IL-6 Protein Release Research Articles

Dexamethasone, Remdesivir and Azithromycin modulate ACE2 and IL-6 in Lung Epithelial Cells.

The optimal use of steroids in COVID-19 patients remains challenging. Current S3-guidelines "Recommendations for patients with COVID-19" recommend dexamethasone (DEX) for patients requiring respiratory support, remdesivir (RD) in the early disease phase and azythromycin (AZ) is no longer recommended. We investigated effects of DEX, RD and AZ in a lipopolysaccharide induced inflammation in lung cells in vitro and analyzed publicly available datasets with a focus on the Angiotensin-converting enzyme 2 (ACE2) to better understand drugs' mechanisms of action. human bronchial (Calu) and alveolar (A549) lung epithelial cells were treated with DEX, AZ or RDV in the presence of lipopolysaccharides (LPS). Gene expression (GE) of ACE2, IL-6 and the IL-6 protein release were measured. Publicly available GE data from lung tissues of COVID-19 patients and from lung cells treated with DEX were analyzed for the GE of ACE2. DEX increased and RDV and AZ reduced the GE of ACE2 in LPS-stimulated bronchial and alveolar epithelial cells. Only DEX significantly reduced LPS-induced IL-6 releases in alveolar cells substantially. The database analyses showed an, albeit not always significant, increase in ACE2 for lung tissue or cell lines treated with DEX. Lung tissue from patients after COVID-19 infection as well as bronchial cell cultures after COVID-19 infection showed lower GEs of ACE2. DEX can increase ACE2 expression in vitro and thereby the portal of entry of SARS-CoV-2 into lung cells during an LPS induced inflammation. Simultaneously the inflammatory marker IL-6 is reduced. Comparative database analyses indicate that these processes can also take place in vivo.

In vitro evidence of antioxidant and anti-inflammatory effects of a new nutraceutical formulation explains benefits in a clinical setting of COPD patients.

Background and Aim: Increased oxidative stress within the airways is associated to epithelial damage and amplification of inflammatory responses that in turn contribute to Chronic Obstructive Pulmonary Disease (COPD) progression. This study was aimed to identify whether a new formulation of N-acetylcisteine (NAC), carnitine, curcumin and B2 vitamin could counteract oxidative stress and downstream pro-inflammatory events promoted by cigarette smoke extract (CSE) exposure in primary bronchial epithelial cells (PBEC), both submerged/undifferentiated (S-PBEC) and cultured at the air-liquid interface (ALI-PBEC). Methods: PBEC were exposed to CSE with/without the new formulation or NAC alone and ROS production, IL-8 and IL-6 gene expression and protein release were evaluated. Results: CSE increased ROS, IL-8 and IL-6 gene expression and protein release and the new formulation counteracted these effects. NAC alone was not effective on IL-8 and IL-6 release. The effects of a similar nutraceutical formulation were evaluated in COPD patients treated for six months. The results showed that the treatment reduced the concentration of IL-8 in nasal wash and improved quality of life. Conclusion: The tested formulation, exerting antioxidant and anti-inflammatory effects, can preserve airway epithelial homeostasis and improve clinical symptoms in COPD.

INTERVERTEBRAL DISC CELLS IN VIVO INTERNALIZE GRAM-POSITIVE BACTERIA: IMPLICATIONS FOR INTERVERTEBRAL DISC DEGENERATION

A key cause of low back pain is the degeneration of the intervertebral disc (IVD). Causality between infection of the IVD and its degenerative process gained great interest over the last decade. Granville Smith et al. (2021) identified 36 articles from 34 research studies investigating bacteria in human IVDs. Bacteria was identified in 27 studies, whereas 9 attributed bacterial presence to contamination. Cutibacterium acnes was the most abundant, followed by coagulase-negative staphylococcus. However, whether bacteria identified were present in vivo or represent perioperative contamination remains unclear. This study investigated whether bacteria are present in IVDs and what potential effects they may have on native disc cells.Immunohistochemical staining for Gram positive bacteria was performed on human IVD tissue to identify presence and characterise bacterial species. Nucleus pulposus (NP) cells in monolayer and 3D alginate were stimulated with LPS and Peptidoglycan (0.1-50 µg/ml) for 48hrs. Following stimulation qPCR for factors associated with disc degeneration including matrix genes, matrix degrading enzymes, cytokines, neurotrophic factors and angiogenic factors and conditioned media collected for ELISA and luminex analysisGram positive bacteria was detected within human IVD tissue. Internalisation of bacteria by NP cells influenced the cell and nuclei morphology. Preliminary results of exposure of NP cells to bacterial components indicate that LPS as well as Peptidoglycan increase IL-8 and ADAMTS-4 gene expression following 48 hours of stimulation with a dose response seen for IL-8 induction by peptidoglycan compared to the control group. Underlining these results, IL-8 protein release was increased for treated groups compared to non-treated control. Further analysis is underway investigating other output measures and additional biological repeats.This study has demonstrated bacteria are present within IVD cells within IVD tissue removed from degenerate IVD and is determining the potential influence of these on disc degeneration.

Grape Pomace Polyphenols as a Source of Compounds for Management of Oxidative Stress and Inflammation—A Possible Alternative for Non-Steroidal Anti-Inflammatory Drugs?

