Interleukin-6 Treatment Research Articles

Investigating the Impact of IL-6 and CXCL8 on Neurodegeneration and Cognitive Decline in Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder primarily affecting the elderly, characterized by severe cognitive impairment and memory loss. Emerging evidence suggests that neuroinflammation plays a significant role in AD pathogenesis, with cytokines like interleukin-6 (IL-6) and C-X-C motif chemokine ligand 8 (CXCL8) contributing to the disease progression. We utilized GEO datasets to identify IL-6 and CXCL8 as pivotal inflammatory markers in AD. In vitro experiments were conducted using SK-N-BE(2)-M17 and THP-1 cell lines treated with IL-6 and CXCL8 to model AD. Additionally, in vivo tests on Amyloid Precursor Protein/Presenilin 1 (APP/PS1) AD mouse models were performed to assess the impact of these cytokines on cognitive functions and brain pathology. The results indicated a significant decrease in cell viability, increased apoptosis, and elevated inflammatory factor secretion following IL-6 and CXCL8 treatment in vitro. In vivo, AD mouse models treated with these cytokines exhibited exacerbated emotional distress, decreased social interaction, impaired cognitive functions, and increased amyloid protein deposition in neural tissues. The study highlights the detrimental effects of IL-6 and CXCL8 on neuronal health and cognitive functions in AD. These findings suggest that targeting these cytokines could offer potential therapeutic interventions for improving patient outcomes in Alzheimer's disease.

Evaluation of drug resistance for EGFR-TKIs in lung cancer via multicellular lung-on-a-chip

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.

Interleukin-6 Modulates Action Potential Generation in Freshly Isolated Rat Dorsal Root Ganglia Neurons And its Stimulating Effect Is Exacerbated in Polycystic Kidney Disease

Our laboratory has demonstrated a central role for increased afferent nerve renal activity (ARNA) in the progression of cystogenesis in the PCK rat model of polycystic kidney disease (PKD). The underlying cause for the pathological increase in ARNA is unknown. Inflammatory cell infiltration and increased proinflammatory cytokine content, such as interleukin 6 (IL-6), have been reported in PKD. We hypothesized inflammatory cytokines directly increase sensory nerve activity, and this response is exacerbated in PKD. We measured action potential generation (AP) in freshly isolated dorsal root ganglion (DRG) neurons in PCK rats and non-cystic controls, and their response to IL-6. DRGs (T10-L1) were isolated from Sprague-Dawley (SD) or PCK rat (Age: 10-26 weeks) spinal cords, enzymatically digested and mechanically dissociated. Neurons were plated on coverslips, and AP were recorded within 24-hours (whole-cell patch-clamp). We tested the effects of IL-6 (0.04 μg/ml) and bradykinin (BK; 40μg/ml) on AP under 100, 200 pA, and 300 pA current clamp steps. IL-6 elicited either an excitatory response or inhibitory response in AP. In excitatory cells (EC), AP was normalized to BK peak response at +300 pA. In inhibited cells (IC), AP was normalized to baseline at +300 pA. PCK and IL-6 effects were assessed by two-way ANOVA (Bonferroni post hoc test;*p<.05); data as mean ± SEM. Proportion of ECs and ICs did not differ between SD and PCK rats. IL-6 increased AP at 300 pA from 0.48±0.28 (baseline) to 0.95±0.05 (p<.05) in SD ECs cells. BK further increased AP, showing no cell fatigue by IL-6 exposure. In PCK ECs, IL-6 increased AP at 100 pA (0.07±0.05 vs. 0.52±0.25), at 200 pA (0.47±0.27 vs. 1.39±0.81), and 300 pA (0.54±0.25 vs. 1.11±0.47). BK further increased AP at 100 pA to 0.70± 0.28 but decreased AP at 200 pA to 1.21±0.40 and at +300 pA to 1.00±0.00, indicating that neurons activity was likely plateaued by IL-6 at +200 and +300 pA. In both SD and PCK ICs, there was a main effect of IL-6 and BK treatment across all current steps (p<0.05). In SD ICs, IL-6 decreased AP at 200 pA (1.03±0.67 vs. 0.28±0.24) and 300 pA (1.00±0.00 vs. 0.28±0.24). BK had no effect at +200 pA and partially reversed the effect of IL-6 at +300 pA (AP increase to 0.53±0.27). In PCK ICs, IL-6 decreased AP at +200 pA (0.21±0.08 vs. 0.11±0.09) and +300 pA (1.00±0.00 vs. 0.42±0.24). BK increased AP at +200 pA (0.67±0.37) and +300 pA (0.71±0.33). These data partially support our hypothesis that IL-6 increases sensory nerve discharge. This effect was not observed uniformly across cells, as there were inhibitory and excitatory responses in both PCK and SD rats. We conclude that pro-inflammatory cytokine IL-6 can increase a subset of DRG neuron activity and responsiveness, an effect potentiated in PKD. This neuro-immune interaction might contribute to the underlying increase in ARNA and cystogenesis in PKD. Further experimentation is underway to test neuronal sensitivity to IL-6 and other inflammatory cytokines, as well as the posited potentiated effects in PKD. Supported in part by the PKD Foundation Research Award 1022534 (CB); NIDDK/PKD RRC Pilot and Feasibility Award U24DK126110 (CB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Investigating the Impact of IL6 on Insulin Secretion: Evidence from INS-1 Cells, Human Pancreatic Islets, and Serum Analysis

