged cartilage tissue in the arthritic joint. High levels of proinflammatory cytokines present at arthritic joints can cause cartilage destruction and instability of the engineered cartilage tissue, and thus it is critical to engineer strong and stable cartilage that is resistant to the inflammatory environment. In this study, we demonstrate that scaffolding materials with different pore sizes and fabrication methods influence the microenvironment of chondrocytes and the response of these cells to proinflammatory cytokines, interleukin-1beta, and tumor necrosis factor alpha. Silk scaffolds prepared using the organic solvent hexafluoroisopropanol as compared to an aqueous-based method, as well as those with larger pore sizes, supported the deposition of higher cartilage matrix levels and lower expression of cartilage matrix degradation-related genes, as well as lower expression of endogenous proinflammatory cytokines IL-1β in articular chondrocytes. These biochemical properties could be related to the physical properties of the scaffolds such as the water uptake and the tendency to leach or adsorb proinflammatory cytokines. Thus, scaffold structure may influence the behavior of chondrocytes by influencing the microenvironment under inflammatory conditions, and should be considered as an important component for bioengineering stable cartilage tissues.

Biomimetic Interfaces Reveal Activation Dynamics of C‐Reactive Protein in Local Microenvironments

CRP is an acute-phase reactant secreted by hepatocytes in the liver upon stimulation with endogenous proinfl ammatory cytokines and reaches a level, 100–1000-fold compared to normal conditions (0.8 mg L −1 ) in blood. [ 1 ] CRP levels are clinically measured to monitor the severity of infection, infl ammatory conditions, and cardiovascular diseases. Circulating CRP specifi cally binds to phospholipids as well as immunoglobulins in damaged tissue, [ 2 ] which enhances innate immune responses by recruiting complement components. Generally, CRP forms a pentraxin (115 kDa), a non-covalent symmetrical arrangement of fi ve identical subunits (23 kDa). [ 3 ] Each subunit possesses a PC-binding site coordinated by two calcium ions. The binding of CRP to PCs as a molecular interaction mechanism for activating CRP at the site of infection is intriguing. Classically, two Ca 2+ are believed to have a common equilibrium dissociation constant ( K D = 0.06 × 10 −3 M ) to the PC-binding pocket in CRP. [ 4 ] Recent advances revealed an intricate profi le of the CRP–Ca 2+ affi nities in which each Ca 2+ is taken up in a stepwise manner ( K D,high = 0.03 × 10 −3 M , K D,low = 5.45 × 10 −3 M ). [ 5 ]

520-d Isolation and confinement simulating a flight to Mars reveals heightened immune responses and alterations of leukocyte phenotype

During interplanetary exploration, chronic stress caused by long term isolation and confinement in the spacecraft is one of the major concerns of physical and psychological health of space travelers. And for human on Earth, more and more people live in an isolated condition, which has become a common social problem in modern western society. Collective evidences have indicated prolonged chronic stress could bring big influence to human immune function, which may lead to a variety of health problems. However, to what extent long-term isolation can affect the immune system still remains largely unknow. A simulated 520-d Mars mission provided an extraordinary chance to study the effect of prolonged isolation. Six healthy males participated in this mission and their active neuroendocrine and immune conditions were studied with saliva and blood samples from all participants on chosen time points during the isolation period. As a typical neuroendocrine parameter, stress hormone cortisol was measured in the morning saliva samples. Immune phenotype changes were monitored through peripheral leukocyte phenotype analysis. Using an ex vivo viral infection simulation assay we assessed the immune response changes characterized by the ability to produce representative endogenous pro-inflammatory cytokines. The results of this study revealed elevated cortisol levels, increased lymphocyte amount and heightened immune responses, suggesting that prolonged isolation acting as chronic stressors are able to trigger leukocyte phenotype changes and poorly controlled immune responses.