Flavonoids, stilbenes, lignans, and phenolic acids, classes of polyphenols found in grape pomace (GP), were investigated as an important alternative source for active substances that could be used in the management of oxidative stress and inflammation. The benefic antioxidant and anti-inflammatory actions of GP are presented in the literature, but they are derived from a large variety of experimental in vitro and in vivo settings. In these in vitro works, the decrease in reactive oxygen species, malondialdehyde, and thiobarbituric acid reactive substances levels and the increase in glutathione levels show the antioxidant effects. The inhibition of nuclear factor kappa B and prostaglandin E2 inflammatory pathways and the decrease of some inflammatory markers such as interleukin-8 (IL-8) demonstrate the anti-inflammatory actions of GP polyphenols. The in vivo studies further confirmed the antioxidant (increase in catalase, superoxide dismutase and glutathione peroxidase levels and a stimulation of endothelial nitric oxide synthase -eNOS gene expression) and anti-inflammatory (inhibition of IL-1𝛼, IL-1β, IL-6, interferon-𝛾, TNF-α and C-reactive protein release) activities. Grape pomace as a whole extract, but also different individual polyphenols that are contained in GP can modulate the endogenous pathway responsible in reducing oxidative stress and chronic inflammation. The present review analyzed the effects of GP in oxidative stress and inflammation, suggesting that it could become a valuable therapeutic candidate capable to reduce the aforementioned pathological processes. Grape pomace extract could become an adjuvant treatment in the attempt to reduce the side effects of the classical anti-inflammatory medication like non-steroidal anti-inflammatory drugs (NSAIDs).

Nerve Growth Factor (NGF) as Partaker in the Modulation of UV-Response in Cultured Human Conjunctival Fibroblasts.

Corroborating data sustain the pleiotropic effect of nerve growth factor (NGF) in the protection of the visual system from dangerous stimuli, including ultraviolet (UV). Since UV exposure might promote ocular surface changes (conjunctival inflammation and matrix rearrangement), as previously reported from in vivo studies sustaining some protective NGF effects, in vitro cultures of human conjunctival fibroblasts (FBs) were developed and exposed to a single UV exposure over 15 min (0.277 W/m2), either alone or supplemented with NGF (1–10–100 ng/mL). Conditioned media and cell monolayers were collected and analyzed for protein release (ELISA, ELLA microfluidic) and transcript expression (real-time PCR). A specific “inflammatory to remodeling” pattern (IL8, VEGF, IL33, OPN, and CYR61) as well as a few epigenetic transcripts (known as modulator of cell differentiation and matrix-remodeling (DNMT3a, HDAC1, NRF2 and KEAP1)) were investigated in parallel. UV-exposed FBs (i), showed no proliferation or significant cytoskeleton rearrangement; (ii), displayed a trkANGFR/p75NTR phenotype; and (iii), synthesized/released IL8, VEGF-A, IL33, OPN, and CYR61, as compared to unexposed ones. NGF addition counteracted IL8, IL33, OPN, and CYR61 protein release merely at lower NGF concentrations but not VEGF. NGF supplementation did not affect DNMT3a or HDAC1 transcripts, while it significantly upregulated NRF2 at lowest NGF doses and did not change KEAP1 expression. Taken together, a single UV exposure activated conjunctival FBs to release pro-inflammatory/fibrogenic factors in association with epigenetic changes. The effects were selectively counteracted by NGF supplementation in a dose-dependent fashion, most probably accountable to the trkANGFR/p75NTR phenotype. Further in vitro studies are underway to better understand this additional NGF pleiotropic effect. Since UV-shield impairments represent a worldwide alert and UV radiation can slowly affect ocular surface homeostasis (photo-ageing, cataract) or might exacerbate ocular diseases with a preexisting fibrosis (pterygium, VKC), these findings on NGF modulation of UV-exposed FBs might provide additional information for protecting the ocular surface (homeostasis) from low-grade long-lasting UV insults.

Biological Response-Enhancing Activity with Antigens in A549 Cells Exposed to Representative Polycyclic Aromatic Hydrocarbons.

The question of what kinds of airborne particles, including diesel exhaust particles and their adherent chemical constituents, exacerbate the activity of allergic and inflammatory respiratory diseases has not been elucidated in detail. Therefore, chemicals that have amplifying actions on Dermatophagoides farinae (Df) body extract-induced IL-8, the inflammatory cytokines of the innate immune system, were comprehensively examined using commonly used human alveolar epithelial cells, A549, as simple screening for 17 polycyclic aromatic hydrocarbons (PAHs), which are representative organic constituents in atmospheric samples. The significant amplifying actions of two PAHs, dibenzo[a,l]pyrene (DB[a,l]P) at 50 nM and dibenzo[a,i]pyrene (DB[a,i]P) at 2 μM for 48 h, for IL-8 protein release induced by mite antigens in epithelial cells were observed for the first time. In contrast, the enhancement of IL-8 was not observed in protein levels for these PAHs without the antigens. Meanwhile, the significant synergistic amplifying effect of DB[a,l]P at 50 nM on proinflammatory actions was measured in gene expression (i.e., IL-8, IL-6, ICAM-1, and TNF-α) levels in the experimental setting; for the results, the induction of TNF-α may have been the essential factor that enhanced the amplifying activity of DB[a,l]P for IL-8 gene expression and protein release. Examining the exacerbating effect on allergic pathophysiological states for DB[a,l]P is planned for further study.

