Overexpression Of Interleukin-6 Research Articles

Suppression of miR-143 contributes to overexpression of IL-6, HIF-1α and NF-κB p65 in Cr(VI)-induced human exposure and tumor growth

Hexavalent chromium [Cr(VI)] is a known occupational and environmental contaminant and carcinogen, but new mechanisms of Cr(VI)-induced carcinogenesis remain to be elucidated. In this study, we found that expression of miR-143 is decreased, whereas that of Interleukin 6 (IL-6) is increased in blood samples of Cr(VI)-exposing workers compared with corresponding unexposed workers. In addition, IL-6 was increased in human bronchial epithelial cells (BEAS-Cr) exposed to Cr(VI) compared with unexposed BEAS-2B cells. To further investigate the mechanisms by which Cr(VI) promotes these changes, we assessed the effects of miR-143 on gene expression and found that miR-143 suppressed expression of IL-6, HIF-1α and NF-κB p65, and that inhibiting miR-143 promoted expression of IL-6, HIF-1α and NF-κB p65. Interestingly, IL-6 regulated expression of HIF-1α, and HIF-1α transcriptionally regulated expression of IL-6. Experiments in animals showed that miR-143 inhibited tumor growth and angiogenesis by regulating IL-6/HIF-1α and downstream signaling pathways in vivo. These outcomes support the hypothesis that the miR-143/IL-6/HIF-1α pathway functions to regulate Cr(VI)-induced carcinogenesis.

Significance of IL-6 Deficiency in Recognition Memory in Young Adult and Aged Mice

Chronic peripheral elevation of interleukin 6 (IL-6) in humans is associated with cognitive deficits. 4- and 24-month-old IL-6-deficient C57BL/6J (IL-6KO) and reference wild-type (WT) mice were tested in an object recognition test. Discrimination ratios and recognition indexes were significantly lower in 4-month-old IL-6KO and in 24-month-old WT mice vs 4-month-old WT animals. Their discrimination ratios had negative values and recognition indexes were below 50% indicating inability to differentiate the novel from the familiar object after 1-hour delay. In 24-month-old IL-6KO mice recognition index reached 53.17% indicating that their recognition memory was not worsened with age in comparison with younger IL-6-deficient animals. Results of holeboard and elevated plus maze indicated that this effect was memory specific. Inborn IL-6 deficiency attenuated recognition memory in 4-month-old mice and did not altered recognition memory in aged animals. IL-6 signalling may constitute a target for development of the protection against memory disturbances connected with IL-6 overexpression.

Vascular endothelial growth factor is up-regulated by leukemia inhibitory factor and interleukin-6 in human endometriotic stromal cells

Objective: To compare the expression of leukemia inhibitory factor (LIF), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in tissue and fluid samples from patients with endometriosis, and investigate whether LIF and IL-6 regulate VEGF in human endometriotic stromal cells (ESC). Methods: The levels of VEGF, LIF, IL-6 in serum, peritoneal fluid of patients with and without endometriosis were measured by ELISA. The mRNA of these three factors in the ectopic and eutopic endometrial tissue and stromal cells were measured by real-time PCR. ESC derived from ovarian endometriomas were cultured using the method of primary cell culture with LIF and IL-6, and the level of VEGF mRNA and protein were measured by the method of real-time PCR and ELISA respectively. Results: VEGF and IL-6 concentration were 1.2 and 1.3 times higher in the serum of patients with endometriosis than in the control group [(94±19) versus (78±17) ng/L; (45±14) versus (35±9) ng/L; all P<0.05]. VEGF and IL-6 concentration were 1.2 and 1.4 times higher in the peritoneal fluid of patients with endometriosis than in the control group [(110±25) versus (91±21) ng/L; (69±20) versus (49±15) ng/L; all P<0.05]. VEGF and IL-6 concentrations in peritoneal fluid of patients with endometriosis were 1.2 and 1.5 times higher than in serum (all P<0.01). VEGF, LIF and IL-6 mRNA expression were 2.2, 8.6, 44.7 times higher in ESC compared with the matching eutopic endometrial stromal cells (all P<0.01). LIF and IL-6 mRNA were 2.0 and 64.8 times higher in ectopic endometrial tissue than the matching eutopic endometrial tissue (all P<0.05).ESC cultured with LIF, IL-6 and LIF+IL-6 induce VEGF protein secretion [(106±18), (124±30), (140±27) ng/L] by 1.3, 1.5 and 1.7 times (all P<0.05). Conclusion: Overexpression of LIF and IL-6 may synergistically contribute to upregulation of VEGF in ESC and promote development of endometriosis.