Interleukin-6 (IL6) is a pleiotropic cytokine implicated in metabolic disorders and inflammation, yet its precise influence on insulin secretion and glucose metabolism remains uncertain. This study examined IL6 expression in pancreatic islets from individuals with/without diabetes, alongside a series of functional experiments, including siRNA silencing; IL6 treatment; and assessments of glucose uptake, cell viability, apoptosis, and expression of key β-cell genes, which were conducted in both INS-1 cells and human islets to elucidate the effect of IL6 on insulin secretion. Serum levels of IL6 from Emirati patients with type 2 diabetes (T2D) were measured, and the effect of antidiabetic drugs on IL6 levels was studied. The results revealed that IL6 mRNA expression was higher in islets from diabetic and older donors compared to healthy or young donors. IL6 expression correlated negatively with PDX1, MAFB, and NEUROD1 and positively with SOX4, HES1, and FOXA1. Silencing IL6 in INS-1 cells reduced insulin secretion and glucose uptake independently of apoptosis or oxidative stress. Reduced expression of IL6 was associated with the downregulation of Ins, Pdx1, Neurod1, and Glut2 in INS-1 cells. In contrast, IL6 treatment enhanced insulin secretion in INS-1 cells and human islets and upregulated insulin expression. Serum IL6 levels were elevated in patients with T2D and associated with higher glucose, HbA1c, and triglycerides, regardless of glucose-lowering medications. This study provides a new understanding of the role of IL6 in β-cell function and the pathophysiology of T2D. Our data highlight differences in the response to IL6 between INS-1 cells and human islets, suggesting the presence of species-specific variations across different experimental models. Further research is warranted to unravel the precise mechanisms underlying the observed effects of IL-6 on insulin secretion.

Mitochondrial fission enhances IL-6-induced metastatic potential in ovarian cancer via ERK1/2 activation.

Ovarian cancer is a lethal gynecologic cancer mostly diagnosed in an advanced stage with an accumulation of ascites. Interleukin-6 (IL-6), a pro-inflammatory cytokine is highly elevated in malignant ascites and plays a pleiotropic role in cancer progression. Mitochondria are dynamic organelles that undergo fission and fusion in response to external stimuli and dysregulation in their dynamics has been implicated in cancer progression and metastasis. Here, we investigate the effect of IL-6 on mitochondrial dynamics in ovarian cancer cells (OVCs) and its impact on metastatic potential. Treatment with IL-6 on ovarian cancer cell lines (SKOV3 and PA-1) led to an elevation in the metastatic potential of OVCs. Interestingly, a positive association was observed between dynamin-related protein 1 (Drp1), a regulator of mitochondrial fission, and IL-6R in metastatic ovarian cancer tissues. Additionally, IL-6 treatment on OVCs was linked to the activation of Drp1, with a notable increase in the ratio of the inhibitory form p-Drp1(S637) to the active form p-Drp1(S616), indicating enhanced mitochondrial fission. Moreover, IL-6 treatment triggered the activation of ERK1/2, and inhibiting ERK1/2 mitigated IL-6-induced mitochondrial fission. Suppressing mitochondrial fission through siRNA transfection and a pharmacological inhibitor reduced the IL-6-induced migration and invasion of OVCs. This was further supported by 3D invasion assays using patient-derived spheroids. Altogether, our study suggests the role of mitochondrial fission in the metastatic potential of OVCs induced by IL-6. The inhibition of mitochondrial fission could be a potential therapeutic approach to suppress the metastasis of ovarian cancer.