Anti-inflammatory evaluation of Scurrula ferruginea (jack) danser parasitizing on Tecoma stans (L.) H.B.K. in LPS/IFN-γ-induced RAW 264.7 macrophages

Ethnopharmacological relevanceScurrula ferruginea (Jack) Danser (locally known as ‘Dedalu’ or ‘dian nan ji sheng’ in Malaysia and China) is a hemi-parasitic shrub that is widely used as herbal medicine to treat inflammation, rheumatism, and stroke. However, the scientific basis of its anti-inflammatory function and mechanism remain to be proven.Aim of the study: To evaluate the anti-inflammatory activity as well as the preliminary mechanism of S. ferruginea parasitizing on Tecoma stans. Materials and methodsThe anti-inflammatory capability of freeze-dried stem aqueous extract was assessed via inhibition of inflammatory cytokines interleukin- (IL-) 1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) production in lipopolysaccharide (LPS) and interferon-γ (IFN-γ) stimulated RAW 264.7 macrophages. The underlying anti-inflammatory mechanism was deciphered through reverse transcriptase and real time quantitative polymerase chain reactions (RT-PCR and qPCR) for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), IL-1β, and TNF-α mRNA expression. ResultsThe results exhibited that aqueous extract of freeze-dried S. ferruginea stem sample concentration-dependently inhibited IL-1β protein production along with the down regulation of iNOS and IL-1β mRNA expression. Moreover, it significantly suppressed the protein release of IL-6 and IL-10 in a concentration-dependent manner. However, it slightly reduced TNF-α at higher sample concentration (250 μg/mL) without affecting the mRNA expression levels of COX-2 and TNF-α. ConclusionsThis study suggests that S. ferruginea parasitizing on Tecoma stans exerted anti-inflammatory capability attributed to inhibition of iNOS and IL-1β mRNA expression, NO creation, IL-1β, IL-6, IL-10, and TNF-α protein production, indicating this plant might be a useful plant-derived candidate against inflammation.

Malvidin prevents lipopolysaccharide-induced oxidative stress and inflammation in human peripheral blood mononuclear cells.

It is indicated that malvidin has anti-inflammatory and antioxidant effects on various cells, although the function of malvidin in preventing inflammatory reactions caused by lipopolysaccharide (LPS) in peripheral blood mononuclear cells (PBMCs) is still not known. The objective of this study was to examine the impact of malvidin on inflammatory responses and oxidative stress in PBMCs as caused by LPS. The present findings showed that LPS significantly increased the expression of IL-6, TNF-α, IL-1β, and COX-2 mRNA and protein release from PBMCs 22 hr after treatments. It was also revealed that increased levels in cytokine expression coincided with increased phosphorylation of JNK, P65-NF-κB, and IKKα/IKKβ. Also, the expression of IL-6, TNF-α, IL-1β, and COX-2 mRNA induced by LPS as well as secretion of protein in PBMC has been significantly decreased by pretreatment of malvidin. Importantly, pretreatment of the cells with malvidin completely abrogated the phosphorylation of P65-NF-κB, JNK, and IKKα/IKKβ in LPS treated cells. Malvidin protection against LPS-induced inflammation was coupled with a decline in the levels of nitric oxide metabolite and malondialdehyde, along with an increase in the ferric reducing antioxidant power, total thiol activity, and also superoxide dismutase and glutathione peroxidase activity. In accordance with this finding, malvidin may represent a promising therapeutic agent for the prevention of inflammation in PBMCs.

P002 Specific genome editing to model hypermethylation of the SP140 gene that associates to Crohn’s disease

Abstract Background SP140 (Speckled 140 KDa) encodes an epigenetic reader protein with an immune restricted expression, that binds to epigenetically modified (acetylated and methylated) histones and thereby regulates expression of large gene sets, including pro-inflammatory cytokines, in innate immune cells. SP140 is implicated in CD because single nucleotide polymorphisms, as well as defective protein function are associated with CD and marks anti-TNF response. Through a genome-wide methylation screen of Crohn’s disease (CD) patients peripheral blood, we identified two hypermethylated positions in SP140 locus associated with CD patients. We hypothesise that this DNA hypermethylation at the SP140 locus controls SP140 expression in CD patients contributing to their colitis development. Methods To address the role of SP140 DNA methylation, we used CRISPR ‘dead’ Cas9 (dCas9) epigenome-editing for specifically adding methyl groups (dCas9-DNMT) or removing methyl groups (dCas9-TET) in monocyte cell line THP1. We developed guide RNAs complementary to the gene expression regulatory region of the SP140 gene. With lentiviral delivery, we transduced THP-1 cells with guide RNA-lentiviruses, and with dCas9-DNMT or dCas9-TET lentiviruses. We assessed the level of SP140 methylation using bisulphite Sanger sequencing and the effect of methylation intervention of SP140 using qPCR and ELISA for SP140, IL-6, TNFα, IL-1β. Results We observed that SP140 gene in THP-1 cells under control conditions contained little methylated CpG sites. We induced sp140 hypermethylation through transduction of dCas9-DNMT. We validated hypermethylation of the two SP140 CpGs in transduced THP1 cells, thus mimicking the observed hypermethylation in CD patients cells. SP140 hypermethylation in THP1 cells polarised into M1 macrophages and stimulated with lipoteichoic acid (TLR-2 ligand), displayed a decrease of TNFα and (p = 0.042) protein levels. Similarly, we showed a decrease of TNFα (p = 0.02) and IL-6 (p = 0.03) protein release after transduction dCas9-DNMT and stimulation with LPS or Zymosan (TLR-2/4 ligand). Conclusion In this study, we demonstrated that editing SP140 gene methylation through CRISPR-dCAS9 technology allows modelling of the relevance of epigenetic marks for CD aetiology. Through methylome editing, we could affect the expression of CD-associated pro-inflammatory genes. Our dCas9 technique will allow us to investigate the role of DNA-methylation in the aetiology of CD.

