Proinflammatory Cytokines IL-1beta Research Articles

Polychlorinated Biphenyls (PCBS)-induced oxidative stress and inflammation in human thyrocytes: involvement of AhR and NRF-2/HO-1 pathway.

In this in vitro study, we investigated the effects of polychlorinated biphenyls (PCBs) on human thyrocytes, with a focus on the involvement of AhR, a key player in xenobiotic response, and the anti-oxidant Nrf-2/HO-1 pathway. Primary cultured thyrocytes were exposed to the dioxin-like congeners PCB118 and PCB126 at 2.5 and 5 µM concentrations. mRNA expression was assessed by real-time PCR, and protein expression by Western Blot and ELISA, while protein quantification was assessed by densitometric analysis. In cultured thyrocytes, PCB118 and PCB126 induced a significant (P < 0.01) increase of mRNA and protein levels of the pro-inflammatory cytokines IL-1beta and IL-6, while reducing those of thyroglobulin (TG) and NIS (p < 0.05), indicating down-regulation of these thyroid-specific genes in PCB-induced inflammation. ROS production also increased (p < 0.001). mRNA levels of AhR and the downstream molecules cytochrome P4501A, Nrf-2/HO-1 increased (p < 0.001), as well as related protein levels (p < 0.01), suggesting the activation of AhR and Nrf-2 pathways in response to PCBs exposure. AhR silencing decreased AhR-related gene expression and restored NIS and TG expression, while reducing inflammatory cytokines and oxidative stress markers (p < 0.05). Dioxin-like PCBs (PCB118 and PCB126) may promote inflammation and oxidative stress in thyrocytes, impairing the expression of genes that are key players of thyroid function. These effects can be partially attributed to the activation of the AhR and Nrf-2 pathways. These data may contribute to explain the mechanisms underlying thyroid toxicity of PCBs, highlighting the potential role of these pollutants as a trigger of autoimmune thyroid inflammation and damage.

Lipid from electronic cigarette-aerosol both with and without nicotine induced pro-inflammatory macrophage polarization and disrupted phagocytosis

Clinical cases and experimental evidence revealed that electronic cigarettes (ECIG) induce serious adverse health effects, but underlying mechanisms remain to be fully uncovered. Based on recent exploratory evidence, investigating the effects of ECIG on macrophages can broadly define potential mechanisms by focusing on the effect of ECIG exposure with or without nicotine. Here we investigated the effect of ECIG-aerosol exposure on macrophages (MQ) phenotype, inflammatory response, and function of macrophages.MQ were cultured at air liquid interface and exposed to ECIG-aerosol. Oxidative stress was determined by reactive oxygen species (ROS), heat shock protein 60 (HSP60), glutathione peroxidase (GPx) and heme oxygenase1 (HMOX1). Lipid accumulation and lipid peroxidation were defined by lipid staining and level of malondialdehyde (MDA) respectively. MQ polarization was identified by surface expression markers CD86, CD11C and CD206 as well as pro-inflammatory and anti-inflammatory cytokines in gene and protein level. Phagocytosis of E. coli by MQ was investigated by fluorescence-based phagocytosis assay.ECIG-aerosol exposure in presence or absence of nicotine induced oxidative stress evidenced by ROS, HSP60, GPx, GPx4 and HMOX1 upregulation in MQ. ECIG-aerosol exposure induced accumulation of lipids and the lipid peroxidation product MDA in MQ. Pro-inflammatory MQ (M1) markers CD86 and CD11C but not anti-inflammatory MQ (M2) marker CD206 were upregulated in response to ECIG-aerosol exposure. In addition, ECIG induced pro-inflammatory cytokines IL-1beta and IL-8 in gene level and IL-6, IL-8, and IL-1beta in protein level whereas ECIG exposure downregulated anti-inflammatory cytokine IL-10 in protein level. Phagocytosis activity of MQ was downregulated by ECIG exposure. shRNA mediated lipid scavenger receptor ‘CD36’ silencing inhibited ECIG-aerosol-induced pro-inflammatory MQ polarization and recovered phagocytic activity of MQ.ECIG exposure alters lung lipid homeostasis and thus induced inflammation by inducing M1 type MQ and impair phagocytic function, which could be a potential cause of ECIG-induced lung inflammation in healthy and inflammatory exacerbation in disease condition.

