Overexpression Of Interleukin-6 Research Articles

Expression of Interleukin-8, Interleukin-12 and Interleukin-13 in Esophageal Squamous Cell Carcinoma: Biomarker Potentiality and Prognostic Significance.

Interleukin-8 (IL8), Interleukin-12 (IL12) and Interleukin-13 (IL13) are cytokines that play regulatory role in cancer pathogenesis. We analysed their expression profile to evaluate as molecular biomarkers of esophageal squamous cell carcinoma (ESCC) and their association with different parameters and patient survival. Expression analysis was performed by Real time quantitative polymerase chain reaction and receiver operating characteristic (ROC) curve analysis was done. The expression profiles were associated with different clinicopathological and dietary factors. Survival and hazard analysis were also performed. IL8 expression showed upregulation in tissue (p = 0.000) and blood samples (p = 0.481), IL12 expression showed downregulation in tissue samples (p = 0.064) and upregulation in blood samples (p = 0.689) and IL13 expression showed upregulation in tissue (p = 0.000) and blood samples (p = 0.006). IL13 expression in tissue showed the highest area under the curve (AUC) value (0.773) for ESCC diagnosis, followed by IL8 expression in tissue (0.704) and IL13 expression in blood (0.643). This study also reveals the correlation of studied cytokines in tissue and blood level. Different clinicopathological and dietary factors showed significant association (p < 0.05) with IL8, IL12 and IL13 expression and with survival of ESCC patients. IL8 expression in blood and IL12 expression in tissue and blood showed significant association (p < 0.05) with patient survival. Altered expression of IL8, IL12 and IL13 may be associated with ESCC progression. Overexpression of IL8 and IL13 in tissue samples may be potential biomarkers for ESCC screening. Additionally, both survival and hazard analysis data indicate the effects of different parameters on the prognosis of ESCC patients.

Suppressive effect of isofraxidin on the overexpression of IL-6 and its molecular mechanism in a TPA-treated human hepatocellular carcinoma cell line, HuH-7.

Interleukin-6 (IL-6) is a pleiotropic cytokine that has many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes. Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. The expression of IL-6 mRNA was measured using quantitative real-time PCR, and intracellular signaling molecules were detected using western blotting. When the HuH-7 human hepatocellular carcinoma cell line and HepG2 human hepatoblastoma cell line were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose-dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.

IL-10 Overexpression Reduces the Protective Response of an Experimental Chlamydia abortus Vaccine in a Murine Model.

In ovine populations, the enzootic nature of Chlamydia abortus (C. abortus) is attributed to its capacity to establish persistent intracellular infections, which necessitate a cellular immune response mediated by interferon-gamma (IFN-γ) for effective resolution. In both natural hosts and murine models, interleukin-10 (IL-10) has been demonstrated to modulate the cellular immune response crucial for the eradication of C. abortus. During gestation, it has also been shown to play a role in preventing inflammatory damage to gestational tissues and foetal loss through the downregulation of pro-inflammatory cytokines. This paradigm can be key for events leading to a protective response towards an infectious abortion. Previous research successfully established a mouse model of chronic C. abortus infection using transgenic mice overexpressing IL-10 (IL-10tg), simulating the dynamics of chronic infection observed in non-pregnant natural host. This study aims to evaluate the efficacy of an experimental inactivated vaccine against C. abortus and to elucidate the immune mechanisms involved in protection during chronic infection using this model. Transgenic and wild-type (WT) control mice were immunized and subsequently challenged with C. abortus. Vaccine effectiveness and immune response were assessed via immunohistochemistry and cytokine serum levels over a 28-day period. Morbidity, measured by daily weight loss, was more pronounced in non-vaccinated transgenic IL-10 mice, though no mortality was observed in any group. Vaccinated control mice eliminated the bacterial infection by day 9 post-infection (p.i.), whereas presence of bacteria was noted in vaccinated transgenic IL-10 mice until day 28 p.i. Vaccination induced an early post-infection increase in IFN-γ production, but did not alter IL-10 production in transgenic mice. Histological analysis indicated suboptimal recruitment of inflammatory cells in vaccinated transgenic IL-10 mice compared to WT controls. In summary, the findings suggest that IL-10 overexpression in transgenic mice diminishes the protective efficacy of vaccination, confirming that this model can be useful for validating the efficacy of vaccines against intracellular pathogens such as C. abortus that require robust cell-mediated immunity.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Lung-specific interleukin 6 mediated transglutaminase 2 activation and cardiopulmonary fibrogenesis.

