Upregulation Of Interleukin-8 Expression Research Articles

Toll-like receptor 2-mediated ERK activation significantly upregulates interleukin-6 expression in M2-polarized macrophages

Abstract Objectives M2-polarized macrophages and interleukin (IL)-6 significantly alter the tumor microenvironment and promote the malignant behaviors of tumor cells. This study aimed to establish M2-type macrophages from THP-1 cells, which are human leukemia monocytes, and investigate the significance of toll-like receptor 2 (TLR2) signaling in IL-6 production. Methods THP-1 cells were treated with phorbol 12-myristate 13-acetate, IL-4, and IL-13 to stimulate their differentiation into M2 macrophages. Cell differentiation was confirmed by cytokine production, marker expression, and morphological alterations. Treatment with TLR agonists induced TLR stimulation in M2 macrophages. Subsequently, secretion and expression levels of IL-6 in M2 macrophages were measured using enzyme-linked immunosorbent assay, quantitative reverse transcription-polymerase chain reaction, and western blotting. Results Myeloid differentiation factor 88, tumor necrosis factor-associated factor 6, and IL-1 receptor-associated kinase-1/4 signaling pathways contributed to IL-6 production upon TLR2 activation in M2 macrophages. While both TLR2 and TLR4 activated NF-κB in M2 macrophages, IL-6 production was mainly dependent on TLR2, not TLR4, suggesting the involvement of major mechanisms other than NF-κB in IL-6 production. Notably, TLR2-stimulated extracellular signal-regulated kinase (ERK) was necessary for abundant IL-6 production, indicating that TLR2-mediated ERK signaling plays an essential role in M2 macrophages. Conclusions These results highlight the significance of TLR2 signaling in IL-6 production by M2 macrophages and provide insights into the underlying regulatory mechanisms.

Acute myeloid leukemia (AML)-derived mesenchymal stem cells induce chemoresistance and epithelial-mesenchymal transition-like program in AML through IL-6/JAK2/STAT3 signaling.

Acute myeloid leukemia (AML) has a high rate of treatment failure due to increased prevalence of therapy resistance. Mesenchymal stem cells (MSCs) in the leukemia microenvironment contribute to chemoresistance in AML, but the specific mechanism remains unclear. The critical role of the epithelial-mesenchymal transition (EMT)-like profile in AML chemoresistance has been gradually recognized. However, there is no research to suggest that the AML-derived bone marrow mesenchymal stem cells (AML-MSCs) induce the EMT program in AML thus far. We isolated AML-MSCs and cocultured them with AML cells. We found that AML-MSCs induced a significant mesenchymal-like morphology in drug-resistant AML cells, but it was scarce in parental AML cells. The AML-MSCs promoted growth of AML cells in the presence or absence of chemotherapeutics in vitro and in vivo. Acute myeloid leukemia MSCs also induced EMT marker expression in AML cells, especially in chemoresistant AML cells. Mechanistically, AML-MSCs secreted abundant interleukin-6 (IL-6) and upregulated IL-6 expression in AML cells. Acute myeloid leukemia cells upregulated IL-6 expression in AML-MSCs in turn. Meanwhile, AML-MSCs activated the JAK2/STAT3 pathway in AML cells. Two JAK/STAT pathway inhibitors counteracted the AML-MSCs induced morphology change and EMT marker expression in AML cells. In conclusion, AML-MSCs not only promote the emergence of chemoresistance but also enhance it once AML acquires chemoresistance. AML-MSCs induce EMT-like features in AML cells; this phenotypic change could be related to chemoresistance progression. AML-MSCs induce the EMT-like program in AML cells through IL-6/JAK2/STAT3 signaling, which provides a therapeutic target to reverse chemoresistance in AML.

EBV Rta-induced IL-6 Promotes Migration of Bystander Tumor Cells Through IL-6R/JAK/STAT3 Pathway In Vitro.