Abstract T P76: Human Neural Stem Cells Rapidly Ameliorate Symptomatic Inflammation In Ischemic-Reperfusion Cerebral Injury

Ischemic stroke injury results not only from ischemia but also from reperfusion. Stroke causes a loss of cerebral parenchyma and its associated cells, disruption of neuronal connections, compromise of the blood-brain barrier, and inflammation. We tested whether exogenously engrafted human neural stem cells (hNSCs) could migrate rapidly and extensively to damaged regions following intracranial transplantation and counteract a number of these pathological processes. We used a stroke mouse model with middle cerebral artery occlusion (MCAO) to induce focal ischemia followed by reperfusion. We transplanted hNSCs 24 hours after MCAO to correspond to the upregulation of endogenous proinflammatory cytokines, which can act as stem cell-recruiting factors. We found that 24 hrs post-transplantation, engrafted hNSCs had migrated extensively to the lesion, brain infarct volume was reduced, and behavioral function was significantly improved compared to MCAO controls. Because the time course of this repair was too short for cell replacement, we hypothesized that the mechanisms of therapeutic action are anti-inflammatory. Microglial activation and proinflammatory gene expression (TNF-α, IL-6, and IL-1β) were reduced, and blood-brain barrier damage was ameliorated in hNSC-transplanted mice. Our findings demonstrate clear benefits of transplanting hNSCs during the early stages of ischemic stroke.

Disorders of Hemostasis in Overt and Subclinical Hypercortisolism

Glucocorticoids are a group of hormones of a particular impact on hemostasis. Epidemiological studies show an approximately severalfold greater incidence of thromboembolic events in hypercortisolemic patients compared to those without hormonal disorders. The prothrombotic action of this steroid class is caused by both the direct impact of hypercortisolism on the activation of coagulation and the inhibition of fibrinolysis, as well as, the pathology of hemostasis due to metabolic disorders, which occur in this endocrinopathy. The aim of this study was to discuss the hemostasis abnormalities that occur in patients with overt and subclinical hypercortisolism with a particular emphasis on plasmatic coagulation, endogenous anticoagulation system, homocysteine and proinflammatory cytokines.

Abstract 1484: Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis.

Abstract Background and aims: Colorectal cancer (CRC) represents the third most common form of cancer worldwide and is associated with chronic colonic inflammation. Inflammation is partially regulated by the Corticotropin Releasing Hormone (CRH) family of neuropeptides and their receptors CRHR1 and CRHR2 which are expressed in CNS and periphery including the gastrointestinal tract. However, the effect of CRH system on tumorigenesis is poorly studied and controversial. Up-to-date there are no studies implicating the CRH family of peptides in CRC development and progression. Here, we aim to identify the role the CRH family members play in CRC growth and metastasis in vitro and in vivo and explore the underlying molecular mechanism. Methods: mRNA levels of CRH family members were monitored by qRT-PCR in 46 and 56 normal human colon and CCA biopsies, respectively, as well as in 7 CCA cell lines. CCA cell lines were lentivirally transduced for CRHR2 expression (CRHR2+). The parental and CRHR2+ cells were tested for expression of UCN2, IL-6 and IL-6R using qRT-PCR. Tumor progression was assessed in vitro by proliferation, invasion and migration assays after treatment with IL-6, astressin 2B or Ucn2. Single cell colony formation was assessed by soft agar assay. Expression of pSTAT3 and EMT markers was determined by western blot analysis. An orthotopic transplantation mouse model was used for validation of metastasis in vivo. Results: CCA biopsies and cell lines had almost lost expression of CRHR2 and elevated levels of its selective agonist UCN2, compared to their normal counterparts. CRHR2+ clones had reduced endogenous UCN2 and pro-inflammatory cytokine mRNA levels including IL-6 and its receptor IL-6R. These effects were more pronounced in highly metastatic CRC cell lines and after exogenous administration of UCN2. CRHR2+ clones with downregulated IL-6R levels showed a significantly reduced pSTAT3 levels compared to parental cells, followed by decreased cell proliferation, colony formation, invasion and migration. The above events were able to be reversed by the selective CRHR2 antagonist, astressin 2B, indicating that they were CRHR2 specific. EMT markers were also significantly dysregulated in CRHR2+ clones, suggesting EMT as a downstream target of CRHR2 signaling in CRC. The decreased metastatic potential of CRHR2+ clones, observed in vitro is currently validated in vivo. Conclusions: This is the first demonstration of the protective role of CRHR2 in preventing CRC growth rates and aggressiveness and a novel molecular evidence linking chronic inflammation and CRC through modulation of the Stat3 signaling pathway. Furthermore, monitoring CRHR2 and Ucn2 levels in CRC tissues may benefit early diagnosis of metastasis, and might serve as a novel drug target for prevention and treatment of advanced stages of CRC. Citation Format: Stavroula Baritaki, Jorge Rodriguez, Dimitrios Iliopoulos, Charalabos Pothoulakis. Effects of UCN2 and its receptor CRHR2 in colorectal cancer growth and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1484. doi:10.1158/1538-7445.AM2013-1484