CANOPY-A: A phase III study of canakinumab as adjuvant therapy in patients with surgically resected non-small cell lung cancer (NSCLC).

TPS8570 Background: Overexpression of interleukin (IL)-1β has been described in solid tumors, including lung. IL-1β can promote angiogenesis, tumor invasiveness, and induce tumor-associated immunosuppression through myeloid-derived suppressor cell (MDSC) accumulation in tumors. Pre-clinical data has shown that IL-1β inhibition stably reduces tumor growth, by limiting inflammation and inducing the maturation of MDSCs into M1 macrophages. Canakinumab is a human monoclonal antibody with high affinity and specificity for IL-1β. Recently, it was found that canakinumab was associated with a significant and dose-dependent reduction in incidence and mortality from lung cancer based on CANTOS study. Methods: CANOPY-A is a phase III, randomized, double-blind, placebo-controlled study designed to evaluate efficacy and safety of adjuvant canakinumab versus placebo in patients with surgically resected NSCLC. This trial will enroll adult patients, with completely resected (R0) AJCC/UICC v.8 stages II−IIIA and IIIB (T &gt;5 cm and N2) NSCLC, who have completed standard-of-care adjuvant treatments, including cisplatin-based chemotherapy and mediastinal radiation therapy (if applicable). Prior treatment with neoadjuvant chemotherapy or neoadjuvant radiotherapy is not permitted. Approximately 1500 patients will be randomized 1:1 to receive canakinumab (200 mg Q3W, s.c) or placebo (Q3W, s.c.) for 18 cycles or until disease recurrence, unacceptable toxicity, treatment discontinuation at the discretion of the investigator or patient, death, or loss to follow-up. Randomization will be stratified by AJCC/UICC v.8 stage, tumor histology, and region. The primary objective is disease-free survival, per investigator assessment. Secondary objectives include overall survival (key secondary objective), lung cancer-specific survival, safety, pharmacokinetics and immunogenicity of canakinumab, and patient-reported outcomes. Enrollment is ongoing. Clinical trial information: NCT03447769.

IL-10 inhibits apoptosis in brain tissue around the hematoma after ICH by inhibiting proNGF.

To explore the roles of interleukin-10 (IL-10), proNGF and p75NTR in apoptosis of brain tissues induced by intracerebral hemorrhage (ICH). According to the time of sample collection after ICH, brain tissue samples were divided into < 6 h group, 6-24 h group (including 24 h), 24-72 h group (including 72 h) and > 72 h group. Meanwhile, 10 tissues that dropped from the beginning at the cortical stoma (distal part of the hematoma) were harvested as controls. AI in brain tissues around the hematoma after ICH was calculated based on TUNEL staining. Expression levels of IL-10, proNGF and p75NTR in brain tissues were determined by quantitative Real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Protein expressions of Bcl-2 and Bax were detected by Western blot. Rat cortical astrocytes were harvested and cultured in vitro. After transfection of IL-10 overexpression plasmid, expression levels of IL-10, proNGF and p75NTR were detected by Western blot. AI increased in 6-24 h group, 24-72 h group and > 72 h group compared with < 6 h group and control group, which achieved the peak at 24-72 h. However, no significant difference in AI was observed between < 6 h group and control group. With the prolongation of ICH, IL-10 level gradually decreased and achieved the lowest level at 24-72 h. After 72 h, IL-10 level began to increase. Additionally, mRNA and protein levels of proNGF and p75NTR started to upregulate within 6 h of ICH, achieveing the peak at 24-72 h. Bcl-2 level gradually decreased after 6 h of ICH, while Bax level increased. We did not found significant difference in mRNA and protein levels of IL-10 in brain tissues around hematoma between < 6 h group and control group. With the prolongation of ICH, IL-10 level gradually decreased and achieved the lowest level at 24-72 h. After 72 h, IL-10 level began to increase. Transfection with IL-10 overexpression plasmid in rat astrocytes markedly downregulated protein levels of proNGF and p75NTR compared with those of controls. IL-10 expression is downregulated in brain tissues around the hematoma after ICH. IL-10 alleviates inflammation and apoptosis by inhibiting levels of proNGF, p75NTR and Bax/Bcl-2, thus protecting brain tissue after ICH.