Rta, a transactivator of Epstein-Barr virus, is associated with progression of nasopharyngel carcinoma (NPC); however, its mechanism of contribution to the pathogenesis of NPC remains unclear. Interleukin-6 (IL-6), a tumor promoter, is detected in NPC. This in vitro study examined whether and how Rta promotes NPC progression by up-regulating IL-6. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, ELISA, immunoblotting assays, reporter gene assays, and transwell migration assays were performed. In NPC cells, Rta up-regulated IL-6 expression at the mRNA and protein levels, and the Rta's C-terminus was essential for promoter activation and expression of IL-6. The induction of IL-6 by Rta also required activation of extracellular signal-regulated kinase 1/2 and activator protein-1. Furthermore, IL-6 secreted from Rta-expressing NPC cells promoted migration of Rta-negative NPC cells by activating IL-6 receptor/Janus kinase/signal transducer and activator of transcription 3 pathway. Rta contributes to progression of NPC cells through induction of IL-6 in vitro.

Regulation of stimulus-induced interleukin-8 gene transcription in human adrenocortical carcinoma cells – Role of AP-1 and NF-κB

Stimulation of H295R adrenocortical carcinoma cells with angiotensin II or cytokines induces the secretion of the chemokine interleukin-8 (IL-8). Here, we have analyzed the molecular mechanism of stimulus-induced IL-8 expression. IL-8 expression and IL-8 promoter activity increased in H295R cells expressing an activated Gαq-coupled designer receptor. H295R cells stimulated with either interleukin-1β (IL-1β) or phorbol ester also showed elevated IL-8 mRNA levels and higher IL-8 promoter activities. Deletion and point mutations of the IL-8 promoter revealed that the AP-1 binding site within the IL-8 promoter is essential to connect designer receptor stimulation with the transcriptional activation of the IL-8 gene. Expression of a constitutively active mutant of c-Jun, or expression of constitutively active mutants of the protein kinases MEKK1 and MKK6 confirmed that the IL-8 gene is a bona fide target of AP-1 in adrenocortical carcinoma cells. Upregulation of IL-8 expression in IL-1β-treated H295R cells required NF-κB while the phorbol ester TPA used both the AP-1 and NF-κB sites of the IL-8 gene to stimulate IL-8 expression. These data were corroborated in experiments with chromatin-embedded AP-1 or NF-κB-responsive reporter genes. While stimulation of Gαq-coupled designer receptors increased the AP-1 activity in the cells, IL-1β specifically stimulated NF-κB-regulated transcription. Stimulation of the cells with TPA increased both AP-1 and NF-κB activities. We conclude that stimulation of Gαq-coupled designer receptors or IL-1 receptors triggers distinct signaling pathways in H295R cells leading to the activation of either AP-1 or NF-κB. Nevertheless, both signaling cascades converge to the IL-8 gene, inducing IL-8 gene transcription.

Effects of SIRT1/Akt pathway on chronic inflammatory response and lung function in patients with asthma.

Asthma is the most common chronic airway inflammatory disease. Sirtuin 1 (SIRT1) exerts a crucial effect on regulating chronic inflammatory responses. Therefore, this study aims to explore the effect of SIRT1 on the pathogenesis of asthma. Serum level of SIRT1 in asthma patients and healthy controls was detected by Western blot. Correlation between SIRT1 level and pulmonary function in asthma patients was analyzed. Subsequently, asthma model in mouse was established. Primary airway epithelial cells were extracted from asthma mice and control mice to detect SIRT1 level. Furthermore, relative levels of Akt and interleukin 6 (IL-6) were detected in 16HBE cells. Regulatory effects of Akt on SIRT1 in 16HBE cells were determined as well. SIRT1 was highly expressed in serum of asthma patients, which was negatively correlated with FEV1/FVC (r=-0.27, **p<0.01). Both mRNA and protein levels of SIRT1 were downregulated in primary airway epithelial cells extracted from asthma mice compared with those from controls. SIRT1 knockdown in 16HBE cells upregulated IL-6 expression, which was reversed by Akt inhibitors. SIRT1 regulates IL-6 level via Akt pathway, thereafter affecting pulmonary function in asthma patients.

