Interleukin-6 Signaling Pathway Research Articles

Immunotherapy in metastatic sarcomatoid renal cell carcinoma: A single institution experience

IntroductionImmune checkpoint inhibitors (CPIs) were recently approved in advanced clear cell renal cell carcinoma (RCC) and could be a promising option for metastatic RCC with sarcomatoid differentiation (sRCC) which otherwise carry a poor prognosis. We sought to compare outcomes between patients who received immunotherapy (IO) including CPIs or high dose interleukin-2 (HD IL2) for metastatic sRCC versus those who did not. Patients and MethodsWe performed a single-center retrospective data analysis of 44 consecutive sRCC patients with any percentage of sarcomatoid differentiation from our institutional RCC database of whom 34 received IO and 10 patients did not. ResultsBaseline variables between the two groups were not significantly different except for a greater percentage of patients with ≥40% sarcomatoid differentiation in the non-IO cohort. At a median follow-up of 27.6 months, patients treated with IO had a median overall survival of 57.6 months compared to 6.6 months in patients not treated with IO (p = 0.0002). Overall response rates (ORR) between the IO and non-IO group were 35.3% and 0% respectively (p = 0.06). When IO was given in the 1st line setting, the ORR was 25.0%, as compared to 44.4% in the 2nd line setting and beyond though limitations of small sample sizes apply. Immune-related adverse events (IRAE) occurred in 38.2% of patients in the IO group, with grade 3 events (mostly gastrointestinal) in 20.6% with no grade 4 or 5 events. IRAEs led to interruption or discontinuation of immunotherapy in 26.5%. ConclusionOur results support IO as an effective therapeutic option for patients with metastatic sarcomatoid RCC. Further study of various IO regimens, including those affecting the interleukin-2 signaling pathway, and their efficacy in neoadjuvant and adjuvant settings are warranted in sRCC.

Bv8-Like Toxin from the Frog Venom of Amolops jingdongensis Promotes Wound Healing via the Interleukin-1 Signaling Pathway.

Prokineticins are highly conserved small peptides family expressed in all vertebrates, which contain a wide spectrum of functions. In this study, a prokineticin homolog (Bv8-AJ) isolated from the venom of frog Amolops jingdongensis was fully characterized. Bv8-AJ accelerated full-thickness wounds healing of mice model by promoting the initiation and the termination of inflammatory phase. Moreover, Bv8-AJ exerted strong proliferative effect on fibroblasts and keratinocytes isolated from newborn mice by activating interleukin (IL)-1 production. Our findings indicate that Bv8 is a potent wound healing regulator and may reveal the mechanism of rapid wound-healing in amphibian skins.

Mucus obstruction and inflammation in early cystic fibrosis lung disease: Emerging role of the IL-1 signaling pathway.

Mucus plugging constitutes a nutrient-rich nidus for a bacterial infection that has long been recognized as a potent stimulus for neutrophilic airway inflammation driving progressive lung damage in people with cystic fibrosis (CF). However, mucus plugging and neutrophilic inflammation are already present in many infants and young children with CF even in the absence of detectable bacterial infection. A series of observational studies in young children with CF, as well as investigations in animal models with CF-like lung disease support the concept that mucus plugging per se can trigger inflammation before the onset of airways infection. Here we review emerging evidence suggesting that activation of the interleukin-1 (IL-1) signaling pathway by hypoxic epithelial cell necrosis, leading to the release of IL-1α in mucus-obstructed airways, may be an important mechanistic link between mucus plugging and sterile airway inflammation in early CF lung disease. Furthermore, we discuss recent data from preclinical studies demonstrating that treatment with the IL-1 receptor (IL-1R) antagonist anakinra has anti-inflammatory as well as mucus modulating effects in mice with CF-like lung disease and primary cultures of human CF airway epithelia. Collectively, these studies support an important role of the IL-1 signaling pathway in sterile neutrophilic inflammation and mucus hypersecretion and suggest inhibition of this pathway as a promising anti-inflammatory strategy in patients with CF and potentially other muco-obstructive lung diseases.

IRAK2 is associated with systemic lupus erythematosus risk.