Ex-Vivo Human Whole Blood Assay for Evaluating the Pharmacodynamics of HT-6184: Reduction in NLRP3 Inflammasome Mediated Cytokine Production

The NLRP3 inflammasome is an intracellular multiprotein complex that plays a crucial role in the innate immune response by mediating the maturation and release of pro-inflammatory cytokines IL-1 beta, and IL-18. The release of these cytokines leads to the downstream release of TNF-alpha, IL-6, which propagate a robust inflammatory response. Dysregulation of the NLRP3 inflammasome pathway has been linked to various inflammatory diseases, suggesting the possibility for novel therapeutic strategies targeting NLRP3 activation. NLRP3 has been linked to regulating hematopoietic stem progenitor cell (HSPCs) development, expansion, release, and mobility. In addition, various hematological disorders such as myelodysplastic syndrome, myeloproliferative neoplasms, acute and chronic leukemias, and graft-versus-host disease (GvHD) have all been shown to involve NLRP3 inflammasome activation. Given its significant role in these conditions, inhibiting the NLRP3 inflammasome may be a novel therapeutic strategy in hematology and hemato-oncology This study utilized an ex-vivo human whole blood assay to investigate the pharmacodynamics of HT-6184, an allosteric Nek7 inhibitor. We used LPS and ATP to prime and activate the NLRP3 inflammasome and examined the effects of pre-treatment with various concentrations of HT-6184 (starting at 1 mM) on the production of critical pro-inflammatory cytokines, such as IL-1b, IL-18, IL-6 and TNF-a. Notably, preincubation with HT-6184 at concentrations as low as 4.1 nM led to a significant reduction in cytokine production (p&amp;lt;0.0001). Specifically, TNF-α and IL-6 were reduced by 38% and 58%, respectively, while IL-1β and IL-18 were decreased by 44% and 53% respectively. These results provide further evidence of the ability of HT-6184 to modulate the activity of the NLRP3 inflammasome in a complex biological milieu. Our study suggests that HT-6184 has significant therapeutic potential in treating a broad spectrum of inflammasome-mediated diseases. In addition, this assay has the potential to be used to measure the pharmacodynamic properties and the efficacy of HT-6184 and other inflammasome inhibitors in future healthy volunteer clinical trials. In conclusion, this study highlights the importance of innovative and physiologically relevant assay systems in identifying and evaluating potential therapeutic agents for inflammatory diseases.

12/15-lipoxygenase inhibition attenuates neuroinflammation by suppressing inflammasomes.

Lipoxygenases (LOXs) have essential roles in stroke, atherosclerosis, diabetes, and hypertension. 12/15-LOX inhibition was shown to reduce infarct size and brain edema in the acute phase of experimental stroke. However, the significance of 12/15-LOX on neuroinflammation, which has an essential role in the pathophysiology of stroke, has not been clarified yet. In this study, ischemia/recanalization (I/R) was performed by occluding the proximal middle cerebral artery (pMCAo) in mice. Either the 12/15-LOX inhibitor (ML351, 50 mg/kg) or its solvent (DMSO) was injected i.p. at recanalization after 1 h of occlusion. Mice were sacrificed at 6, 24, and 72-h after ischemia induction. Infarct volumes were calculated on Nissl-stained sections. Neurological deficit scoring was used for functional analysis. Lipid peroxidation was determined by the MDA assay, and the inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-10, and TGF-beta were quantified by ELISA. The inflammasome proteins NLRP1 and NLRP3, 12/15-LOX, and caspase-1 were detected with immunofluorescence staining. Infarct volumes, neurological deficit scores, and lipid peroxidation were significantly attenuated in ML351-treated groups at 6, 24, and 72-h. ELISA results revealed that the pro-inflammatory cytokines IL-1beta, IL-6, and TNF-alpha were significantly decreased at 6-h and/or 24-h of I/R, while the anti-inflammatory cytokines IL-10 and TNF-alpha were increased at 24-h or 72-h of ML351 treatment. NLRP1 and NLRP3 immunosignaling were enhanced at three time points after I/R, which were significantly diminished by the ML351 application. Interestingly, NLRP3 immunoreactivity was more pronounced than NLRP1. Hence, we proceeded to study the co-localization of NLRP3 immunoreactivity with 12/15-LOX and caspase-1, which indicated that NLRP3 was co-localized with 12/15-LOX and caspase-1 signaling. Additionally, NLRP3 was found in neurons at all time points but in non-neuronal cells 72 h after I/R. These results suggest that 12/15-LOX inhibition suppresses ischemia-induced inflammation in the acute and subacute phases of stroke via suppressing inflammasome activation. Understanding the mechanisms underlying lipid peroxidation and its associated pathways, like inflammasome activation, may have broader implications for the treatment of stroke and other neurological diseases characterized by neuroinflammation.