TRPM2-L Participates in the Interleukin-6 Pathway to Enhance Tumor Growth in Prostate Cancer by Hypoxia-Inducible Factor-1α.

Interleukin-6 (IL-6) can promote cell proliferation in prostate cancer (PCa). Full-length transient receptor potential melastatin 2 (TRPM2-L) is highly expressed in PCa. However, the association between IL-6 and TRPM2-L in PCa is unclear. Here, human PCa cell lines, PC-3 and DU-145, were treated with 10 μg/mL tocilizumab, an IL-6 receptor (IL-6R) inhibitor, and the TRPM2-L protein expression in cells was significantly decreased. Cells were stably transfected with TRPM2 short-interfering RNA (siRNA) and cell survival clearly declined. Recombinant IL-6 treatment weakened the effects of TRPM2-siRNA on cell survival. TRPM2-L binds directly to IL-6R in PC-3 and DU-145 cells. The protein expression of hypoxia-inducible factor-1α was suppressed by reduction with TRPM2-L in PC-3 and DU-145 cells. Human umbilical vein endothelial cells (HUVECs) were indirectly cocultured with PCa cells, and the invasion and angiogenic activity of HUVECs were enhanced after coculture with PCa cells. However, TRPM2-L reduction in PCa cells significantly decreased the invasion and angiogenic activity of HUVECs compared to the control coculture. In vivo, xenograft tumors were induced using PC-3 cells. Tocilizumab treatment or TRPM2-L reduction clearly suppressed tumor growth. Meanwhile, the injection of mouse recombinant IL-6 weakened the antitumor effects of TRPM2-L reduction. These data demonstrate that the IL-6/TRPM2-L axis in PCa tumor growth is important, and interference of the IL-6/TRPM2-L axis may be a novel approach for PCa therapy.

Role of interleukin 6 and its soluble receptor on the diffusion barrier dysfunction of alveolar tissue.

During respiratory infection, barrier dysfunction in alveolar tissue can result from "cytokine storm" caused by overly reactive immune response. Particularly, interleukin 6 (IL-6) is implicated as a key biomarker of cytokine storm responsible for and further progression to pulmonary edema. In this study, alveolar-like tissue was reconstructed in a microfluidic device with: (1) human microvascular lung endothelial cells (HULEC-5a) cultured under flow-induced shear stress and (2) human epithelial cells (Calu-3) cultured at air-liquid interface. The effects of IL-6 and the soluble form of its receptor (sIL-6R) on the permeability, electrical resistance, and morphology of the endothelial and epithelial layers were evaluated. The diffusion barrier properties of both the endothelial and epithelial layers were significantly degraded only when IL-6 treatment was combined with sIL-6R. As suggested by recent review and clinical studies, our results provide unequivocal evidence that the barrier dysfunction occurs through trans-signaling in which IL-6 and sIL-6R form a complex and then bind to the surface of endothelial and epithelial cells, but not by classical signaling in which IL-6 binds to membrane-expressed IL-6 receptor. This finding suggests that the role of both IL-6 and sIL-6R should be considered as important biomarkers in developing strategies for treating cytokine storm.

Interleukin-6 Facilitates Acute Myeloid Leukemia Chemoresistance via Mitofusin 1-Mediated Mitochondrial Fusion.

IL6 treatment induces MFN1-mediated mitochondrial fusion, promotes OXPHOS, and confers chemoresistance in AML cells. Targeting IL6 regulation in mitochondria is a promising therapeutic strategy to enhance the chemosensitivity of AML.