Interleukin-1 receptor-associated kinases (IRAKs) are serine-threonine kinases involved in toll-like receptor and interleukin-1 signaling pathways. They play a key role in inflammation and innate immunity. IRAKs have been previously incriminated in autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis and inhibition of IRAKs has been recently regarded as a potential therapeutic strategy for SLE. The aim of the present study was to test the association between IRAK2 rs708035 and rs3844283 with SLE. IRAK2 rs708035 and rs3844283 were genotyped by mutagenically separated polymerase chain reaction (MS-PCR) in 142 SLE patients and 149 age- and gender-matched controls. The hyperfunctional IRAK2 rs708035 A allele was more frequent among SLE patients than controls (62.9% versus 54.7%, p = 0.046). IRAK2 rs3844283 C allele was present in 66.5% of patients and 75.5% of controls. The CC genotype was the most frequently exhibited genotype. It was carried by 45.1% of patients with SLE and 57.7% of controls. The G allele was associated with an increased risk of SLE (OR = 1.54, 95%, CI = 1.07-2.22, p = 0.017). IRAK2 rs708035 and IRAK2 rs3844283 were in linkage disequilibrium (D' = 0.64). The AG haplotype was more frequently observed in SLE patients than in controls (0.292 versus 0.194, p = 0.008). This study for the first time ever reveals the association of IRAK2 rs708035 and IRAK2 rs3844283 and the corresponding haplotypes with SLE. Our findings give additional rationale to target IRAKs in the treatment of SLE.Key Points• IRAK2 rs708035 A allele is more frequent in SLE patients than in controls and IRAK2 rs3844283 G allele is associated with SLE susceptibility.• These two alleles are in linkage disequilibrium.• The AG haplotype is associated with SLE.

Dynamic Regulation of miRNA Expression by Functionally Enhanced Placental Mesenchymal Stem Cells PromotesHepatic Regeneration in a Rat Model with Bile Duct Ligation.

Placenta-derived mesenchymal stem cells (PD-MSCs) were highlighted as therapeutic sources in several degenerative diseases. Recently, microRNAs (miRNAs)were found to mediate one of the therapeutic mechanisms of PD-MSCs in regenerative medicine. To enhance the therapeutic effects of PD-MSCs, we established functionally enhanced PD-MSCs with phosphatase of regenerating liver-1 overexpression (PRL-1(+)). However, the profile and functions of miRNAs induced by PRL-1(+) PD-MSCs in a rat model with hepatic failure prepared by bile duct ligation (BDL) remained unclear. Hence, the objectives of the present study were to analyze the expression of miRNAs and investigate their therapeutic mechanisms for hepatic regeneration via PRL-1(+) in a rat model with BDL. We selected candidate miRNAs based on microarray analysis. Under hypoxic conditions, compared with migrated naïve PD-MSCs, migrated PRL-1(+) PD-MSCs showed improved integrin-dependent migration abilitythrough Ras homolog (RHO) family-targeted miRNA expression (e.g., hsa-miR-30a-5p, 340-5p, and 146a-3p). Moreover, rno-miR-30a-5p and 340-5p regulated engraftment into injured rat liver by transplantedPRL-1(+) PD-MSCs through the integrin family. Additionally, an increase inplatelet-derived growth factor receptor A (PDGFRA) by suppressing rno-miR-27a-3p improved vascular structure in rat liver tissues after PRL-1(+) PD-MSC transplantation. Furthermore, decreased rno-miR-122-5p was significantly correlated with increased proliferation of hepatocytes in liver tissues by PRL-1(+) PD-MSCs byactivating the interleukin-6 (IL-6) signaling pathway through the repression of rno-miR-21-5p. Taken together, these findings improve the understandingof therapeutic mechanisms based on miRNA-mediated stem-cell therapy in liver diseases.

Two Distinct Subtypes Revealed in Blood Transcriptome of Breast Cancer Patients With an Unsupervised Analysis.