Inflammatory response and biomechanical properties of coaxial scaffolds for engineered skin in vitro and post-grafting

Engineered skin (ES) offers many advantages over split-thickness skin autografts for the treatment of burn wounds. However, ES, both in vitro and after grafting, is often significantly weaker, less elastic and more compliant than normal human skin. Biomechanical properties of ES can be tuned in vitro using electrospun co-axial (CoA) scaffolds. To explore the potential for coaxial scaffold-based ES use in vivo, two CoA scaffolds were fabricated with bioactive gelatin shells and biodegradable synthetic cores of polylactic acid (PLA) and polycaprolactone (PCL), and compared with gelatin monofilament scaffolds. Fibroblast and macrophage production of inflammatory cytokines interleukin 6 (IL-6) and transforming growth factor β-1 was significantly higher when cultured on PLA and PCL monofilament scaffolds compared to gelatin monofilament scaffolds. The core-shell fiber configuration significantly reduced production of pro-inflammatory cytokines to levels similar to those of gelatin monofilament scaffolds. In vitro, ES mechanical properties were significantly enhanced using CoA scaffolds; however, after grafting CoA- and gelatin-based ES to full-thickness excisional wounds on athymic mice, the in vitro mechanical advantage of CoA grafts was lost. A substantially increased inflammatory response to CoA-based ES was observed, with upregulation of IL-6 expression and a significant M2 macrophage presence. Additionally, expression of matrix metalloproteinase I was upregulated and collagen type I alpha 1 was downregulated in CoA ES two weeks after grafting. These results suggest that while coaxial scaffolds provide the ability to regulate biomechanics in vitro, further investigation of the inflammatory response to core materials is required to optimize this strategy for clinical use. Statement of SignificanceEngineered skin has been used to treat very large burn injuries. Despite its ability to heal these wounds, engineered skin exhibits reduced biomechanical properties making it challenging to manufacture and surgically apply. Coaxial fiber scaffolds have been utilized to tune the mechanical properties of engineered skin while maintaining optimal biological properties but it is not known how these perform on a patient especially with regards to their inflammatory response. The current study examines the biomechanical and inflammatory properties of coaxial scaffolds and uniaxial scaffolds in vitro and in vivo. The results show that the biological response to the scaffold materials is a critical determinant of tissue properties after grafting with reduced inflammation and rapid scaffold remodeling leading to stronger skin.

Inhibition of Proteasome Activity Upregulates IL-6 Expression in RPE Cells through the Activation of P38 MAPKs.

Purpose As far as we know, during the development of age-related macular degeneration (AMD), the activity of proteasome in retinal pigment epithelium cells (RPE) gradually decreases. And a lot of research has shown that age-related macular degeneration is closely related to inflammation and autoimmune. Moreover, there are many cytokines (CKs) involved in the course of inflammation. In this study, we are going to investigate how the decrease of proteasome activity affects the production of interleukin-6 (IL-6) in human retinal pigment epithelium cells (ARPE-19). Methods Cultured ARPE-19 was treated with or without MG132, a proteasome inhibitor, and the levels of IL-6 mRNA (messenger ribonucleic acid) in RPE at 1 h, 4 h, 8 h, and IL-6 protein in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were measured by real-time polymerase chain reaction (real-time PCR) and enzyme-linked immunosorbent assay (ELISA). The protein levels of MCP-1 (monocyte chemoattractant protein-1) in the culture medium at 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h were also measured by ELISA. Then we tested which of cell signal pathways regulating the production of IL-6 were activated when we added MG132 into the medium by Western blot and electrophoretic mobility shift assays (EMSA). After that, we put the inhibitors of these activated cell signal pathways into the medium individually to see which inhibitor can counteract the effect of upregulating the levels of IL-6 in the culture medium of RPE. Results MG132 decreased the secretion of MCP-1 in the culture medium of RPE, but it increased the expression of IL-6 mRNA in RPE and IL-6 protein level in the culture medium of RPE. MG132 treatment was also found to enhance the level of phosphorylated p38 mitogen-activated protein kinases (MAPKs) and c-Jun N-terminal Kinase (JNK) by Western blotting. More importantly, the effect of MG132 on upregulating the levels of IL-6 was inhibited by SB203580, an inhibitor of P38 MAP kinases. But the JNK inhibitor, SP600125, cannot prevent the effect of upregulating the levels of IL-6 by MG132 in the RPE culture medium. Conclusions We concluded that the proteasome inhibitor, MG132, upregulates IL-6 production in RPE cells through the activation of P38 MAPKs.