Pulmonary hypertension (PH) pathogenesis is driven by inflammatory and metabolic derangements as well as glycolytic reprogramming. Induction of both interleukin 6 (IL6) and transglutaminase 2 (TG2) expression participates in human and experimental cardiovascular diseases. However, little is known about the role of TG2 in these pathologic processes. The current study aimed to investigate the molecular interactions between TG2 and IL6 in mediation of tissue remodeling in PH. A lung-specific IL6 over-expressing transgenic mouse strain showed elevated right ventricular (RV) systolic pressure as well as increased wet and dry tissue weights and tissue fibrosis in both lungs and RVs compared to age-matched wild-type littermates. In addition, IL6 over-expression induced the glycolytic and fibrogenic markers, hypoxia-inducible factor 1α, pyruvate kinase M2 (PKM2), and TG2. Consistent with these findings, IL6 induced the expression of both glycolytic and pro-fibrogenic markers in cultured lung fibroblasts. IL6 also induced TG2 activation and the accumulation of TG2 in the extracellular matrix. Pharmacologic inhibition of the glycolytic enzyme, PKM2 significantly attenuated IL6-induced TG2 activity and fibrogenesis. Thus, we conclude that IL6-induced TG2 activity and cardiopulmonary remodeling associated with tissue fibrosis are under regulatory control of the glycolytic enzyme, PKM2.

Chronic Expression of Interleukin-10 Transgene Modulates Cardiac Sympathetic Ganglion Resulting in Reduced Ventricular Arrhythmia.

The cardiac autonomic nervous system (CANS) is intimately connected to the regulation of electrophysiology and arrhythmogenesis in cardiac systems. This work aimed at investigating whether interleukin-10 (IL-10) could effectively modulate CANS and suppress ischemia-induced ventricular arrhythmia (VA) through chronically acting on the cardiac sympathetic ganglion (CSG). Using an adeno-associated virus (AAV), we achieved local chronic overproduction of IL-10 in the CSG, left stellate ganglion (LSG). As a result, in the IL-10 group, we observed a decreased number of tyrosine hydroxylase-positive (TH+) cells in the LSG. IL-10 markedly downregulated the nerve growth factor, synaptophysin, as well as growth-associated protein 43 expression. In vivo, results from ambulatory electrocardiography showed that IL-10 overexpression significantly inhibited the cardiac sympathetic nervous system activity and improved heart rate variability. Meanwhile, we observed decreased LSG function as well as prolonged ventricular effective refractory period and suppressed VA after myocardial infarction (MI) in the IL-10 group. In addition, IL-10 overexpression attenuated inflammation and decreased norepinephrine levels in the myocardium after acute MI. In conclusion, our data suggest that chronic IL-10 overexpression modulates cardiac sympathetic nerve remodeling and suppresses VA induced by MI. Neuromodulation through AAV-mediated IL-10 overexpression may have the characteristics of and advantages as a potential neuroimmunotherapy for preventing MI-induced VAs.

Gold Nanoparticles Downregulate IL-6 Expression/Production by Upregulating microRNA-26a-5p and Deactivating the RelA and NF-κBp50 Transcription Pathways in Activated Breast Cancer Cells.