High Mobility Group Box 1/Toll-like Receptor Danger Signaling Increases Brain Neuroimmune Activation in Alcohol Dependence

Innate immune gene expression is regulated in part through high mobility group box 1 (HMGB1), an endogenous proinflammatory cytokine, that activates multiple members of the interleukin-1/Toll-like receptor (TLR) family associated with danger signaling. We investigated expression of HMGB1, TLR2, TLR3, and TLR4 in chronic ethanol-treated mouse brain, postmortem human alcoholic brain, and rat brain slice culture to test the hypothesis that neuroimmune activation in alcoholic brain involves ethanol activation of HMGB1/TLR danger signaling. Protein levels were assessed using Western blot, enzyme-linked immunosorbent assay, and immunohistochemical immunoreactivity (+IR), and messenger RNA (mRNA) levels were measured by real time polymerase chain reaction in ethanol-treated mice (5 g/kg/day, intragastric, 10 days + 24 hours), rat brain slice culture, and postmortem human alcoholic brain. Ethanol treatment of mice increased brain mRNA and +IR protein expression of HMGB1, TLR2, TLR3, and TLR4. Postmortem human alcoholic brain also showed increased HMGB1, TLR2, TLR3, and TLR4 +IR cells that correlated with lifetime alcohol consumption, as well as each other. Ethanol treatment of brain slice culture released HMGB1 into the media and induced the proinflammatory cytokine, interleukin-1 beta (IL-1β). Neutralizing antibodies to HMGB1 and small inhibitory mRNA to HMGB1 or TLR4 blunted ethanol induction of IL-1β. Ethanol-induced HMGB1/TLR signaling contributes to induction of the proinflammatory cytokine, IL-1β. Increased expression of HMGB1, TLR2, TLR3, and TLR4 in alcoholic brain and in mice treated with ethanol suggests that chronic alcohol-induced brain neuroimmune activation occurs through HMGB1/TLR signaling.

Prevention of methamphetamine-induced microglial cell death by TNF-α and IL-6 through activation of the JAK-STAT pathway

BackgroundIt is well known that methamphetamine (METH) is neurotoxic and recent studies have suggested the involvement of neuroinflammatory processes in brain dysfunction induced by misuse of this drug. Indeed, glial cells seem to be activated in response to METH, but its effects on microglial cells are not fully understood. Moreover, it has been shown that cytokines, which are normally released by activated microglia, may have a dual role in response to brain injury. This led us to study the toxic effect of METH on microglial cells by looking to cell death and alterations of tumor necrosis factor-alpha (TNF-α) and interleukine-6 (IL-6) systems, as well as the role played by these cytokines.MethodsWe used the N9 microglial cell line, and cell death and proliferation were evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and incorporation of bromodeoxyuridine, respectively. The TNF-α and IL-6 content was quantified by enzyme-linked immunosorbent assay, and changes in TNF receptor 1, IL-6 receptor-alpha, Bax and Bcl-2 protein levels by western blotting. Immunocytochemistry analysis was also performed to evaluate alterations in microglial morphology and in the protein expression of phospho-signal transducer and activator of transcription 3 (pSTAT3).ResultsMETH induced microglial cell death in a concentration-dependent manner (EC50 = 1 mM), and also led to significant morphological changes and decreased cell proliferation. Additionally, this drug increased TNF-α extracellular and intracellular levels, as well as its receptor protein levels at 1 h, whereas IL-6 and its receptor levels were increased at 24 h post-exposure. However, the endogenous proinflammatory cytokines did not contribute to METH-induced microglial cell death. On the other hand, exogenous low concentrations of TNF-α or IL-6 had a protective effect. Interestingly, we also verified that the anti-apoptotic role of TNF-α was mediated by activation of IL-6 signaling, specifically the janus kinase (JAK)-STAT3 pathway, which in turn induced down-regulation of the Bax/Bcl-2 ratio.ConclusionsThese findings show that TNF-α and IL-6 have a protective role against METH-induced microglial cell death via the IL-6 receptor, specifically through activation of the JAK-STAT3 pathway, with consequent changes in pro- and anti-apoptotic proteins.