Follistatin-like protein 1 plays a tumor suppressor role in clear-cell renal cell carcinoma

BackgroundWe previously showed that the expression of follistatin-like protein 1 (FSTL1) was significantly down-regulated in metastatic clear-cell renal cell carcinoma (ccRCC). In this study, we aimed to characterize the role of FSTL1 in the development of ccRCC.MethodsThe effects of FSTL1 on cell activity and cell cycle were investigated in ccRCC cell lines with altered FSTL1 expression. Gene expression microarray assays were performed to identify the major signaling pathways affected by FSTL1 knockdown. The expression of FSTL1 in ccRCC and its effect on postoperative prognosis were estimated in a cohort with 89 patients.ResultsFSTL1 knockdown promoted anchorage-independent growth, migration, invasion, and cell cycle of ccRCC cell lines, whereas FSTL1 overexpression attenuated cell migration. FSTL1 knockdown up-regulated nuclear factor-κB (NF-κB) and hypoxia-inducible factor (HIF) signaling pathways, increased epithelial-to-mesenchymal transition, up-regulated interleukin-6 expression, and promoted tumor necrosis factor-α-induced degradation of NF-κB inhibitor (IκBα) in ccRCC cell lines. FSTL1 immunostaining was selectively positive in epithelial cytoplasm in the loop of Henle, and positive rate of FSTL1 was significantly lower in ccRCC tissues than in adjacent renal tissues (P < 0.001). The multivariate Cox regression analysis showed that the intratumoral FSTL1 expression conferred a favorable independent prognosis with a hazard ratio of 0.325 (95% confidence interval 0.118–0.894). HIF-2α expression was negatively correlated with FSTL1 expression in ccRCC specimens (r = − 0.229, P = 0.044). Intratumoral expression of HIF-2α, rather than HIF-1α, significantly predicted an unfavorable prognosis in ccRCC (log-rank, P = 0.038).ConclusionsFSTL1 plays a tumor suppression role possibly via repressing the NF-κB and HIF-2α signaling pathways. To increase FSTL1 expression might be a candidate therapeutic strategy for metastatic ccRCC.

CCAR2 negatively regulates IL-8 production in cervical cancer cells.

Cell cycle and apoptosis regulator 2 (CCAR2) is a multifaceted protein that controls diverse cellular functions; however, its function in cancer is unclear. To better understand its potential role in cancer, we examined gene expression patterns regulated by CCAR2 in cervical cancer cells. Cytokine and chemokine production by CCAR2-deficient cells increased under oxidative conditions. In particular, H2O2-treated CCAR2-depleted cells showed a significant increase in interleukin-8 (IL-8) production, indicating a negative regulation of IL-8 by CCAR2. Upregulation of IL-8 expression in CCAR2-deficient cells occurred via activation of transcription factor AP-1. The negative correlation between CCAR2 and IL-8 expression was confirmed by examining mRNA and protein levels in tissues from cervical cancer patients. Furthermore, CCAR2-regulated IL-8 expression is associated with a shorter survival of cervical cancer patients. Overall, the data suggest that CCAR2 plays a critical role in controlling both the cancer secretome and cancer progression.

Treatment with Mountain-Cultivated Ginseng Alleviates Trimethyltin-Induced Cognitive Impairments in Mice via IL-6-Dependent JAK2/STAT3/ERK Signaling.

Panax ginseng is the most widely used herbal medicine for improving cognitive functions. The pharmacological activity and underlying mechanisms of mountain-cultivated ginseng, however, have yet to be clearly elucidated, in particular, against trimethyltin-induced cognitive dysfunction. We previously reported that interleukin-6 plays a protective role against trimethyltin-induced cognitive dysfunction. Because of this, we have implemented a study system that uses interleukin-6 null (-/-) and wild-type mice. Interestingly, mountain-cultivated ginseng significantly upregulated interleukin-6 expression. With this study, we sought to determine whether the interleukin-6-dependent modulation of the Janus kinase 2/signal transducer activator of transcription 3 and extracellular signal-regulated kinase signaling network is also associated with the pharmacological activity of mountain-cultivated ginseng against trimethyltin-induced cognitive dysfunction. Trimethyltin treatment (2.4 mg/kg, intraperitoneal) causes the downregulation of Janus kinase 2/signal transducer activator of transcription 3, extracellular signal-regulated kinase signaling, and impairment of the cholinergic system. We found that mountain-cultivated ginseng treatment (50 mg/kg, intraperitoneal) significantly attenuated cognitive impairment normally induced by trimethyltin by upregulating p-Janus kinase 2/signal transducer activator of transcription 3, p-extracellular signal-regulated kinase signaling, and the cholinergic system. Trimethyltin-induced cognitive impairments were more pronounced in interleukin-6 (-/-) mice than wild-type mice, and they were markedly reduced by treatment with either mountain-cultivated ginseng or recombinant interleukin-6 protein (6 ng, intracerebroventricular). Additionally, treatment with either AG490 (20 mg/kg, intraperitoneal), a Janus kinase 2/signal transducer activator of transcription 3 inhibitor, or U0126 (2 µg/head, intracerebroventricular), an extracellular signal-regulated kinase inhibitor, reversed the effects of mountain-cultivated ginseng treatment. The effects of mountain-cultivated ginseng treatment were comparable to those of recombinant interleukin-6 protein in interleukin-6 (-/-) mice. Our results, therefore, suggest that mountain-cultivated ginseng acts through interleukin-6-dependent activation of Janus kinase 2/signal transducer activator of transcription 3/extracellular signal-regulated kinase signaling in order to reverse cognitive impairment caused by trimethyltin treatment.