First Report on Ex Vivo Delivery of Paracrine Active Human Mesenchymal Stromal Cells to Liver Grafts During Machine Perfusion.

Machine perfusion is rapidly becoming the standard for graft preservation and provides new opportunities for organ salvage, reconditioning, and repair. Based on their immune-modulatory and regenerating properties,1 arguable mesenchymal stromal cells (MSCs) are the most promising cell therapy for graft repair on the pump, though proof of concept is still lacking.2 Here, we show the feasibility of delivering clinically relevant numbers of human MSCs (hMSCs; 5–10 × 106/kg) during 30-minute hypothermic oxygenated machine perfusion in porcine liver grafts (n = 8; Figure 1). Full methods are described in Supplemental Materials and Methods (SDC, https://links.lww.com/TP/B821). To track the biodistribution of hMSC, cells were genetically labeled with click beetle red luciferase.3 As shown in Figure 2, bioluminescent imaging of both the anterior and posterior side of the liver showed a wide range and patchy distribution of hMSC which retain throughout the liver after 30 minutes of perfusion. No significant difference between arterial (n = 4) or venous infusion (n = 4) was observed. Histological and RNA-expression analysis confirmed the delivery of the hMSC throughout the liver grafts. About 104–105 hMSC were estimated to be still present per kilogram of liver graft. Importantly, the hMSC retained their paracrine activity after infusion. Using both Luminex and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, increasing levels of human-specific interleukin-6 and interleukin-8 released in porcine blood were shown during 4-hour normothermic perfusion. In conclusion, this is the first report showing an effective delivery of hMSC in a liver machine perfusion model. Evidence of paracrine activity of hMSC after delivery indicates regenerative and immune-modulatory effects during normothermic graft perfusion, though this needs further research in a transplantation model.FIGURE 1.: Graphical abstract of the procedure. The timeline indicates the details of each time point. Pig livers are procured after a warm ischemia period of 30 min and infused with 5 × 106–107 click beetle red luciferase (CBred2)-labeled human mesenchymal stromal cell (hMSC) either via the portal vein or hepatic artery. The distribution of hMSC is visualized by bioluminescence imaging (BLI) immediately (time point 0) after infusion using an IVIS Imaging System 200. The liver is then placed on a purpose-made perfusion device in which it is perfused with cold (10°C) oxygenated University of Wisconsin (UW)-organ preservation fluid. After 30 min of perfusion, the distribution of the hMSC is again imaged using BLI. After imaging, the livers were perfused for 4 h with their own, heparinized, full blood at normothermic temperature (37°C) by ex vivo machine perfusion after which biopsies were collected for further analysis. The BLI signal is quantified, presence of hMSC was assessed in biopsies taken from left and right periphery and center of the liver using human-specific antibodies, proteomic and genetic analysis. HOPE, hypothermic oxygenated machine perfusion; qPCR, quantitative polymerase chain reaction.FIGURE 2.: Effective delivery of paracrine active human mesenchymal stromal cells (hMSCs) during normothermic hepatic machine perfusion. Live imaging of the livers (n = 8) infused with click beetle red luciferase (CBred2)-transduced hMSC in the hepatic artery (n = 4; A, B, E, F) or portal vein (n = 4; C, D, G, H) using the IVIS Imaging System 200, directly after infusion (directly after infusion, A, B, C, D) and after 30 min of perfusion (E, F, G, H). Images are representative images showing bright luciferin-based luminescence at the posterior (A, C, E, G) and anterior side (B, D, F, H) of the liver, demonstrating whole organ coverage of the infused hMSC. The presence of hMSC was confirmed by immunohistochemical staining for copepod green fluorescent protein (copGFP) (I arrows; ×40 objective) at end of perfusion, whereas no staining was observed in liver biopsies before infusion (J). Representative pictures are shown. Human-specific B2M gene expression was determined by nested quantitative polymerase chain reaction (qPCR) to confirm the presence of hMSC in the pig liver at the genetic level (M). Shown are the raw cycle threshold (Ct) values of B2M expression of 3 pigs. B2M expression was measured in the pig samples that were perfused with hMSC, (pig 1–3) and in nonperfused pig livers (n = 3). Luminex-based analysis using human-specific cytokines showed that interleukin (IL)-6 (O) and IL-8 (P) was secreted in the liver perfusate in 2 of the 4 pigs that were infused via the hepatic artery (filled symbols). In pig livers that were not infused with hMSC, no IL-6 or IL-8 was detected (open symbols). Human-specific IL-6 and IL-8 protein release was confirmed in the same 2 pig livers by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis (data not shown). nd, not detectable.

Exogenous Clara cell protein 16 attenuates silica particles-induced inflammation in THP-1 macrophages by down-regulating NF-κB and caspase-1 activation.