MicroRNAs (miRNAs) are involved in the modulation of pathogenic genes by binding to their mRNA sequences' 3' untranslated regions (3'UTR). Interleukin-6 (IL-6) is known to promote cancer progression and treatment resistance. In this study, we aimed to explore the therapeutic effects of gold nanoparticles (GNP) against IL-6 overexpression and the modulation of miRNA-26a-5p in breast cancer (BC) cells. GNP were synthesized using the trisodium citrate method and characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). To predict the binding of miR-26a-5p in the IL-6 mRNA's 3'UTR, we utilized bioinformatics algorithms. Luciferase reporter clone assays and anti-miRNA-26a-5p transfection were employed to validate the binding of miR26a-5p in the IL-6 mRNA's 3'UTR. The activity of RelA and NF-κBp50 was assessed and confirmed using Bay 11-7082. The synthesized GNP were spherical with a mean size of 28.3 nm, exhibiting high stability, and were suitable for BC cell treatment. We found that miR-26a-5p directly regulated IL-6 overexpression in MCF-7 cells activated with PMA. Treatment of MCF-7 cells with GNP resulted in the inhibition of IL-6 overexpression and secretion through the increase of miR26a-5p. Furthermore, GNP deactivated NF-κBp65/NF-κBp50 transcription activity. The newly engineered GNP demonstrated safety and showed promise as a therapeutic approach for reducing IL-6 overexpression. The GNP suppressed IL-6 overexpression and secretion by deactivating NF-κBp65/NF-κBp50 transcription activity and upregulating miR-26a-5p expression in activated BC cells. These findings suggest that GNP have potential as a therapeutic intervention for BC by targeting IL-6 expression and associated pathways.

Interleukin-6-elicited chronic neuroinflammation may decrease survival but is not sufficient to drive disease progression in a mouse model of Leigh syndrome

BackgroundMitochondrial diseases (MDs) are genetic disorders characterized by dysfunctions in mitochondria. Clinical data suggest that additional factors, beyond genetics, contribute to the onset and progression of this group of diseases, but these influencing factors remain largely unknown. Mounting evidence indicates that immune dysregulation or distress could play a role. Clinical observations have described the co-incidence of infection and the onset of the disease as well as the worsening of symptoms following infection. These findings highlight the complex interactions between MDs and immunity and underscore the need to better understand their underlying relationships.ResultsWe used Ndufs4 KO mice, a well-established mouse model of Leigh syndrome (one of the most relevant MDs), to test whether chronic induction of a neuroinflammatory state in the central nervous system before the development of neurological symptoms would affect both the onset and progression of the disease in Ndufs4 KO mice. To this aim, we took advantage of the GFAP-IL6 mouse, which overexpresses interleukin-6 (IL-6) in astrocytes and produces chronic glial reactivity, by generating a mouse line with IL-6 overexpression and NDUFS4 deficiency. IL-6 overexpression aggravated the mortality of female Ndufs4 KO mice but did not alter the main motor and respiratory phenotypes measured in any sex. Interestingly, an abnormal region-dependent microglial response to IL-6 overexpression was observed in Ndufs4 KO mice compared to controls.ConclusionOverall, our data indicate that chronic neuroinflammation may worsen the disease in Ndufs4 KO female mice, but not in males, and uncovers an abnormal microglial response due to OXPHOS dysfunction, which may have implications for our understanding of the effect of OXPHOS dysfunction in microglia.

MiR-26a-5p restoration via EZH2 silencing blocks the IL-6/STAT3 axis to repress the growth of prostate cancer