Immune Regulatory Markers in Clinical Cases of COVID-19

Abstract The objective of this research study is to profile anti-viral and pro-inflammatory cytokines, major histocompatibility complex (MHC) class I &amp; II molecules and cell death markers in clinical cases of sepsis. Research study design included three patient cohorts namely, COVID-19 with sepsis (C19wSepsis, N=17), COVID-19 with no sepsis (C19NoSepsis, N=17) and sepsis alone (N=24). Healthy subjects served as controls (N=16). Serum samples derived from patient cohorts and control subjects were analyzed by ELISA for anti-viral and pro-inflammatory cytokines (IFN-gamma and IL-1beta), MHC class I (MHCA) &amp; MHC class II (MHCDRb1) molecules and cell death markers. Data and statistical analyses were performed using GraphPad Prism 7.0. ELISA of serum samples identified a significant increase in serum IFN-gamma, MHCA and MHCDRb1 levels in C19wSepsis cohort compared to C19NoSepsis cohort and control subjects [p &amp;lt; 0.0001]. Although there was a significant increase in IL-1beta levels in serum samples of C19wSepsis compared to control subjects and sepsis alone cohort [p &amp;lt; 0.0005], the levels were comparable between C19wSepsis and C19NoSepsis cohorts [p &amp;gt;= 0.05]. Cell death markers, Caspase-3 and p62/SQSTM1, showed significantly altered levels between the three patient cohorts when compared to controls [p &amp;lt; 0.01]. Representative data from flow cytometry confirm ELISA results. Results from this study identify key immune regulatory markers in clinical cases of COVID-19.

Extracellular vesicles from immortalized mesenchymal stromal cells protect against neonatal hypoxic-ischemic brain injury

BackgroundHuman mesenchymal stromal cell (MSC)-derived extracellular vesicles (EV) revealed neuroprotective potentials in various brain injury models, including neonatal encephalopathy caused by hypoxia–ischemia (HI). However, for clinical translation of an MSC-EV therapy, scaled manufacturing strategies are required, which is challenging with primary MSCs due to inter- and intra-donor heterogeneities. Therefore, we established a clonally expanded and immortalized human MSC line (ciMSC) and compared the neuroprotective potential of their EVs with EVs from primary MSCs in a murine model of HI-induced brain injury. In vivo activities of ciMSC-EVs were comprehensively characterized according to their proposed multimodal mechanisms of action.MethodsNine-day-old C57BL/6 mice were exposed to HI followed by repetitive intranasal delivery of primary MSC-EVs or ciMSC-EVs 1, 3, and 5 days after HI. Sham-operated animals served as healthy controls. To compare neuroprotective effects of both EV preparations, total and regional brain atrophy was assessed by cresyl-violet-staining 7 days after HI. Immunohistochemistry, western blot, and real-time PCR were performed to investigate neuroinflammatory and regenerative processes. The amount of peripheral inflammatory mediators was evaluated by multiplex analyses in serum samples.ResultsIntranasal delivery of ciMSC-EVs and primary MSC-EVs comparably protected neonatal mice from HI-induced brain tissue atrophy. Mechanistically, ciMSC-EV application reduced microglia activation and astrogliosis, endothelial activation, and leukocyte infiltration. These effects were associated with a downregulation of the pro-inflammatory cytokine IL-1 beta and an elevated expression of the anti-inflammatory cytokines IL-4 and TGF-beta in the brain, while concentrations of cytokines in the peripheral blood were not affected. ciMSC-EV-mediated anti-inflammatory effects in the brain were accompanied by an increased neural progenitor and endothelial cell proliferation, oligodendrocyte maturation, and neurotrophic growth factor expression.ConclusionOur data demonstrate that ciMSC-EVs conserve neuroprotective effects of primary MSC-EVs via inhibition of neuroinflammation and promotion of neuroregeneration. Since ciMSCs can overcome challenges associated with MSC heterogeneity, they appear as an ideal cell source for the scaled manufacturing of EV-based therapeutics to treat neonatal and possibly also adult brain injury.

Cotinus coggygria Scop. Attenuates Acetic Acid-Induced Colitis in Rats by Regulation of Inflammatory Mediators.