Neutrophil and cytokine dysregulation in hyperinsulinemic obese horses

Equine metabolic syndrome is characterized by obesity and regional adiposity coupled with evidence of recurrent laminitis. Although inflammation has been well characterized in several experimental models of acute laminitis, the inflammatory events associated with endocrinopathic laminitis are not well documented. The aim of this study was to characterize selected markers of inflammation in horses with clinical evidence of equine metabolic syndrome (EMS). Neutrophil phagocytosis and oxidative burst, as well as endogenous and stimulated cytokine expression were evaluated. A marked increase in neutrophil reactive oxygen species production upon phagocytosis was observed in horses with EMS that was strongly correlated to the blood insulin concentration. Increased oxidative burst activity of neutrophils in hyperinsulinemic horses may predispose horses with metabolic syndrome to laminitis. In contrast, peripheral blood cells of obese hyperinsulinemic horses showed decreased endogenous proinflammatory cytokine gene expression (IL-1 and IL-6) and similar cytokine response following immune stimulation compared to that of control horses. This may suggest that, unlike in people, cytokine-mediated inflammation does not increase in direct response to obesity or insulin resistance in horses. This species-specific disparity may explain the difference in clinical outcomes observed in obese horses compared to obese people.

Differential regulation of endogenous pro-inflammatory cytokine genes by medroxyprogesterone acetate and norethisterone acetate in cell lines of the female genital tract

Background Medroxyprogesterone acetate (MPA) and norethisterone (NET) and its derivatives are widely used in female reproductive therapy, but little is known about their mechanisms of action via steroid receptors in the female genital tract. MPA used as a contraceptive has been implicated in effects on local immune function. However, the relative effects of progesterone (Prog), MPA and norethisterone acetate (NET-A) on cytokine gene expression in the female genital tract are unknown. Study Design Using two epithelial cell lines generated from normal human vaginal (Vk2/E6E7) and ectocervical (Ect1/E6E7) cells as in vitro cell culture model systems for mucosal immunity of the female cervicovaginal environment, we investigated steroid receptor expression and activity as well as regulation of cytokine/chemokine genes by MPA and NET-A, as compared to the endogenous hormone Prog. Results We show that the Prog, androgen, glucocorticoid and estrogen receptors (PR, AR, GR and ER, respectively) are expressed in both the Vk2/E6E7 and Ect1/E6E7 cell lines, and that the GR and AR are transcriptionally active. This study is the first to show ligand-, promoter- and cell-specific regulation of IL-6, IL-8 and RANTES (regulated-upon-activation, normal T cell expressed and secreted) gene expression by Prog, MPA and NET-A in these cell lines. Moreover, we show that the repression of the TNF-α-induced RANTES gene by MPA in the Ect1/E6E7 cell line is mediated by the AR. Conclusion Collectively, these data demonstrate that cell lines from different anatomical sites of the female genital tract respond differently to Prog and the synthetic progestins, most likely due to differential actions via different steroid receptors. The results highlight the importance of choice of progestins for immune function in the cervicovaginal environment. They further suggest that choice of progestins in endocrine therapy may have implications for women's risk of susceptibility to infections due to differential actions on genes involved in inflammation and immune function.