Bmpr2 Mutant Rats Develop Pulmonary and Cardiac Characteristics of Pulmonary Arterial Hypertension.

Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH. The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels. Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca2+), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca2+, decreased [Ca2+] transient amplitude, reduced sarcoplasmic reticulum Ca2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats. We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

HDAC2-mediated upregulation of IL-6 triggers the migration of osteosarcoma cells.

Understanding the mechanisms of advanced metastasis in osteosarcoma (OS) cell is important for the targeted treatment and drug development. Emerging evidence shows that epigenetic factors such as histone deacetylases (HDACs) are involved in the progression and chemoresistance of OS, while their roles are largely unknown. Our data showed that the expression of HDAC2, while not HDAC1, 4, or 8, was significantly increased in OS cells and tissues. OS patients with increased expression of HDAC2 showed reduced overall survival with p value of 0.0001. Targeted inhibition of HDAC2 suppressed the in vitro migration and invasion of OS cells. Our data showed that the inhibition of HDAC2 can decrease the expression and transcription of interleukin-6 (IL-6) in OS cells. Overexpression of IL-6 can reverse si-HDAC2-induced suppression of cell migration. Mechanistical studies showed that inhibition of HDAC2 decreased the phosphorylation and nuclear accumulation of p65, the key factor of NF-κB complex responsible for the transcription of IL-6. This was due to that HDAC2 can activate the transcription of IKK-β in OS cells. Collectively, our data showed that HDAC2-activated NF-κB can increase the expression of IL-6 in OS cells, which resulted in the promotion of cell migration. It suggested that targeted inhibition of HDAC2/NF-κB/IL-6 might be a potential approach for OS therapy.

Puerarin pre-conditioning on the expression levels of CK-MB, cTnI and inflammatory factors in patients undergoing cardiac valve replacement.

Effect of puerarin preconditioning on the expression levels of nuclear factor κB (NF-κB), interleukin 6 (IL-6), interleukin 8 (IL-8), troponin I (cTnI), and creatine kinase isoenzyme MB (CK-MB) in the neutrophils of patients undergoing cardiac valve replacement under cardiopulmonary bypass (CPB) was evaluated. We enrolled 50 patients scheduled for cardiac valve replacement and assigned them randomly divided into either a puerarin or a control group. Puerarin was dissolved in 10 ml normal saline before CPB, and administered by intravenous infusion to patients in the puerarin group. The control group was administered an equivalent amount of saline. We used flow cytometry to determine the expression levels of NF-κB, IL-6 and IL-8 in neutrophils and an auto chemistry analyzer to determine the serum levels of cTnI and CK-MB before anesthesia induction (T0), 30 min after aortic declamping (T1), 4 h after aortic declamping (T2), and 8 h after aortic declamping (T3). We found the mean serum cTnI and CK-MB levels of the puerarin group tended to decrease with time. The positive rates of NF-κB, IL-6 and IL-8 at different time-points were lower in patients of the puerarin group than in those of the control group (and the differences at T3 were statistically significant). The clinical manifestations of patients in the puerarin group after operation were better than those in the control group (P<0.05). We found that the expression levels of NF-κB, IL-6 and IL-8 were positively correlated with the levels of CK-MB and cTnI (P<0.05). Puerarin preconditioning can reduce the NF-κB activation and the overexpression of IL-6 and IL-8 in neutrophils, and it inhibits the release of myocardial enzyme cTnI and CK-MB reflecting myocardial cell protection. Puerarin seems to improve safety and efficacy of valvular replacement operations.

The regulation of skeletal muscle fatigability and mitochondrial function by chronically elevated interleukin-6.