ABSTRACT Background Interleukin-6 (IL-6) is involved in the activation of several oncogenic pathways in prostate cancer. However, its upstream trans-signaling pathway remains largely unknown. This work proposes a mechanistic explanation of IL-6’s upstream effectors in prostate carcinogenesis. Research design & methods Samples were harvested to validate the expression of EZH2, miR-26a-5p, and IL-6. Moreover, the protein and its phosphorylation of STAT3 (signal transducer and transcription activator 3) were assessed in prostate cancer cells. We explored the effects of these effectors on malignant phenotypes in vitro and tumor growth in vivo using functional assays. Bioinformatics analysis, dual-luciferase reporter gene assays, and chromatin immunoprecipitation (ChIP) assays were used to determine their binding relationships. Results Overexpression of EZH2 and IL-6, and under expression of miR-26a-5p was observed in prostate cancer. Silencing IL-6 repressed STAT3 to suppress the malignant phenotypes of prostate cancer cells. Mechanistically, EZH2 inhibited miR-26a-5p expression by promoting H3K27 histone methylation, and miR-26a-5p restricted the malignant phenotypes of prostate cancer by targeting IL-6. Ectopic EZH2 expression reduced xenograft growth by inhibiting miR-26a-5p and activating the IL-6/STAT3 axis. Conclusion EZH2 May potentially be involved in regulating its expression by recruiting H3K27me3 to the miR-26a-5p promoter region, which could further impact the IL6/STAT3 pathway.

Chronic interleukin-6 mediated neuroinflammation decreases anxiety, and impaires spatial memory in aged female mice

IntroductionNeuroinflammation is a common feature of many psychiatric disorders as well as a common underlying mechanism of neurodegenerative diseases. Sex has been shown to strongly influence the development as well as the clinical expression of these pathologies. However, there is still a neglect regarding the consideration of sex effects in rodent experiments, and a substantial underrepresentation of females in studies. This work set out to expand our knowledge of neuroinflammatory mechanisms in female mice, at both a behavioral and molecular level.MethodsThis study used GFAP-IL6 mice, a model of chronic neuroinflammation, in which interleukin-6 (IL6) is overexpressed in the central nervous system under the control of the glial fibrillary acidic protein (GFAP) promoter. We evaluated aged (11-15-month-old) wild type-like (WT) and GFAP-IL6 female mice in behavioral tests assessing anxiety (elevated plus-maze, EPM, Light/dark box), and spatial learning and memory (Y-maze, YM and Barnes Maze, BM) and associative learning (fear conditioning, FC). We also examined gene expression of markers linked to neuroinflammation, neurodegeneration and neurotransmission via RT-qPCR in brain regions involved in motor control, anxiety, learning and memory.ResultsFemale GFAP-IL6 mice exhibited reduced anxiety-like behavior in the EPM, and hypolocomotion in the light-dark test and EPM. Short-term memory impairment was evident in the YM but associative learning in FC was intact in GFAP-IL6 mice, suggesting domain-specific cognitive deficits in female GFAP-IL6 mice. In the BM, all mice showed intact learning and memory, but GFAP-IL6 mice exhibited higher latencies to enter the escape hole than WT mice. We analyzed the search strategy and found differences in the way GFAP-IL6 mice searched for the escape hole compared to WTs. RT-qPCR showed increased mRNA levels for molecules involved in pro-inflammatory pathways in the cerebellum, motor cortex, hippocampus, and amygdala in GFAP-IL6 mice. Of the regions examined, the cerebellum and the hippocampus showed upregulation of neuroinflammatory makers as well as dysregulation of glutamatergic and GABAergic neurotransmission gene expression in GFAP-IL6 mice compared to WTs.ConclusionIn conclusion, we showed that chronic neuroinflammation via IL6 overexpression in aged female mice led to a less anxious-like phenotype, hypolocomotion and impaired intermediate-term spatial learning and memory in the YM.

Overexpression of Interleukin-8 Promotes the Progression of Fatty Liver to Nonalcoholic Steatohepatitis in Mice.