Background: Breast cancer (BC) is a highly heterogeneous cancer. The interaction between immune system and BC is complex, widespread yet unclear. In this study, we aimed to reveal the heterogeneity of host systemic immune response to BC and understand the possible mechanisms that may drive the heterogeneity using transcriptomic data from peripheral blood mononuclear cells (PBMCs).Methods: Transcriptome-wide gene expressions of PBMCs in 33 BC patients were generated by RNA sequencing. An unsupervised clustering algorithm was employed to discover PBMC transcriptome subtypes among BC patients. Association analysis between PBMC subtypes and age, clinical stage, abundance of immune cells, and other clinical factors was performed to understand the underlying biological processes that may drive this heterogeneity. Immune gene signature identification and in silico survival analysis were performed to investigate the potential clinical implications of these PBMC subtypes. The findings were validated using the whole blood transcriptomes of an independent cohort.Results: We observed that established BC subtypes were not associated with PBMC gene expression profiles. Instead, we discovered and validated two new BC subtypes using PBMC transcriptome, which have distinct immune cell proportions, especially for lymphocytes (P = 5.22 × 10−12) and neutrophils (P = 1.13 × 10−14). Enrichment analysis of differentially expressed genes revealed that these two subtypes had distinct patterns of immune responses, including osteoclast differentiation and interleukin-10 signaling pathway. We developed two immune gene signatures that can differentiate these two BC PBMC subtypes. Further analysis suggested they had the ability to predict the clinical outcome of BC patients.Conclusions: PBMC transcriptome profiles can classify BC patients into two distinct subtypes. These two subtypes are mainly shaped by different immune cell abundance, which may have implications on clinical outcomes.

P5505Inflammation implicated in the aetiology of major vascular and non-vascular diseases in East Asians

Abstract Background Mendelian randomisation (MR) studies using genetic variants in the IL6R gene encoding the interleukin-6 (IL-6) receptor have demonstrated that IL-6 plays a causal role in the aetiology of coronary heart disease (CHD) in European populations, with implications for the development of drugs targeting inflammation-related pathways. It is less clear whether IL-6 signalling plays a causal role in vascular disease or major non-vascular diseases in East Asians. Purpose Using an MR approach, we investigated associations of altered IL-6 signalling with subtypes of CHD, stroke, cancer and respiratory disease in a large East Asian cohort. Methods In approximately 150,000 Chinese adults from the China Kadoorie Biobank, we investigated associations of rs7529229 (in strong linkage disequilibrium, r2=0.99, with the IL6R Asp358Ala variant rs2228145) with blood biomarkers and selected disease events in which inflammation has previously been implicated. First, we used linear regression to quantify the per-allele association of rs7529229 with levels in plasma of log-transformed C-reactive protein (CRP) (n=17,866), fibrinogen (n=9,255), and IL-6 protein (n=633). Second, we used logistic regression to evaluate the association of rs7529229 with incidence of CHD, stroke, cancer, and respiratory disease events. All models were adjusted for age, age-squared, sex (except breast cancer), and case ascertainment (for CRP and fibrinogen), and stratified by recruitment region. We assessed significance at a 5% false discovery rate. Results IL6R rs7529229 C-allele was associated with lower log CRP (–0.11 SDs per C-allele; p=4.9x10–25) and log fibrinogen (–0.07 SDs; p=2.2x10–7), and higher log IL-6 (0.15 SDs; p=0.011) (Figure 1), mimicking therapeutic blockade of IL6R. IL6R rs7529229 was associated with a lower risk of acute myocardial infarction (n=4,047 cases; OR: 0.92 [95% CI 0.88–0.96] per C-allele; p=2.8x10–4), with the association similar for fatal and non-fatal cases (Figure 2). There was no evidence of association with ischaemic stroke (n=18,315; OR: 1.00 [0.98–1.03]; p=0.90) or intracerebral haemorrhage (n=7,372; OR: 1.03 [0.99–1.07]; p=0.10). For non-cardiovascular diseases, the IL6R rs7529229 was associated with a lower risk of oesophageal (n=824; OR: 0.88 [0.79–0.97] per C-allele; p=0.013) and colorectal (n=1,151; OR: 0.89 [0.82–0.97]; p=8.3x10–3) cancers, but a higher risk of tuberculosis (n=1,017; OR: 1.15 [1.05–1.26]; p=2.4x10–3). Conclusion The results of the present study are consistent with a causal role for the IL-6 signalling pathway in the aetiology of myocardial infarction and some cancers, but not of stroke. These findings provide further genetic support for drug development targeting inflammation in the prevention and treatment of coronary and selected cancer outcomes. Acknowledgement/Funding Kadoorie Charitable Foundation, UK Wellcome Trust, Chinese Ministry of Science and Technology, BHF, CRUK, NIHR

SPOP inhibits mice pancreatic stellate cell activation by promoting FADD degradation in cerulein-induced chronic pancreatitis