Inhalation of silica particles leads to pulmonary inflammatory responses. Clara cell protein 16 (CC16) has been reported to played a protective role in inflammatory lung diseases. However, its role on silica particles-induced inflammation has not been fully clarified. In this study, THP-1 macrophages were exposed to 75 μg/cm2 silica particles with or without 2 μg/mL exogenous CC16 (recombinant CC16, rCC16) for 24 hr. The production of inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6, in the cell supernatants of different groups was detected through ELISA kits and real-time RT-PCR, respectively. The nuclear translocation of nuclear factor (NF)-κB, protein levels of pro-IL-1β, the nucleotide-binding domain-like receptor protein 3 (NLRP3) and caspase-1 were evaluated via immunofluorescence or western blot. Results showed that, at 75 μg/cm2 silica particle concentration, the treatment of rCC16 significantly decreased IL-1β, TNF-α and IL-6 protein release and mRNA levels in THP-1 macrophages. Compared to those only exposed to silica particles, THP-1 macrophages exposed to both silica particles and rCC16 showed significantly lower nuclear levels and higher cytosol levels of NF-κB p65, as well as lower co-localization coefficients through immunofluorescence. Additionally, the administration of rCC16 significantly attenuated the increase of pro-IL-1β, NLRP3 and caspase-1 levels induced by silica particle exposure. Our results suggested that exogenous CC16 could inhibit silica particles-induced inflammation in THP-1 macrophages, mainly through suppressing NF-κB pathway and caspase-1 activation.

Wnt-3a Induces Cytokine Release in Human Mast Cells

Mast cells are well known for their detrimental effects in allergies and asthma, and Wnt signaling has recently been implicated in asthma and other airway diseases. However, it is not known if or how Wnts affect human mast cells. Since Wnt expression is elevated in individuals with asthma and is linked to a Th2 profile, we hypothesized that mast cells could be affected by Wnts in the context of asthma. We therefore sought to investigate the role of Wnt signaling in human mast cell development and activation. We first examined the expression of the 10 main Wnt receptors, Frizzled 1–10 (FZD1–10), and found expression of several FZDs in human mast cells. Treatment with purified recombinant Wnt-3a or Wnt-5a did not affect the proliferation or maturation of CD34+ progenitors into mast cells, as indicated by cellular expression of CD117 and FcεRI, activation by FcεRI crosslinking, and histamine and tryptase release. Furthermore, Wnt treatment did not change the phenotype from MCT to MCTC, since MrgX2 expression, compound 48/80-mediated activation, and carboxypeptidase A3 content were not affected. However, Wnt-3a activated WNT/β-catenin signaling in mature human mast cells, as revealed by stabilization of β-catenin, upregulation of IL-8 and CCL8 mRNA expression, and release of IL-8 protein. Thus, our data suggest that Wnt-3a activation of mast cells could contribute to the recruitment of immune cells in conditions associated with increased Wnt-3a expression, such as asthma.

Anti-inflammatory effects of interleukin-4 on intervertebral disc cells

BACKGROUND CONTEXTInflammation has been associated with a number of pathological conditions including intervertebral disc (IVD) degeneration, increased risks of low back pain and other spinal diseases. Downregulating disc inflammation may be a strategy to reduce degeneration and more importantly back pain. Interleukin (IL)-4 was first discovered as a T-cell secreted factor that enhanced the proliferation of anti-IgM stimulated B cells and is now known as a cytokine that can stimulate cell proliferation and differentiation, tissue regeneration and neurological functions. IL-4 has been shown to be effective in inhibiting inflammatory pathways in chondrocytes. Immunohistochemical studies have shown that disc tissues are immunopositive for IL-4 receptor α (IL-4Rα) and IL-4. Yet, the roles of IL-4 and IL-4R in disc biology remain unknown. PURPOSEThe purpose of this study is to understand the roles of IL-4 and IL-4Rα in IVDs and to determine if IL-4 can function to inhibit inflammation in IVD cells. STUDY DESIGN/SETTINGIn vitro experiment. METHODSDeidentified patient IVD tissues were collected after surgery under the Orthopedic Information, Tissue and Implant Repository (ORA L00011021). IVD cells were isolated and cultured in monolayer. IL-4R protein expression was analyzed using immunocytochemistry. To test if the IL-4R was responsive to its ligand, signal transducer and activator of transcription 6 (STAT6) phosphorylation was analyzed on cell lysates of IVD cells treated with recombinant human IL-4 for 30 minutes using enzyme linked immunosorbent assay kit. Gene expression analysis of IL-4 up- and downregulated genes were analyzed using real-time RT-PCR. Anti-inflammatory effects of IL-4 were determined by cotreating disc cells with lipopolysaccharide (LPS) and IL-4 and measuring gene expression and protein release of inflammatory markers, IL-6 and IL-8. The significance of differences among means of data on gene expression and protein analyses were analyzed by one-way analysis of variance or student t test. Differences were considered significant when the p value was below 0.05. RESULTSImmunocytochemistry staining for IL-4Rα in primary IVD cells (n=8) showed the majority of immunopositive staining was intracellular. After IVD cells (n=3–7) were treated with different concentrations of recombinant human IL-4 (0.1–100 ng/mL) for 30 minutes, phospho-STAT6 levels significantly increased by two- to four-fold at all concentrations tested compared with untreated cells. Gene expression of IL-4Rα and IL-6 increased significantly in cells undergoing IL-4 treatment for 24 hours compared with control treated IVD cells (n=5–10). LPS stimulated inflammatory gene expression of interferon (IFN)β, IL-12, IL-6, and IL-8 were downregulated significantly in the presence of IL-4 (n=7). Lastly, protein release of IL-6 and IL-8 were reduced significantly in cells treated with IL-4 and LPS compared with those treated with LPS alone (n=7). CONCLUSIONSThis study was the first to explore the function of IL-4 and IL-4R in IVD cells. Immunocytochemistry studies confirmed that the majority of cells isolated from patient IVDs expressed IL-4Rα at the protein level. Also, IVD cells can respond to IL-4 by up-regulating IL-4Rα and IL-6 genes and inhibiting inflammatory genes and proteins induced by LPS. Further studies to test the anti-inflammatory effects of IL-4 in the IVD would be needed in animal models. CLINICAL RELEVANCEBiological therapies which include intradiscal delivery of cells, anti-inflammatories or growth factors are being investigated to treat disc degeneration and back pain in animal models and in the clinic. Based on our findings that IL-4 has anti-inflammatory effects on IVD cells, the results of this study suggest including recombinant IL-4 delivery into the intervertebral disc may be a beneficial therapeutic strategy to treat patients with back pain by reducing disc inflammation.