Nonalcoholic steatohepatitis (NASH) is an advanced stage of fatty liver disease characterized by liver damage, inflammation, and fibrosis. Although neutrophil infiltration is consistently observed in the livers of patients with NASH, the precise role of neutrophil-recruiting chemokines and infiltrating neutrophils in NASH pathogenesis remains poorly understood. Here, we aimed to elucidate the role of neutrophil infiltration in the transition from fatty liver to NASH by examining hepatic overexpression of interleukin-8 (IL8), a major chemokine responsible for neutrophil recruitment in humans. Mice fed a high-fat diet (HFD) for 3 months developed fatty liver without concurrent liver damage, inflammation, and fibrosis. Subsequent infection with an adenovirus overexpressing human IL8 for an additional 2 weeks increased IL8 levels, neutrophil infiltration, and liver injury in mice. Mechanistically, IL8-induced liver injury was associated with the upregulation of components of the NADPH oxidase 2 complex, which participate in neutrophil oxidative burst. IL8-driven neutrophil infiltration promoted macrophage aggregate formation and upregulated the expression of chemokines and inflammatory cytokines. Notably, IL8 overexpression amplified factors associated with fibrosis, including collagen deposition and hepatic stellate cell activation, in HFD-fed mice. Collectively, hepatic overexpression of human IL8 promotes neutrophil infiltration and fatty liver progression to NASH in HFD-fed mice.

Gene expression of Interleukin-10 and Foxp3 as critical biomarkers in rheumatoid arthritis patients

Introduction and Aim: Forkhead box P3 (FOXP3) and interleukin-10 (IL-10) are the key regulators controlling the activity of Treg cells, which are crucial for maintaining immune tolerance and reducing autoimmune reactions. The objective of this study was to investigate the potential utility of elevated levels of FOXP3 and IL-10 gene expression as a diagnostic indicator in patients with rheumatoid arthritis (RA). Materials and Methods: The study used quantitative polymerase chain reaction (qPCR) to examine the expression levels of FOXP3 and IL-10 transcripts in whole blood samples from Iraqi patients with rheumatoid arthritis. A group of healthy control subjects were also included in the study. Results: In blood samples taken from Iraqi patients diagnosed with rheumatoid arthritis, a statistically significant decrease (P 0.01) in the expression levels of the FOXP3 gene and a statistically significant elevation (P 0.01) of IL-10 expression were seen in contrast to the healthy control group. Conclusion: Rheumatoid arthritis patients in Iraq may benefit from FOXP3 and IL-10 gene expression tests. IL-10 and FOXP3 overexpression promotes T cell and immune system immunoreactivity.

A V2CTx Mxene-Based Immune Tag for the Electrochemical Detection of Interleukin 6 (IL-6) from Breast Cancer Cells

Interleukin 6 (IL-6) is a cytokine that can act in an anti-inflammatory or pro-inflammatory role1 and over-expression of IL-6 in the tumor microenvironment (TME) has been linked to numerous tumors including breast cancer 2. The quantitative detection of IL-6 is critical for the diagnosis of tumors and biological research. Although there are several methods for the detection of IL-6, an enzyme-linked immunosorbent assay (ELISA) is the most standard and conventional approach 3. Conventional methods have the disadvantages of complex operation, time-consuming, usage of expensive or bulky instruments and need for trained personnel, which make them unadaptable for point-of-care (POC) tests. Therefore, the development of a reliable, rapid and on-site detection platform for the quantitative analysis of IL-6 is needed.Electrochemical (EC) biosensors are an alternative technique for detection of IL-6 owing to their advantages of a quick readout, low cost, ultra-sensitivity, high selectivity, and simplicity. Furthermore, electrochemical biosensors are easier to be miniaturized and mass fabricated, which makes them more desirable for POC applications 4. MXenes possessing high hydrophilicity, metallic conductivity and excellent plasmon resonance in the visible range are a novel family of 2D materials and are a suitable nanomaterial for enhancing EC signal 5.Herein, a sensitive EC platform was proposed and developed based on a 2D vanadium carbide (V2CTx) MXene-based immune tag. V2CTx@ Fe₄[Fe(CN)6]₃ (Prussian Blue) @Au NPs was prepared by an in-situ generation method. Then, the anti-IL-6 antibody was fixed on the V2CTx@ PB @Au NPs. The prepared V2CTx MXene-based immune tag and EC platform was characterized by electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), differential pulse voltammetry (DPV), scanning electron microscope (SEM), x-ray diffraction (XRD) method, x-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR). The in-situ generated V2CTx@PB@Au NPs showed stronger signal than electrostatic attracted Au NPs on V2CTx@PB. The linearity range were obtained as 0.005-0.5 ng/mL. The V2CTx@PB@Au maintained an EC signal of ~80% after 10 days and also performed well in real breast cancer cell samples. Our study thus suggests that the EC with V2CTx MXene-based immune tag is an effective platform for detecting cytokines secreted by breast cancer cells cultured in confined chips. References S. A. Jones and B. J. Jenkins, Nature Reviews Immunology, 18 (12), 773-789 (2018). J. Milovanović, N. Todorović-Raković, and M. Radulovic, Cytokine, 118 93-98 (2019). M. Rahbar, Y. Wu, J. A. Subramony, and G. Liu, Frontiers in Bioengineering and Biotechnology, 9 (2021). F. Cui, Z. Zhou, and H. S. Zhou, Journal of The Electrochemical Society, 167 (3), 037525 (2019). Y. Zhang, W. Lu, J. Zhou, D. Sun, and H. Li, Journal of Alloys and Compounds, 929 167276 (2022). Figure 1