What is the central question of this study? Interleukin-6 has been associated with muscle mass and metabolism in both physiological and pathological conditions. A causal role for interleukin-6 in the induction of fatigue and disruption of mitochondrial function has not been determined. What is the main finding and its importance? We demonstrate that chronically elevated interleukin-6 increased skeletal muscle fatigability and disrupted mitochondrial content and function independent of changes in fibre type and mass. Interleukin-6 (IL-6) can initiate intracellular signalling in skeletal muscle by binding to the IL-6-receptor and interacting with the transmembrane gp130 protein. Circulating IL-6 has established effects on skeletal muscle mass and metabolism in both physiological and pathological conditions. However, the effects of circulating IL-6 on skeletal muscle function are not well understood. The purpose of this study was to determine whether chronically elevated systemic IL-6 was sufficient to disrupt skeletal muscle force, fatigue and mitochondrial function. Additionally, we examined the role of muscle gp130 signalling during overexpression of IL-6. Systemic IL-6 overexpression for 2weeks was achieved by electroporation of an IL-6 overexpression plasmid or empty vector into the quadriceps of either C57BL/6 (WT) or skeletal muscle gp130 knockout (KO) male mice. Tibialis anterior muscle in situ functional properties and mitochondrial respiration were determined. Interleukin-6 accelerated in situ skeletal muscle fatigue in the WT, with a 18.5% reduction in force within 90s of repeated submaximal contractions and a 7% reduction in maximal tetanic force after 5min. There was no difference between fatigue in the KO and KO+IL-6. Interleukin-6 reduced WT muscle mitochondrial respiratory control ratio by 36% and cytochromec oxidase activity by 42%. Interleukin-6 had no effect on either KO respiratory control ratio or cytochromec oxidase activity. Interleukin-6 also had no effect on body weight, muscle mass or tetanic force in either genotype. These results provide evidence that 2weeks of elevated systemic IL-6 is sufficient to increase skeletal muscle fatigability and decrease muscle mitochondrial content and function, and these effects require muscle gp130 signalling.

Interleukin-6 Interweaves the Bone Marrow Microenvironment, Bone Loss, and Multiple Myeloma.

The immune system is strongly linked to the maintenance of healthy bone. Inflammatory cytokines, specifically, are crucial to skeletal homeostasis and any dysregulation can result in detrimental health complications. Interleukins, such as interleukin 6 (IL-6), act as osteoclast differentiation modulators and as such, must be carefully monitored and regulated. IL-6 encourages osteoclastogenesis when bound to progenitors and can cause excessive osteoclastic activity and osteolysis when overly abundant. Numerous bone diseases are tied to IL-6 overexpression, including rheumatoid arthritis, osteoporosis, and bone-metastatic cancers. In the latter, IL-6 can be released with growth factors into the bone marrow microenvironment (BMM) during osteolysis from bone matrix or from cancer cells and osteoblasts in an inflammatory response to cancer cells. Thus, IL-6 helps create an ideal microenvironment for oncogenesis and metastasis. Multiple myeloma (MM) is a blood cancer that homes to the BMM and is strongly tied to overexpression of IL-6 and bone loss. The roles of IL-6 in the progression of MM are discussed in this review, including roles in bone homing, cancer-associated bone loss, disease progression and drug resistance. MM disease progression often includes the development of drug-resistant clones, and patients commonly struggle with reoccurrence. As such, therapeutics that specifically target the microenvironment, rather than the cancer itself, are ideal and IL-6, and its myriad of downstream signaling partners, are model targets. Lastly, current and potential therapeutic interventions involving IL-6 and connected signaling molecules are discussed in this review.

The role of the interleukin (IL)-6/IL-6 receptor axis in cancer.

Interleukin-6 (IL-6) is a pleiotropic cytokine that activates a classic signalling pathway upon binding to its membrane-bound receptor (IL-6R). Alternatively, IL-6 may 'trans-signal' in a manner that is facilitated by its binding to a soluble derivative of the IL-6 receptor (sIL-6R). Resultant signal transduction is, respectively, driven by the association of IL-6/IL-6R or IL-6/sIL-6R complex with the membrane-associated signal transducer, gp130 (Glycoprotein 130). Distinct JAK (Janus tyrosine kinase)/STAT (signal transducers and activators of transcription) and other signalling pathways are activated as a consequence. Of translational relevance, overexpression of IL-6 has been documented in several neoplastic disorders, including but not limited to colorectal, ovarian and breast cancer and several haematological malignancies. This review attempts to summarise our current understanding of the role of IL-6 in cancer development. In short, these studies have shown important roles for IL-6 signalling in tumour cell growth and survival, angiogenesis, immunomodulation of the tumour microenvironment, stromal cell activation, and ultimate disease progression. Given this background, we also consider the potential for therapeutic targeting of this system in cancer.

IL-17 Receptor Signaling Negatively Regulates the Development of Tubulointerstitial Fibrosis in the Kidney.