Pancreatic stellate cells (PSCs) have been recognized as key mediators of pancreatic fibrosis, a characteristic feature of chronic pancreatitis (CP). As a cullin-based E3 ubiquitin ligase, speckle-type POZ protein (SPOP) has been identified to participate in tumorigenesis and organ development. However, its biological role in CP remains unknown. Therefore, this study sought to investigate the changed expression of SPOP in CP and to examine the effect on mice PSCs activation of SPOP. We found that SPOP was downregulated in the pancreatic tissues of cerulein-induced CP mice. siRNA-mediated knockdown of SPOP led to significant promotion in primary PSCs activity by activating the nuclear factor-kappaB (NF-κB)/interleukin-6 (IL-6) signaling pathway. In addition, we examined the effects of Fas-associated death domain (FADD), a proven SPOP substrate that activates NF-κB, on the regulation of PSCs activation. We found that FADD was downregulated by SPOP via interaction-mediated degradation, and was upregulated during PSCs activation. The promotion of PSCs activation in knocking down SPOP with siSPOP-1 were counteracted by knocking down FADD. The results suggest that the SPOP-induced inhibition of PSCs activation partially depended on FADD. These results highlight the importance of SPOP in CP and provide a potential target for therapeutic intervention.

Gene Regulatory Networks in Peripheral Mononuclear Cells Reveals Critical Regulatory Modules and Regulators of Multiple Sclerosis

Multiple sclerosis (MS) is a complex, demyelinating disease with the involvement of autoimmunity and neurodegeneration. Increasing efforts have been made towards identifying the diagnostic markers to differentiate the classes of MS from other similar neurological conditions. Using a systems biology approach, we constructed four types of gene regulatory networks (GRNs) involved in peripheral blood mononuclear cells (PBMCs). The regulatory strength of each GRN across primary progressive MS (PPMS), relapsing-remitting MS (RRMS), secondary progressive MS (SPMS), and control were evaluated by an integrity algorithm. Among the constructed GRNs (referred as TF_gene_miRNA), POU3F2_CDK6_hsa-miR-590-3p, MEIS1_CASC3_hsa-miR-1261, STAT3_OGG1_hsa-miR-298, and TCF4_FMR1_hsa-miR-301b were top-ranked and differentially regulated in all classes of MS compared to control. These GRNs showed potential involvement in regulating various molecular pathways such as interleukin, integrin, glypican, sphingosine phosphate, androgen, and Wnt signaling pathways. For validation, the qPCR analysis of the GRN components (TFs, gene, and miRNAs) in PBMCs of healthy controls (n = 30), RRMS (n = 14), PPMS (n = 13) and SPMS (n = 12) were carried out. Real-time expression analysis of GRNs showed a similar regulatory pattern as derived from our systems biology approach. Also, our study provided several novel GRNs that regulate unique and common molecular mechanisms between MS conditions. Hence, these regulatory components of GRNs will help to understand the disease mechanism across MS classes and further insight may though light towards diagnosis.

Autocrine activin A signalling in ovarian cancer cells regulates secretion of interleukin 6, autophagy, and cachexia.

BackgroundThe majority of patients with advanced cancer develop cachexia, a weight loss syndrome that severely reduces quality of life and limits survival. Our understanding of the underlying mechanisms that cause the condition is limited, and there are currently no treatment options that can completely reverse cachexia. Several tumour‐derived factors and inflammatory mediators have been suggested to contribute to weight loss in cachectic patients. However, inconsistencies between studies are recurrent. Activin A and interleukin 6 (IL‐6) are among the best studied factors that seem to be important, and several studies support their individual role in cachexia development.MethodsWe investigated the interplay between activin A and IL‐6 in the cachexia‐inducing TOV21G cell line, both in culture and in tumours in mice. We previously found that the human TOV21G cells secrete IL‐6 that induces autophagy in reporter cells and cachexia in mice. Using this established cachexia cell model, we targeted autocrine activin A by genetic, chemical, and biological approaches. The secretion of IL‐6 from the cancer cells was determined in both culture and tumour‐bearing mice by a species‐specific ELISA. Autophagy reporter cells were used to monitor the culture medium for autophagy‐inducing activities, and muscle mass changes were evaluated in tumour‐bearing mice.ResultsWe show that activin A acts in an autocrine manner to promote the synthesis and secretion of IL‐6 from cancer cells. By inhibiting activin A signalling, the production of IL‐6 from the cancer cells is reduced by 40–50% (up to 42% reduction on protein level, P = 0.0048, and 48% reduction on mRNA level, P = 0.0308). Significantly reduced IL‐6 secretion (P < 0.05) from the cancer cells is consistently observed when using biological, chemical, and genetic approaches to interfere with the autocrine activin A loop. Inhibiting activin signalling also reduces the ability of the cancer cells to accelerate autophagy in non‐cancerous cells (up to 43% reduced autophagy flux, P = 0.0006). Coherent to the in vitro data, the use of an anti‐activin receptor 2 antibody in cachectic tumour‐bearing mice reduces serum levels of cancer cell‐derived IL‐6 by 62% (from 417 to 159 pg/mL, P = 0.03), and, importantly, it reverses cachexia and counteracts loss of all measured muscle groups (P < 0.0005).ConclusionsOur data support a functional link between activin A and IL‐6 signalling pathways and indicate that interference with activin A‐induced IL‐6 secretion from the tumour has therapeutic potential for cancer‐induced cachexia.