Enhanced production and single step purification of biologically active recombinant anti-IL6 scFv from Escherichia coli inclusion bodies

Interleukin 6 (IL-6) is a proinflammatory cytokine that plays a critical role in immune responses and inflammation. Overexpression of IL-6 serves as a biomarker for various diseases. Therefore, it is vital to develop IL-6-specific antibodies for the diagnosis and treatment of inflammatory diseases. Alternatively, high-yield production of single-chain variable fragments (scFv) in prokaryotes reduces the time and cost required while attaining a similar specificity. However, overexpression of eukaryotic proteins in bacterial expression systems often leads to the formation of inclusion bodies. Here, a codon-optimized anti-IL6 scFv was expressed in the E. coli SHuffle strain, and the effects of inclusion body solubilisation methods on the product yield were investigated. A high concentration (23 mg/L) of biologically active and pure (>95 %) scFv was obtained without any need for a refolding step following solubilisation with a non-denaturing solubilisation agent, N-lauroyl sarcosine. Activity assays showed that anti-IL6 scFv could specifically bind to its antigen. Overall, we present reproducible and efficient production of anti-IL6 scFv in E. coli by using a one-step purification method. While the biologically active and pure antibody fragment can be used for diagnostic studies of the IL-6 cytokine, the developed method can be applied to other recombinant proteins expressed in E. coli.

Abstract P3059: Recombinant Adeno-associated Viral Vector 2/8 Model Of Interleukin 6 Mediated Chronic Inflammation