MiR-21 upregulation increases IL-8 expression and tumorigenesis program in airway epithelial cells exposed to cigarette smoke.

Cigarette smoke exposure, increasing Toll-like receptor 4 (TLR4) and reactive oxygen species (ROS), promotes inflammatory responses in airway epithelial cells. Chronic inflammation, microRNA (miRNA), and oxidative stress are associated with cancer development. The present study was aimed to explore whether cigarette smoke exposure, altering miR-21 expression, promoted inflammatory responses and tumorigenesis processes in airway epithelial cells. Airway normal and cancer epithelial cells (16HBE and A549) were exposed to cigarette smoke extracts (CSE) or with/without agomiR-21, and then it was assessed: a) miR-21 expression; b) signal transducer and activator of transcription 3 (STAT3) nuclear protein expression and ERK1/2 activation; c) IL-8 gene expression and protein release. An antagonist of TLR4 (CLI-095) and the antioxidant flavonoid, apigenin, were also included to evaluate miR-21 expression in CSE exposed cells. It was demonstrated that: a) A549 cells constitutively expressed higher levels of miR-21 and IL-8; b) CSE increased STAT3 nuclear expression in 16HBE; c) in both cell lines, CSE and agomiR-21 increased: miR-21 expression; ERK1/2 activation and IL-8 gene expression and protein release; d) TLR4 inhibition counteracted the effects of CSE on miR-21 in A549; e) apigenin reduced miR-21 and IL-8 gene expression in both cell lines. Data herein provided identified for the first time new mechanisms supporting the crucial role of cigarette smoke-induced miR-21 expression in the amplification of inflammatory responses and in tumorigenesis processes within the airways.

14-3-3E, a new alarmin candidate, elicits a catabolic and proinflammatory effect involving innate immunity through TLR signaling in osteoarthritis