T-cell acute lymphoblastic leukemia displays autocrine production of Interleukin-7

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by an accumulation of immature T cells. Although patient outcomes have improved, novel targeted therapies are needed to reduce the intensity of chemotherapy and improve the prognosis of high-risk patients. Interleukin-7 (IL-7) modulates the survival and proliferation of normal and malignant T cells. Targeting the IL-7 signaling pathway is thus a potentially effective therapeutic strategy. To achieve such aim, it is essential to first understand how the IL-7 signaling pathway is activated. Although IL-7 production has been observed from multiple stromal tissues, T-ALL autocrine IL-7 secretion has not yet been described. Interestingly, using T-ALL cell lines, primary and patient-derived xenotransplanted (PDX) T-ALL cells, we demonstrate that T-ALL cells produce IL-7 whereas normal T cells do not. Finally, using knock down of IL7 gene in T-ALL cells, we describe to what extent IL-7 autocrine secretion is involved in the T-ALL cells propagation in bone marrow and how it affects the number of leukemia-initiating cells in PDX mice. Together, these results demonstrate how the autocrine production of the IL-7 cytokine mediated by T-ALL cells can be involved in the oncogenic development of T-ALL and offer novel insights into T-ALL spreading.

Interleukin-6 signaling pathway involved in major depressive disorder: selective serotonin reuptake inhibitor regulates IL-6 pathway

Abstract Background Evidence indicates that pro-inflammatory Th17 and Th1 cells are involved in major depressive disorder (MDD) pathogenesis. Development of Th17 and Th1 are regulated by IL-6 and IFN-γ, respectively. In this study, the levels of IL-6 and IFN-γ, and mRNA expression of related signaling components and, Th17/Th1 transcription factors were investigated in MDD patients with/without selective serotonin reuptake inhibitor (SSRI) medication. Materials and methods Forty-six patients and 38 healthy controls (HCs) were recruited. Twenty patients were received the SSRI (sertraline 50–200 mg/day) for at least 1 year, and 26 patients were not received medication. Expression of IL-6R, IFN-γR, JAK1, JAK2, TYK2, STAT1, STAT3, T-bet and RORγt were assessed with Real-Time-PCR. Serum and supernatant levels of IL-6 and IFN-γ were determined using ELISA. Results and discussion The serum and supernatant levels of IL-6 were increased in patients without (SSRI−) compared with HCs, while its levels was reduced in SSRI+. Elevated expressions of IL-6R, STAT3 and RORγt were observed in SSRI− compared with HCs. Expressions of IL-6R, STAT3, RORγt and IFN-γR, were decreased in SSRI+ compared to SSRI− patients. Conclusion Increased IL-6 involved in MDD, and SSRI regulates IL-6 pathway and IL-6 production. MDD patients may benefit from IL-6/IL-6R targeted therapeutic intervention.