Chronic inflammation has an important role in the development and progression of most fibrotic diseases, for which no effective treatments exist. Tubulointerstitial fibrosis (TF) is characterized by irreversible deposition of fibrous tissue in chronic kidney diseases. Prolonged injurious stimuli and chronic inflammation regulate downstream events that lead to TF. In recent years, interleukin-17 (IL-17) has been strongly linked to organ fibrosis. However, the role of IL-17 receptor signaling in TF is an active area of debate. Using the unilateral ureteral obstruction (UUO) mouse model of TF, we show that IL-17 receptor A-deficient mice (Il17ra−/−) exhibit increased TF in the obstructed kidney. Consequently, overexpression of IL-17 restored protection in mice with UUO. Reduced renal expression of matrix-degrading enzymes results in failure to degrade ECM proteins, thus contributing to the exaggerated TF phenotype in Il17ra−/− mice. We demonstrate that the antifibrotic kallikrein-kinin system (KKS) is activated in the obstructed kidney in an IL-17-dependent manner. Accordingly, Il17ra−/− mice receiving bradykinin, the major end-product of KKS activation, prevents TF development by upregulating the expression of matrix-degrading enzymes. Finally, we show that treatment with specific agonists for bradykinin receptor 1 or 2 confers renal protection against TF. Overall, our results highlight an intriguing link between IL-17 and activation of KKS in protection against TF, the common final outcome of chronic kidney conditions leading to devastating end-stage renal diseases.

The significant role of interleukin-6 and its signaling pathway in the immunopathogenesis and treatment of breast cancer.

Despite remarkable improvements in cancer treatment approaches, breast cancer is still the main cause of cancer-related death in women. Its principal cause is the resistance of the cancer cells against conventional anticancer therapeutics, mainly in advanced disease stages. It has been shown that chronic inflammation in the tumor microenvironment facilitates tumor growth and induces resistance toward chemo- and radiotherapy. Overexpression of interleukin-6 (IL-6) cytokine in the tumor microenvironment has been demonstrated in numerous tumors including breast cancer. Tumor cells and tumor-associated fibroblasts are the major sources of IL-6 secretion in the tumor microenvironment. Several studies have demonstrated the immunopathogenic function of IL-6 and its signaling in the tumor growth, metastasis, and therapeutic resistance in the breast cancer. Therefore, it seems that targeting IL-6 and/or its receptor in combination with other potent anticancer therapies may be a potent therapeutic approach for breast cancer therapy.

Overexpression of miR-574-3p suppresses proliferation and induces apoptosis of chronic myeloid leukemia cells via targeting IL6/JAK/STAT3 pathway.

The present study aimed to elucidate the potential roles and regulatory mechanism of microRNA (miR)-574-3p in the development of chronic myeloid leukemia (CML). The expression of miR-574-3p in peripheral blood obtained from patients with CML was examined. Subsequently, miR-574-3p was overexpressed and suppressed in CML K562 cells to further investigate the effects of miR-574-3p on cell proliferation, and apoptosis. Furthermore, a luciferase reporter assay was performed to investigate whether interleukin-6 (IL-6) was a target of miR-574-3p. In addition, the regulatory association between miR-574-3p and the IL-6/Janus kinase (JNK)/signal transducer and activator of transcription-3 (STAT3) signaling pathway was explored. The expression of miR-574-3p in the peripheral blood obtained from patients with CML was significantly lower compared with that in healthy controls. Overexpression of miR-574-3p significantly inhibited the proliferation and induced the apoptosis of K562 cells, whereas suppression of miR-574-3p exhibited opposite effects. In addition, IL-6 was identified to be a direct target of miR-574-3p. Overexpression of IL-6 significantly promoted the proliferation and inhibited the apoptosis of K562 cells. Furthermore, overexpression of miR-574-3p inhibited the activation of the JAK/STAT3 signaling pathway, which was rescued by overexpression of IL-6. The results of the current study indicate that miR-574-3p overexpression may serve an important role in inhibiting proliferation and inducing apoptosis of K562 cells via suppression of IL-6/JAK/STAT3 signaling pathway activation. miR-574-3p may serve as a potential therapeutic target for CML.

Targeting Interleukin-6 (IL-6) Sensitizes Anti-PD-L1 Treatment in a Colorectal Cancer Preclinical Model.