In traditional medicine, many medicinal plants are used in the treatment of various diseases caused by inflammation. The objective of the present study is to elucidate for the first time the effects of Cotinus coggygria (CC) ethanol extract (CCE) on colonic structure and inflammation of acetic acid-induced ulcerative colitis in rats. Colonic damage was assessed using disease activity index score, enzyme-linked immunosorbent assay, and hematoxylin-eosin staining. Also, in vitro antioxidant activity of CCE was investigated by ABTS methods. Total phytochemical content of CCE was measured spectroscopically. Acetic acid caused colonic damage according to disease activity index and macroscopic scoring. CCE significantly reversed these damages. While the levels of proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and TGF-1beta increased in tissue with UC, IL-10 level decreased. CCE increased inflammatory cytokine levels to values close to the sham group. At the same time, while markers indicating disease severity such as VEGF, COX-2, PGE2, and 8-OHdG indicated the disease in the colitis group, these values returned to normal with CCE. Histological research results support biochemical analysis. CCE exhibited significant antioxidant against ABTS radical. Also, CCE was found to have a high content of total polyphenolic compounds. These findings provide evidence that CCE might be benefit as a promising novel therapy in the treatment of UC in humans due to high polyphenol content and justify the use of CC in folkloric medicine for treatment of inflamed diseases.

A preliminary study on the association between pro-inflammatory cytokine IL-1 beta polymorphisms and susceptibility to hepatitis C infection in Malay male Malay drug abusers

Introduction: Hepatitis C virus (HCV) infection frequently leads to liver complications, such as fibrosis, cirrhosis, and hepatocellular carcinoma. The incidence of HCV infection transmission among drug abusers is concerning. Interleukin-1 beta (IL-1b) is a pro-inflammatory cytokine secreted during innate and adaptive immune responses and plays a pivotal role in chronic inflammatory diseases. Functional single nucleotide polymorphisms in IL-1b cause it to play different roles in disease susceptibility and progression. This study aimed to investigate the association between genetic polymorphisms of pro-inflammatory cytokines (IL-1b) and HCV infection susceptibility in Malay male drug abusers. Methods: In total, 48 male Malay drug abusers were included in this retrospective case-control study. Genomic DNA was extracted from whole blood samples and analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the IL-1b rs16944 and rs1143634 polymorphisms. Results: Analysis of IL-1b rs1143634 revealed that the C/C genotype was common in both the case and control groups; however, no statistical significance was observed (p = 0.068, c2 = 3.755). Genotyping of IL-1b demonstrated that all samples were of the homozygous mutant type (T/T). Conclusion: There was no association between IL-1b polymorphism (rs1143634 and rs16944) and hepatitis C infection susceptibility among Malay male drug abusers.

Evaluation of Antibacterial, Antioxidant and Anti-inflammatory Properties of Methanol Extract of Varthemia iphionoides

Medicinal plants represent an essential source of active biological compounds. The current study examined the methanol extract of V. iphionoides for its antibacterial, antioxidant, and antiinflammatory activities. The antibacterial activity was evaluated against six bacterial strains (Staphylococcus xyloses, Escherichia coli, Klebsiella oxytoca, Enterobacter aerogenes, Achromabacter xylosoxidans, and Pseudomonas aerugenosa). V. iphionoides extract showed good antibacterial activity against Staphylococcus xylosus followed by, pseudomonas aeruginosa and Klebsiella oxytoca, and the minimum inhibitory concentrations (MICs) were 33, 33, and 11 µg/ml, respectively. However, the extract did not show any antibacterial activity against the other bacterial strains. The antioxidant activity of the plant extract was evaluated using ABTS and DPPH radical scavenging assays, and the Trolox equivalents were 93±0.31 and 84.8±0.28 mg Trolox/g plant extract, respectively. Anti-inflammatory activity in LPS-induced mice was investigated by measuring the concentration of pro-inflammatory cytokines TNF-alpha, IL 1beta, and antioxidant enzyme catalase and glutathione peroxidase. The results demonstrated that the highest concentration of V. iphionoides extract was the most efficient. Anti-inflammatory activity was exhibited by reducing the concentrations of pro-inflammatory TNF-α and IL 1 beta and reducing the concentrations of antioxidant enzyme catalase and glutathione peroxidase compared to the negative control group.