Purpose: Osteoarthritis (OA) is now considered as a whole-joint disease characterized by cartilage degradation, synovial inflammation and bone remodeling. Recent studies have shown the implication of the innate immune system and low-grade inflammation in OA progression. Our team identified 14-3-3ε as a novel soluble mediator critical in the communication between subchondral bone and cartilage in OA. This protein acts as a potent MMP-3 and MMP-13-stimulatory factor in chondrocytes leading to catabolic phenotype. 14-3-3ε seems to share common characteristics with alarmins, endogenous molecules with intracellular functions that are released in the extracellular media after infection or tissue damage. Alarmins (such as S100 and HMGB1 involved in cartilage degradation) bind to receptors expressed on the surface of cells engaged in host defense and tissue repair. These receptors include the Toll-like receptors, TLR2 and TLR4. Moreover, innate alarmins can stimulate the activation of resident immune cells of the synovium, particularly macrophages leading to synovitis. As well as alarmins, 14-3-3ε could determine macrophage polarization. Our aim is to investigate the interactions between 14-3-3ε and its potential receptors, TLR2 and TLR4. Methods: Primary cultures of chondrocytes were performed from murine cartilage of the C57BL/6J mice (5 days old), and from human cartilage samples, obtained from OA patients undergoing total joint replacement surgery. The receptors TLR2 and TLR4 were blocked by monoclonal antibodies and pharmacologic inhibitors in chondrocytes (15mn) and then stimulated by recombinant 14-3-3ε during 24h. The stimulation by 14-3-3ε was also done on primary cultures of chondrocytes from TLR4 KO or TLR2 KO mice pups. Stimulation by 14-3-3ε has been performed with addition of Polymyxin B to avoid endotoxin effects. Immunocytochemistry was realized on murine chondrocytes with receptors antibodies. Chondrocyte gene expression and release of MMP-3, MMP-13 and IL-6 were evaluated by RT-PCR and ELISA. The direct interaction of 14-3-3ε with TLR2 was measured using Surface plasmonic resonance (SPR) on a Biacore 3000 instrument. Their molecular interaction was modeled in silico using GrammX and Rosetta programs to model the interaction and optimize the predicted conformations, respectively. The best complexes of 14-3-3ε with TLR2 were visually analyzed using PyMOL. M1 polarization of macrophages was performed by activating THP1 cells (ATCC) with PMA during 24h and and followed by 24h stimulation with LPS or 14-3-3ε. Different markers of the M1 type were analysed by ELISA and RT-qPCR to identify the polarisation of macrophages (M1 : TNF, IL-6, IL-1β, MCP1, Nos2). Results: TLR2 and TLR4 receptors were found on murine articular chondrocytes by immunocytochemistry using monoclonal antibodies. Invalidation of TLR2 and TLR4 receptors by blocking antibody in articular chondrocytes reduced significantly mRNA expression and protein release of MMP-3, MMP-13 and IL-6 induced by 14-3-3ε. For MMP3 mRNA expression, the inhibition rate reached 78% with blocking antibodies and 98% with OxPAPC, a TLR2/TLR4 inhibitor. The inhibition rate reached 65% (blocking antibodies), 98% (OxPAPC) for MMP13 and 74% (blocking antibodies) and 93% (OxPAPC) for IL-6. ELISA experiments showed an inhibition rate of 84% (blocking antibodies) and 97% (OxPAPC) for MMP3. Similar rates were observed for IL-6 secretion (83% and 97%). Moreover, the effect of 14-3-3ε on MMP3, MMP13 and IL6 expression was reduced in TLR4-KO and abolished in TLR2-KO chondrocytes. In the Biacore analysis, human recombinant TLR2 and TLR4 were covalently immobilized on CM5 chip. Results demonstrated a direct interaction between recombinant TLR2 and 14-3-3ε (83 RU) and between TLR4 and 14-3-3ε (41 RU). Computational structural analysis identified in silico a putative region of interaction, centered on a phosphorylation site in TLR2. This region of binding is consistent as 14-3-3ε is known to accommodate specifically phosphorylated residues. The affinity measurements between 14-3-3ε and peptides of TLR2 designed accordingly to this phospho-site are currently in progress. Finally, polarization of macrophages stimulated by recombinant 14-3-3ε seems to polarize them toward a M1 inflammatory phenotype. Conclusions: We have discovered 14-3-3ε as a new catabolic factor, produced by the subchondral bone and able to induce a catabolic phenotype on chondrocytes. These effects are mediated, at least in part, by TLR2 or TLR4 signalling, leading to the activation of innate immunity. Taken together, our results designate 14-3-3ε as a novel alarmin in OA, triggering catabolic and inflammatory effects through TLRs signaling. This new alarmin candidate could represent a new target either for therapeutic and/or prognostic purposes.

S100A8 & S100A9: Alarmin mediated inflammation in tendinopathy

Alarmins S100A8 and S100A9 are endogenous molecules released in response to environmental triggers and cellular damage. They are constitutively expressed in immune cells such as monocytes and neutrophils and their expression is upregulated under inflammatory conditions. The molecular mechanisms that regulate inflammatory pathways in tendinopathy are largely unknown therefore identifying early immune effectors is essential to understanding the pathology. Based on our previous investigations highlighting tendinopathy as an alarmin mediated pathology we sought evidence of S100A8 & A9 expression in a human model of tendinopathy and thereafter, to explore mechanisms whereby S100 proteins may regulate release of inflammatory mediators and matrix synthesis in human tenocytes. Immunohistochemistry and quantitative RT-PCR showed S100A8 & A9 expression was significantly upregulated in tendinopathic tissue compared with control. Furthermore, treating primary human tenocytes with exogenous S100A8 & A9 significantly increased protein release of IL-6, IL-8, CCL2, CCL20 and CXCL10; however, no alterations in genes associated with matrix remodelling were observed at a transcript level. We propose S100A8 & A9 participate in early pathology by modulating the stromal microenvironment and influencing the inflammatory profile observed in tendinopathy. S100A8 and S100A9 may participate in a positive feedback mechanism involving enhanced leukocyte recruitment and release of pro-inflammatory cytokines from tenocytes that perpetuates the inflammatory response within the tendon in the early stages of disease.

Effect of Phyllanthus emblica L. fruit on improving regulation of methylglyoxal-induced insulin resistance in 3T3-L1 cells

The increasing methylglyoxal (MG) level of body has been found in people with obesity and insulin resistance, resulting from their dietary style and abnormal metabolic functions. MG promotes inflammation, oxidative stress, glycation, and all of which are closely related to insulin resistance and chronic diseases. Phyllanthus emblica L. fruit has various bioactivities such as anti-inflammation, anti-diabetes, anti-nonalcoholic fatty liver, and anti-dyslipidemia. Therefore, this study was aimed to investigate the effects of water extract of P. emblica (WEPE) and its enriched compound, ellagic acid, on MG-induced inflammation, insulin resistance, and lipogenesis in 3T3-L1 cells. The results showed that MG activated the peroxisome proliferator activated receptor-gamma (PPARγ) and CCAAT/ enhancer-binding protein alpha (C/EBPα), which can increase adipogenesis in adipocytes. In addition, MG enhanced pro-inflammatory cytokine IL-6 protein expression and release through the activation of MAPK and NF-κB signaling pathway, as well as increasing the monocyte chemoattractant protein-1 expression to cause macrophage infiltration. MG also significantly reduced glucose uptake, indicating that insulin resistance in obese patients may be related to MG generation. WEPE and ellagic acid effectively decreased IL-6 protein expression and cytokine release through inactivation of JNK and p65 pathways. WEPE and ellagic acid significantly increased glucose uptake and reduced insulin resistance by MG treatment. WEPE also decreased the protein-tyrosine phosphatase 1B to reduce insulin resistance and inhibited MG-induced fat accumulation related proteins such as PPARγ, C/EBPα, FAS, and p-ACC. Therefore, WEPE may have the potential to ameliorate MG-induced inflammation, increase glucose uptake, and decrease fat accumulation.