A directly GP130-targeting small molecule ameliorates collagen-induced arthrites by inhibiting IL-6/GP130 signalling and Th17 differentiation

Abstract Rheumatoid arthritis is a chronic inflammatory disease that is characterized by joint destruction and inflammation by T helper 17 (Th17) cells. Interleukin-6 (IL-6) is secreted in cells such as macrophage or synovial fibroblast and induces the differentiation and function of Th17 cells that increase lymphocytic infiltration in the joint. LMT-28 suppresses IL-6 signaling through direct binding to glycoprotein-130, and alleviates inflammatory disease (such as rheumatoid arthritis, inflammatory bowel disease) in previous study. The purpose of this study is to assess whether LMT-28 would potently inhibit Th17 differentiation, and the mechanism of LMT-28 that attenuates rheumatoid arthritis through IL-6 signaling pathway. LMT-28 reduced the arthritis score and showed protective effects against bone and cartilage destruction in collagen induced arthritis (CIA) mice. In mice with CIA, LMT-28 markedly decreased the serum levels of IL-6, TNF, and IL-1β compared with each vehicle treated group. Moreover, LMT-28 attenuated Th17 cell activation in lymph nodes of CIA mice. We demonstrated that LMT-28 suppressed differentiation of Th17 in mouse splenocytes and human PBMCs. Additionally, LMT-28 inhibited the phosphorylation of GP130, STAT3, and ERK induced by Hyper IL-6 in human fibroblast-like synoviocytes(FLS). Collectively, these results suggested that LMT-28 inhibits differentiated/activated-Th17 cells in rheumatoid arthritis by blocking activation of the STAT3 pathway.

Meet the First Authors.

Meet the First Authors.

Interleukin 6 signaling maintains the stem-like properties of bladder cancer stem cells.

BackgroundThe relapse and metastasis of bladder cancer are due to its strong resistance to chemotherapeutic drugs after surgery as a result of the expansion and self-renewal of cancer stem cells (CSCs). However, the molecular mechanisms underlying the biology of bladder CSCs are unknown. This study aimed to investigate the role of interleukin 6 (IL6)/IL6 receptor (IL6R) in the stem-like characteristics of bladder CSCs.MethodsEnzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect IL6 expression in the supernatant and cells of bladder CSCs, respectively. Following that, self-renewal, stem cell-associated gene expression, invasion, metastasis, and tumorigenicity were assessed by sphere-forming assay, qRT-PCR, invasion and transwell assays, and tumor-forming experiment in NOD/SCID mice, respectively. Finally, Western blot and qRT-PCR were employed to examine the IL6 signaling pathway in regulating the stem-like properties of bladder CSCs.ResultsThe spheres, originating from the bladder cancer cell lines RT4 and J82, possessed a higher expression of stem-associated genes. The expression levels of IL6 were elevated in the supernatant and cells of the bladder CSCs. IL6R was also up-regulated in the bladder CSCs. Recombinant IL6 promoted the stem-like properties of the bladder CSCs, including self-renewal, expression of stem-associated genes, invasion, migration, and tumorigenicity. Mechanistically, IL6 exerted its biological effects by binding to IL6R, which enhanced the phosphorylation of STAT3 and triggered its activation. Furthermore, these effects were alleviated by the FDA-approved drug tocilizumab.ConclusionsOur findings demonstrate that IL6/IL6R/STAT3 maintains the stem-like properties of bladder CSCs. Furthermore, IL6R may serve as a potential therapeutic target for CSCs in bladder cancer.

Interleukin 6 signaling maintains the stem-like properties of bladder cancer stem cells

Background: The relapse and metastasis of bladder cancer are due to its strong resistance to chemotherapeutic drugs after surgery as a result of the expansion and self-renewal of cancer stem cells (CSCs). However, the molecular mechanisms underlying the biology of bladder CSCs are unknown. This study aimed to investigate the role of interleukin 6 (IL6)/IL6 receptor (IL6R) in the stem-like characteristics of bladder CSCs. Methods: Enzyme-linked immunosorbent assay (ELISA) and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect IL6 expression in the supernatant and cells of bladder CSCs, respectively. Following that, self-renewal, stem cell-associated gene expression, invasion, metastasis, and tumorigenicity were assessed by sphere-forming assay, qRT-PCR, invasion and transwell assays, and tumor-forming experiment in NOD/SCID mice, respectively. Finally, Western blot and qRT-PCR were employed to examine the IL6 signaling pathway in regulating the stem-like properties of bladder CSCs. Results: The spheres, originating from the bladder cancer cell lines RT4 and J82, possessed a higher expression of stem-associated genes. The expression levels of IL6 were elevated in the supernatant and cells of the bladder CSCs. IL6R was also up-regulated in the bladder CSCs. Recombinant IL6 promoted the stem-like properties of the bladder CSCs, including self-renewal, expression of stem-associated genes, invasion, migration, and tumorigenicity. Mechanistically, IL6 exerted its biological effects by binding to IL6R, which enhanced the phosphorylation of STAT3 and triggered its activation. Furthermore, these effects were alleviated by the FDA-approved drug tocilizumab. Conclusions: Our findings demonstrate that IL6/IL6R/STAT3 maintains the stem-like properties of bladder CSCs. Furthermore, IL6R may serve as a potential therapeutic target for CSCs in bladder cancer.