A phytotherapic blend immunity-6™ inhibits myeloid leukemic cells 2 activation involving purinergic signaling

Leukemia is among the most common types of hematological cancers and the use of herbal medicines to prevent and treat leukemia are under quick development. Among several molecular pathways involved in leukemia pathogenesis and exacerbations, purinergic signaling is revealed as a key component. In the present study, the effects of two doses (5 ug/mL and 10 ug/mL) of Immunity-6™, a phytocomplex composed by beta-glucan, green tea (Camelia sinensis), chamomile (Matricaria chamomilla), and ascorbic acid (vitamin C) was tested in vitro, using chronic myelogenous leukemia cell line (K-562; 5 ×104/mL/well), which were challenged with lipopolysaccharide (LPS; 1 ug/mL) for 24 h. The results demonstrated that both doses of Immunity-6™ inhibited ATP release (p < 0.001) and P2×7 receptor at mRNA levels expression (p < 0.001). Purinergic inhibition by Immunity-6™ was followed by reduced release of proinflammatory cytokines IL-1beta (p < 0.001) and IL-6 (p < 0.001), while only 5 ug/mL of Immunity-6™ reduced the release of TNF-alpha (p < 0.001). Beyond to inhibit the release of pro-inflammatory cytokines, both doses of Immunity-6™ induced the release of anti-inflammatory cytokine IL-10 (p < 0.001), while only the higher dose (10 ug/mL) of Immunity-6™ induced the release of anti-inflammatory IL-1ra (p < 0.05) and klotho (p < 0.001). Thus, Immunity-6™ may be a promising adjuvant in the treatment of leukemia and further clinical trials are guaranteed.

The Use of Agro-Industrial Waste Rich in Omega-3 PUFA during the Weaning Stress Improves the Gut Health of Weaned Piglets

(1) Background: The weaning period is a very important stage in the pig life, as during weaning, the animals are very susceptible to pathogens and develop postweaning diarrhoea. The aim of our study was to counteract weaning stress and to improve piglets’ gut health by using a nutritional intervention consisting of a mix of agro-industrial wastes (grapeseed, flaxseed and sea blackthorn meals) rich in omega-3 PUFA. (2) Methods: Twelve cross-bred TOPIG hybrid piglets with an average body weight of 11.25 kg were randomly distributed to one of the two experimental groups: a control group fed basic corn soybean diet (control diet) and an experimental group fed a diet with a 10% mixture of grapeseed, flaxseed and sea buckthorn meals in a ratio of 3:4:1 (GFS diet). (3) Results: the GFS diet had no effect on the performance, biochemical parameters or the total antibody synthesis. GFS diet was able to significantly reduce the concentration of proinflammatory cytokines IL-1 beta and TNF-alpha and to significantly increases the expression of junction proteins (occludin, claudin 4, claudin 7 and extracellular protein matrix) at the gene or protein level as compared with control. The presence of GFS in the diet increased the abundance of Bifidobacterium and Lactobacillus species in the colonic content as well as the concentration of propionic and butyric acids. (4) Conclusions: Taken together, our results showed that agro-industrial wastes rich in omega-3 PUFA can be used as an ecological, environmentally friendly nutritional intervention for improving the negative effects associated with the weaning stress.

Antioxidant and Anti-Inflammatory Activity of Star Anise (Illicium Verum) in Murine Model

Star anise (Illicium verum) is a medium-sized plant that is native to Asia as well as one of the most important medicinal plants used in Chinese herbal medicine. Star anise has bioactive compounds having antioxidant and anti-inflammatory properties. The antioxidant activity of Star anise (Illicium verum) methanolic extract was studied by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′azinobis-(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assay. The current study aims to investigate the effect of Star anise (Illicium verum) methanolic extract as ant-inflammatory by reducing the concentration of pro-inflammatory cytokines TNF-α and IL 1beta and reducing the oxidative stress by acting as a scavenger because inflammation and oxidative stress can induce each other. The highest free radical scavenging activities were exerted by the APTS method (95.1±0.33 Trolox/g) whereas, the free radical scavenging activities were exerted by ABTS was 77.7±0.30 Trolox/g. The anti-inflammatory activity of the Star anise (Illicium verum) methanolic extract was studied by its ability to inhibit pro-inflammatory cytokines productions (Tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β)) and reduce oxidative stress at different concentrations. Star anise (Illicium verum) methanolic extract significantly reduce the pro-inflammatory TNF-α and IL-1β production (p&lt;0.05) compared with negative control which is treated with lipopolysaccharide (LPS) and has a similar effect in reducing pro-inflammatory cytokines production similar to the positive control which treated with ascorbic acid. Star anise (Illicium verum) methanolic extract significantly reduces oxidative stress (p&lt;0.05) by reducing antioxidant enzyme activity catalase and glutathione-peroxidase compared with the LPS treated group. In conclusion Star anise (Illicium verum) methanolic extract act as strong antioxidant and anti-inflammatory medicinal plant.