Role of Suppressor of Cytokine Signaling 3 (SOCS3) in Altering Activated Microglia Phenotype in APPswe/PS1dE9 Mice.

In response to changes of the central nervous system environment, microglia are capable of acquiring diverse phenotypes for cytotoxic or immune regulation and resolution of injury. Alzheimer's disease (AD) pathology also induces several microglial activations, resulting in production of pro-inflammatory cytokines and reactive oxygen species or clearance of amyloid-β (Aβ) through phagocytosis. We previously demonstrated that microglial activation and increase in oxidative stress started from the middle age in APPswe/PS1dE9 mice, and hypothesized that M1 activation occurs in middle-aged AD mice by Aβ stimulation. In the present study, we analyzed in vivo expressions of pro-inflammatory cytokines (M1 microglial markers), M2 microglial markers, and suppressor of cytokine signaling (SOCS) family, and examined the microglial phenotypic profile in APPswe/PS1dE9 mice. Then we compared the in vitro gene expression patterns of Aβ- and lipopolysaccharide (LPS)-stimulated primary-cultured microglia. Microglia in APPswe/PS1dE9 mice exhibited an M1-like phenotype, expressing tumor necrosis factor α (TNFα) but not interleukin 6 (IL6). Aβ-stimulated primary-cultured microglia also expressed TNFα but not IL6, whereas LPS-stimulated primary-cultured microglia expressed both pro-inflammatory cytokines. Furthermore, both microglia in APPswe/PS1dE9 mice and Aβ-stimulated primary-cultured microglia expressed SOCS3. Reduction of SOCS3 expression in Aβ-challenged primary-cultured microglia resulted in upregulation of IL6 expression. Our findings indicate that SOCS3 suppresses complete polarization to M1 phenotype through blocking IL6 production, and Aβ-challenged primary-cultured microglia replicate the in vivo gene expression pattern of microglia in APPswe/PS1dE9 mice. Aβ may induce the M1-like phenotype through blocking of IL6 by SOCS3.

SN38 increases IL-8 expression through the MAPK pathways in HCT8 cells.

The overexpression of interleukin-8(IL-8) is closely associated with poor tumor differentiation, metastasis and tumor progression. This study aimed to examine the effects and mechanisms of action of SN38(a metabolite of the camptothecin derivative, CPT-11) on IL-8 expression in HCT8 cells, using ELISA, CCK-8 and western blot analysis. Among jatrorrhizine, evodiamine, 5-fluorouracil and SN38, SN38 was found to inhibit the proliferation of HCT8 cells in a dose-dependent manner, but to increase IL-8 secretion from HCT8 cells. Of the other agents, evodiamine was found to inhibit both IL-8 secretion and cell proliferation, and jatrorrhizine was found to increase IL-8 secretion without any obvious inhibitory effect on cell proliferation. Further experiments revealed that the increased activation of p38mitogen-activated protein kinase(MAPK), extracellular signal-regulated kinase(ERK)1/2 and c-Jun N-terminal kinase(JNK) by SN38 contributed to the decreased cell proliferation and to the overexpression of IL-8 induced by SN38. Our results suggested that the MAPK pathways are activated by SN38, resulting in the upregulation of IL-8 expression and in the inhibition of cell proliferation in an IL-8-independent manner. Thus, the potential benefit of the use of a combination of camptothecin-11 with other chemical drugs with inhibitory effects on IL-8 expression, should be paid more attention in treating colon cancer.