Background: Interleukin 6 (IL6) is a pleiotropic cytokine mostly known as a proinflammatory cytokine. There is a need for a reliable in vivo model to study IL6 mediated chronic inflammation. Hypothesis: The aim of our study was to create a mouse model mimicking the chronic inflammatory state as observed in cancer and inflammatory diseases. We opted for a recombinant adeno-associated viral vector (rAAV) to mimic the acute initiation of chronic inflammation later in life. Methods: We opted for a rAAV2/8 subtype, human EF1a short (EFS) promoter and firefly luciferase led by an internal ribosome entry site (IL6-IRES-fLuc) DNA cassette to create a stable low-grade expression of IL6 in liver and serum, with minimal expression in other organs, particularly the heart. rAAV2/8_EFS-IL6-IRES-fLuc and the control vector rAAV2/8_EFS-MCS-IRES-fLuc were generated and different dosages were tested. Results: All mice treated with high dose AAV2/8-IL6 vector (8.8* 10 10 genome copies (GC)/animal) showed severe weight loss and hepatosplenomegaly, starting between week 4-6 post injection. Histological examination of the liver showed features of chronic hepatitis. Infiltrating cells were found in both the liver and the heart. In the spleen, the histological structure of red and white pulp was disturbed and more giant cells could be detected. Cardiac function remained normal at 6 weeks post injection. Assuming that the previous results were due to an overexpression of IL6 due to too high amounts of rAAV2/8_EFS-IL6-IRES-fLuc, we subsequently challenged mice with a rAAV2/8_EFS-IL6-IRES-fLuc dilution series to test which vector dose could be used for reliable long-term low grade IL6 overexpression. After extensive testing, the ideal dose for a 12 weeks IL6 overexpression model was between 2.2 and 2.93* 10 10 GC. Using this dose range, we saw stable overexpression of IL6 without weight loss. Hepatosplenomegaly, disturbed anatomy of the spleen and infiltrating cells in the liver were still present yet less pronounced. Conclusion: We successfully generated and characterized a mouse model of IL6 overexpression to mimic chronic IL6 mediated inflammation. To the best of our knowledge this is the first model employing IL6 overexpression using a rAAV viral vector.

IL-8-induced CXCR2 down-regulation in circulating monocytes in hepatocellular carcinoma is partially dependent on MAGL

BackgroundCXC-chemokine receptor 2 (CXCR2) expression was found to be down-regulated on circulating monocytes of cancer patients. Here, we analyze the percentage of CD14+CXCR2+ monocyte subsets in hepatocellular carcinoma (HCC) patients, and investigate the mechanisms that regulate CXCR2 surface expression on monocytes and its biological function.MethodsFlow cytometry was used to analyze the proportion of the CD14+CXCR2+ subset from the total circulating monocytes of HCC patients. Interleukin 8 (IL-8) levels were measured from serum and ascites, and their correlation with the CD14+CXCR2+ monocyte subset proportion was calculated. THP-1 cells were cultured in vitro and treated with recombinant human IL-8 and CXCR2 surface expression was analyzed. CXCR2 was knocked down to examine how it affects the antitumor activity of monocytes. Finally, a monoacylglycerol lipase (MAGL) inhibitor was added to analyze its effect on CXCR2 expression.ResultsA decrease in the proportion of the CD14+CXCR2+ monocyte subset was observed in HCC patients compared with healthy controls. CXCR2+ monocyte subset proportion was associated with the AFP value, TNM stage, and liver function. Overexpression of IL-8 was observed in the serum and ascites of HCC patients, and negatively correlated with CXCR2+ monocyte proportion. IL-8 decreased CXCR2 expression in THP-1 cells, contributing to decreased antitumor activity toward HCC cells. MAGL expression in THP-1 cells was up-regulated after IL-8 treatment, and the MAGL inhibitor partially reversed the effects of IL-8 on CXCR2 expression.ConclusionsOverexpression of IL-8 drives CXCR2 down-regulation on circulating monocytes of HCC patients, which could be partially reversed by a MAGL inhibitor.

Creatine supplementation increases postnatal growth and strength and prevents overexpression of pro-inflammatory interleukin 6 in the hippocampus in an experimental model of cerebral palsy

ABSTRACT Objectives: The aim of this study was thus to evaluate the effect of Cr supplementation on morphological changes and expression of pro-inflammatory cytokines in the hippocampus and on developmental parameters. Methods: Male Wistar rat pups were submitted to an experimental model of CP. Cr was administered via gavage from the 21st to the 28th postnatal day, and in water after the 28th, until the end of the experiment. Body weight (BW), food consumption (FC), muscle strength, and locomotion were evaluated. Expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) were assessed in the hippocampus by quantitative real-time polymerase chain reaction. Iba1 immunoreactivity was assessed by immunocytochemistry in the hippocampal hilus. Results: Experimental CP caused increased density and activation of microglial cells, and overexpression of IL-6. The rats with CP also presented abnormal BW development and impairment of strength and locomotion. Cr supplementation was able to reverse the overexpression of IL-6 in the hippocampus and mitigate the impairments observed in BW, strength, and locomotion. Discussion: Future studies should evaluate other neurobiological characteristics, including changes in neural precursor cells and other cytokines, both pro- and anti-inflammatory.