CCL2 Inhibition of Pro-Resolving Mediators Potentiates Neuroinflammation in Astrocytes.

The chemokine CCL2 participates in multiple neuroinflammatory processes, mainly through the recruitment of glial cells. However, CCL2 has also been proven to exert different types of actions on these cells, including the modification of their response to inflammatory stimuli. In the present study we analyzed the effect of CCL2 on the resolution of inflammation in astrocytes. We observed that genetic removal of CCL2 increases the expression of the enzymes responsible for the synthesis of specialized pro-resolving mediators arachidonate 15-lipoxygenase and arachidonate 5-lipoxygenase in the brain cortex of 5xFAD mice. The expression of FPR2 receptor, known to mediate the activity of pro-resolving mediators was also increased in mice lacking CCL2.The downregulation of these proteins by CCL2 was also observed in cultured astrocytes. This suggests that CCL2 inhibition of the resolution of inflammation could facilitate the progression of neuroinflammatory processes. The production of the pro-inflammatory cytokine IL-1beta by astrocytes was analyzed, and allowed us to confirm that CCL2 potentiates the activation of astrocytes trough the inhibition of pro-resolving pathways mediated by Resolvin D1. In addition, the analysis of the expression of TNFalpha, MIP1alpha and NOS2 further confirmed CCL2 inhibition of inflammation resolution in astrocytes.

Prenatal and postnatal exposure to PFAS and cardiometabolic factors and inflammation status in children from six European cohorts.

Developing children are particularly vulnerable to the effects of exposure to per- and polyfluoroalkyl substances (PFAS), a group of endocrine disrupting chemicals. We hypothesized that early life exposure to PFASs is associated with poor metabolic health in children. We studied the association between prenatal and postnatal PFASs mixture exposure and cardiometabolic health in children, and the role of inflammatory proteins. In 1,101 mothers-child pairs from the Human Early Life Exposome project, we measured the concentrations of PFAS in blood collected in pregnancy and at 8years (range=6-12years). We applied Bayesian Kernel Machine regression (BKMR) to estimate the associations between exposure to PFAS mixture and the cardiometabolic factors as age and sex- specific z-scores of waist circumference (WC), systolic and diastolic blood pressures (BP), and concentrations of triglycerides (TG), high-density lipoprotein (HDL-C) and low-density lipoprotein (LDL-C) cholesterol. We measured thirty six inflammatory biomarkers in child plasma and examined the underlying role of inflammatory status for the exposure-outcome association by integrating the three panels into a network. Exposure to the PFAS mixture was positively associated with HDL-C and systolic BP, and negatively associated with WC, LDL-C and TG. When we examined the independent effects of the individual chemicals in the mixture, prenatal PFHxS was negatively associated with HDL-C and prenatal PFNA was positively associated with WC and these were opposing directions from the overall mixture. Further, the network consisted of five distinct communities connected with positive and negative correlations. The selected inflammatory biomarkers were positively, while the postnatal PFAS were negatively related with the included cardiometabolic factors, and only prenatal PFOA was positively related with the pro-inflammatory cytokine IL-1beta and WC. Our study supports that prenatal, rather than postnatal, PFAS exposure might contribute to an unfavorable lipidemic profile and adiposity in childhood.

P.161: Small Molecule Inhibitor of Toll-like Receptor-4, TAK-242, Attenuates Allogenic Immune Responses In Vitro