Citrullination of histone H3 drives IL-6 production by bone marrow mesenchymal stem cells in MGUS and multiple myeloma

Multiple myeloma (MM), an incurable plasma cell malignancy, requires localisation within the bone marrow. This microenvironment facilitates crucial interactions between the cancer cells and stromal cell types that permit the tumour to survive and proliferate. There is increasing evidence that the bone marrow mesenchymal stem cell (BMMSC) is stably altered in patients with MM—a phenotype also postulated to exist in patients with monoclonal gammopathy of undetermined significance (MGUS) a benign condition that precedes MM. In this study, we describe a mechanism by which increased expression of peptidyl arginine deiminase 2 (PADI2) by BMMSCs in patients with MGUS and MM directly alters malignant plasma cell phenotype. We identify PADI2 as one of the most highly upregulated transcripts in BMMSCs from both MGUS and MM patients, and that through its enzymatic deimination of histone H3 arginine 26, PADI2 activity directly induces the upregulation of interleukin-6 expression. This leads to the acquisition of resistance to the chemotherapeutic agent, bortezomib, by malignant plasma cells. We therefore describe a novel mechanism by which BMMSC dysfunction in patients with MGUS and MM directly leads to pro-malignancy signalling through the citrullination of histone H3R26.

A homeobox protein, NKX6.1, up-regulates interleukin-6 expression for cell growth in basal-like breast cancer cells

Among breast cancer subtypes, basal-like breast cancer is particularly aggressive, and research on the molecules involved in its pathology might contribute to therapy. In this study, we found that expression of NKX6.1, a homeobox transcription factor, is higher in basal-like breast cancer than in other subtypes. In loss-of-function experiments on basal-like breast cancer cell lines, NKX6.1-depleted cells exhibited reduced cell growth. Because cytokine interleukin-6 (IL-6) is expressed in basal-like breast cancer, and increases cell growth, we analyzed expression levels of IL6, an IL-6 gene, and observed reduced IL6 expression in NKX6.1-depleted cells. In a reporter assay, IL6 promoter activity was reduced by loss of NKX6.1 function. A pull-down assay showed that NKX6.1 binds to the proximal region in IL6 promoter. These results indicate that NKX6.1 directly up-regulates IL6 expression. To investigate further, we established cells with forced expression of IL-6. We observed that exogenous IL-6 expression restored the reduced cell growth of NKX6.1-depleted cells. Furthermore, orthotopic xenografts showed that NKX6.1-depleted cells lost the capacity for tumor formation. We therefore conclude that NKX6.1 is a factor for IL-6-regulated growth and tumor formation in basal-like breast cancer. Our findings facilitate profound understanding of basal-like breast cancer, and the development of suitable therapy.

Oxidative stress and endocytosis are involved in upregulation of interleukin-8 expression in airway cells exposed to PM2.5.

Inhaled PM2.5 (particulate matter with an aerodynamic diameter of 2.5 μm or less) can induce lung inflammation through released inflammatory mediators from airway cells, such as interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α). However, the mechanisms underlying PM2.5-induced IL-8 gene expression have not been fully characterized. BEAS-2B cells (a human bronchial epithelial cell line) and THP-1 cells (a human macrophage-like cell line) were used as the in vitro models to investigate the underlying mechanism in this study. IL-8 expression was increased in the cells treated with PM2.5 in a dose-dependent manner. The water-soluble and insoluble fractions of PM2.5 suspension were both shown to induce IL-8 expression. PM2.5 exposure could obviously induce ROS (reactive oxygen species) generation, indicative of oxidative stress. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) potently inhibited PM2.5-induced IL-8 expression. Employment of the transition metal chelators including TPEN (N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine) or DFO (desferrioxamine) inhibited IL-8 expression induced by PM2.5 by over 20% in BEAS-2B cells, but had minimal effect in THP-1 cells. Pretreatment with the endocytosis inhibitor CytD markedly blocked IL-8 expression induced by PM2.5 in both BEAS-2B and THP-1 cells. In summary, exposure to PM2.5 induced IL-8 gene expression through oxidative stress induction and endocytosis in airway cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1869-1878, 2016.