BackgroundLimited efficacy of immune checkpoint blockades was observed in clinical trials in colorectal (CRC) patients, especially in the microsatellite-stable patients. Interleukin-6 (IL-6) is critical in modeling immune responses in cancers. However, the effects of targeting IL-6 in combination with immune checkpoint blockades is unknown in CRC.Material/MethodsIn the present study, we investigated the profile of IL-6 expression in tumor tissues of CRC patient and we established CRC mouse models with various IL-6 expression levels using CT26 cells and MC38 cells. Effects of anti-IL-6 and anti-PD-L1 combination treatment were tested in these models.ResultsA total of 105 CRC patients were included in this study, with 41 (39%) females and 64 (61%) males. Sixty patients showed IL-6 high expression and 45 patients showed IL-6 low expression. The patients with IL-6 high expression tended to have shorter survival (median survival time of 25.5 months) than the patients with IL-6 low expression (median survival time of 46 months, P value=0.013). In the CRC mouse models, tumors with IL-6 overexpression tended to grow faster than the tumors with IL-6 knockout. The numbers of CD8+ T cells and CD4+ T cells were decreased in IL-6 overexpressed tumors. On the contrary, myeloid-derived suppressor cells and regulatory/suppressor T cells were more numerous in tumors with IL-6 overexpression. PD-L1 expression was upregulated in the tumors with IL-6 overexpression. Importantly, an IL-6 blockade reversed the anti-PD-L1 resistance and prolonged tumor-bearing mouse survival.ConclusionsOur study indicates that IL-6 induces strong immunosuppression in the CRC microenvironment by recruiting immunosuppression cells and impairing T cell infiltration. Inhibition of IL-6 enhanced the efficacy of anti-PD-L1 in CRC, providing a novel strategy to overcome anti-PD-L1 resistance in CRC.

Gene Modification of Transforming Growth Factor β (TGF-β) and Interleukin 10 (IL-10) in Suppressing Mt Sonicate Induced Osteoclast Formation and Bone Absorption

BackgroundOsteoarticular tuberculosis is an osteolytic lesion caused by Mycobacterium tuberculosis (MTB). Inflammatory factors such as TNF-α play a critical role in anti-tuberculosis immunity by regulating osteoblast and osteoclast functions. Both TGF-β and IL-10 have immune suppression effects to downregulate secretion and release of inflammatory factors, such as TNF-α, that play roles in regulating osteoblast and osteoclast functions. This study thus investigated the effects of osteoclast with modified TGF-β and IL-10 gene expression on MTB-induced osteoclast formation and bone absorption.Material/MethodsBone marrow mononuclear cells were induced to differentiate into osteoblasts and osteoclasts in vitro to generate a co-culture system. MTB powder lysed by ultrasound (Mt sonicate) were added in gradients to observe osteoblast formation and osteoclast absorption. Cell apoptosis was measured by flow cytometry, while ELISA was used assess TNF-α, TGF-β, and IL-10. Viral vectors carrying TGF-β or IL-10 gene were used to transfect osteoclasts, followed by ELISA assay. Bone absorption and osteoblast apoptosis were compared among groups.ResultsMt sonicate significantly facilitated osteoclast formation and bone formation. It upregulated contents of TNF-α, TGF-β, and IL-10, induced osteoblast apoptosis, enhanced RANKL expression in osteoblasts, and decreased OPG expression. Overexpression of TGF-β and/or IL-10 significantly decreased its upregulation effect on TNF-α by Mt sonicate, and hindered Mt sonicate-induced osteoblast apoptosis, osteoclast formation, and bone absorption.ConclusionsOverexpression of TGF-β and IL-10 significantly inhibits TMB-induced TNF-α synthesis and release, suppresses osteoblast apoptosis, and hinders osteoclast formation and bone absorption.

Abstract 1065: IL-10 as an indicator of metastatic potential in colorectal carcinoma