Effects of bromodomain and extra-terminal inhibitor JQ1 and interleukin-6 on breast cancer cells.

Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

Interleukin-6 stimulates in vitro development of late-stage ovine embryos

The effect of interleukin-6 (IL-6) supplementation during the different phases of in vitro embryo culturing (IVC) on embryo development and embryonic gene expression was studied in ovine. IL-6 was added to IVC medium during the late phases (72–192 h; 5, 10, and 25 ng/ml IL-6) or entire period (0–192 h; 10 ng/ml IL-6) of IVC to determine its effect on embryo development. Further, the effect of IL-6 (10 ng/ml) supplementation at the 72 h of IVC on gene expressions associated with JAK/STAT signalling and pluripotency in 8–16 cell embryos (1 h post-supplementation) and compact morulae (48 h post-supplementation), and apoptosis and primitive endoderm (PrE) development in compact morulae was investigated. The supplementation of 10 ng/ml IL-6 during the late phases of IVC significantly (P < 0.05) increased blastocyst formation (35.2 ± 1.52%) compared to the control (21.1 ± 1.11%), and 5 ng/ml (25.9 ± 2.98%) or 25 ng/ml (16.5 ± 0.73%) IL-6 groups. Conversely, IL-6 (10 ng/ml) treatment throughout the IVC period significantly (P < 0.05) decreased the rate of cleavage (55.4 ± 1.57%) and blastocyst formation (14.5 ± 1.28%) compared to the control group (65.8 ± 1.35% and 21.5 ± 0.97%, respectively). In 8–16 cell embryos and compact morulae, the IL-6 treatment significantly (P < 0.05) affected the expression of genes associated with JAK/STAT signalling and pluripotency. Further, the treatment significantly (P < 0.05) downregulated BAX and CASP3, and upregulated GATA6 expression in compact morulae. In conclusion, IL-6 supplementation affected the in vitro development of ovine embryos in a dose- and time-dependent manner. The beneficial effect of IL-6 on the development of late-stage embryos was mediated through the changes in gene expressions associated with JAK/STAT signalling, pluripotency, apoptosis and PrE development.

Early Interleukin-6 Mediated Activation of SGK1 is Primarily via Variant 6

Hypertension is associated with inflammation, including elevated interleukin-6 (IL-6) levels. IL-6 is an inflammatory cytokine, which we have previously shown to activate the mineralocorticoid receptor (MR) and the downstream sodium (Na) transporters- Na-chloride cotransporter (NCC) and the epithelial Na channel (ENaC). However, how IL-6 activates the MR is still unknown. IL-6 is also linked to Serum and Glucocorticoid-regulated Kinase 1 (SGK1), which is a downstream effector of the MR. SGK1 regulates both NCC and ENaC, increasing activity and tubular reabsorption of Na. This increased Na reabsorption favors water retention, thus increasing blood pressure. Previously, IL-6 has also been linked with SGK1 activation. We hypothesize that IL-6 increases SGK1 mRNA and protein expression in renal epithelial cells. To test this, we used in vitro renal epithelial cell models of both the late distal convoluted tubule (DCT2) and inner medullary collecting duct (IMCD), mDCT15 and IMCD3, respectively. Cells were treated with an IL-6 time-course (100ng/ml for 1, 3 and 6 hours). There are 6 known transcriptional variants for SGK1. We designed variant-specific PCR primers and, a pilot study based on regular PCR concluded that expression levels of only variants 5 and 6 (V5, V6) are significant. Using Real Time quantitative RT-PCR, we were then able to quantify transcript levels of both SGK1 V5 and V6 variants. With IL-6 treatment, we observed a 160%-211% increase at early time points (30mins-1hr) in SKG1 V6 and a lesser response (26%-88% increase) with V5 as compared to vehicle treated mDCT15 cells. Since SGK1 has been observed in the membrane fraction and in the cytoplasm, protein extracts from cells were fractionated (mPER kit) to assess membrane and cytosolic SGK1 protein levels using immunoblots. Densitometric analysis of immunoblots demonstrated a 5.5- and 3.1-fold increase in SGK1 protein expression at 1hr in mDCT15 and IMCD3 cells, respectively. These data suggest that IL-6 induces increases in SGK1 transcription and translation. Interestingly, we also found that majority of SGK1 was in the membrane fraction and the level was consistent. Together, these data suggest that SGK1 V6 may be responsible for the increased sodium reabsorption in response to IL-6 stimulation in renal epithelial cells. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Src-FAK Signaling Mediates Interleukin 6-Induced HCT116 Colorectal Cancer Epithelial–Mesenchymal Transition