GNAI1 and GNAI3 Reduce Colitis-Associated Tumorigenesis in Mice by Blocking IL6 Signaling and Down-regulating Expression of GNAI2

Interleukin 6 (IL6) and tumor necrosis factor contribute to the development of colitis-associated cancer (CAC). We investigated these signaling pathways and the involvement of G protein subunit alpha i1 (GNAI1), GNAI2, and GNAI3 in the development of CAC in mice and humans. B6;129 wild-type (control) or mice with disruption of Gnai1, Gnai2, and/or Gnai3 or conditional disruption of Gnai2 in CD11c+ or epithelial cells were given dextran sulfate sodium (DSS) to induce colitis followed by azoxymethane (AOM) to induce carcinogenesis; some mice were given an antibody against IL6. Feces were collected from mice, and the compositions of microbiomes were analyzed by polymerase chain reactions. Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) isolated from spleen and colon tissues were analyzed by flow cytometry. We performed immunoprecipitation and immunoblot analyses of colon tumor tissues, MDSCs, and mouse embryonic fibroblasts to study the expression levels of GNAI1, GNAI2, and GNAI3 and the interactions of GNAI1 and GNAI3 with proteins in the IL6 signaling pathway. We analyzed the expression of Gnai2 messenger RNA by CD11c+ cells in the colonic lamina propria by PrimeFlow, expression of IL6 in DCs by flow cytometry, and secretion of cytokines in sera and colon tissues by enzyme-linked immunosorbent assay. We obtained colon tumor and matched nontumor tissues from 83 patients with colorectal cancer having surgery in China and 35 patients with CAC in the United States. Mouse and human colon tissues were analyzed by histology, immunoblot, immunohistochemistry, and/or RNA-sequencing analyses. GNAI1 and GNAI3 (GNAI1;3) double-knockout (DKO) mice developed more severe colitis after administration of DSS and significantly more colonic tumors than control mice after administration of AOM plus DSS. Development of increased tumors in DKO mice was not associated with changes in fecal microbiomes but was associated with activation of nuclear factor (NF) κB and signal transducer and activator of transcription (STAT) 3; increased levels of GNAI2, nitric oxide synthase 2, and IL6; increased numbers of CD4+ DCs and MDSCs; and decreased numbers of CD8+ DCs. IL6 was mainly produced by CD4+/CD11b+, but not CD8+, DCs in DKO mice. Injection of DKO mice with a blocking antibody against IL6 reduced the expansion of MDSCs and the number of tumors that developed after CAC induction. Incubation of MDSCs or mouse embryonic fibroblasts with IL6 induced activation of either NF-κB by a JAK2-TRAF6-TAK1-CHUK/IKKB signaling pathway or STAT3 by JAK2. This activation resulted in expression of GNAI2, IL6 signal transducer (IL6ST, also called GP130) and nitric oxide synthase 2, and expansion of MDSCs; the expression levels of these proteins and expansion of MDSCs were further increased by the absence of GNAI1;3 in cells and mice. Conditional disruption of Gnai2 in CD11c+ cells of DKO mice prevented activation of NF-κB and STAT3 and changes in numbers of DCs and MDSCs.Colon tumor tissues from patients with CAC had reduced levels of GNAI1 and GNAI3 and increased levels of GNAI2 compared with normal tissues. Further analysis of a public human colorectal tumor DNA microarray database (GSE39582) showed that low Gani1 and Gnai3 messenger RNA expression and high Gnai2 messenger RNA expression were significantly associated with decreased relapse-free survival. GNAI1;3 suppresses DSS-plus-AOM-induced colon tumor development in mice, whereas expression of GNAI2 in CD11c+ cells and IL6 in CD4+/CD11b+ DCs appears to promote these effects. Strategies to induce GNAI1;3, or block GNAI2 and IL6, might be developed for the prevention or therapy of CAC in patients.