Targeted blockade of interleukin-8 negates metastasis and chemoresistance via Akt/Erk-NFκB axis in oral cancer

BackgroundThe tumor microenvironment plays a significant role in tumor growth, metastasis and chemoresistance via dysregulated signaling pathways. Toward this, an inflammatory chemokine, interleukin-8 (IL-8), is overexpressed in various cancers and is involved in tumor progression and chemoresistance. However, the mechanistic role of IL-8 in mediating metastasis and chemoresistance in oral squamous cell carcinoma (OSCC) is not known. Methods and resultsIn the present study, we evaluated the effect of IL-8 in regulating metastasis as well as chemoresistance in OSCC cell lines. For this, IL-8 was blocked exogenously using neutralizing IL-8 monoclonal antibody and IL-8 levels were enhanced by exogenous supply of recombinant human IL-8 (rhIL-8) to OSCC cells. The epithelial-to-mesenchymal transition (EMT) was evaluated using qPCR, migration by scratch/wound healing assay and invasion ability using transwell assay. rIL-8 induced chemoresistance was studied by apoptosis assay and the nuclear localization of NFκB using immunocytochemistry. IL-8 was significantly overexpressed in OSCC patients and cell lines. While exogenous blockade of IL-8 significantly reduced EMT, migration and invasion potential in OSCC cells, IL-8 overexpression upregulated these cellular traits thereby confirming the role of IL-8 in OSCC metastasis. Exogenous blockade of IL-8 also reversed chemoresistance in cisplatin resistant OSCC subline via NFκB signaling. ConclusionIL-8 plays a crucial role in OSCC metastasis and its targeted blockade can help in management of cisplatin resistance.

Lnc001776 Affects CPB2 Toxin-Induced Excessive Injury of Porcine Intestinal Epithelial Cells via Activating JNK/NF-kB Pathway through ssc-let-7i-5p/IL-6 Axis

Piglet diarrhea caused by Clostridium perfringens (C. perfringens) type C (CpC) seriously endangers the development of the pig production industry. C. perfringens beta2 (CPB2) toxin is a virulent toxin produced by CpC. Long non-coding RNAs (lncRNAs) are key regulators in the immune inflammatory response to bacterial infection. Nevertheless, the functional mechanism of lncRNAs in bacterial piglet diarrhea is unclear. Herein, a novel lncRNA lnc001776 expression was confirmed to be substantially elevated in the ileum tissue of CpC-infected diarrhea piglets and in CPB2 toxin-treated porcine small intestinal epithelial cells (IPEC-J2). lnc001776 knockdown restrained CPB2 toxin-induced apoptosis, inflammatory injury, barrier dysfunction and activation of JNK/NF-kB pathway in IPEC-J2 cells. Additionally, ssc-let-7i-5p was identified as sponge for lnc001776. Overexpression of ssc-let-7i-5p repressed CPB2-induced injury in IPEC-J2 cells. Interleukin 6 (IL-6), a target gene of ssc-let-7i-5p, was enhanced in CPB2 toxin-treated IPEC-J2 cells. Rescue experiments demonstrated that a ssc-let-7i-5p mimic reversed the effect of lnc001776 overexpression on CPB2 toxin-induced IPEC-J2 cell injury and JNK/NF-kB pathway, whereas IL-6 overexpression partially restored the impact of lnc001776. Overall, lnc001776 overexpression exacerbated CPB2 toxin-induced IPEC-J2 cell damage by sponging ssc-let-7i-5p to regulate IL-6 to activate JNK/NF-kB pathway, indicating that lnc001776 could be a key target for piglet resistance to CpC-induced diarrhea.