Tenascin-C May Accelerate Cardiac Fibrosis by Activating Macrophages via the Integrin αVβ3/Nuclear Factor–κB/Interleukin-6 Axis

Tenascin-C (TN-C) is an extracellular matrix protein not detected in normal adult heart, but expressed in several heart diseases closely associated with inflammation. Accumulating data suggest that TN-C may play a significant role in progression of ventricular remodeling. In this study, we aimed to elucidate the role of TN-C in hypertensive cardiac fibrosis and underlying molecular mechanisms. Angiotensin II was administered to wild-type and TN-C knockout mice for 4 weeks. In wild-type mice, the treatment induced increase of collagen fibers and accumulation of macrophages in perivascular areas associated with deposition of TN-C and upregulated the expression levels of interleukin-6 and monocyte chemoattractant protein-1 as compared with wild-type/control mice. These changes were significantly reduced in TN-C knockout/angiotensin II mice. In vitro, TN-C accelerated macrophage migration and induced accumulation of integrin αVβ3 in focal adhesions, with phosphorylation of focal adhesion kinase (FAK) and Src. TN-C treatment also induced nuclear translocation of phospho-NF-κB and upregulated interleukin-6 expression of macrophages in an NF-κB-dependent manner; this being suppressed by inhibitors for integrin αVβ3 and Src. Furthermore, interleukin-6 upregulated expression of collagen I by cardiac fibroblasts. TN-C may enhance inflammatory responses by accelerating macrophage migration and synthesis of proinflammatory/profibrotic cytokines via integrin αVβ3/FAK-Src/NF-κB, resulting in increased fibrosis.

Ox-LDL Upregulates IL-6 Expression by Enhancing NF-κB in an IGF2-Dependent Manner in THP-1 Macrophages.

Interleukin 6 (IL-6) is a pro-inflammatory cytokine that is well established as a vital factor in determining the risk of coronary heart disease and pathogenesis of atherosclerosis. Moreover, accumulating evidences have shown that oxidized low-density lipoprotein (ox-LDL) can promote IL-6 expression in macrophages. Nevertheless, the underlying mechanism of how ox-LDL upregulates IL-6 expression remains largely unexplained. We found that the expression of insulin-like growth factor 2 (IGF2), nuclear factor kappa B (NF-κB), and IL-6 was upregulated at both the messenger RNA (mRNA) and protein levels in a dose-dependent manner when treated with 0, 25, 50, or 100μg/mL of ox-LDL for 48h in THP-1 macrophages. Moreover, overexpression of IGF2 significantly upregulated NF-κB and IL-6 expressions in THP-1 macrophages. However, the upregulation of NF-κB and IL-6 expressions induced by ox-LDL were significantly abolished by IGF2 small interfering RNA (siRNA) in THP-1 macrophages. Further studies indicated the upregulation of IL-6 induced by ox-LDL could be abolished when treated with NF-κB siRNA in THP-1 macrophages. Ox-LDL might upregulate IL-6 in the cell and its secretion via enhancing NF-κB in an IGF2-dependent manner in THP-1 macrophages.

Upregulated interleukin-6 expression contributes to erlotinib resistance in head and neck squamous cell carcinoma

Despite the role of epidermal growth factor receptor (EGFR) signaling in head and neck squamous cell carcinoma (HNSCC) development and progression, clinical trials involving EGFR tyrosine kinase inhibitors (TKIs) have yielded poor results in HNSCC patients. Mechanisms of acquired resistance to the EGFR TKI erlotinib was investigated by developing erlotinib-resistant HNSCC cell lines and comparing their gene expression profiles with their parental erlotinib-sensitive HNSCC cell lines using microarray analyses and subsequent pathway and network analyses. Erlotinib-resistant HNSCC cells displayed a significant upregulation in immune response and inflammatory pathways compared to parental cells. Interleukin-6 (IL-6) was one of thirteen genes that was significantly differentially expressed in all erlotinib-resistant HNSCC cell lines, which was validated using RT-PCR and ELISA. Blockade of IL-6 signaling using the IL-6 receptor antagonist tocilizumab, was able to overcome erlotinib-resistance in erlotinib-resistant SQ20B tumors in vivo. Overall, erlotinib-resistant HNSCC cells display elevated IL-6 expression levels compared to erlotinib-sensitive HNSCC cells and blockade of the IL-6 signaling pathway may be an effective strategy to overcome resistance to erlotinib and possibly other EGFR TKIs for HNSCC therapy.