Colorectal cancer is one of the most prevalent and lethal malignancies, affecting approximately 900,000 individuals each year worldwide. Patients with colorectal cancer are found with elevated serum interleukin-6 (IL-6), which is associated with advanced tumor grades and is related to their poor survival outcomes. Although IL-6 is recognized as a potent inducer of colorectal cancer progression, the detail mechanisms underlying IL-6-induced colorectal cancer epithelial-mesenchymal transition (EMT), one of the major process of tumor metastasis, remain unclear. In the present study, we investigated the regulatory role of IL-6 signaling in colorectal cancer EMT using HCT116 human colorectal cancer cells. We noted that the expression of epithelial marker E-cadherin was reduced in HCT116 cells exposed to IL-6, along with the increase in a set of mesenchymal cell markers including vimentin and α-smooth muscle actin (α-SMA), as well as EMT transcription regulators-twist, snail and slug. The changes of EMT phenotype were related to the activation of Src, FAK, ERK1/2, p38 mitogen-activated protein kinase (p38MAPK), as well as transcription factors STAT3, κB and C/EBPβ. IL-6 treatment has promoted the recruitment of STAT3, κB and C/EBPβ toward the Twist promoter region. Furthermore, the Src-FAK signaling blockade resulted in the decline of IL-6 induced activation of ERK1/2, p38MAPK, κB, C/EBPβ and STAT3, as well as the decreasing mesenchymal state of HCT116 cells. These results suggested that IL-6 activates the Src-FAK-ERK/p38MAPK signaling cascade to cause the EMT of colorectal cancer cells. Pharmacological approaches targeting Src-FAK signaling may provide potential therapeutic strategies for rescuing colorectal cancer progression.

Estrogen-related receptor γ (ERRγ) is a key regulator of lysyl oxidase gene expression in mouse hepatocytes

Lysyl oxidase (LOX), the copper-dependent extracellular enzyme, plays a critical role in the regulation of protein cross-linking in the extracellular matrix (ECM). It is also involved in liver regeneration and liver fibrosis. However, the mechanism of LOX regulation in mouse hepatocytes is still unclear. Here, we identify a molecular mechanism showing that orphan nuclear receptor estrogen-related receptor γ (ERRγ) regulates LOX gene expression in the presence of the pro-inflammatory cytokine, interleukin 6 (IL6). IL6 significantly stimulated the expression of ERRγ and LOX in mouse hepatocytes. Overexpression of ERRγ increased LOX mRNA and protein levels. Moreover, knockdown of ERRγ attenuated IL6-mediated LOX gene expression at mRNA and protein levels. Overexpression of ERRγ or IL6 treatment upregulated LOX gene promoter activity, while knockdown of ERRγ decreased the IL6-induced LOX promoter activity. Furthermore, GSK5182, a specific ERRγ inverse agonist, inhibited the induction effect of IL6 on LOX promoter activity and gene expression in mouse hepatocytes. Overall, our study elucidates the mechanism involved in the LOX gene regulation by nuclear receptor ERRγ in response to IL6 in mouse hepatocytes, suggesting that, in conditions such as chronic inflammation, IL6 may contribute to liver fibrosis via inducing LOX gene expression. Thus, LOX gene regulation by the inverse agonist of ERRγ can be applied to improve liver fibrosis.

Interleukin-6 and Hypoxia Synergistically Promote EMT-Mediated Invasion in Epithelial Ovarian Cancer via the IL-6/STAT3/HIF-1α Feedback Loop.