Introduction: Development of innate and adaptive immune response against transplanted islets has a major impact on the poor long term survival of allogenic islet transplants. Our previous studies have shown that TAK-242, a small molecule inhibitor of toll-like receptor-4 either in a soluble form or covalently linked to the islet surface can effectively protect islets from damage inflicted by sterile inflammation in vitro and also prolongs survival of syngeneic islet transplants. Based on recent reports indicating crosstalk between TLR-4 and major histocompatibility complex molecules, we hypothesized that TAK-242 can inhibit proliferation of lymphocytes in response to allogenic stimulation. Methods: Human peripheral blood mononuclear cells labeled with carboxyfluorescein succinimidyl ester were tested for proliferation in a one-way mixed lymphocyte reaction against mitomycin treated, CellTrace Violet-labeled allogenic splenocytes as stimulators. Proliferation of CD4+ and CD8+ T cells was analyzed by flow cytometry. Concentrations of pro-inflammatory cykokines in the culture supernatant was measured by Luminex Multiplex Bead Assay. Varying concentrations (0–30 µM) of TAK242 or MAP84, an inactive structure analog of TAK242, were added to the culture medium. Results: Addition of TAK-242 to the mixed lymphocyte reaction dose dependently inhibited proliferation of both CD4+ and CD8+ T cells (Fig. 1A). The inhibition of proliferation was optimal on day 5. Proliferation of CD4+ and CD8+ T cells was not affected by the addition of MAP84 at similar concentrations. Cytokine measurements showed that IL-2 concentrations were not inhibited by TAK-242. However, proinflammatory cytokines IL-1beta, IL-6, IP-10 and TNF-alpha were significantly inhibited specifically by TAK-242 (Fig. 1B). These results mirrored our previous data showing TAK-242 inhibition of pro-inflammatory cytokines by islets stimulated with TLR-4 ligands. Conclusion: The present data has shown for the first time that blockade of toll-like receptor-4 by TAK-242 is an effective method to attenuate development of T cell response against allogenic targets in addition to the inhibition of inflammation. Crosstalk between TLR pathway with other critical receptors involved the development of immune response is likely to play a role in this inhibition. Further experiments to decipher the mechanism underlying this unique immunosuppressive property of TAK-242 are in progress.

Synchrotron fluorescence imaging of individual mouse beta-cells reveals changes in zinc, calcium, and iron in a model of low-grade inflammation.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158± 57.7 femtograms (fg) to 65.7± 29.7 fg; calcium decreased from 216± 67.4 to 154.3± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4± 12.2 to 47.2± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Exposure to cytokines IL‐6 and IL‐1beta changes intracellular distributions and total content of calcium, zinc and iron in mouse primary pancreatic beta‐cells

Pancreatic beta-cells secrete insulin, a hormone that maintains blood glucose homeostasis. The dysfunction of beta-cells leads to development of type2 diabetes (T2D). The causes of beta-cell dysfunction in T2D are complex. In obese individuals, increases in circulating proinflammatory cytokines IL-6 and IL-1beta are linked to a higher risk of developing diabetes, suggesting that these cytokines can be diabetes-promoting. When used at circulating concentrations found in obese individuals, these cytokines have been shown to alter calcium homeostasis in mouse beta-cells. In addition, changes in homeostasis of zinc and iron have been reported in diabetic individuals. The central hypothesis of this study: exposure to diabetes-promoting cytokines, at concentrations found in obese individuals, leads to changes in metal intracellular concentrations or distributions in beta-cells. Mouse primary beta-cells were exposed for 48 hours to cytokines: 10pg/mL IL-1beta and 20pg/mL IL-6. After cytokine exposure the cells were chemically fixed and synchrotron X-Ray fluorescence (SXRF) was employed, to investigate intracellular distributions and concentrations of zinc, calcium and iron. SXRF estimated the total cellular iron content to be 30.44 ± 12.18 (fg), and after acute exposure to cytokines the iron content was slightly increased to 47.21 ± 36.44 (fg) (mean ± S.D.). High concentrations of iron were localized in puncta structures, which were observed throughout the cytosol in all beta-cells and further analysis of the iron puncta revealed that iron was found at higher density in puncta of cells after they were exposed to cytokines, suggesting iron accumulation in these structures. The total cellular zinc content was 158.69 ± 57.68 (fg) in control cells and was significantly decreased to 65.73 ± 29.65 (fg) in cells after exposure to cytokines (mean ± S.D.) (Welch's t-test, t(28.97) = 6.655, P<0.0001). Zinc was localized to a perinuclear space of the cells in control cells and was evenly distributed after exposure to cytokines, suggesting that cytokines had a significant effect on zinc homeostasis in beta-cells. Calcium was also observed in perinuclear space of beta-cells before cytokine exposure and was localized to similar cellular compartments as zinc. Synchrotron X-ray fluorescence estimated total cellular calcium to be 216.10 ± 67.37 femtograms (fg) control cells and was significantly decreased to 154.28 ± 68.66 (fg) after the exposure to cytokines (mean ± S.D.), (Two sample t-test, t(43)=3.040, P=0.0040). In conclusion, the synchrotron X-ray fluorescence identified significant changes to zinc, calcium and iron metallomes of pancreatic beta-cells after exposure to cytokines, which warrant further investigation.