Enhanced invasion of lung adenocarcinoma cells after co-culture with THP-1-derived macrophages via the induction of EMT by IL-6

Lung cancer is the leading cause of cancer mortality worldwide, and the cause of death is metastasis. The epithelial-to-mesenchymal transition (EMT) plays a key role in the process of metastasis. Macrophages within the lung cancer microenvironment release cytokines, such as interleukin-6 (IL-6), and promote lung cancer cell invasion and metastasis. However, the interaction between macrophages and lung cancer cells and the effect of this interaction on the expression of IL-6, EMT, and the invasiveness of lung cancer cells remain unclear. Therefore, we established an in vitro co-culture model of human lung adenocarcinoma A549 or H1299 cells with THP-1-derived macrophages to illuminate the important role of macrophages in the invasion of lung cancer. In this study, we demonstrated that the concentrations of IL-6 in the co-culture supernatants were significantly increased compared with controls. Thus, a complex chemical cross-talk is induced by the indirect cell-to-cell contact between lung cancer cells and THP-1-derived macrophages. THP-1-derived macrophages appeared to play an important initiator role in the process. The analysis of the mRNA expression profiles of the sorted cells from the co-culture system revealed that the co-cultured lung cancer cells are the main source of the observed increase in IL-6 secretion. In addition, the interactions between lung cancer cells and THP-1-derived macrophages are bidirectional. The THP-1-derived macrophages underwent differentiation towards the M2-macrophage phenotype during the co-culture process. The expression of IL-6 was correlated with the induction of EMT, which contributed to a significant increase in the invasiveness of the A549 and H1299 cells in vitro. In addition, the addition of an anti-IL-6 antibody reversed these changes. In summary, we demonstrated that the in vitro co-culture of A549 or H1299 cells with THP-1-derived macrophages upregulates IL-6 expression, which increases the invasion ability of the A549 and H1299 cells through the EMT pathway. The THP-1-derived macrophages that interacted with the lung cancer cells differentiated towards the M2-macrophage phenotype. Thus, the inhibition of IL-6 or of the interactions between lung cancer cells and macrophages may be an effective target for anti-cancer therapy in patients with non-small cell lung cancer.

Regulatory effect of miR-149 on interleukin-6 expression in silica-induced pulmonary fibrosis

To investigate the regulatory effect of miR-149 on interleukin-6 (IL-6) expression in silica-induced pulmonary fibrosis. A mouse model of pulmonary fibrosis was established using silica dust; the level of miR-149 in the lung tissues of mice with silica-induced pulmonary fibrosis was measured by quantitative real-time polymerase chain reaction (qRT-PCR), while the protein expression of IL-6 was measured by immunohistochemistry and Western blot. Type II alveolar epithelial cells (A549) and bronchial epithelial cells (HBE) were exposed to silica dust to establish a model; the level of miR-149 was measured by qRT-PCR, while the protein expression of IL-6 was measured by Western blot. A549 cells were transfected with miR-149 mimics and inhibitor in vitro, and the cellular expression of IL-6 was measured by Western blot. Serum samples from patients with coal workers' pneumoconiosis were examined by double-antibody sandwich ELISA to measure the protein expression of IL-6. At three time points after silica treatment, the miR-149 expression in lung tissues was significantly down-regulated while an evident increase in IL-6 expression was observed in lung tissues (P < 0.01). Silica-stimulated epithelial cell (A549 and HBE) had up-regulated IL-6 expression and down-regulated miR-149 expression (P < 0.01). Increased levels of miR-149 attenuated IL-6 expression, whereas adverse results were found when miR-149 was inhibited. Compared with that in control group, serum level of IL-6 was significantly increased in patients with stage II and III coal workers' pneumoconiosis (P < 0.01). Down-regulation of miR-149 and up-regulation of IL-6 might be involved in the progression of silica-induced pulmonary fibrosis; miR-149 could negatively regulate IL-6 expression.