Abstract The purpose of this study was to evaluate the expression of TGF-β and IL-10 in primary and metastatic tumors to determine the contribution that these cytokines may have to metastasis. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that inhibits antigen-presenting cells and the secretion of pro-inflammatory cytokines. Because tumors typically thrive in a pro-inflammatory microenvironment, the anti-inflammatory properties of IL-10 have been shown to decrease the metastatic ability of tumors. IL-10 has also been shown to suppress the anti-tumor immune response, thereby increasing metastatic potential. These conflicting conclusions led us to investigate the possible correlation IL-10 expression may have on the metastatic capabilities of colorectal cancer. TGF-β was also chosen for this study because of the observed connection between cytokine expression and metastasis. In order to evaluate cytokine expression, we utilized standard immunohistochemistry procedures. Human tissues were stained with antibodies against TGF-β and IL-10, along with GAPDH as a positive control and a universal negative isotype control. We tested colon adenocarcinoma (n=30), metastatic colon adenocarcinoma (n=30), cancer adjacent normal tissue (n=20), normal colon tissue (n=10), and tubular adenoma (n=10). Using ImageJ software, samples were analyzed by converting tissue images to a grayscale and then assigning a threshold to ensure analysis was not affected by negative space within the tissue. Gray intensity values above 115 were determined to be ‘negative' for expression, while samples with gray intensity values below 115 were ‘positive' for expression. Our results show that IL-10 expression was significantly higher in patients with metastatic colon cancer when compared to patients with non-metastatic colon cancer, suggesting a connection between the over expression of IL-10 and metastatic capability. In colon adenocarcinoma samples, 20% of patients expressed high levels of IL-10 (average gray intensity = 111.46), while 80% of patients expressed low levels of IL-10 (average gray intensity = 125.51). In metastatic colon adenocarcinoma samples the proportion of high cytokine expression was higher, with 53% of patients expressing high levels of IL-10 and 47% of patients expressing low levels of IL-10. Additionally, IL-10 levels were significantly greater (p&amp;lt;.0001) in patients with metastatic colorectal carcinoma, with an average gray intensity of 106.42 in Metastatic IL-10 High patients and 119.95 in Metastatic IL-10 Low patients. TGF-β expression appeared to remain consistent between normal and cancerous tissues, showing no significant variation between primary tumor and metastatic tissue samples. We have concluded that while TGF-β expression showed little variability between primary and metastatic tumors, IL-10 may be an effective biomarker for characterizing tumors and determining their metastatic potential. Citation Format: Michelle H. Townsend, Taylor P. Cox, Abigail M. Felsted, Steven R. Piccolo, Richard A. Robison, Kim L. O'Neill. IL-10 as an indicator of metastatic potential in colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1065.

Effect of interleukin-6 on myocardial regeneration in mice after cardiac injury

Our aim was to investigate the role of interleukin-6 (IL-6) in myocardial regeneration from mice after cardiac injury. The newborn mice were divided into the following 4 groups (16 in each group): sham group, model group, IL-6-/- group (IL-6 knockout) and IL-6 group (IL-6 overexpression). Electrocardiography was performed on all mice and found higher LVEDD, LVESD and IVST and lower LVEF and LVFS in the IL-6 group compared with the sham group. Using HE staining, severe myocardial injury combined with infarction and fibrosis were observed in the IL-6-/- group, while the damaged myocardial tissue was repaired to some extent in the IL-6 group. The expression of IL-6 in the IL-6 group were significantly up-regulated. BrdU immunofluorescence found that the IL-6-/- group had the least number of BrdU positive cells, while the IL-6 group had more BrdU positive cells than the model group and the IL-6-/- group. Expressions of IL-6, cyclinD1 and Bcl-2 in the IL-6 group were up-regulated compared with other groups. In conclusion, IL-6 overexpression could enhance cardiomyocyte proliferation and relevant protein expression in mice myocardium, thus promoting cardiac regeneration.

Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites.

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman's disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients' blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL-6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.

TIPE2, an inflammation transfer switch, regulates the on-and-off immunosuppressive function of MDSCs in tumors

Abstract Inhibition of the tumor-promoting inflammation of myeloid-derived suppressor cells (MDSCs) is an important determinant of successful clinical immunotherapies. However, the critical checkpoints governing MDSC function in cancer remain poorly characterized. We report here that the critical role of the essential negative regulator of inflammation tumor necrosis factor-α–induced protein 8-like 2 (TIPE2) in the accumulation and immune inhibitory function of MDSCs. TIPE2-deficient MDSCs decreased immune-regulatory activity and enabled the activation of T cell function and the induction antitumor responses. TIPE2 expression in MDSCs was induced by tumor-derived reactive oxygen species (ROS). Importantly, TIPE2 promoted CCAAT/enhancer-binding protein-β (C/EBPβ) by negative feedback inhibiting phospho-AKT/phospho-GSK-3β/phospho-C/EBPβ signaling axis, leading to decreased interleukin-6 (IL-6) and IL-10 production and STAT3 phosphorylation. IL-6 overexpression restored immune-suppressive activity of TIPE2-deficient MDSCs. These findings suggest a critical role of TIPE2 in tumor-induced tolerance and a novel therapeutic strategy of targeting TIPE2 in MDSCs to enhance the potential of cancer immunotherapy.