Targeting of IL-6-Relevant Long Noncoding RNA Profiles in Inflammatory and Tumorous Disease.

Interleukin-6 (IL-6) is a critical cytokine with a diverse repertoire of physiological functions. Dysregulation of IL-6 signaling is associated with inflammatory disorders as well as cancers. However, blockade of IL-6 activity via antibodies directed against the IL-6 signaling pathway may compromise the efficacy of the immune system; therefore, patients may not have a uniformly satisfactory response to treatment. Long noncoding RNAs (lncRNAs) have been discovered to be evolutionary conserved transcripts of noncoding DNA sequences and have emerged as biomarkers with great predictive and prognostic value, further employed as a targeted anticancer therapy. LncRNAs have been recently implicated in the regulation of IL-6-related signaling and function; they are tightly linked to the development of a range of IL-6 dysregulated diseases. Here, we will highlight those lncRNAs involved in IL-6 signaling, with an emphasis on the mechanisms of lncRNAs that interact with IL-6. Targeting of such lncRNAs related to IL-6 regulation could be, in the near future, a promising therapeutic strategy in the treatment of inflammatory- and tumor-related diseases.

Endonuclease and redox activities of human apurinic/apyrimidinic endonuclease 1 have distinctive and essential functions in IgA class switch recombination

The base excision repair (BER) pathway is an important DNA repair pathway and is essential for immune responses. In fact, it regulates both the antigen-stimulated somatic hypermutation (SHM) process and plays a central function in the process of class switch recombination (CSR). For both processes, a central role for apurinic/apyrimidinic endonuclease 1 (APE1) has been demonstrated. APE1 acts also as a master regulator of gene expression through its redox activity. APE1's redox activity stimulates the DNA-binding activity of several transcription factors, including NF-κB and a few others involved in inflammation and in immune responses. Therefore, it is possible that APE1 has a role in regulating the CSR through its function as a redox coactivator. The present study was undertaken to address this question. Using the CSR-competent mouse B-cell line CH12F3 and a combination of specific inhibitors of APE1's redox (APX3330) and repair (compound 3) activities, APE1-deficient or -reconstituted cell lines expressing redox-deficient or endonuclease-deficient proteins, and APX3330-treated mice, we determined the contributions of both endonuclease and redox functions of APE1 in CSR. We found that APE1's endonuclease activity is essential for IgA-class switch recombination. We provide evidence that the redox function of APE1 appears to play a role in regulating CSR through the interleukin-6 signaling pathway and in proper IgA expression. Our results shed light on APE1's redox function in the control of cancer growth through modulation of the IgA CSR process.

Long noncoding RNA expression profile from cryptococcal meningitis patients identifies DPY19L1p1 as a new disease marker.

SummaryAimsLncRNAs play a vital role in the pathological and physiological process. This study aimed to explore the involvement of lncRNAs in cryptococcal meningitis.MethodsMicroarray was performed in cryptococcal meningitis patients, and then, GO and KEGG pathways were analyzed. Coexpression relationship between lncRNA and mRNA was explored. The expressions of the lncRNAs and mRNAs, and their changes after treatment were detected by PCR.ResultsA total of 325 mRNAs (201 upregulated and 124 downregulated) and 497 lncRNAs (263 upregulated and 234 downregulated) were identified. The top three enriched GO terms for the mRNAs were arachidonic acid binding, activin receptor binding, and replication fork protection complex. The top three pathways in KEGG were asthma, one carbon pool by folate, and allograft rejection. A total of 305 coexpression relationships were found between 108 lncRNAs and 87 mRNAs. LncRNA‐DPY19L1p1 was significantly increased in patients and decreased after treatment. ROC analysis revealed DPY19L1p1 was a potential diagnostic marker (AUCROC = 0.9389). Furthermore, the target genes of DPY19L1p1 in cis or trans regulation were mainly involved in immune‐related pathways like the interleukin signaling pathway.ConclusionsThis study analyzed the differential lncRNA profile in cryptococcal meningitis patients and revealed DPY19L1p1 could be used for treatment evaluation and disease diagnosis.