Telomere length dependent regulation of IL1R1 (Interleukin 1Receptor type I) by TRF2 (Telomere repeat binding factor 2) is crucial to TAM (tumor associated macrophage) infiltration in cancer cells

TRF2 (Telomere Repeat Binding Factor 2) is a known component of the mammalian shelterin complex and is critical for telomere maintenance. However, recent findings suggest that TRF2 can associate with DNA outside of telomeres including several gene promoters. Our work shows that TRF2 can modulate transcription by inducing local epigenetics in a telomere length dependent manner. Herein, we present the first evidence that TRF2 can occupy the IL1R1 (Interleukin 1 Receptor Type 1) promoter by associating with promoter G-quadruplexes. TRF2 activates the IL1R1 promoter by primarily inducing H3K27ac by recruiting p300 Histone acetyl transferase to the promoter. As a consequence of TRF2 mediated activation of IL1R1, cells with high TRF2 have enhanced sensitivity to the pro-inflammatory cytokine IL1 beta and downstream activation of NF kappa B targets like IL6, IL8 and TNFα. As suggested by previous studies, the activation of the IL1 pathway might have implications in cancer progression by inducing the production of pro-inflammatory cytokines in the tumour microenvironment. Recent reports suggest the IL1R1- IL1 beta axis of paracrine and autocrine signaling are crucial to the maintenance of Tumor associated macrophages (TAM) and consequent suppression of immune response against cancer cells. Our work goes on to explore how TRF2 mediated upregulation of IL1R1 in cancer cells might have crucial implications in recruitment and maintenance of TAMs in the tumor micro-environment and its connection to telomere length of cancer cells.

Additive Antidepressant Effects of Combined Administration of Ecitalopram and Caloric Restriction in LPS-Induced Neonatal Model of Depression in Rats

Abstract Background and aims: Increasing evidence indicates that inflammation in the periphery and neuroinflammation in the brain might be involved in the pathophysiology of depressive symptoms in humans. Relatively little is known about the effects of selective serotonin re-uptake inhibitors (SSRI) on individuals exposed to differential dietary regimens, like caloric restriction (CR). The aim of the current study is to assess the antidepressant and antineuroinflammatory effects of CR in single administration and combined with SSRIsantidepressant escitalopram in LPS-induced model of depression in Wistar rats. Materials and methods: For this purpose, we used 36 Wistar rats and applied 3 behavioral tests for depression (FST, SPT and NSFT) in animals and an ELISA-method for measurement of brain IL-1beta levels. Results: Behavioral assessment and results from ELISA-method have shown that CR not only augments the effect of the antidepressant escitalopram on forced swim test (FST) and sucrose preference test (SPT), but also reduces the brain levels of proinflammatory cytokine IL-1beta. Combined with escitalopram, CR enhances antidepressant and antinflamatory properties of this SSRI. Discussion and conclusion: These results show that the response to antidepressive treatment depends on the diverse dietary regimens, especially low-caloric diet. We suggest that the background of this is augmentation of anidepressant and antineuronflammatory properties of some antidepressants by CR. Manipulation of dietary regimens is attractive and new strategy for the management of pharmacoresistant depression.

Chronic Inflammatory-Modulating Potential of Cassia auriculata with Proinflammatory Cytokine IL-1beta and Its Anticancer Effect on Lung Cancer Cell Line.

Inflammation is a key element in tumor progression, over time, persistent inflammation causes damage to DNA and leads to cancer. The relationship between chronic inflammation and tumor development is well established, blocking of which can help in cancer prevention and treatment in the future. Hence, with this background, the present study aims to evaluate the anti-inflammatory and anticancer potential of Cassia auriculata (CA) solvent fractions through in silico and in vitro means, respectively. Generally, inflammatory mediators play a key task in chronic inflammation, following its inflection was chosen for their interactions with nine structurally varied phytoconstituents of CA identified through GCMS. The ethanolic extract of CA was assessed for its apoptotic effects on A549 lung cancer cells by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, JC-10 staining, DNA fragmentation assay and quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The interactions between bioactive components and target protein revealed that important molecules like 5,7-dihydroxy-2-[2-nethoxyphenyl]- 4H-1-Benzopyran-4-one, a flavonoid, and three other components can bind target interleukin 1-beta associated with lung cancer. In vitro data also confirmed that the diverse active components of CA extract might follow the intrinsic mitochondrial pathway to provoke cancer cell death. Hence, these findings strongly propose that Cassia auriculata (CA) solvent fractions could be exploited in the future to design ligands for obtaining novel leads for treating chronic inflammation linked with lung cancer, and also the extracts of CA can be recommended as a potential agent for lung cancer chemotherapy.