Extensive peritoneal spread and capacity for distant metastasis account for the majority of mortality from epithelial ovarian cancer (EOC). Accumulating evidence shows that interleukin-6 (IL-6) promotes tumor invasion and migration in EOC, although the molecular mechanisms remain to be fully elucidated. Meanwhile, the hypoxic microenvironment has been recognized to cause metastasis by triggering epithelial-mesenchymal transition (EMT) in several types of cancers. Here, we studied the synergy between IL-6 and hypoxia in inducing EMT in two EOC cell lines, A2780 cells and SKOV3 cells. Exogenous recombination of IL-6 and autocrine production of IL-6 regulated by plasmids both induced EMT phenotype in EOC cells characterized by downregulated E-cadherin as well as upregulated expression of vimentin and EMT-related transcription factors. The combined effects of IL-6 and hypoxia were more significant than those of either one treatment on EMT. Suppression of hypoxia-inducible factor-1α (HIF-1α) before IL-6 treatment inhibited the EMT phenotype and invasion ability of EOC cells, indicating that HIF-1α occupies a key position in the regulatory pathway of EMT associated with IL-6. EMT score was found positively correlated with mRNA levels of IL-6, signal transducer and activator of transcription 3 (STAT3), and HIF-1α, respectively, in 489 ovarian samples from The Cancer Genome Atlas dataset. Next, blockade of the abovementioned molecules by chemical inhibitors reversed the alteration in the protein levels of EMT markers induced by either exogenous or endogenous IL-6. These findings indicate a positive feedback loop between IL-6 and HIF-1α, and induce and maintain EMT phenotype through STAT3 signaling, which might provide a novel rationale for prognostic prediction and therapeutic targets in EOC.

Interleukin 6 mediated activation of the mineralocorticoid receptor in the aldosterone-sensitive distal nephron.

Hypertension is characterized by increased sodium (Na+) reabsorption along the aldosterone-sensitive distal nephron (ASDN) as well as chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na+ reabsorption within the ASDN does not always correlate with increases in aldosterone (Aldo), the primary hormone that modulates Na+ reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased ASDN Na+ reabsorption may occur independent of Aldo stimulation is critical. Here, we show that IL-6 can activate the MR by activating Rac1 and stimulating the generation of reactive oxygen species (ROS) with a consequent increase in thiazide-sensitive Na+ uptake. Using an in vitro model of the distal convoluted tubule (DCT2), mDCT15 cells, we observed nuclear translocation of eGFP-tagged MR after IL-6 treatment. To confirm the activation of downstream transcription factors, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs; then treated with vehicle, Aldo, or IL-6. Aldosterone or IL-6 treatment increased luciferase activity that was reversed with MR antagonist cotreatment, but IL-6 treatment was reversed by Rac1 inhibition or ROS reduction. In both mDCT15 and mpkCCD cells, IL-6 increased amiloride-sensitive transepithelial Na+ current. ROS and IL-6 increased 22Na+ uptake via the thiazide-sensitive sodium chloride cotransporter (NCC). These results are the first to demonstrate that IL-6 can activate the MR resulting in MRE activation and that IL-6 increases NCC-mediated Na+ reabsorption, providing evidence for an alternative mechanism for stimulating ASDN Na+ uptake during conditions where Aldo-mediated MR stimulation may not occur.

Activating IL-6/STAT3 Enhances Protein Stability of Proteasome 20S α+β in Colorectal Cancer by miR-1254.