The phosphodiesterase 5 inhibitor sildenafil decreases the proinflammatory chemokine IL-8 in diabetic cardiomyopathy: in vivo and in vitro evidence

PurposeInterleukin (IL)-8 is a proinflammatory C-X-C chemokine involved in inflammation underling cardiac diseases, primary or in comorbid condition, such diabetic cardiomyopathy (DCM). The phosphodiesterase type 5 inhibitor sildenafil can ameliorate cardiac conditions by counteracting inflammation. The study aim is to evaluate the effect of sildenafil on serum IL-8 in DCM subjects vs. placebo, and on IL-8 release in human endothelial cells (Hfaec) and peripheral blood mononuclear cells (PBMC) under inflammatory stimuli.MethodsIL-8 was quantified: in sera of (30) DCM subjects before (baseline) and after sildenafil (100 mg/day, 3-months) vs. (16) placebo and (15) healthy subjects, by multiplatform array; in supernatants from inflammation-challenged cells after sildenafil (1 µM), by ELISA.ResultsBaseline IL-8 was higher in DCM vs. healthy subjects (149.14 ± 46.89 vs. 16.17 ± 5.38 pg/ml, p < 0.01). Sildenafil, not placebo, significantly reduced serum IL-8 (23.7 ± 5.9 pg/ml, p < 0.05 vs. baseline). Receiver operating characteristic (ROC) curve for IL-8 was 0.945 (95% confidence interval of 0.772 to 1.0, p < 0.01), showing good capacity of discriminating the response in terms of drug-induced IL-8 decrease (sensitivity of 0.93, specificity of 0.90). Sildenafil significantly decreased IL-8 protein release by inflammation-induced Hfaec and PBMC and downregulated IL-8 mRNA in PBMC, without affecting cell number or PDE5 expression.ConclusionSildenafil might be suggested as potential novel pharmacological tool to control DCM progression through IL-8 targeting at systemic and cellular level.

Inflammatory cytokine oncostatin M induces endothelial activation in macro- and microvascular endothelial cells and in APOE*3Leiden.CETP mice.

AimsEndothelial activation is involved in many chronic inflammatory diseases, such as atherosclerosis, and is often initiated by cytokines. Oncostatin M (OSM) is a relatively unknown cytokine that has been suggested to play a role in both endothelial activation and atherosclerosis. We comprehensively investigated the effect of OSM on endothelial cell activation from different vascular beds and in APOE*3Leiden.CETP mice.Methods and resultsHuman umbilical vein endothelial cells, human aortic endothelial cells and human microvascular endothelial cells cultured in the presence of OSM express elevated MCP-1, IL-6 and ICAM-1 mRNA levels. Human umbilical vein endothelial cells and human aortic endothelial cells additionally expressed increased VCAM-1 and E-selectin mRNA levels. Moreover, ICAM-1 membrane expression is increased as well as MCP-1, IL-6 and E-selectin protein release. A marked increase was observed in STAT1 and STAT3 phosphorylation indicating that the JAK/STAT pathway is involved in OSM signaling. OSM signals through the LIF receptor alfa (LIFR) and the OSM receptor (OSMR). siRNA knockdown of the LIFR and the OSMR revealed that simultaneous knockdown is necessary to significantly reduce MCP-1 and IL-6 secretion, VCAM-1 and E-selectin shedding and STAT1 and STAT3 phosphorylation after OSM stimulation. Moreover, OSM administration to APOE*3Leiden.CETP mice enhances plasma E-selectin levels and increases ICAM-1 expression and monocyte adhesion in the aortic root area. Furthermore, Il-6 mRNA expression was elevated in the aorta of OSM treated mice.ConclusionOSM induces endothelial activation in vitro in endothelial cells from different vascular beds through activation of the JAK/STAT cascade and in vivo in APOE*3Leiden.CETP mice. Since endothelial activation is an initial step in atherosclerosis development, OSM may play a role in the initiation of atherosclerotic lesion formation.

Curcumin ameliorates palmitate-induced inflammation in skeletal muscle cells by regulating JNK/NF-kB pathway and ROS production.

Curcumin, a natural polyphenol compound, has the beneficial effects on several diseases such as metabolic syndrome, cancer, and diabetes. The anti-inflammatory property of curcumin has been demonstrated in different cells; however, its role in prevention of palmitate-induced inflammation in skeletal muscle C2C12 cells is not known. In this study, we examined the effect of curcumin on the inflammatory responses stimulated by palmitate in C2C2 cells. The results showed that palmitate upregulated the mRNA expression and protein release of IL-6 and TNF-α cytokines in C2C12 cells, while pretreatment with curcumin was able to attenuate the effect of palmitate on inflammatory cytokines. The anti-inflammatory effect of curcumin was associated with the repression of phosphorylation of IKKα-IKKβ, and JNK. Palmitate also caused an increase in reactive oxygen species (ROS) level that curcumin abrogated it. Collectively, these findings suggest that curcumin may represent a promising therapy for prevention of inflammation in skeletal muscle cells.