A widely recognized feature of colorectal cancer (CRC) is an increase in cytokine levels, which result in an inflammatory environment in the tumor. Interleukin-6 (IL-6) is a robust protumor cytokine. Several studies suggest that IL-6 plays a role in the development of tumors. Most intracellular protein breakdown occurs in eukaryotes via the ubiquitin-proteasome pathway; this mechanism may also be involved in cancer pathogenesis. The tumor tissues and paracancerous tissues were collected from 90 patients with colorectal cancer. The expressions of pSTAT3, proteasome 20S α+β, miR-1254, and PSMD1 in tissues were detected by immunohistochemistry, ELISA, and qRT-PCR, and the effects of pSTAT3 and proteasome 20s α+β expressions on the survival of patients were studied. HCT116 and HCT116-R cells were cultured and added IL-6, AG490, STAT3 plasmid, or overexpression/knockdown of miR-1254 in cells. Immunofluorescence, western blot, qRT-PCR, double luciferase gene reporter assay, and flow cytometry were used to detect the expression of pSTAT3, STAT3, proteasome 20s α+β, miR-1254, and PSMD1 and cell cycle. The nude mouse xenograft model was constructed and divided into 3 groups: PBS group, IL-6 treatment group, and IL-6+miR-1254 mimic group. After 28 days, the tumor tissues were collected, and the expressions of miR-1254, pSTST3, proteasome 20s α+β, and PSMD1 in the tissues were detected by qRT-PCR and immunohistochemistry, respectively. Our study discovered that the level of proteasome 20S α+β had a strong connection with pSTAT3 in CRC patients. They were also linked to the development and clinical outcome of CRC. In addition, we found that IL-6 dramatically increased the expression of proteasome 20S α+β and pSTAT3; however, it did not affect the proteasome 20S α+β mRNA synthesis. Circulating proteasome concentration correlated with tumor tissue proteasome 20S α+β. STAT3 could occupy the miR-1254 promoter to inhibit transcription, and it could suppressed miR-1254 which targeted PSMD10, promoting proteasome 20S α+β protein stability. This is a prospective target for developing a new colorectal cancer therapy strategy.

IL-6 Promotes Hepatocellular Carcinoma Invasion by Releasing Exosomal miR-133a-3p.

Interleukin-6 (IL-6), an important inflammatory cytokine, is a key factor regulating cancer metastasis. Cancer cells can modulate their tumorigenic abilities by sorting specific microRNAs (miRNAs) as exosomes into the tumor microenvironment. The relationship between IL-6 and exosomal miRNAs related to hepatocellular carcinoma (HCC) metastasis remains to be elucidated. We examined the metastatic ability of HCC cells after IL-6 treatment and found that miR-133a-3p was sorted into exosomes after IL-6 stimulation and was subsequently released into the tumor microenvironment. In vitro analysis confirmed that exosomal miR-133a-3p acted as a tumor suppressor in HCC. Bioinformatic analysis revealed several signaling pathways and hub genes (CREB1, VCP, CALM1, and YES1) regulated by miR-133a-3p. Survival curves further verified the important roles of hub genes in the prognosis of patients with HCC. It is envisaged that the IL-6/miR-133a-3p axis may be related to the activation of CREB1, VCP, CALM1, and YES1. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression under inflammatory conditions.

Angiopathic activity of LRG1 is induced by the IL-6/STAT3 pathway

Leucine-rich α-2-glycoprotein 1 (LRG1) is a secreted glycoprotein that under physiological conditions is produced predominantly by the liver. In disease, its local induction promotes pathogenic neovascularisation while its inhibition leads to reduced dysfunctional angiogenesis. Here we examine the role of interleukin-6 (IL-6) in defective angiogenesis mediated by LRG1. IL-6 treatment induced LRG1 expression in endothelial cells and ex vivo angiogenesis cultures and promoted vascular growth with reduced mural cell coverage. In Lrg1−/− explants, however, IL-6 failed to stimulate angiogenesis and vessels exhibited improved mural cell coverage. IL-6 activated LRG1 transcription through the phosphorylation and binding of STAT3 to a conserved consensus site in the LRG1 promoter, the deletion of which abolished activation. Blocking IL-6 signalling in human lung endothelial cells, using the anti-IL6 receptor antibody Tocilizumab, significantly reduced LRG1 expression. Our data demonstrate that IL-6, through STAT3 phosphorylation, activates LRG1 transcription resulting in vascular destabilisation. This observation is especially timely in light of the potential role of IL-6 in COVID-19 patients with severe pulmonary microvascular complications, where targeting IL-6 has been beneficial. However, our data suggest that a therapy directed towards blocking the downstream angiopathic effector molecule LRG1 may be of greater utility.

Simvastatin Combined with Resistance Training Improves Outcomes in Patients with Chronic Heart Failure by Modulating Mitochondrial Membrane Potential and the Janus Kinase/Signal Transducer and Activator of Transcription 3 Signaling Pathways.

Background Chronic heart failure (CHF) is the end stage of cardiac disease with a 5-year mortality rate reaching 50%. Simvastatin is an antioxidant with lipid-lowering effects, which is commonly used to treat CHF. Resistance training is a nondrug treatment for CHF and exerts a positive effect on both the myocardial structure and function. Objective This study is aimed at exploring the effects and outcomes of simvastatin combined with resistance training on the mitochondrial membrane potential (MMP) of peripheral blood lymphocytes and the Janus kinase/signal transducer and activator of the transcription 3 (JAK/STAT3) signaling pathway in patients with CHF. Methods One hundred and eleven patients with CHF were allocated to the control group (CNG) (n = 55) and intervention group (IG) (n = 56) using the random number table method. The CNG received simvastatin treatment only, whereas the IG received simvastatin treatment plus resistance training. Treatment efficacy, diastolic interventricular septal thickness (IVST), left ventricular ejection fraction (LVEF), left ventricular end-diastolic diameter (LVDD), MMP fluorescence intensity, JAK mRNA and STAT3 mRNA relative expression levels, serum C-reactive protein (CRP), galectin-3, interleukin-6 (IL-6), N-terminal–probrain natriuretic peptide (NT-proBNP), high-sensitivity cardiac troponin T (hs-cTnT), and heart-type fatty acid-binding protein (H-FABP) levels were compared in both groups. Results After 6 months of treatment, diastolic IVST, LVDD, and serum levels of CRP, galectin-3, IL-6, NT-proBNP, hs-cTnT, and H-FABP decreased in both groups and were lower in the IG than in the CNG (P < 0.05), whereas LVEF, JAK and STAT3 mRNA relative expression levels, and MMP fluorescence intensity of peripheral blood lymphocytes were higher in the IG than in the CNG (P < 0.05). Conclusion Simvastatin combined with resistance training improves heart function and reduces myocardial damage as well as the occurrence of adverse cardiac events compared with simvastatin alone. The mechanism may be related to the increase of expression of MMP, JAK, and STAT3, the regulation of MMP and JAK/STAT3 signaling pathways in peripheral lymphocytes, the alleviation of mitochondrial damage, and the inhibition of inflammatory response.

814 Differences in Human, Pig, and Rat Spinal Cord Stem Cells in Response to Inflammatory and Regenerative Factors In Vitro

INTRODUCTION: The translation of preclinical science to human applications, including regenerative strategies for spinal cord injury, has been poor. Historically, animal models, such as rats and pigs, have been used to model human spinal cord given the difficulty in obtaining live human spinal cord. METHODS: To mimic post-injury inflammation, primary-derived NSPCs from adult humans (n=8), rats (n=6), and pigs (n=5) were treated with interleukin-6 (IL-6), tumor necrosis factor- α (TNFα), or transforming growth factor- β (TGFβ). To direct regeneration, NSPCs were treated with retinoic acid (RA), platelet-derived growth factor (PDGFα), or bone morphogenic protein-4 (BMP4) to induce neurons, oligodendrocytes or astrocytes, respectively. NSPCs were treated for 7 or 14 days and characterized by immunocytochemistry (GFAP, β-iii tubulin, O4). RESULTS: IL-6, TNFα and TGFβ induced astrogenesis of rat NSPCs (3.9±0.7, 5.0±0.9, and 4.0±0.6 fold, respectively) and reduced neurogenesis (0.14±0.90, 0.07±0.04, 0.07±0.05 fold, respectively) after 7 days. Pig NSPCs increased astrogenesis (1.38±0.04, 1.26±0.05, and 1.45±0.04 fold, respectively) after 14 days. Human NSPCs had reduced astrogenesis (0.14±0.07, 0.6±0.2, and 0.12±0.07 fold, respectively) over 14 days, but generated more neurons with IL-6 and TGFβ treatments (1.23±0.05 and 1.34±0.04 fold, respectively). RA increased neurogenesis of human and rat NSPCs, PDGFα increased oligodendrocyte differentiation of rat NSPCs, and BMP4 increased astrogenesis of human and rat NSPCs at low (40ng/mL) and high (100ng/mL) concentrations, respectively. CONCLUSIONS: For the first time, we have compared primary-derived adult spinal cord NSPCs from humans, pigs, and rats and identified differences in their response to pathophysiological and regenerative factors which would impact successful translation of stem cell-based therapies.